SMART MODE:

NORMAL GENOMIC

• van Gestel J, Vlamakis H, Kolter R
• New tools for comparing microscopy images: quantitative analysis of cell types in Bacillus subtilis.
• J Bacteriol. 2015; 197: 699-709
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• Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy images. We show how image data can be converted to distribution data. These data can be subjected to a cluster analysis that makes it possible to objectively compare microscopy images. The distribution data can further be analyzed using distribution fitting. We illustrate our methods by scrutinizing two independently acquired data sets, each containing microscopy images of a doubly labeled Bacillus subtilis strain. For the first data set, we examined the expression of srfA and tapA, two genes which are expressed in surfactin-producing and matrix-producing cells, respectively. For the second data set, we examined the expression of eps and tapA; these genes are expressed in matrix-producing cells. We show that srfA is expressed by all cells in the population, a finding which contrasts with a previously reported bimodal distribution of srfA expression. In addition, we show that eps and tapA do not always have the same expression profiles, despite being expressed in the same cell type: both operons are expressed in cell chains, while single cells mainly express eps. These findings exemplify that the quantification and comparison of microscopy data can yield insights that otherwise would go unnoticed.

• Okada M
• Post-translational isoprenylation of tryptophan.
• Biosci Biotechnol Biochem. 2011; 75: 1413-7
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• Bacillus subtilis and related bacilli produce a post-translationally modified oligopeptide, ComX pheromone, that stimulates natural genetic competence controlled by quorum sensing. The ComX pheromones are formed by geranylation or farnesylation on a tryptophan residue at the 3 position of its indole ring. This results in the formation of a tricyclic structure including, a newly formed five-membered ring, similar to proline. Isoprenylation of ComX to form ComX pheromones is essential for pheromonal activity, and is functionally more crucial than its amino acid sequence. The ComX pheromone is the first example of isoprenoidal modifiations of tryptophan residues in living organisms and post-translational isoprenylation of any amino acid in prokaryotes. Because the presence of geranylated compounds is unusual in primary and secondary metabolites outside the plant kingdom, post-translational geranylation in bacilli is unprecedented in nature.

• Wang YK et al.
• Site-directed mutations of thermostable direct hemolysin from Grimontia hollisae alter its arrhenius effect and biophysical properties.
• Int J Biol Sci. 2011; 7: 333-46
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• Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 degrees C but is reactivated by additional heating above 80 degrees C. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDH(WT)) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDH(Y53H/T59I) and Gh-rTDH(T59I/S63T) double-mutants and the Gh-rTDH(Y53H/T59I/S63T) triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDH(WT). Circular dichroism measurements of Gh-rTDH(WT) and Gh-rTDH(mut) showed a conspicuous change from a beta-sheet to alpha-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy.

• Zaghloul TI, Embaby AM, Elmahdy AR
• Key determinants affecting sheep wool biodegradation directed by a keratinase-producing Bacillus subtilis recombinant strain.
• Biodegradation. 2011; 22: 111-28
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• OVAT (one variable at a time) approach was applied in this study to screen the most important physicochemical key determinants involved in the process of sheep wool biodegradation. The process was directed by a keratinase-producing Bacillus subtilis DB 100 (p5.2) recombinant strain. Data indicate that, sheep wool could be degraded efficiently in cultures incubated at 30 degrees C, with initial pH of 7 with agitation at 150 rpm. Two times autoclaved alkali treated and undefatted chopped sheep wool is more accessible to biodegradation. B. subtilis recombinant cells could utilize sheep wool as a sole source of carbon and nitrogen. Sheep wool-based modified basal medium II, lacking NH(4)Cl and yeast extract, could greatly support the growth of these bacterial cells. Sheep wool biodegradation was conducted efficiently in the absence of kanamycin consequently; high stability of the recombinant plasmid (p5.2) represents a great challenge upon scaling up this process. Three key determinants (sheep wool concentration, incubation time and inoculum size) imposing considerable constraints on the process are highlighted. Sheep wool-based tap water medium and sheep wool-based distilled water medium were formulated in this study. High levels of released end products, produced from sheep wool biodegradation are achieved upon using these two sheep wool-based water media. Data indicate that, sheep wool hydrolysate is rich in some amino acids, such as tyrosine, phenylalanine, lysine, proline, isoleucine, leucine, valine, aspartic acid and glutamic acid. Moreover, the resulting sheep wool hydrolysate contains soluble proteins of high and intermediate molecular weights. The present study demonstrates a feasible, cheap, reproducible, efficient and rapid biotechnological approach towards utilization of raw sheep wool waste through a recombinant bacterium.

• Yssel A, Reva O, Tastan Bishop O
• Comparative structural bioinformatics analysis of Bacillus amyloliquefaciens chemotaxis proteins within Bacillus subtilis group.
• Appl Microbiol Biotechnol. 2011; 92: 997-1008
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• Chemotaxis is a process in which bacteria sense their chemical environment and move towards more favorable conditions. Since plant colonization by bacteria is a multifaceted process which requires a response to the complex chemical environment, a finely tuned and sensitive chemotaxis system is needed. Members of the Bacillus subtilis group including Bacillus amyloliquefaciens are industrially important, for example, as bio-pesticides. The group exhibits plant growth-promoting characteristics, with different specificity towards certain host plants. Therefore, we hypothesize that while the principal molecular mechanisms of bacterial chemotaxis may be conserved, the bacterial chemotaxis system may need an evolutionary tweaking to adapt it to specific requirements, particularly in the process of evolution of free-living soil organisms, towards plant colonization behaviour. To date, almost nothing is known about what parts of the chemotaxis proteins are subjected to positive amino acid substitutions, involved in adjusting the chemotaxis system of bacteria during speciation. In this novel study, positively selected and purified sites of chemotaxis proteins were calculated, and these residues were mapped onto homology models that were built for the chemotaxis proteins, in an attempt to understand the spatial evolution of the chemotaxis proteins. Various positively selected amino acids were identified in semi-conserved regions of the proteins away from the known active sites.

• Jost BH, Lucas EA, Billington SJ, Ratner AJ, McGee DJ
• Arcanolysin is a cholesterol-dependent cytolysin of the human pathogen Arcanobacterium haemolyticum.
• BMC Microbiol. 2011; 11: 239-239
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• BACKGROUND: Arcanobacterium haemolyticum is an emerging human pathogen that causes pharyngitis, wound infections, and a variety of occasional invasive diseases. Since its initial discovery in 1946, this Gram positive organism has been known to have hemolytic activity, yet no hemolysin has been previously reported. A. haemolyticum also displays variable hemolytic activity on laboratory blood agar that is dependent upon which species the blood is derived. RESULTS: Here we describe a cholesterol-dependent cytolysin (CDC) secreted by A. haemolyticum, designated arcanolysin (aln), which is present in all strains (n = 52) tested by DNA dot hybridization. Among the known CDCs, ALN is most closely related to pyolysin (PLO) from Trueperella (formerly Arcanobacterium) pyogenes. The aln probe, however, did not hybridize to DNA from T. pyogenes. The aln open reading frame has a lower mol %G+C (46.7%) than the rest of the A. haemolyticum genome (53.1%) and is flanked by two tRNA genes, consistent with probable acquisition by horizontal transfer. The ALN protein (~ 64 kDa) contains a predicted signal sequence, a putative PEST sequence, and a variant undecapeptide within domain 4, which is typically important for function of the toxins. The gene encoding ALN was cloned and expressed in Escherichia coli as a functional recombinant toxin. Recombinant ALN had hemolytic activity on erythrocytes and cytolytic activity on cultured cells from human, rabbit, pig and horse origins but was poorly active on ovine, bovine, murine, and canine cells. ALN was less sensitive to inhibition by free cholesterol than perfringolysin O, consistent with the presence of the variant undecapeptide. CONCLUSIONS: ALN is a newly identified CDC with hemolytic activity and unique properties in the CDC family and may be a virulence determinant for A. haemolyticum.

• Murphy TM, Nilsson AY, Roy I, Harrop A, Dixon K, Keshavarz T
• Enhanced intracellular Ca2+ concentrations in Escherichia coli and Bacillus subtilis after addition of oligosaccharide elicitors.
• Biotechnol Lett. 2011; 33: 985-91
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• Elicitation can lead to overproduction of secondary metabolites in plants and microbes. Potential changes in cytosolic Ca(2+) levels in bacteria were studied in response to elicitation. We report, for the first time, the effect of oligosaccharide elicitors on intracellular Ca(2+) levels. The apoaequorin gene was cloned into Escherichia coli DH5alpha and Bacillus subtilis 1604 cultures. Addition of elicitors, oligoguluronate and mannan oligosaccharides, to the cultures caused up to 11-fold increase in cytosolic Ca(2+) in E. coli and tenfold increase in B. subtilis. These increases in Ca(2+) levels could therefore contribute to the enhancement of secondary metabolite levels.

• Takenaka S, Sato T, Koshiya J, Murakami S, Aoki K
• Gene cloning and characterization of a deaminase from the 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d.
• FEMS Microbiol Lett. 2009; 298: 93-8
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• The 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d produces a deaminase that catalyzes the deamination of 2-amino-5-carboxymuconic 6-semialdehyde. A gene encoding the deaminase, ahdB, was cloned and expressed in Escherichia coli; ahdB is located downstream from the previously reported genes encoding 4-amino-3-hydroxybenzoate 2,3-dioxygenase (ahdA) and a LysR-type regulator. The deduced amino acid sequence of ahdB shows 30-33% identity to those of previously reported 2-aminomuconate deaminases. We identified a region (RAGDFLXVSG) conserved in AhdB and three other deaminases. The recombinant enzyme AhdB was purified to homogeneity. After a coupled enzyme assay with purified AhdA, AhdB, and the substrate 4-amino-3-hydroxybenzoate, the final product, formed by the action of AhdA, AhdB, and by nonenzymatic decarboxylation, was identified by HPLC, MS, and (1)H-nuclear magnetic resonance analyses as 2-hydroxymuconic 6-semialdehyde.

• Kapralou S, Fabbretti A, Garulli C, Gualerzi CO, Pon CL, Spurio R
• Characterization of Bacillus stearothermophilus infA and of its product IF1.
• Gene. 2009; 428: 31-5
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• Bacillus stearothermophilus infA encoding translation initiation factor IF1 was cloned and expressed in Escherichia coli and its transcript and protein product characterized. Although the functional properties of B. stearothermophilus and E. coli IF1, compared in several translational tests in the presence of both homologous and heterologous components, are not entirely identical, the two proteins are interchangeable in an in vitro translational system programmed with a natural mRNA. The availability of purified B. stearothermophilus IF1 now allows us to analyze the translation initiation pathway using efficient in vitro tests based entirely on purified components derived from this thermophilic Gram-positive bacterium.

• Obata F, Kitami M, Inoue Y, Atsumi S, Yoshizawa Y, Sato R
• Analysis of the region for receptor binding and triggering of oligomerization on Bacillus thuringiensis Cry1Aa toxin.
• FEBS J. 2009; 276: 5949-59
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• The determination of the receptor-binding region of Cry toxins produced by Bacillus thuringiensis is expected to facilitate an improvement in their insecticidal ability through protein engineering. We analyzed the region on Cry1Aa molecules involved in interactions with the cadherin-like protein receptor BtR175 using cysteine-substituted mutant toxins and several synthetic peptides corresponding to the loops in domain 2. In addition, the region necessary to trigger oligomerization was analyzed using these mutant toxins. The mutant toxins were modified by two types of molecule, i.e. digested fragments of the Cry1Aa precursor with an average molecular mass of 2 kDa and 5-iodoacetamidofluorescein, which has a molecular mass of 515 kDa. We examined whether these modifications interfere with the toxin-BtR175 interaction as a result of steric hindrance. 5-Iodoacetamidofluorescein modification of R311C, N376C and G442C revealed steric hindrance effects, indicating that R311 on loop 1, N376 on loop 2 and G442 on loop 3 are on the contact face of the toxin-BtR175 interface when Cry1Aa binds to BtR175. Loop 2 is thought to interact with BtR175 directly, as a peptide corresponding to the N-terminal half of loop 2, (365)LYRRIILG(372), has the potential to bind to BtR175 fragments. Meanwhile, mutant toxins with cysteine substitutions in loops 1 and 2 were oligomerized by the binding of digested fragments in the activation process without receptor interaction, and the wild-type toxin formed oligomers by interaction with BtR175 fragments. These observations suggest that loops 1 and 2 form both a binding region and a sensor region, which triggers toxin oligomer formation. Structured digital abstract: * MINT-7259673, MINT-7259722, MINT-7259737, MINT-7259757, MINT-7259774, MINT-7259791, MINT-7259808, MINT-7259685, MINT-7259707, MINT-7259830: btr175 (uniprotkb:Q9XY09) binds (MI:0407) to cry1Aa (uniprotkb:P0A366) by surface plasmon resonance (MI:0107).

• Sena JA, Hernandez-Rodriguez CS, Ferre J
• Interaction of Bacillus thuringiensis Cry1 and Vip3A proteins with Spodoptera frugiperda midgut binding sites.
• Appl Environ Microbiol. 2009; 75: 2236-7
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• Vip3Aa, Vip3Af, Cry1Ab, and Cry1Fa were tested for their toxicities and binding interactions. Vip3A proteins were more toxic than Cry1 proteins. Binding assays showed independent specific binding sites for Cry1 and Vip3A proteins. Cry1Ab and Cry1Fa competed for the same binding sites, whereas Vip3Aa competed for those of Vip3Af.

• Lin L et al.
• Improved catalytic efficiency of endo-beta-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution.
• Appl Microbiol Biotechnol. 2009; 82: 671-9
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• Bacillus subtilis endo-beta-1,4-glucanase (Cel5A) hydrolyzes cellulose by cleavage of the internal bonds in the glucose chains, producing new ends randomly. Using directed evolution techniques of error-prone polymerase chain reaction (PCR) and DNA shuffling, several Cel5A variants with improved catalytic activity had been screened from the mutant library, which contained 71,000 colonies. Compared with the wild-type enzyme, the variants (M44-11, S75 and S78) showed 2.03 to 2.68-fold increased activities toward sodium carboxymethyl cellulose (CMC), while the M44-11 also exhibited a wider pH tolerance and higher thermostability. Structural models of M44-11, S75, S78, and WT proteins revealed that most of the substitutions were not located in the strictly conserved regions, except the mutation V255A of S75, which was closed to the nucleophile Glu257 in the catalytic center of the enzyme. Moreover, V74A and D272G of M44-11, which were not located in the substrate binding sites and the catalytic center, might result in improved stability and catalytic activity. These results provided useful references for directed evolution of the enzymes that belonged to the glycoside hydrolase family 5 (GH5).

• Val G, Marin S, Mellado RP
• A sensitive method to monitor Bacillus subtilis and Streptomyces coelicolor-related [corrected] bacteria in maize rhizobacterial communities: the use of genome-wide microarrays.
• Microb Ecol. 2009; 58: 108-15
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• Commercially available DNA microarrays containing genome-wide spotted oligonucleotides encompass the soil bacteria Bacillus subtilis or Streptomyces coelicolor genomes. These have been used to analyse potential differences in rhizobacterial communities of transgenic maize engineered to express the Bacillus thuringensis Cry toxin (Bt maize) in three different agricultural soils. No differences in hybridisation were observed between genetically and non-genetically modified maize rhizobacteria from two Bt lines with a detection sensitivity of five copies of a particular gene above the background. Soil-specific hybridisation results were obtained when rhizobacterial DNA was compared to the corresponding genomic DNA spotted in the microarrays suggesting that the use of genome-wide DNA arrays could serve as a useful tool for the molecular monitoring of rhizobacterial communities.

• Galopin S, Cattoir V, Leclercq R
• A chromosomal chloramphenicol acetyltransferase determinant from a probiotic strain of Bacillus clausii.
• FEMS Microbiol Lett. 2009; 296: 185-9
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• The mechanism of resistance to chloramphenicol was studied in four strains of Bacillus clausii included in a probiotic mixture, which is administered to humans for prevention of gastrointestinal side effects due to oral antibiotic therapy. By cloning experiments, a chloramphenicol acetyltransferase (CAT) gene, cat(Bcl), coding for a putative 228-amino acid CAT protein was identified in B. clausii SIN. The deduced amino acid sequence displayed from 31% to 85% identity with 56 CAT proteins from other Gram-positive bacterial strains. The cat(Bcl) gene was also detected by PCR in the three other B. clausii strains resistant to chloramphenicol, whereas it was absent in the three control strains susceptible to chloramphenicol. Pulse-field gel electrophoresis of total DNA digested by I-CeuI followed by hybridization with a cat-specific probe as well as unsuccessful repeated attempts of in vitro transfer of chloramphenicol resistance to various recipient cells indicated that cat(Bcl) was chromosomally located in all four resistant B. clausii strains.

• Leaver M, Dominguez-Cuevas P, Coxhead JM, Daniel RA, Errington J
• Life without a wall or division machine in Bacillus subtilis.
• Nature. 2009; 457: 849-53
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• The cell wall is an essential structure for virtually all bacteria, forming a tough outer shell that protects the cell from damage and osmotic lysis. It is the target of our best antibiotics. L-form strains are wall-deficient derivatives of common bacteria that have been studied for decades. However, they are difficult to generate and typically require growth for many generations on osmotically protective media with antibiotics or enzymes that kill walled forms. Despite their potential importance for understanding antibiotic resistance and pathogenesis, little is known about their basic cell biology or their means of propagation. We have developed a controllable system for generating L-forms in the highly tractable model bacterium Bacillus subtilis. Here, using genome sequencing, we identify a single point mutation that predisposes cells to grow without a wall. We show that propagation of L-forms does not require the normal FtsZ-dependent division machine but occurs by a remarkable extrusion-resolution mechanism. This novel form of propagation provides insights into how early forms of cellular life may have proliferated.

• Sanada H, Nakanishi T, Inoue H, Kitamura M
• Cloning and expression of the MutM gene from obligate anaerobic bacterium Desulfovibrio vulgaris (Miyazaki F).
• J Biochem. 2009; 145: 525-32
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• The gene encoding a MutM from Desulfovibrio vulgaris (Miyazaki F) was cloned and expressed in Escherichia coli. A 5.9-kb DNA fragment, isolated from D. vulgaris (Miyazaki F) by XhoI and PvuII, contained a MutM gene and other open reading frames. The nucleotide sequence of the MutM gene indicated that the protein was composed of 336 amino acids. The amino-acid sequence deduced from the MutM gene was highly homologous with the MutM of other bacteria; however an additional insert consisted of 64 amino acids. An expression system for the MutM gene under the control of the T7 promoter was constructed in E. coli. From the kinetic analysis results, the purified His-tagged MutM showed 8-oxoguanine-DNA glycosylase activity comparable with that of MutM from E. coli. In this study, the amounts of mRNA and protein for MutM were scant in the D. vulgaris (Miyazaki F). MutM activity may be induced by oxidative stress. However, its induction may not be frequently generated because sulfate-reducing bacteria generally grow in anaerobic conditions. MutM might play a role in the protection against the mutagenicity of oxygen when oxygen stress exceeded the capacity of the defense systems against oxygen toxicity.

• Hua G, Zhang R, Bayyareddy K, Adang MJ
• Anopheles gambiae alkaline phosphatase is a functional receptor of Bacillus thuringiensis jegathesan Cry11Ba toxin.
• Biochemistry. 2009; 48: 9785-93
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• Alkaline phosphatases (ALPs, EC 3.1.3.1) isolated from lepidopteran and dipteran species are identified as receptors for Cry1Ac and Cry11Aa toxins, respectively [Jurat-Fuentes, J. L., and Adang, M. J. (2004) Eur. J. Biochem. 7, 3127-3135; Fernandez, L. E., et al. (2006) Biochem. J. 396, 77-84]. In our study, an alkaline phosphatase cDNA (AgALP1) was cloned from the midgut of Anopheles gambiae larvae. The encoded 63 kDa protein has a predicted glycosylphosphatidylinositol (GPI) anchor omega-site ((526)Asp), an N-glycosylation site ((239)Asn-Leu-Thr), and an O-glycosylation site ((312)Ser). AgALP1(t) was expressed in Escherichia coli and used to prepare antiserum and to analyze the interaction of AgALP with mosquitocidal Cry11Ba toxin. Anti-AgALP serum localized AgALP to the apical brush border in the anterior and posterior midgut of larvae and detected a 65 kDa species on a blot of brush border membrane vesicles (BBMVs) protein prepared from larvae. ALP activity was released from larval BBMVs prepared by phosphatidylinositol-specific phospholipase C (PIPLC) treatment, and after separation by two-dimensional gel electrophoresis and blotting, a chain of doublet spots at 65 kDa was detected by anti-AgALP. A subset of these doublet spots bound Cry11Ba on a reprobed blot. Heterologously expressed AgALP1(t) bound [(125)I]Cry11Ba on dot blots and reduced the level of binding of [(125)I]Cry11Ba to brush border membrane vesicles by 41%, a percentage comparable to that of unlabeled Cry11Ba and aminopeptidase AgAPN2(t1) peptide. AgALP1(t) binds Cry11Ba toxin with a high affinity (23.9 nM) and shares a binding site on Cry11Ba with AgAPN2(t1). In bioassays against An. gambiae larvae, the presence of AgALP1(t) reduced larval mortality from 78 to 8%. We conclude that AgALP1 is a binding protein and a functional receptor for Cry11Ba toxin.

• Kim ES, Lee HJ, Bang WG, Choi IG, Kim KH
• Functional characterization of a bacterial expansin from Bacillus subtilis for enhanced enzymatic hydrolysis of cellulose.
• Biotechnol Bioeng. 2009; 102: 1342-53
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• Expansin is a plant protein family that induces plant cell wall-loosening and cellulose disruption without exerting cellulose-hydrolytic activity. Expansin-like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a beta-expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a cellulose substrate, the recombinant protein exhibited both cellulose-binding and cellulose-weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7-fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non-eukaryotic source.

• Fantino JR, Barras F, Denizot F
• Sposensor: a whole-bacterial biosensor that uses immobilized Bacillus subtilis spores and a one-step incubation/detection process.
• J Mol Microbiol Biotechnol. 2009; 17: 90-5
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• A generic whole-cell bacterial sensor called sposensor was developed with immobilized spores from engineered Bacillus subtilis. Sposensor contains two different types of spores: reporting spores that contain a reporter gene fused to a promoter responding to a compound to be detected, and control spores use to monitor cell germination and viability. A one-step incubation/detection process was developed to meet the constraints of on-site analysis. Spores were directly incubated with culture medium containing the compound to be detected. beta-Galactosidase was chosen as a reporter protein in both cases and its activity followed by a colorimetric assay. Results showed that sposensor was efficient in detecting two different compounds, a metal (Zn(2+)) and a peptidic antibiotic (bacitracin). Owing to the stability and robustness of spores, sposensor is a very efficient and easy tool to manipulate for analyzing the presence of toxic compounds in natural settings.

• Dammak M, Tounsi S, Hamadou DB, Abdelkafi L, Schultz P, Jaoua S
• Restoration of the crystallization of altered delta-endotoxins Cry1Ac, by the promotion of their in vivo integration into the Bacillus thuringiensis native crystals.
• FEMS Microbiol Lett. 2009; 292: 268-73
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• Cry1Ac is one of the most-studied Bacillus thuringiensisdelta-endotoxins. Structurally, it is divided into two domains: the N-terminal half corresponding to the toxic component and the C-terminal half corresponding to the region responsible for the crystal formation. We engineered Cry1Ac delta-endotoxins modified in their N-terminal part and studied the effect of such modifications on crystallization and delta-endotoxin production. When expressed in an acrystalliferous B. thuringiensis strain, Cry1Ac(*) and Cry1AcDelta, variants with four point mutations and a deletion, respectively, could not form crystals. However, when expressed in a crystalliferous strain, these altered proteins were shown to interact with the endogenous delta-endotoxins and cocrystallize with them, forming atypical crystals observed by electron microscopy. This cocrystallization of the altered delta-endotoxins with the endogenous ones led to a decrease in delta-endotoxin production (27%) by the corresponding recombinant B. thuringiensis strains. This ability of altered delta-endotoxins containing an intact C-terminal part to cocrystallize with native ones could be exploited to promote the crystallization of foreign proteins by fusing them with the C-terminal part of Cry1A delta-endotoxins.

• Berne S, Lah L, Sepcic K
• Aegerolysins: structure, function, and putative biological role.
• Protein Sci. 2009; 18: 694-706
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• Aegerolysins, discovered in fungi, bacteria and plants, are highly similar proteins with interesting biological properties. Certain aegerolysins possess antitumoral, antiproliferative, and antibacterial activities. Further possible medicinal applications include their use in the prevention of atherosclerosis, or as vaccines. Additional biotechnological value of fungal aegerolysins lies in their involvement in development, which could improve cultivation of commercially important edible mushrooms. Besides, new insights on microheterogeneity of raft-like membrane domains could be gained by using aegerolysins as specific markers in cell and molecular biology. Although the exact function of aegerolysins in their producing organisms remains to be explained, they are biochemically well characterized all-beta structured proteins sharing the following common features: low isoelectric points, similar molecular weights (15-17 kDa), and stability in a wide pH range.

• Sasaki H, Kawamoto E, Tanaka Y, Sawada T, Kunita S, Yagami K
• Identification and characterization of hemolysin-like proteins similar to RTX toxin in Pasteurella pneumotropica.
• J Bacteriol. 2009; 191: 3698-705
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• Pasteurella pneumotropica is an opportunistic pathogen that causes lethal pneumonia in immunodeficient rodents. The virulence factors of this bacterium remain unknown. In this study, we identified the genes encoding two RTX toxins, designated as pnxI and pnxII, from the genomic DNA of P. pneumotropica ATCC 35149 and characterized with respect to hemolysis. The pnxI operon was organized according to the manner in which the genes encoded the structural RTX toxin (pnxIA), the type I secretion systems (pnxIB and pnxID), and the unknown orf. The pnxII gene was involved only with the pnxIIA that coded for a structural RTX toxin. Both the structural RTX toxins of deduced PnxIA and PnxIIA were involved in seven of the RTX repeat and repeat-like sequences. By quantitative PCR analysis of the structural RTX toxin-encoding genes in P. pneumotropica ATCC 35149, the gene expression of pnxIA was found to have increased from the early log phase, while that of pnxIIA increased from the late log to the early stationary phase. As expressed in Escherichia coli, both the recombinant proteins of PnxIA and PnxIIA showed weak hemolytic activity in both sheep and murine erythrocytes. On the basis of the results of the Southern blotting analysis, the pnxIA gene was detected in 82% of the isolates, while the pnxIIA gene was detected in 39%. These results indicate that the products of both pnxIA and pnxIIA were putative associations of virulence factors in the rodent pathogen P. pneumotropica.

• Kashimoto T, Ueno S, Ehara H, Fukudome S, Komai M, Susa N
• Oligomerization is essential for apoptotic activity of Vibrio vulnificus hemolysin.
• J Vet Med Sci. 2009; 71: 1403-6
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• Vibrio vulnificus hemolysin (VVH), a pore forming toxin, is thought to be a virulence factor of this bacterium. It is well known that VVH induces apoptosis as well as cell lysis in susceptible target cells. Although pore formation is an essential step in cell lysis, it is unknown whether this step is necessary for VVH-induced apoptosis. In this study, Chinese hamster ovary (CHO) cells were exposed to non-oligomerized mutant F334I, in which phenylalanine 334 was replaced by isoleucine. The rate of apoptosis caused by the wild type VVH (VVH wt) was 41.5 +/- 6.4 %, whereas that caused by F334I was 0.4 +/- 0.8% at the same concentration. Our results clearly showed that oligomerization is essential for the cell lytic activity as well as apoptotic activity of this toxin.

• Fujisawa M, Wada Y, Tsuchiya T, Ito M
• Characterization of Bacillus subtilis YfkE (ChaA): a calcium-specific Ca2+/H+ antiporter of the CaCA family.
• Arch Microbiol. 2009; 191: 649-57
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• YfkE, a protein from Bacillus subtilis, exhibits homology to the Ca(2+):Cation Antiporter (CaCA) Family. In a fluorescence-based assay of everted membrane vesicles prepared from Na(+)(Ca(2+))/H(+) antiporter-defective mutant Escherichia coli KNabc, YfkE exhibited robust Ca(2+)/H(+) antiport activity, with a K (m) for Ca(2+) estimated at 12.5 muM at pH 8.5 and 113 muM at pH 7.5. Neither Na(+) nor K(+) served as a substrate. Mg(2+) also did not serve as a substrate, but inhibited the Ca(2+)/H(+) antiporter activity. The Ca(2+) transport capability of YfkE was also observed directly by transport assays in everted membrane vesicles using radiolabeled (45)Ca(2+). Transcriptional analysis from the putative yfkED operon using beta-garactosidase activity as a reporter revealed that both of the yfkE and yfkD genes are regulated by forespore-specific sigma factor, SigG, and the general stress response regulator, SigB. These results suggest that YfkE may be needed for Ca(2+) signaling in the sporulation or germination process in B. subtilis. ChaA is proposed as the designation for YfkE of B. subtilis.

• Ni Y et al.
• Expression of arginine deiminase from Pseudomonas plecoglossicida CGMCC2039 in Escherichia coli and its anti-tumor activity.
• Curr Microbiol. 2009; 58: 593-8
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• Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent in clinical trials for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. The arcA gene encoding ADI was cloned from a recently isolated strain Pseudomonas plecoglossicida CGMCC2039. The nucleotide sequence of ADI comprises an ORF of 1,254 bp encoding 417 amino acids. The deduced ADI protein sequence has a calculated molecular weight of 46.5 kDa and shows 97% and 85% identity to ADIs from P. putida and P. aeruginosa, respectively. The arcA from P. plecoglossicida CGMCC2039 was expressed in Escherichia coli BL21 with a N-terminal His6-tag, and purified to homogeneity. A molecular mass of approximate 49 kDa was confirmed by SDS-PAGE analysis and specific activity was determined to be 4.76 U/mg (pH 6.0 and 37 degrees C). In vivo activity study showed that the rADI could effectively inhibit H22 tumor growth at a total dose of 5 U/mouse over a 2-week dosing period.

• Eiamphungporn W, Soonsanga S, Lee JW, Helmann JD
• Oxidation of a single active site suffices for the functional inactivation of the dimeric Bacillus subtilis OhrR repressor in vitro.
• Nucleic Acids Res. 2009; 37: 1174-81
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• Bacillus subtilis OhrR is a dimeric repressor that senses organic peroxides and regulates the expression of the OhrA peroxiredoxin. Derepression results from oxidation of an active site cysteine which ultimately results in formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to the sulfinic or sulfonic acids. We expressed a single-chain OhrR (scOhrR) in which the two monomers were connected by a short amino-acid linker. scOhrR variants containing only one active site cysteine were fully functional as repressors and still responded, albeit with reduced efficacy, to organic peroxides in vivo. Biochemical analyses indicate that oxidation at a single active site is sufficient for derepression regardless of the fate of the active site cysteine. scOhrR with only one active site cysteine in the amino-terminal domain is inactivated at rates comparable to wild-type whereas when the active site is in the carboxyl-terminal domain the protein is inactivated much more slowly. The incomplete derepression noted for single active site variants of scOhrR in vivo is consistent with the hypothesis that protein reduction regenerates active repressor and that, in the cell, oxidation of the second active site may also contribute to derepression.

• Hasegawa H, Hase CC
• The extracellular metalloprotease of Vibrio tubiashii directly inhibits its extracellular haemolysin.
• Microbiology. 2009; 155: 2296-305
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• Vibrio tubiashii is a re-emerging pathogen of molluscs that secretes a variety of extracellular products (ECPs), including a metalloprotease and a cytolysin/haemolysin. Previously, we reported that the V. tubiashii haemolysin locus consists of two ORFs (vthB and vthA), similar to that of the homologous haemolysin genes (vvhB and vvhA) found in Vibrio vulnificus. Here, we demonstrate that the concomitant expression of both V. tubiashii genes resulted in significantly higher haemolytic activity than the vthA gene alone. In addition, we created a VthAB- mutant strain of V. tubiashii that was virtually devoid of haemolytic activity in liquid media. Interestingly, significant production of an additional haemolysin(s) was observed on blood plates. Moreover, we have previously reported that in V. tubiashii, proteolytic and haemolytic activities are inversely produced during bacterial growth. Here, we study this correlation in more detail and present evidence that the VtpA metalloprotease inhibits haemolytic activity in culture supernatants, based on the following evidence: (i) loss of metalloprotease activity by either mutation or EDTA inhibition resulted in increased haemolytic activity; (ii) overexpression of the vtpA gene resulted in decreased haemolytic activity; (iii) purified VtpA metalloprotease directly diminished haemolytic activity by purified VthA haemolysin. Importantly, we found not only that vthAB gene expression remained high throughout growth but also that there were no dramatic differences in vthAB gene expression between the parent and VtpA- mutant strains. Thus, our results strongly suggest that the V. tubiashii metalloprotease directly targets its haemolysin.

• Lauber MA, Running WE, Reilly JP
• B. subtilis ribosomal proteins: structural homology and post-translational modifications.
• J Proteome Res. 2009; 8: 4193-206
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• Ribosomal proteins of the model gram-positive bacterium B. subtilis 168 were extensively characterized in a proteomic study. Mass spectra of the 52 proteins expected to be constitutive components of the 70S ribosome were recorded. Peptide MS/MS analysis with an average sequence coverage of 85% supported the identification of these proteins and facilitated the unambiguous assignment of post-translational modifications, including the methylation of S7, L11, and L16 and the N-terminal acetylation of S9. In addition, the high degree of structural homology between B. subtilis and other eubacterial ribosomal proteins was demonstrated through chemical labeling with S-methylthioacetimidate. One striking difference from previous characterizations of bacterial ribosomal proteins is that dozens of protein masses were found to be in error and not easily accounted for by post-translational modifications. This, in turn, led us to discover an inordinate number of sequencing errors in the reference genome of B. subtilis 168. We have found that these errors have been corrected in a recently revised version of the genome.

• Mikhailova EO, Mardanova AM, Balaban NP, Rudenskaya GN, Ilyinskaya ON, Sharipova MR
• Biochemical properties of Bacillus intermedius subtilisin-like proteinase secreted by a Bacillus subtilis recombinant strain in its stationary phase of growth.
• Biochemistry (Mosc). 2009; 74: 308-15
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• Biochemical properties of Bacillus intermedius subtilisin-like proteinase (AprBi) secreted by a B. subtilis recombinant strain in the early and late stationary phases of growth have been determined. Protein structure was analyzed and its stability estimated. It was noted that the enzyme corresponding to different phases of bacterial growth retains activity in the presence of reducing and oxidizing agents (C2H5OH and H2O2). Different effects of bivalent metal ions on activity of two proteinase fractions were found. Calcium ions more efficiently activate proteinase secreted in the late stationary phase. Unlike the first enzyme fraction, the second forms catalytically active dimers.

• Emptage CD, Knox RJ, Danson MJ, Hough DW
• Nitroreductase from Bacillus licheniformis: a stable enzyme for prodrug activation.
• Biochem Pharmacol. 2009; 77: 21-9
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• 5-aziridinyl-2,4-dinitrobenzamide (CB1954) has potential applications in enzyme/prodrug targeted anti-cancer therapies since it can be activated by nitroreductases to form a cytotoxic, bifunctional hydroxylamine derivative. A nitroreductase that can activate CB1954 has been previously isolated from Escherichia coli, but its usefulness is limited by its poor stability and low catalytic efficiency for CB1954. We now report the identification and characterization of a nitroreductase enzyme from the thermophilic bacterium Bacillus licheniformis. Although there is only 28% amino acid sequence identity between this enzyme and the previously isolated E. coli nitroreductase, the two enzymes have a number of characteristics in common. Both enzymes have been shown to reduce both CB1954 and menadione in the presence of NADH and NADPH. However, whereas E. coli nitroreductase produces equimolar amounts of the 2- and 4- hydroxylamine derivative of CB1954, the B. licheniformis enzyme produces only the desired 4-hydroxylamine derivative. It has a preference for NADPH as cosubstrate, and is also active with a range of CB1954 derivatives as substrate and reduced pyridinium cofactor analogues. Moreover, the enzyme is much more thermostable than the E. coli nitroreductase and shows maximum activity at 30 degrees C. These characteristics suggest that the B. licheniformis nitroreductase may be a possible candidate enzyme for enzyme/prodrug therapies due to its bacterial origin, the high activity observed with CB1954 and its enhanced stability.

• Moran S, Rai DK, Clark BR, Murphy CD
• Precursor-directed biosynthesis of fluorinated iturin A in Bacillus spp.
• Org Biomol Chem. 2009; 7: 644-6
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• Some iturin A-producing strains of Bacillus subtilis will elaborate the novel fluorinated analogue when incubated with 3-fluoro-L-tyrosine. The activity of iturin A is dependent on the D-tyrosine residue and the presence of fluorotyrosine may result in an improvement of the biological properties of this lipopeptide. The fluorinated iturin might also be used as a probe for studying its interaction with biological membranes.

• Shu C et al.
• Characterization of two novel cry8 genes from Bacillus thuringiensis strain BT185.
• Curr Microbiol. 2009; 58: 389-92
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• Two novel cry8-type genes, cry8Ea1 and cry8Fa1, obtained from a Holotrichia parallela-specific Bacillus thuringiensis strain, BT185, were characterized. Findings showed that cry8Ea1 and cry8Fa1 encoded polypeptides of 1164 and 1174 amino acid residues, respectively. The deduced amino acid sequences of both Cry8Ea1 and Cry8Fa1 polypeptides are the most similar to that of Cry8Ba1. Eight conserved blocks (blocks 1-8) exist in Cry8Ea1 and Cry8Fa1 polypeptides compared with known Cry proteins. Cry8Ea1 and the Cry8Fa1 toxins could form spheric crystals when they were expressed in the acrystalliferous mutant strain HD73(-). The spores and crystals from the recombinant strain containing cry8Ea1 were toxic to Holotrichia parallela, with an LC(50) of 0.0875 x 10(8) colony-forming units (CFU)/g. However, Cry8Fa1 expressed in the recombinant strain was not toxic to H. parallela, Anomala corpulenta, or H. oblita.

• Perera OP, Willis JD, Adang MJ, Jurat-Fuentes JL
• Cloning and characterization of the Cry1Ac-binding alkaline phosphatase (HvALP) from Heliothis virescens.
• Insect Biochem Mol Biol. 2009; 39: 294-302
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• Membrane-bound alkaline phosphatases (mALPs, EC 3.1.3.1) in the insect midgut have been reported as functional receptors for Cry toxins from the bacterium Bacillus thuringiensis. We previously reported the identification of HvALP in the midgut of Heliothis virescens larvae as a Cry1Ac-binding protein that is down-regulated in Cry1Ac-resistant insects. To further characterize HvALP, we localized mALP protein to foregut and midgut tissues using anti-mALP serum and then cloned five mALPs from H. virescens larval midgut. All five clones displayed high levels of sequence identity (above 90%), suggesting that they may represent allelic variants, and grouped with other lepidopteran mALPs in sequence alignments. All these cloned ALPs were predicted to contain a glycosylphosphatidylinositol (GPI) anchor and were named HvmALP1-5. We expressed two of the most diverse HvmALPs in a heterologous system to test binding of Cry1Ac and recognition by HvALP cross-reacting antiserum. Our data highlight the importance of glycosylation for Cry1Ac binding to HvALP and suggest that, depending on glycosylation, all the identified HvmALPs may be synonymous with HvALP, the Cry1Ac-binding phosphatase identified in H. virescens midgut epithelium.

• Ulbegi-Mohyla H et al.
• Synergistic and antagonistic hemolytic activities of bacteria of genus Arcanobacterium and CAMP-like hemolysis of Arcanobacterium phocae and Arcanobacterium haemolyticum with Psychrobacter phenylpyruvicus.
• Res Vet Sci. 2009; 87: 186-8
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• A total of 57 bacteria representing eight species of genus Arcanobacterium (A.) were investigated for hemolytic properties on blood agar containing sheep and rabbit blood and for CAMP-like reactions. An enhanced hemolysis on blood agar containing rabbit blood compared to sheep blood could be observed for A. haemolyticum, less pronounced for A. hippocoleae and A. pluranimalium. A synergistic hemolytic reaction with staphylococcal beta-hemolysin appeared to be constantly visible for A. hippocoleae, A. pluranimalium and A. pyogenes, with Streptococcus agalactiae for A. phocae and A. haemolyticum, with Rhodococcus equi for A. phocae, A. haemolyticum, A. pluranimalium and A. pyogenes and with A. haemolyticum for A. hippocoleae, A. pluranimalium and A. pyogenes, respectively. A reverse CAMP-reaction in the zone of staphylococcal beta-hemolysin could be observed for A.phocae and A.haemolyticum. In addition, a novel CAMP-like reaction could be noted between Psychrobacter phenylpyruvicus, identified by 16S rDNA sequencing, and A. phocae and A. haemolyticum. These synergistic or antagonistic hemolytic properties could possibly be used as additional criteria for identification of bacteria of genus Arcanobacterium.

• Ceragioli M, Cangiano G, Esin S, Ghelardi E, Ricca E, Senesi S
• Phagocytosis, germination and killing of Bacillus subtilis spores presenting heterologous antigens in human macrophages.
• Microbiology. 2009; 155: 338-46
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• Bacillus subtilis is a Gram-positive spore-bearing bacterium long used as a probiotic product and more recently regarded as an attractive vehicle for delivering heterologous antigens to be used for mucosal vaccination. This report describes the in vitro interaction between human macrophages and B. subtilis spores displaying the tetanus toxin fragment C or the B subunit of the heat-labile toxin of Escherichia coli on their surface in comparison to spores of the parental strain. Recombinant and parental B. subtilis spores were similarly internalized by human macrophages, at a frequency lower than 2.5%. Inside macrophages, nearly all spores germinated and were killed within 6 h. Using germination-defective spores and inhibiting spore germination inside macrophages, evidence was produced that only germinated spores were killed by human macrophages and that intracellular spore germination was mediated by an alanine-dependent pathway. The germinated spores were killed by macrophages before any round of cell duplication, as estimated by fluorescence microscopy analysis of macrophages infected with spores carrying the gfp gene fused to abrB, a B. subtilis gene shown here to be expressed at the transition between outgrowth and vegetative growth. Monitoring of macrophage infection never revealed cytotoxic effects being exerted by B. subtilis spores. These in vitro data support the hypothesis that B. subtilis spores may potentially be used as a suitable and safe vehicle for administering heterologous antigens to humans.

• Kodgire P, Rao KK
• hag expression in Bacillus subtilis is both negatively and positively regulated by ScoC.
• Microbiology. 2009; 155: 142-9
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• In Bacillus subtilis, motility and chemotaxis require the expression of hag, which encodes flagellin. This gene is transcribed by the sigma(D) form of RNA polymerase and is regulated by a group of proteins called transition state regulators (TSRs). Our studies show that hag transcription is negatively regulated by the transition state regulator ScoC, by binding to its promoter. Furthermore, ScoC, indirectly, also positively regulates hag by increasing the availability of sigma(D) by downregulating the levels of the anti-sigma(D)-factor FlgM. We further show that the positive regulation by ScoC predominates over the negative regulation.

• Kodgire P, Rao KK
• A dual mode of regulation of flgM by ScoC in Bacillus subtilis.
• Can J Microbiol. 2009; 55: 983-9
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• In Bacillus subtilis, the transition state regulator ScoC indirectly, negatively regulates the anti-sigmaD factor FlgM in a SinR-dependent pathway leading to an increased availability of sigmaD. In addition to the SinR-dependent pathway, ScoC negatively regulates FlgM via directly repressing flgM transcription by binding to two sites in the promoter region of the flgM operon. Our studies also show that the regulation of FlgM by SinR is not at the transcriptional or translational levels. Thus, ScoC shows a dual mode of downregulation of FlgM, via both SinR-dependent and -independent pathways, which eventually results in the increased sigmaD activity.

• Kaufman-Szymczyk A, Wojtasik A, Parniewski P, Bialkowska A, Tkaczuk K, Turkiewicz M
• Identification of the csp gene and molecular modelling of the CspA-like protein from Antarctic soil-dwelling psychrotrophic bacterium Psychrobacter sp. B6.
• Acta Biochim Pol. 2009; 56: 63-9
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• We cloned and sequenced the cspA-like gene from a psychrotrophic Antarctic soil-dwelling bacterial strain Psychrobacter sp. B6. The gene is 213 bp long and shows 99% and 98% sequence identity with the Psychrobacter cryohalolentis K5 gene encoding a cold-shock DNA-binding domain protein and the Psychrobacter arcticus transcriptional regulator-CspA gene, respectively. The protein encoded by the Psychrobacter sp. B6 cspA-like gene shows 100% identity with the two proteins mentioned above, and also 61% sequence identity with CspB from Bacillus subtilis and Csp from Bacillus caldolyticus, and 56% - with Escherichia coli CspA protein. A three-dimensional model of the CspA-like protein from Psychrobacter sp. B6 was generated based on three known structures of cold shock proteins: the crystal structure of the major cold shock protein from Escherichia coli (CspA), the NMR structure of the latter protein, and the NMR structure of Csp from Thermotoga maritima. The deduced structure of the CspA-like protein from Psychrobacter sp. B6 was found to be very similar to these known structures of Csp-like proteins.

• Westers L et al.
• Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome.
• Proteomics. 2008; 8: 2704-13
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• Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.

• Guldan H, Sterner R, Babinger P
• Identification and characterization of a bacterial glycerol-1-phosphate dehydrogenase: Ni(2+)-dependent AraM from Bacillus subtilis.
• Biochemistry. 2008; 47: 7376-84
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• The exclusive presence of glycerol-1-phosphate dehydrogenases (G1PDH) has been postulated to be a key feature that distinguishes archaea from bacteria. However, homologues of G1PDH genes can be found in several bacterial species, among them the hitherto uncharacterized open reading frame araM from Bacillus subtilis. We produced recombinant AraM in Escherichia coli and demonstrate that the purified protein forms a homodimer that reversibly reduces dihydroxyacetone phosphate (DHAP) to glycerol-1-phosphate (G1P) in a NADH-dependent manner. AraM, which constitutes the first identified G1PDH from bacteria, has a similar catalytic efficiency as its archaeal homologues, but its activity is dependent on the presence of Ni (2+) instead of Zn (2+). On the basis of these findings and the analysis of an araM knockout mutant, we propose that AraM generates G1P for the synthesis of phosphoglycerolipids in Gram-positive bacterial species.

• Kouwen TR, Dubois JY, Freudl R, Quax WJ, van Dijl JM
• Modulation of thiol-disulfide oxidoreductases for increased production of disulfide-bond-containing proteins in Bacillus subtilis.
• Appl Environ Microbiol. 2008; 74: 7536-45
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• Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of B. subtilis for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule Escherichia coli PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were approximately 3.5-fold larger than the amounts secreted by the parental strain B. subtilis 168. Our findings indicate that Bacillus strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.

• Blair KM, Turner L, Winkelman JT, Berg HC, Kearns DB
• A molecular clutch disables flagella in the Bacillus subtilis biofilm.
• Science. 2008; 320: 1636-8
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• Biofilms are multicellular aggregates of sessile bacteria encased by an extracellular matrix and are important medically as a source of drug-resistant microbes. In Bacillus subtilis, we found that an operon required for biofilm matrix biosynthesis also encoded an inhibitor of motility, EpsE. EpsE arrested flagellar rotation in a manner similar to that of a clutch, by disengaging motor force-generating elements in cells embedded in the biofilm matrix. The clutch is a simple, rapid, and potentially reversible form of motility control.

• Hara Y, Seki M, Matsuoka S, Hara H, Yamashita A, Matsumoto K
• Involvement of PlsX and the acyl-phosphate dependent sn-glycerol-3-phosphate acyltransferase PlsY in the initial stage of glycerolipid synthesis in Bacillus subtilis.
• Genes Genet Syst. 2008; 83: 433-42
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• The gene responsible for the first acylation of sn-glycerol-3-phosphate (G3P) in Bacillus subtilis has not yet been determined with certainty. The product of this first acylation, lysophosphatidic acid (LPA), is subsequently acylated again to form phosphatidic acid (PA), the primary precursor to membrane glycerolipids. A novel G3P acyltransferase (GPAT), the gene product of plsY, which uses acyl-phosphate formed by the plsX gene product, has recently been found to synthesize LPA in Streptococcus pneumoniae. We found that in B. subtilis growth arrests after repression of either a plsY homologue or a plsX homologue were overcome by expression of E. coli plsB, which encodes an acyl-acylcarrier protein (acyl-ACP)-dependent GPAT, although in the case of plsX repression a high level of plsB expression was required. B. subtilis has, therefore, a capability to use the acyl-ACP dependent GPAT of PlsB. Simultaneous expression of plsY and plsX suppressed the glycerol requirement of a strict glycerol auxotrophic derivative of the E. coli plsB26 mutant, although either one alone did not. Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [14C]-labeled G3P to synthesize [14C]-labeled LPA, whereas those from DeltaplsY cells did not. The results indicate unequivocally that PlsY is an acyl-phosphate dependent GPAT. Expression of plsX corrected the glycerol auxotrophy of a DeltaygiH (the deleted allele of an E. coli homologue of plsY) derivative of BB26-36 (plsB26 plsX50), suggesting an essential role of plsX other than substrate supply for acyl-phosphate dependent LPA synthesis. Two-hybrid examinations suggested that PlsY is associated with PlsX and that each may exist in multimeric form.

• Absalon C et al.
• The GTPase CpgA is implicated in the deposition of the peptidoglycan sacculus in Bacillus subtilis.
• J Bacteriol. 2008; 190: 3786-90
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• Depletion of the Bacillus subtilis GTPase CpgA produces abnormal cell shapes, nonuniform deposition of cell wall, and five- to sixfold accumulation of peptidoglycan precursors. Nevertheless, the inherent structure of the cell wall appeared mostly unchanged. The results are consistent with CpgA being involved in coordinating normal peptidoglycan deposition.

• Liu YH, Lu FP, Li Y, Yin XB, Wang Y, Gao C
• Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600.
• Appl Microbiol Biotechnol. 2008; 78: 85-94
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• Based on the original thermostable alpha-amylase gene from Bacillus licheniformis, two amino acids were site-directed mutagenised by polymerase chain reaction to obtain a new gene. This gene, with Leu134-->Arg and Ser320-->Ala, was substituted for acid-resistant capability previously. To favor purification of the product, high-level expression and secretion of mature, authentic and stable recombinant mutagenised alpha-amylase were achieved with protease-deficient strain Bacillus subtilis WB600 as the host. The recombinant mutagenised alpha-amylase with the activity of 4,700 U/mL was then purified by ammonium sulphate fractionation, anion exchange and gel filtration, consecutively. By multi-step purification, the specific activity of the recombinant protein was up to 916.7 U/mg with a 187.1-fold purification. The mutagenised protein was found to be more acid resistant than the native protein. The optimum pH and stable range of pH with the mutagenised protein was 4.5 and 4.0 to 6.5, respectively, compared with pH 6.5 and 5.5 to 7.0 as the favorite pH and pH stability range of the native protein.

• Nandy SK, Prasad V, Venkatesh KV
• Effect of temperature on the cannibalistic behavior of Bacillus subtilis.
• Appl Environ Microbiol. 2008; 74: 7427-30
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• Bacillus subtilis resorts to cannibalism to delay sporulation under severe nutritional limitation. We report the effect of temperature on the dynamics of cannibalism demonstrated by B. subtilis. A model consisting of a delay differential equation may explain the effect of temperature on the dynamics of cannibalism.

• Pottathil M, Jung A, Lazazzera BA
• CSF, a species-specific extracellular signaling peptide for communication among strains of Bacillus subtilis and Bacillus mojavensis.
• J Bacteriol. 2008; 190: 4095-9
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• ComX and CSF are Bacillus subtilis extracellular signaling peptides. Many different strains of B. subtilis do not communicate due to strain-specific variation of ComX. We demonstrate that CSF is a species-specific signaling molecule that partially compensates for the lack of ComX-mediated communication between different strains of B. subtilis.

• Kim GA et al.
• Characterization of cold-active uracil-DNA glycosylase from Bacillus sp. HJ171 and its use for contamination control in PCR.
• Appl Microbiol Biotechnol. 2008; 80: 785-94
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• In this study, the gene encoding Bacillus sp. HJ171 uracil-DNA glycosylase (Bsp HJ171 UDG) was cloned and sequenced. The Bsp HJ171 UDG gene consists of a 738-bp DNA sequence, which encodes for a protein that is 245-amino-acid residues in length. The deduced amino acid sequence of the Bsp HJ171 UDG had a high sequence similarity with other bacterial UDGs. The molecular mass of the protein derived from this amino acid sequence was 27.218 kDa. The Bsp HJ171 UDG gene was expressed under the control of a T7lac promoter in the pTYB1 plasmid in Escherichia coli BL21 (DE3). The expressed enzyme was purified in one step using the Intein Mediated Purification with an Affinity Chitin-binding Tag purification system. The optimal temperature range, pH, NaCl concentration, and KCl concentration of the purified enzyme was 20-25 degrees C, 8.0, 25 and 25 mM, respectively. The half-life of the enzyme at 40 degrees C and 50 degrees C were approximately 131 and 45 s, respectively. These heat-labile characteristics enabled Bsp HJ171 UDG to control carry-over contamination in the polymerase chain reaction product (PCR) without losing the PCR product.

• Ter Beek A et al.
• Transcriptome analysis of sorbic acid-stressed Bacillus subtilis reveals a nutrient limitation response and indicates plasma membrane remodeling.
• J Bacteriol. 2008; 190: 1751-61
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• The weak organic acid sorbic acid is a commonly used food preservative, as it inhibits the growth of bacteria, yeasts, and molds. We have used genome-wide transcriptional profiling of Bacillus subtilis cells during mild sorbic acid stress to reveal the growth-inhibitory activity of this preservative and to identify potential resistance mechanisms. Our analysis demonstrated that sorbic acid-stressed cells induce responses normally seen upon nutrient limitation. This is indicated by the strong derepression of the CcpA, CodY, and Fur regulon and the induction of tricarboxylic acid cycle genes, SigL- and SigH-mediated genes, and the stringent response. Intriguingly, these conditions did not lead to the activation of sporulation, competence, or the general stress response. The fatty acid biosynthesis (fab) genes and BkdR-regulated genes are upregulated, which may indicate plasma membrane remodeling. This was further supported by the reduced sensitivity toward the fab inhibitor cerulenin upon sorbic acid stress. We are the first to present a comprehensive analysis of the transcriptional response of B. subtilis to sorbic acid stress.

• Ludwig A, von Rhein C, Mischke A, Brade V
• Release of latent ClyA cytolysin from Escherichia coli mediated by a bacteriophage-associated putative holin (BlyA) from Borrelia burgdorferi.
• Int J Med Microbiol. 2008; 298: 473-81
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• Introduction of the Borrelia burgdorferi blyAB locus into Escherichia coli has recently been reported to cause a hemolytic phenotype that is dependent on the E. coli clyA (hlyE, sheA) gene (a cytolysin gene present in many E. coli strains, including E. coli K-12, which is repressed under standard in vitro growth conditions). The blyA gene product has been suggested to be a prophage-encoded holin, but the processes triggered in E. coli by the expression of blyA and/or blyB, which lead to the hemolytic phenotype, remained unclear. Here we show that expression of blyA in E. coli causes damage to the E. coli cell envelope and a clyA-dependent hemolytic phenotype, regardless whether blyB is present or absent. The expression of blyB in E. coli, on the other hand, did not have obvious phenotypic effects. Transcriptional studies demonstrated that the clyA gene is not induced in E. coli cells expressing blyA. Furthermore, protein analyses suggested that the impairment of the E. coli cell envelope by BlyA is responsible for the emergence of the hemolytic activity as it allows latent intracellular ClyA protein, derived from basal-level expression of the clyA gene, to leak into the medium and to lyse erythrocytes. These findings are compatible with the presumption that BlyA functions as a membrane-active holin.

• Scheffers DJ
• The effect of MinC on FtsZ polymerization is pH dependent and can be counteracted by ZapA.
• FEBS Lett. 2008; 582: 2601-8
• Display abstract

• The min system prevents polar cell division in bacteria. Here, the biochemical characterization of the interaction of MinC and FtsZ from a Gram-positive bacterium, Bacillus subtilis, is reported. B. subtilis MinC inhibits FtsZ polymerization in a pH-dependent manner by preventing the formation of lateral associations between filaments. The inhibitory effect of MinC on FtsZ polymerization is counteracted by the presence of ZapA, a protein that promotes FtsZ filament bundling.

• Itaya M, Fujita K, Kuroki A, Tsuge K
• Bottom-up genome assembly using the Bacillus subtilis genome vector.
• Nat Methods. 2008; 5: 41-3
• Display abstract

• We established a protocol to construct complete recombinant genomes from their small contiguous DNA pieces and obtained the genomes of mouse mitochondrion and rice chloroplast using a B. subtilis genome (BGM) vector. This method allows the design of any recombinant genomes, valuable not only for fundamental research in systems biology and synthetic biology but also for various applications in the life sciences.

• Promdonkoy B, Rungrod A, Promdonkoy P, Pathaichindachote W, Krittanai C, Panyim S
• Amino acid substitutions in alphaA and alphaC of Cyt2Aa2 alter hemolytic activity and mosquito-larvicidal specificity.
• J Biotechnol. 2008; 133: 287-93
• Display abstract

• Cyt2Aa2 produced by Bacillus thuringiensis subsp. darmstadiensis exhibits in vitro cytolytic activity against broad range of cells but shows specific in vivo toxicity against larvae of Dipteran insects. To investigate the role of amino acids in alphaA and alphaC of this toxin, 3 single-point mutants (A61C, S108C and V109A) were generated. All 3 mutant proteins were highly produced as inclusion bodies that could be solubilized and activated by proteinase K similar to that of the wild type. Hemolytic activity of A61C and S108C mutants was significantly reduced whereas the V109A mutant showed comparable hemolytic activity to the wild type. Interestingly, the A61C mutant exhibited high larvicidal activity to both Aedes aegypti and Culex quinquefasciatus. S108C and V109A mutants showed low activity against C. quinquefasciatus but relatively high toxicity to A. aegypti. These results demonstrated for the first time that amino acids in alphaA and alphaC are involved in the selectivity of the Cyt toxin to the targeted organism.

• Berry RM, Armitage JP
• Microbiology. How bacteria change gear.
• Science. 2008; 320: 1599-600

• Tanous C et al.
• The CymR regulator in complex with the enzyme CysK controls cysteine metabolism in Bacillus subtilis.
• J Biol Chem. 2008; 283: 35551-60
• Display abstract

• Several enzymes have evolved as sensors in signal transduction pathways to control gene expression, thereby allowing bacteria to adapt efficiently to environmental changes. We recently identified the master regulator of cysteine metabolism in Bacillus subtilis, CymR, which belongs to the poorly characterized Rrf2 family of regulators. We now report that the signal transduction mechanism controlling CymR activity in response to cysteine availability involves the formation of a stable complex with CysK, a key enzyme for cysteine biosynthesis. We carried out a comprehensive quantitative characterization of this regulator-enzyme interaction by surface plasmon resonance and analytical ultracentrifugation. We also showed that O-acetylserine plays a dual role as a substrate of CysK and as an effector modulating the CymR-CysK complex formation. The ability of B. subtilis CysK to bind to CymR appears to be correlated to the loss of its capacity to form a cysteine synthase complex with CysE. We propose an original model, supported by the determination of the intracellular concentrations of the different partners, by which CysK positively regulates CymR in sensing the bacterial cysteine pool.

• Martinez-Martinez I, Navarro-Fernandez J, Daniel Lozada-Ramirez J, Garcia-Carmona F, Sanchez-Ferrer A
• YesT: a new rhamnogalacturonan acetyl esterase from Bacillus subtilis.
• Proteins. 2008; 71: 379-88
• Display abstract

• YesT, a putative protein from Bacillus subtilis ATCC 6633 that has been provisionally classified as a rhamnogalacturonan acetyl esterase (RGAE) in CE-12 family, was cloned, expressed in Escherichiacoli Rosetta (DE3), and purified. The enzyme is monomeric with a molecular mass of 37 kDa and presents thermophilic properties similar to RGAE from Aspergillus aculeatus, although YesT is more alkaliphilic. The study of inhibitors confirmed the importance of the His and the nucleophilic Ser for the esterase activity, apart from the Asp from the catalytic triad. This enzyme also presents broad substrate specificity, and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, beta-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, YesT achieves a synergistic effect together with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common alpha/beta hydrolase fold. The primary sequence analysis and multiple sequence alignment revealed the lack of a two beta-stranded antiparallel sheet, which results in a clear change in the structure together with the disappearance of one of the three 3(10)-helices presented in RGAE structure. The similarities found in this article among the topological diagrams of RGAE, YesT, and Esterase A from Streptomyces scabies, Platelet-Activating Factor AcetylHydrolase, isoform Ib, alpha subunit [PAF-AH(Ib)alpha(1)], PAF-AH(Ib)alpha(2), the esterase domain from hemagglutinin esterase fusion glycoprotein (HEF1) from Influenza C virus, the thioesterase I (TAP) from E. coli, the hypothetical protein a1r1529 from Nostoc sp., and the hypothetical YxiM precursor that all belong to the SGNH family could indicate a possible divergence of such proteins from a common ancestor.

• Fickers P et al.
• Temperature dependence of mycosubtilin homologue production in Bacillus subtilis ATCC6633.
• Res Microbiol. 2008; 159: 449-57
• Display abstract

• Bacillus subtilis ATCC6633 produces mycosubtilin, a non-ribosomally synthesized lipopeptide of the iturin family which presents antagonistic activities toward various phytopathogens. Different homologues with fatty acid moiety varying from C15 to C17 are usually co-produced, with their biological activities increasing with the number of carbons in the fatty acid chain. In the present report, we highlight that growth temperature modulates both the extent of mycosubtilin production and the relative abundance of the different homologues. A 30-fold increase in mycosubtilin production was observed when the temperature was decreased from 37 degrees C to 25 degrees C for both strain ATCC6633 and its derivative BBG100, a constitutive mycosubtilin overproducer. However, no significant difference in either the expression of the mycosubtilin synthetase encoding genes or in the intracellular synthetase concentration could be found, suggesting that the observed phenotype originated from a higher mycosubtilin synthetase turnover at lower temperature. We also point out that lower growth temperature leads to an increased proportion of odd-numbered fatty acid homologues as a consequence of de novo synthesis of C17 anteiso fatty acid following cell adaptation to low temperatures.

• Aroonsri A, Kitani S, Choi SU, Nihira T
• Isolation and characterization of bamA genes, homologues of the gamma-butyrolactone autoregulator-receptor gene in Amycolatopsis mediterranei, a rifamycin producer.
• Biotechnol Lett. 2008; 30: 2019-24
• Display abstract

• Four genes (bamA1, bamA2, bamA3 and bamA4) encoding homologues of the gamma-butyrolactone autoregulator receptor of Streptomyces were found and cloned from Amycolatopsis mediterranei, a typical non-Streptomyces actinomycetes and a producer of rifamycin, one of the major anti-tuberculosis drugs in clinical treatment. Transcriptional analysis demonstrated that bamA1 and bamA2 are transcribed in a growth-dependent manner, while bamA3 and bamA4 are constitutively transcribed during growth. Binding assays using (3)H-labeled autoregulator analogues as ligands confirmed that all of the recombinant BamA proteins expressed in Escherichia coli have clear binding activity toward several types of Streptomyces autoregulators. The ligand specificity of the recombinant BamA1 protein was identical to that of the crude cell-free lysates of A. mediterranei reported in our previous work. These results suggest that A. mediterranei, which is phylogenetically situated in a distal clade from the genus Streptomyces as non-Streptomyces actinomycetes, has an autoregulator-mediated signaling system.

• Pierce KJ, Salifu SP, Tangney M
• Gene cloning and characterization of a second alanine racemase from Bacillus subtilis encoded by yncD.
• FEMS Microbiol Lett. 2008; 283: 69-74
• Display abstract

• Alanine racemase activity was investigated in Bacillus subtilis. A putative second alanine racemase gene (yncD) was cloned in parallel with the previously identified alanine racemase gene, dal. Each of the B. subtilis genes, dal and yncD complemented the Escherichia coli Alr- DadX- double mutant alanine auxotrophic strain MB2159 in vivo, restoring the prototrophic phenotype. Alanine racemase activity was also detected in vitro in cell-free extracts prepared from cultures of E. coli MB2159 harboring plasmids expressing either of the cloned B. subtilis genes and preliminary characterization of enzyme activity is presented.

• Kitani S, Hoshika M, Nihira T
• Disruption of sscR encoding a gamma-butyrolactone autoregulator receptor in Streptomyces scabies NBRC 12914 affects production of secondary metabolites.
• Folia Microbiol (Praha). 2008; 53: 115-24
• Display abstract

• We report the cloning and sequence analysis of a gamma-butyrolactone autoregulator regulatory island that includes an sscR gene encoding the gamma-butyrolactone autoregulator receptor from Streptomyces scabies NBRC 12914, a plant pathogenic strain. gamma-Butyrolactone autoregulators trigger secondary metabolism, and sometimes morphological differentiation in the Gram-positive genus Streptomyces through binding to a specific autoregulator receptor. This gene cluster showed close similarity to other regulatory islands of Streptomyces origin that are responsible for the control of secondary metabolism. The recombinant SscR protein expressed in Escherichia coli prefers a gamma-butyrolactone autoregulator containing a long C-2 side chain and beta-hydroxyl group at the C-6 position. An inactivation experiment confirmed that this gamma-butyrolactone autoregulator receptor was involved in secondary metabolism but had no effects on the morphological differentiation. In the sscR-deleted mutant, the binding activity of the gamma-butyrolactone autoregulator was completely abolished, suggesting that its primary role is to detect the presence of an autoregulator in the environment. HPLC analysis of the culture broth showed that some peaks disappeared and new peaks that were not present in the broth of the wild-type strain appeared.

• Mak P, Maszewska A, Rozalska M
• The amino acid sequences and activities of synergistic hemolysins from Staphylococcus cohnii.
• FEMS Microbiol Lett. 2008; 287: 230-5
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• Staphylococcus cohnii ssp. cohnii and S. cohnii ssp. urealyticus are a coagulase-negative staphylococci considered for a long time as unable to cause infections. This situation changed recently and pathogenic strains of these bacteria were isolated from hospital environments, patients and medical staff. Most of the isolated strains were resistant to many antibiotics. The present work describes isolation and characterization of several synergistic peptide hemolysins produced by these bacteria and acting as virulence factors responsible for hemolytic and cytotoxic activities. Amino acid sequences of respective hemolysins from S. cohnii ssp. cohnii (named as H1C, H2C and H3C) and S. cohnii ssp. urealyticus (H1U, H2U and H3U) were identical. Peptides H1 and H3 possessed significant amino acid homology to three synergistic hemolysins secreted by Staphylococcus lugdunensis and to putative antibacterial peptide produced by Staphylococcus saprophyticus ssp. saprophyticus. On the other hand, hemolysin H2 had a unique sequence. All isolated peptides lysed red cells from different mammalian species and exerted a cytotoxic effect on human fibroblasts.

• Krasny L, Tiserova H, Jonak J, Rejman D, Sanderova H
• The identity of the transcription +1 position is crucial for changes in gene expression in response to amino acid starvation in Bacillus subtilis.
• Mol Microbiol. 2008; 69: 42-54
• Display abstract

• We identify here a pattern in the transcription start sites (+1A or +1G) of sigma(A)-dependent promoters of genes that are up-/downregulated in response to amino acid starvation (stringent response) in Bacillus subtilis. Upregulated promoters initiate mostly with ATP and downregulated promoters with GTP. These promoters appear to be sensitive to changes in initiating nucleoside triphosphate concentrations. During the stringent response in B. subtilis, when ATP and GTP levels change reciprocally, the identity of the +1 position (A or G) of these promoters is a factor important in their regulation. Mutations that change the identity of position +1 (A for G and vice versa) change the response of the promoter to amino acid starvation.

• Kerff F et al.
• Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization.
• Proc Natl Acad Sci U S A. 2008; 105: 16876-81
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• We solved the crystal structure of a secreted protein, EXLX1, encoded by the yoaJ gene of Bacillus subtilis. Its structure is remarkably similar to that of plant beta-expansins (group 1 grass pollen allergens), consisting of 2 tightly packed domains (D1, D2) with a potential polysaccharide-binding surface spanning the 2 domains. Domain D1 has a double-psi beta-barrel fold with partial conservation of the catalytic site found in family 45 glycosyl hydrolases and in the MltA family of lytic transglycosylases. Domain D2 has an Ig-like fold similar to group 2/3 grass pollen allergens, with structural features similar to a type A carbohydrate-binding domain. EXLX1 bound to plant cell walls, cellulose, and peptidoglycan, but it lacked lytic activity against a variety of plant cell wall polysaccharides and peptidoglycan. EXLX1 promoted plant cell wall extension similar to, but 10 times weaker than, plant beta-expansins, which synergistically enhanced EXLX1 activity. Deletion of the gene encoding EXLX1 did not affect growth or peptidoglycan composition of B. subtilis in liquid medium, but slowed lysis upon osmotic shock and greatly reduced the ability of the bacterium to colonize maize roots. The presence of EXLX1 homologs in a small but diverse set of plant pathogens further supports a role in plant-bacterial interactions. Because plant expansins have proved difficult to express in active form in heterologous systems, the discovery of a bacterial homolog opens the door for detailed structural studies of expansin function.

• Lewin A, Hederstedt L
• Promoted evolution of a shortened variant of heme A synthase in the membrane of Bacillus subtilis.
• FEBS Lett. 2008; 582: 1330-4
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• Bacillus subtilis heme A synthase is a membrane protein with 8 transmembrane segments. By using a two-step mutagenesis approach we have generated and selected a fully functional enzyme protein variant with a seven residue internal deletion. The biochemical properties of the shortened variant are similar to those of the normal enzyme. This could indicate that residue H209 in the mutant protein substitutes for the missing H216 as an axial ligand to the heme iron. Our results provide insight in routes of membrane protein evolution and the structure of heme A synthases.

• Reddick JJ, Williams JK
• The mmgA gene from Bacillus subtilis encodes a degradative acetoacetyl-CoA thiolase.
• Biotechnol Lett. 2008; 30: 1045-50
• Display abstract

• Early in sporulation, the mother cell compartment of Bacillus subtilis transcribes the mother cell metabolic gene (mmg) operon. The gene mmgA was assigned by other workers using sequence homology as an acetyl-CoA acetyltransferase [E.C. 2.3.1.9]. The gene was overexpressed in Escherichia coli, and the protein was purified by Ni(2+)-affinity chromatography. However, the expected MmgA-catalyzed biosynthesis of acetoacetyl-CoA from acetyl-CoA was undetectable by a standard UV assay, HPLC, and mass spectrometry. These methods indicated a preference for the reverse degradative thiolytic reaction, with a k(cat) of 80 s(-1), and a K(m) of 70 and 50 microM for CoA and acetoacetyl-CoA, respectively.

• Lu W et al.
• Cloning and characterization of the genes encoding a glycine betaine ABC-type transporter in Halobacillus trueperi DSM10404T.
• Curr Microbiol. 2007; 54: 124-30
• Display abstract

• By using Southern blot hybridization and inverse polymerase chain reaction, a 5.5-kb DNA fragment was obtained from the genomic DNA of Halobacillus trueperi DSM10404(T). Sequence analysis revealed that it contained a potential operon with high levels of sequence similarity to the opuA operon encoding glycine betaine transporter from Bacillus subtilis, which is a member of the ATP-binding cassette (ABC) substrate binding the protein-dependent transporter superfamily. The potential operon, designated as qatA (quaternary amine transporter), consists of three structural genes, which are predicted to encode an ATP-binding protein (QatAA), a membrane-associated protein (QatAB), and an extracellular substrate-binding protein (QatAC). Moreover, the putative promoter region of the operon was found with close homology to the sigma(A)-dependent promoter of B. subtilis. Reverse transcription (RT)-PCR analysis revealed that qatAA, qatAB, and qatAC genes were transcribed in cells of H. trueperi. Cells of Escherichia coli mutant MKH13 harboring qatA on pAY41 were able to grow on selective M9 salt medium containing glycine betaine and accumulated glycine betaine in the cytoplasm, showing that qatAA, qatAB, and qatAC genes together encode a functional glycine betaine transporter.

• Szurmant H, Fukushima T, Hoch JA
• The essential YycFG two-component system of Bacillus subtilis.
• Methods Enzymol. 2007; 422: 396-417
• Display abstract

• The YycFG two-component system, highly conserved in the low G+C gram positives, is essential for cell viability in most organisms in which it has been studied. The system is organized within an operon that includes at least one but often three to four other genes. Products of two of these genes, yycH and yycI, have been shown to have a regulatory role on this two-component system. Immunofluorescent studies identified YycG kinase localization at the cell division sites consistent with its role in regulating cell divisional processes. The essential nature and operon organization of this system commanded special requirements in studying this system genetically. This chapter presents methods utilized in identifying the regulatory circuit that controls the activity of the YycG kinase in Bacillus subtilis. Most aspects of our approaches are applicable to other two-component systems in B. subtilis and the gram positives. Some are limited to essential systems, such as the YycFG system.

• Erova TE et al.
• Identification of a new hemolysin from diarrheal isolate SSU of Aeromonas hydrophila.
• FEMS Microbiol Lett. 2007; 275: 301-11
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• A clinical strain SSU of Aeromonas hydrophila produces a potent cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. A new gene, which encoded a hemolysin of 439-amino acid residues with a molecular mass of 49 kDa, was identified. This hemolysin (HlyA) was detected based on the observation that the act gene minus mutant of A. hydrophila SSU still had residual hemolytic activity. The new hemolysin gene (hlyA) was cloned, sequenced, and overexpressed in Escherichia coli. The hlyA gene exhibited 96% identity with its homolog found in a recently annotated genome sequence of an environmental isolate, namely the type strain ATCC 7966 of A. hydrophila subspecies hydrophila. The hlyA gene did not exhibit any homology with other known hemolysins and aerolysin genes detected in Aeromonas isolates. However, this hemolysin exhibited significant homology with hemolysin of Vibrio vulnificus as well as with the cystathionine beta synthase domain protein of Shewanella oneidensis. The HlyA protein was activated only after treatment with trypsin and the resulting hemolytic activity was not neutralizable with antibodies to Act. The presence of the hlyA gene in clinical and water Aeromonas isolates was investigated and DNA fingerprint analysis was performed to demonstrate its possible role in Aeromonas virulence.

• Kramer N, Hahn J, Dubnau D
• Multiple interactions among the competence proteins of Bacillus subtilis.
• Mol Microbiol. 2007; 65: 454-64
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• Proteins required for transformation of Bacillus subtilis and other competent bacteria are associated with the membrane or reside in the cytosol. Previous work has shown that RecA, ComGA, ComFA and SsbB are directed to the cell poles in competent cells, and that the uptake of transforming DNA occurs preferentially at the poles. We show that ComGA, ComFA, DprA (Smf), SsbB (YwpH), RecA and YjbF (CoiA) are located at the cell poles, where they appear to colocalize. Using fluorescence resonance energy transfer, we have shown that these six competent (Com) proteins reside in close proximity to one another. This conclusion was supported by the effects of com gene knockouts on the stabilities of Com proteins. Data obtained from the com gene knockout studies, as well as information from other sources, extend the list of proteins in the transformation complex to include ComEC and ComEA. Because ComGA and ComFA are membrane-associated, while DprA, SsbB, RecA and YjbF are soluble, a picture emerges of a large multiprotein polar complex, involving both cytosolic and membrane proteins. This complex mediates the binding and uptake of single-stranded DNA, the protection of this DNA from cellular nucleases and its recombination with the recipient chromosome.

• Jerga A, Lu YJ, Schujman GE, de Mendoza D, Rock CO
• Identification of a soluble diacylglycerol kinase required for lipoteichoic acid production in Bacillus subtilis.
• J Biol Chem. 2007; 282: 21738-45
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• Diacylglycerol kinases (DagKs) are key enzymes in lipid metabolism that function to reintroduce diacylglycerol formed from the hydrolysis of phospholipids into the biosynthetic pathway. Bacillus subtilis is a prototypical Gram-positive bacterium with a lipoteichoic acid structure containing repeating units of sn-glycerol-1-P groups derived from phosphatidylglycerol head groups. The B. subtilis homolog of the prokaryotic DagK gene family (dgkA; Pfam01219) was not a DagK but rather was an undecaprenol kinase. The three members of the soluble DagK protein family (Pfam00781) in B. subtilis were tested by complementation of an E. coli dgkA mutant, and only the essential yerQ gene possessed DagK activity. This gene was dubbed dgkB, and the soluble protein product was purified, and its DagK activity was verified in vitro. Conditional inactivation of dgkB led to the accumulation of diacylglycerol and the cessation of lipoteichoic acid formation in B. subtilis. This study identifies a soluble protein encoded by the dgkB (yerQ) gene as an essential kinase in the diacylglycerol cycle that drives lipoteichoic acid production.

• Huang TC, Huang YW, Hung HJ, Ho CT, Wu ML
• Delta1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline.
• J Agric Food Chem. 2007; 55: 5097-102
• Display abstract

• Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.

• Reeves A, Haldenwang WG
• Isolation and characterization of dominant mutations in the Bacillus subtilis stressosome components RsbR and RsbS.
• J Bacteriol. 2007; 189: 1531-41
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• The general stress response of Bacillus subtilis is controlled by the activity state of the sigma(B) transcription factor. Physical stress is communicated to sigma(B) via a large-molecular-mass (>10(6)-Da) structure (the stressosome) formed by one or more members of a family of homologous proteins (RsbR, YkoB, YojH, YqhA). The positive regulator (RsbT) of the sigma(B) stress induction pathway is incorporated into the complex bound to an inhibitor protein (RsbS). Exposure to stress empowers an RsbT-dependent phosphorylation of RsbR and RsbS, with the subsequent release of RsbT to activate downstream processes. The mechanism by which stress initiates these reactions is unknown. In an attempt to identify changes in stressosome components that could lead to sigma(B) activation, a DNA segment encoding these proteins was mutagenized and placed into B. subtilis to create a merodiploid strain for these genes. Eight mutations that allowed heightened sigma(B) activity in the presence of their wild-type counterparts were isolated. Two of the mutations are missense changes in rsbR, and six are amino acid changes in rsbS. Additional experiments suggested that both of the rsbR mutations and three of the rsbS mutations likely enhance sigma(B) activity by elevating the level of RsbS phosphorylation. All of the mutations were found to be dominant over wild-type alleles only when they are cotranscribed within an rsbR rsbS rsbT operon. The data suggest that changes in RsbR can initiate the downstream events that lead to sigma(B) activation and that RsbR, RsbS, and RsbT likely interact with each other concomitantly with their synthesis.

• Yang CH, Wang HY
• [Cloning, characterization and application of the promoter of alkaline protease gene in Bacillus pumilus].
• Yi Chuan. 2007; 29: 874-80
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• A 797 bp promoter fragment of alkaline protease gene was cloned from the Bacillus pumilus genome by employing TAIL-PCR strategy. The sequence analysis of this promoter fragment showed that the sequence accounting for gene expression was approximately 390bp. Deletion analysis of the fragment defined the minimal required sequence of promoter for initiating transcription lies on a 160 bp region upstream of the start codon. The alkaline protease gene WApQ3 containing the cloned promoter fragment was inserted into the shuttle vector pSUGV4 and the constructed expression plasmid pSUBpWApQ3 was transformed into Bacillus subtilis and B. pumilus. Active alkaline protease was successfully expressed in both host strains. The peak value of extracellular alkaline protease activities of B. subtilis and B. pumilus recombinants reached to 465.5 U/mL and 3060 U/mL, respectively.

• Shaikh FA, Mullegger J, He S, Withers SG
• Identification of the catalytic nucleophile in Family 42 beta-galactosidases by intermediate trapping and peptide mapping: YesZ from Bacillus subtilis.
• FEBS Lett. 2007; 581: 2441-6
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• The mechanism-based inhibitor 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-galactopyranoside (DNP2FGal) was used to inactivate the Family 42 beta-galactosidase (YesZ) from Bacillus subtilis via the trapping of a glycosyl-enzyme intermediate, thereby tagging the catalytic nucleophile in the active site. Proteolytic digestion of the inactivated enzyme and of a control sample of unlabeled enzyme, followed by comparative high-performance liquid chromatography and mass spectrometric analysis identified a labelled peptide of the sequence ETSPSYAASL. These data, combined with sequence alignments of this region with representative members of Family 42, unequivocally identify the catalytic nucleophile in this enzyme as Glu-295, thereby providing the first direct experimental proof of the identity of this residue within Family 42.

• Chen Z, Heng C, Li Z, Liang X, Xinchen S
• Expression and secretion of a single-chain sweet protein monellin in Bacillus subtilis by sacB promoter and signal peptide.
• Appl Microbiol Biotechnol. 2007; 73: 1377-81
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• The sweet protein monellin gene was expressed in Bacillus subtilis under the control of the Bacillus subtilis sacB promoter and signal peptide sequence. A 294-bp DNA fragment, coding for sweet protein monellin, was ligated into the Escherichia coli/B. subtilis shuttle vector pHPC, producing pHPMS, which was subsequently transformed into B. subtilis QB1098, DB104, and DB403. The peptide efficiently directed the secretion of monellin from the recombinant B. subtilis cells. A maximum yield of monellin of 0.29 g protein l(-1) was obtained from the supernatant of B. subtilis DB403 harboring pHPMS. SDS-PAGE confirmed the purity of the recombinant product.

• Zhang Z, Ma C, Pornillos O, Xiu X, Chang G, Saier MH Jr
• Functional characterization of the heterooligomeric EbrAB multidrug efflux transporter of Bacillus subtilis.
• Biochemistry. 2007; 46: 5218-25
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• The Bacillus subtilis genome contains two tandem genes, ebrA and ebrB, which encode two homologues of the SMR family of multidrug efflux transporters. The sequences of EbrA and EbrB are highly similar to each other and to that of EmrE, the prototypical SMR transporter of Escherichia coli. Drug resistance profiling and drug binding experiments showed that the presence of both EbrA and EbrB is required for proper transport function. EbrA and EbrB directly interact and combine to form a functional transporter. They likely form a heterodimer in analogy to the EmrE homodimer. Mutagenesis experiments indicate that the conserved membrane-embedded glutamates in the first transmembrane helices of both EbrA and EbrB are required for multidrug efflux activity. However, the two glutamates are nonequivalent since EbrA E15 is required for substrate binding while EbrB E14 is not. Our studies support a model in which functional residues in EbrAB are relegated to at least two sets that participate in distinct steps of the active drug transport process.

• Schmidt M, Henke E, Heinze B, Kourist R, Hidalgo A, Bornscheuer UT
• A versatile esterase from Bacillus subtilis: cloning, expression, characterization, and its application in biocatalysis.
• Biotechnol J. 2007; 2: 249-53
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• An esterase from Bacillus subtilis DSM402 (BS2) was cloned and functionally expressed in E. coli. The enzyme is active up to 50 degrees C, and the V(max) (1449 mM/min) and K(M) values (119 mM) were determined using p-nitrophenyl acetate as substrate. BS2 belongs to the few hydrolases that can act on tertiary alcohols and was therefore used to resolve racemic acetates of selected tertiary alcohols, but also to selectively remove the tert-butyl ester protecting group from peptides. In addition, the enzyme shows promiscuous amidase activity.

• Marashi SA, Behrouzi R, Pezeshk H
• Adaptation of proteins to different environments: a comparison of proteome structural properties in Bacillus subtilis and Escherichia coli.
• J Theor Biol. 2007; 244: 127-32
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• We compared amino acid solvent accessibilities and helix propensities in data sets of Escherichia coli and Bacillus subtilis proteins. These species reside in very different environments and hold very different physiological properties. From the observations, it was proposed that the cytoplasm of B. subtilis is more ion-rich compared to the cytoplasm of E. coli, which might be more hydrophobic; therefore, during evolution these differences have resulted in different protein folding tracks. Such inherent differences imply that the results of bioinformatic analyses of protein structures might depend on the species from which the proteins are picked. It is also suggested that different cytoplasmic environments cause E. coli and B. subtilis to be appropriate for expression of distinct types of proteins.

• Huseby M et al.
• Structure and biological activities of beta toxin from Staphylococcus aureus.
• J Bacteriol. 2007; 189: 8719-26
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• Beta toxin is a neutral sphingomyelinase secreted by certain strains of Staphylococcus aureus. This virulence factor lyses erythrocytes in order to evade the host immune system as well as scavenge nutrients. The structure of beta toxin was determined at 2.4-A resolution using crystals that were merohedrally twinned. This structure is similar to that of the sphingomyelinases of Listeria ivanovii and Bacillus cereus. Beta toxin belongs to the DNase I folding superfamily; in addition to sphingomyelinases, the proteins most structurally related to beta toxin include human endonuclease HAP1, Escherichia coli endonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 from Bombyx mori. Our biological assays demonstrated for the first time that beta toxin kills proliferating human lymphocytes. Structure-directed active site mutations show that biological activities, including hemolysis and lymphotoxicity, are due to the sphingomyelinase activity of the enzyme.

• Ng B, Nayak S, Gibbs MD, Lee J, Bergquist PL
• Reverse transcriptases: intron-encoded proteins found in thermophilic bacteria.
• Gene. 2007; 393: 137-44
• Display abstract

• A number of thermophilic bacteria have been surveyed for possessing reverse transcriptase genes using a degenerate primer approach derived from an alignment of known group II intron encoded reverse transcriptases (RT) from mesophilic prokaryotes and eukaryotes. Six out of 34 thermophilic isolates gave a PCR product that was indicative of an RT internal fragment on sequencing. A putative RT from Bacillus caldolyticus strain EA1 was isolated by genomic walking and cloned into an Escherichia coli expression vector. The recombinant protein proved to be insoluble and was unable to be recovered from the insoluble fraction of lysates of E. coli. The RT was successfully expressed in a baculovirus vector although yields remained low. We followed RT activity during purification using the poly(rC)*p(dG)(12-18), which specifically detects only RNA-dependent DNA polymerase activity. We could not detect incorporation of dTTP into poly(rC) )*p(dG)(12-18) when using uninfected Sf21 lysates and conclude that the substrate is not a template for DNA-dependent DNA polymerase. Although a high level of RT activity was detected in the total cell protein, when compared to the activity detected in the soluble fraction, only about 10% of the activity was soluble. Sequence comparisons showed significant differences between the EA1-IEP and a Geobacillus RT expressed by others. We conclude that it may be necessary to isolate the IEP RT as a ribonucleoprotein to obtain sufficient material for further analysis.

• Liu Y et al.
• Bacisubin, an antifungal protein with ribonuclease and hemagglutinating activities from Bacillus subtilis strain B-916.
• Peptides. 2007; 28: 553-9
• Display abstract

• An antifungal protein, with a molecular mass of 41.9 kDa, and designated as bacisubin, was isolated from a culture of Bacillus subtilis strain B-916. The isolation procedure consisted of ion exchange chromatography on DEAE-Sepharose Fast Flow, and fast protein liquid chromatography on Phenyl Sepharose 6 Fast Flow and hydroxyapatite columns. The protein was adsorbed on all three chromatographic media. Bacisubin exhibited inhibitory activity on mycelial growth in Magnaporthe grisease, Sclerotinia sclerotiorum, Rhizoctonia solani, Alternaria oleracea, A. brassicae and Botrytis cinerea. The IC50 values of its antifungal activity toward the last four fungal species were 4.01 microM, 0.087 microM, 0.055 microM and 2.74 microM, respectively. Bacisubin demonstrated neither protease activity, nor protease inhibitory activity. However, it manifested ribonuclease and hemagglutinating activities.

• Wang X et al.
• Across genus plasmid transformation between Bacillus subtilis and Escherichia coli and the effect of Escherichia coli on the transforming ability of free plasmid DNA.
• Curr Microbiol. 2007; 54: 450-6
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• The results presented in this article show that direct plasmid transfer from Escherichia coli carrying shuttle plasmid to Bacillus subtilis occurred when close contact between the two species was established by mixing E. coli and B. subtilis onto selective agar plates. The data demonstrate that the production of resistant colonies by plasmid transformation through cell contact was DNase I sensitive and dependent on transformable B. subtilis strains. Furthermore, another observation indicated that the E. coli strain is able to affect the transformation capability of B. subtilis. It is assumed that the donor strain is a momentous factor for taking up plasmid DNA. This conclusion is significant in the assessment of both the possibility of intercellular DNA transfer in natural habitats of micro-organisms and the risk of the application of genetically engineered micro-organisms.

• Liu G et al.
• NMR structure of protein yqbG from Bacillus subtilis reveals a novel alpha-helical protein fold.
• Proteins. 2006; 62: 288-91

• Wisniewski J, Krawczyk-Balska A, Bielecki J
• Associated roles of hemolysin and p60 protein for the intracellular growth of Bacillus subtilis.
• FEMS Immunol Med Microbiol. 2006; 46: 330-9
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• Hemolysin expressing Bacillus subtilis strain (B. subtilis ble/hlA) was used as a carrier for listerial protein p60 to study the impact of this protein on bacterial virulence independent of other gene products of Listeria monocytogenes. Bacillus subtilis ble/hlyA exhibited longer cell chains than B. subtilis ble/hlyA/iap. Recombinant Bacillus strains are able to adhere to the mouse macrophage-like J774 and human epithelial-like Int407 cell lines. The bacterial number of B. subtilis ble/hlyA/iap strain that adhered to the Int407 cell lines was 2.52-fold higher, and its invasion level strain was 2.66-fold higher than that observed for the hemolytic strain. Microscopy analysis of infected monolayers showed that recombinant B. subtilis cells were localized inside the cytoplasm of epithelial cells, near to the nuclei, in cellular compartments with low internal pH. Furthermore, in cells infected with bacteria, the actin structures rapidly changed and accumulation of a fat, wide actin layer around the nucleus zone was observed.

• Irnov, Kertsburg A, Winkler WC
• Genetic control by cis-acting regulatory RNAs in Bacillus subtilis: general principles and prospects for discovery.
• Cold Spring Harb Symp Quant Biol. 2006; 71: 239-49
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• In recent years, Bacillus subtilis, the model organism for gram-positive bacteria, has been a focal point for study of posttranscriptional regulation. In this bacterium, more than 70 regulatory RNAs have been discovered that respond to intracellular proteins, tRNAs, and small-molecule metabolites. In total, these RNA elements are responsible for genetic control of more than 4.1% of the genome-coding capacity. This pool of RNA-based regulatory elements is now large enough that it has become a worthwhile endeavor to examine their general features and to extrapolate these simple observations to the remaining genome in an effort to predict how many more may remain unidentified. Furthermore, both metabolite- and tRNA-sensing regulatory RNAs are remarkably widespread throughout eubacteria, and it is therefore becoming increasingly clear that some of the observations for B. subtilis gene regulation will be generally applicable to many different species.

• Rodkhum C, Hirono I, Crosa JH, Aoki T
• Four novel hemolysin genes of Vibrio anguillarum and their virulence to rainbow trout.
• Microb Pathog. 2005; 39: 109-19
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• Four nucleotide sequences showing homology to known hemolysin genes were cloned and sequenced from V. anguillarum strain H775-3. The four genes, vah2, vah3, vah4 and vah5, have open reading frames encoding polypeptides of 291, 690, 200 and 585 amino acid residues, respectively, with predicted molecular masses of 33, 75, 22 and 66KDa, respectively. VAH2 is most closely related to a putative hemolysin of Vibrio vulnificus YJ016 (89% identity). VAH3 is most closely related to a hemolysin-related protein in Vibrio cholerae O1 (68% identity). VAH4 is most closely related to a thermostable hemolysin in V. cholerae O1 (72% identity). VAH5 is most closely related to a putative hemolysin in V. cholerae O1 (73% identity). The purified hemolysin proteins showed hemolytic activities against erythrocyte of fish, sheep and rabbit. Four strains of V. anguillarum mutants were constructed, each deficient in one of the hemolysin genes. Each mutant was less virulent than V. anguillarum H775-3 to juvenile rainbow trout (Oncorhynchus mykiss), indicating that each hemolysin gene contributes to the virulence of V. anguillarum H775-3.

• Fundoiano-Hershcovitz Y, Rabinovitch L, Shulami S, Reiland V, Shoham G, Shoham Y
• The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase.
• FEMS Microbiol Lett. 2005; 243: 157-63
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• The yet uncharacterized ywad gene from Bacillus subtilis has been cloned and overexpressed in Escherichia coli. The gene product (BSAP) was purified and shown to be an aminopeptidase. The activity of BSAP was optimal at pH 8.4, the enzyme was stable for 20 min at 80 degrees C and its activity was not affected by serine protease and aspartic protease inhibitors, but was completely diminished by the Zn-chelator 1,10-phenanthroline. ZnCl2 was able to restore activity, and the binding stoichiometry of zinc to apo-BSAP indicated two Zn ions per protein molecule. BSAP exhibited high preference toward p-nitroanilide derived Arg, Lys, and Leu synthetic substrates resulting in kcat/Km values of 1-5 x 10(1) s(-1) mM(-1).

• Omura K, Hitosugi M, Zhu X, Ikeda M, Maeda H, Tokudome S
• A newly derived protein from Bacillus subtilis natto with both antithrombotic and fibrinolytic effects.
• J Pharmacol Sci. 2005; 99: 247-51
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• Natto, steamed soybeans fermented by Bacillus subtilis natto, is a traditional Japanese food. We derived a purified protein layer, called NKCP as a trade mark, from B. subtilis natto fermentation. In the present study, we examined the fibrinolytic and antithrombotic effects of NKCP and identified its active component to clarify the fibrinolytic effect of NKCP observed in preliminary clinical trials previously. The active component of NKCP was identified as a 34-kilodalton protein designated bacillopeptidase F. NKCP showed direct degradation of artificial blood clot in saline. The protease activity, accounting for the fibrinolytic effect of NKCP, was examined with a chromogenic substrate for plasmin. Dose-dependent prolongations of both prothrombin time and active partial thromboplastin time were observed in rats with intra-duodenum administration of NKCP. Our in vitro and in vivo studies suggest that NKCP has both a fibrinolytic effect and an antithrombotic effect similar to heparin. Because NKCP is derived from food and has safety data demonstrated by previous animal experiments and preliminary clinical trials, NKCP is considered as safe for clinical use.

• Uechi G, Toma H, Arakawa T, Sato Y
• Molecular cloning and functional expression of hemolysin from the sea anemone Actineria villosa.
• Protein Expr Purif. 2005; 40: 379-84
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• The full-length cDNA that encodes the hemolytic toxin Avt-I, with 226 amino acids, from the venomous sea anemone Actineria villosa has been cloned using the oligo-capping method. The cDNA contains 681bp open reading frame and its predicted amino acid sequences revealed that Avt-I was basic polypeptides without cysteine residues and Arg-Gly-Asp (RGD) motif sequence. The mature Avt-I has a predicted molecular weight of 19.6 kDa and its theoretical isoelectric point is 9.3. The Avt-I revealed 99, 61, 57, and 57% amino acid similarity with hemolytic toxins Pstx20, EqtII, StII, and HmT from Phyllodiscus semoni, Actinia Equina, Stichodactyla helianthus, and Heteractis magnifica, respectively. The characteristic amphiphilic alpha-helix structure was found at the N-terminal region of the mature Avt-I. Recombinant Avt-I (rAvt-I) was expressed in Escherichia coli BL21 (DE3) strain as a biologically active form and purified rAvt-I caused 50% hemolytic activity against 1% sheep erythrocytes at a concentration of 6.3 ng/ml (0.32 nM). M9Y medium led to more than 2-fold increase in rAvt-I yield than cultivation in Luria-Bertani medium.

• Lee Y et al.
• Transthyretin-related proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction.
• FEBS Lett. 2005; 579: 4769-74
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• Purine catabolic pathway in Bacillus subtilis is consisted of more than 14 genes. Among these genes, pucL and pucM are required for uricase activity. While PucL is known to encode the uricase itself, the function of PucM is still unclear although this protein is also indispensable for uric acid decomposition. Here, we provide evidence that PucM, a transthyretin-related protein, functions to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. Based on these results, we propose that transthyretin-related proteins present in diverse organisms are not functionally related to transthyretin but actually function as a hydroxyisourate hydrolase.

• Dehghan-Noude G, Housaindokht M, Bazzaz BS
• Isolation, characterization, and investigation of surface and hemolytic activities of a lipopeptide biosurfactant produced by Bacillus subtilis ATCC 6633.
• J Microbiol. 2005; 43: 272-6
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• Bacillus subtilis ATCC 6633 was grown in BHIB medium supplemented with Mn2+ for 96 h at 37 degrees C in a shaker incubator. After removing the microbial biomass, a lipopeptide biosurfactant was extracted from the supernatant. Its structure was established by chemical and spectroscopy methods. The structure was confirmed by physical properties, such as Hydrophile-Lipophile Balance (HLB), surface activity and erythrocyte hemolytic capacity. The critical micelle concentration (cmc) and erythrocyte hemolytic capacity of the biosurfactant were compared to those of surfactants such as SDS, BC (benzalkonium chloride), TTAB (tetradecyltrimethylammonium bromide) and HTAB (hexadecyltrimethylammonium bromide). The maximum hemolytic effect for all surfactants mentioned was observed at concentrations above cmc. The maximum hemolytic effect of synthetic surfactants was more than that of the biosurfactant produced by B. subtilis ATCC 6633. Therefore, biosurfactant would be considered a suitable surface-active agent due to low toxicity to the membrane.

• Choudhary ML et al.
• Open reading frame yjbI of Bacillus subtilis codes for truncated hemoglobin.
• Protein Expr Purif. 2005; 41: 363-72
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• A hypothetical open reading frame from Bacillus subtilis genome, yjbI [NCBI genome database Accession No. ] having homology to many globin and globin-like proteins from different microbial genomes, was selectively amplified from the chromosomal DNA of B. subtilis strain DB104 based on genome sequence database of B. subtilis strain 168. The gene was cloned and over-expressed in Escherichia coli under the transcriptional control of tandem lambda P(L) and P(R) promoters, and the protein was purified to homogeneity. The single-chain monomeric hemoglobin-like protein is stable to the extent of 5.45 kcal/mol at 25 degrees C, binds carbon mono-oxide, and shows optical spectra characteristic of hemoproteins. The protein also exhibits peroxidase-like activity. This is the first report of a truncated bacterial globin endowed with peroxidase-like activity. The activity is enhanced in the presence of urea and guanidine hydrochloride, more so in the presence of the latter. Presumably, only a small portion of the protein is involved in peroxidase activity, which is exposed with increasing concentration of the denaturants.

• Alegre-Cebollada J, Lacadena V, Onaderra M, Mancheno JM, Gavilanes JG, del Pozo AM
• Phenotypic selection and characterization of randomly produced non-haemolytic mutants of the toxic sea anemone protein sticholysin II.
• FEBS Lett. 2004; 575: 14-8
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• A rapid screening method for haemolytic activity, using blood agar plates, has been developed to analyze randomly produced mutant variants of the pore-forming protein sticholysin II (Stn II). Those exhibiting a reduced activity were selected and the DNA corresponding to each Stn II variant sequenced. Once the mutation produced was determined, protein variants were isolated and characterized in terms of structure (circular dichroism spectra and thermal stability) and haemolytic activity. Three single mutation protein variants, at residues K19, F106 and Y111, showed a significantly decreased haemolytic activity while their thermostability was identical to that of the wild-type protein. Considering the obtained data and based on the three-dimensional structure of the protein, the role of these residues on the mechanism of haemolysis has been analyzed.

• Hsieh FC, Li MC, Lin TC, Kao SS
• Rapid detection and characterization of surfactin-producing Bacillus subtilis and closely related species based on PCR.
• Curr Microbiol. 2004; 49: 186-91
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• The surfactin production genetic locus ( sfp) is responsible for the ability of Bacillus subtilis to produce the lipopeptide biosurfactant, surfactin. This report demonstrates the utility of using PCR of the sfp gene as a means of identifying Bacillus species that produce surfactin. We carried out a hemolysis zone assay, quantitative HPLC and NMR in parallel to ensure that the PCR provided correct results. PCR analyses were performed for the sfp gene on 15 standard strains and 20 field-collected Bacillus spp. isolates native to Taiwan. Among the 15 standard strains, surfactin was produced by seven strains of B. subtilis and two closely related species, B. amyloliquefaciens B128 and B. circulans ATCC 4513. Of the 20 field-collected Bacillus spp. isolates; 16 strains yielded surfactin- positive results with PCR and HPLC. A good correlation was observed. Within the 16 field isolates, B. amyloliquefaciens S13 (452.5 mg/L) and B. subtilis S15 (125.6 mg/L) had high productivity of surfactin. The technique is valuable for finding out potential good yields of surfactin-producing strains. The PCR method we used could also be used to find different species or genera containing homologous genes. This is the first report of the detection of surfactin production by B. amyloliquefaciens and B. circulans based on PCR screening.

• Korepanov AP, Gongadze GM, Garber MB
• General stress protein CTC from Bacillus subtilis specifically binds to ribosomal 5S RNA.
• Biochemistry (Mosc). 2004; 69: 607-11
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• Two recombinant proteins of the CTC family were prepared: the general stress protein CTC from Bacillus subtilis and its homolog from Aquifex aeolicus. The general stress protein CTC from B. subtilis forms a specific complex with 5S rRNA and its stable fragment of 60 nucleotides, which contains internal loop E. The ribosomal protein TL5 from Thermus thermophilus, which binds with high affinity to 5S rRNA in the loop E region, was also shown to replace the CTC protein from B. subtilis in its complexes with 5S rRNA and its fragment. The findings suggest that the protein CTC from B. subtilis binds to the same site on 5S rRNA as the protein TL5. The protein CTC from A. aeolicus, which is 50 amino acid residues shorter from the N-terminus than the proteins TL5 from T. thermophilus and CTC from B. subtilis, does not interact with 5S rRNA.

• Li ZF, Nie J, Dai YC, Li JD, Chen Q, Yu SY
• [Bioinformatic analysis of Vibrio parahaemolyticus thermolabile hemolysin gene].
• Di Yi Jun Yi Da Xue Xue Bao. 2004; 24: 1153-5
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• OBJECTIVE: To carry out bioinformatic analysis of Vibrio parahaemolyticus thermolabile hemolysin gene (tlh) obtained by PCR amplification. METHODS: The tlh gene amplified by PCR was cloned into the vector pET32a(+) and sequenced, followed by analysis of the biological information by with presenting the sequences to the websites of bioinformatics on the Internet. RESULTS AND CONCLUSIONS: The sequenced tlh gene (named tlh14-90) was entered into GenBank with the accession number of AY289609. Tlh14-90 has a length of 1 257 bp with both start and stop codons, having 99% homology with the tlh gene of WP1. Tlh14-90 is predicted to encode a protein containing 418 amino acids (named TLH14-90, with the molecular formula of C(2131)H(3184)N(548)O(649)S(16), molecular weight of 47 392.9, and the theoretical PI of 4.92). This protein consists of 4 Cys and the contents of Ala, Leu and Asn are 11.0%, 7.4% and 7.2%, respectively, having a hydrophobic parameter of -34 calculated using kappa-D method. Predicted as a alpha/protein for its secondary structure, TLH14-90 has not been identified for its tertiary structure.

• Omura K, Hitosugi M, Kaketani K, Zhu X, Maeda H, Tokudome S
• Fibrinolytic and anti-thrombotic effect of NKCP, the protein layer from Bacillus subtilis (natto).
• Biofactors. 2004; 22: 185-7
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• NKCP is the completely refined protein layer from cultured Bacillus subtilis (natto) and is supplied in tablet form. It lacks the distinctive fermented odor and bacteria associated with fresh natto. Almost all vitamin K is removed during the manufacturing process. The main component of NKCP is the active fragment of Bacillopeptidase F, which is a serine protease secreted by Bacillus subtilis. In in vivo studies, NKCP shows fibrinolytic and amidolytic activity on plasmin specific synthetic substrate. Daily oral administration of NKCP in 28 volunteers for two weeks and then in 23 volunteers for several months, was examined. The fibrinolytic effect was demonstrated by a shortened euglobulin lysis time in both trials. Furthermore, we observed an improvement in shoulder stiffness. These results suggest that the oral administration of NKCP can be expected to have a fibrinolytic effect and cause positive changes in local blood flow. Further investigations may demonstrate NKCP to be useful in the prevention of thrombosis.

• Xiao L, Zhang RH, Peng Y, Zhang YZ
• Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of alpha-amylase gene from Bacillus amyloliquefaciens.
• Biotechnol Lett. 2004; 26: 1365-9
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• Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).

• Shimotohno KW, Hidaka T, Morishita T, Endo T
• Molecular cloning of the gene for edeine B1 amidinohydrolase in addition to the agmatinase activity in Bacillus subtilis.
• Biol Pharm Bull. 2003; 26: 262-5
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• A gene with a high-nucleotide sequence homology to the edeine B1 amidinohydrolase gene of Bacillus brevis was identified in the database of the Bacillus subtilis genome. The gene was isolated, expressed in Escherichia coli, and the gene product was analyzed with regard to the characteristics of its enzyme activity. A 32-kDa protein encoded by the ywhG gene showed a 69.8% amino acid sequence-homology to the edeine B1 amidinohydrolase of B. brevis. Among various guanidino-compounds, edeine B1 and agmatine were both efficiently hydrolyzed by the protein encoded by the ywhG gene, although edeine B1 was a more potent substrate than agmatine in this assay system. These data indicate that the protein encoded by the ywhG gene is an agmatinase that is essential for polyamine biosynthesis in B. subtilis.

• Zhang W, Phillips GN Jr
• Structure of the oxygen sensor in Bacillus subtilis: signal transduction of chemotaxis by control of symmetry.
• Structure. 2003; 11: 1097-110
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• Much is now known about chemotaxis signaling transduction for Escherichia coli and Salmonella typhimurium. The mechanism of chemotaxis of Bacillus subtilis is, in a sense, reversed. Attractant binding strengthens the activity of histidine kinase in B. subtilis, instead of an inhibition reaction. The HemAT from B. subtilis can detect oxygen and transmit the signal to regulatory proteins that control the direction of flagella rotation. We have determined the crystal structures of the HemAT sensor domain in liganded and unliganded forms at 2.15 A and 2.7 A resolution, respectively. The liganded structure reveals a highly symmetrical organization. Tyrosine70 shows distinct conformational changes on one subunit when ligands are removed. Our study suggests that disruption of the symmetry of HemAT plays an important role in initiating the chemotaxis signaling transduction cascade.

• Tang M, Tan L, Yu J, Yuan M, Pang Y
• [Cloning and expression of ICP cry1AB16 gene of Bacillus thuringiensis].
• Wei Sheng Wu Xue Bao. 2003; 43: 417-23
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• With two pairs of primers designed on the basis of the sequence of cry1 and cry1Ab gene from Bacillus thuringiensis (Bt), a novel insecticidal crystal protein crylAb gene of Bt was amplified from bacillus strain AC-11 by using Long Template PCR System. Southernblot further confirmed that the novel gene existed in plasmid of the strain. The amplified fragment was sequenced and compared with cry1Ab1 gene in EMBL/GenBank. The results showed that eight nucleotides and seven amino acid residues were different from that of cry1Ab1, suggesting that it was a new crylAb gene, which was designated cry1Ab16 in GenBank (Accession No. AF375608). The cry1Ab16 gene was cloned into the E. coli expression vector pQE30, creating the recombinant plasmid pQCT, which was then transformed into E. coli M15. Westernblot analysis showed that Cry1Ab16 protein, induced by IPTG was expressed in the strain M15 (pQCT) with the molecular mass of approximate 130 kD, but Cry1Ab16 was unstable and was mostly degraded into about 65 kD protein. Bioassay showed that the LC50 of Cry1Ab16 against the third instar lavae of Plutella xylostella with a spreaded method was 258.3 mg/L, and it could also inhibit the growth of Spodoptera larvae.

• Yao S, Gao X, Fuchsbauer N, Hillen W, Vater J, Wang J
• Cloning, sequencing, and characterization of the genetic region relevant to biosynthesis of the lipopeptides iturin A and surfactin in Bacillus subtilis.
• Curr Microbiol. 2003; 47: 272-7
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• Bacillus subtilis B3 was found to produce lipopeptides iturins and fengycin that have activity against several plant pathogens such as Fusarium graminearum, Rhizoctonia solani, Rhizoctonia cerealis, and Pyricularia grisea. A 3642-bp genomic region of B. subtilis B3 comprising srfDB3, aspB3, lpaB3, and yczEB3 genes that resulted in biosynthesis of surfactin in B. subtilis 168 was cloned, sequenced, and characterized. Among them, the srfDB3 gene encodes thioesterase, which is required for biosynthesis of surfactin in B. subtilis; the aspB3 gene encodes a putative aspartate aminotransferase-like protein; the lpaB3 encodes phosphopantetheinyl transferase, which shows high identity to the product of lpa-14 gene regulating the biosynthesis of iturin A and surfactin in B. subtilis RB14; the yczEB3 encodes a YczE-like protein with significant similarities in signal peptide and part of the ABC transport system. The genetic regions between the srfD gene and lpa gene from B. subtilis B3 and B. subtilis A13, which produces iturin A, contain an approximate 1-kb nucleotide fragment encoding an aspartate aminotransferase-like protein; however, the relevant regions from B. subtilis 168 and B. subtilis ATCC21332 producing surfactin comprise an approximately 4-kb nucleotide fragment encoding four unknown proteins. There is 73% identity between the Lpa family and the Sfp family, although both are highly conserved.

• Lee JY, Cho PY, Kim TY, Kang SY, Song KY, Hong SJ
• Hemolytic activity and developmental expression of pore-forming peptide, clonorin.
• Biochem Biophys Res Commun. 2002; 296: 1238-44
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• Peptides pore-forming in cell membrane have been identified from a wide range of animals. A putative pore-forming peptide deduced from a cDNA clone of Clonorchis sinensis (clonorin) was predicted to consist of four amphipathic alpha-helices. Clonorin contained six invariably conserved cysteine residues, identified to form three disulfide bonds. These predicted structural features are highly homologous with pore-forming peptides, the amoebapores. Recombinant clonorin showed hemolytic activity toward rabbit erythrocytes. The hemolytic activity of C. sinensis extract increased dose-dependently and was inhibited by anti-clonorin immune sera. The clonorin was expressed developmentally in juvenile and adult flukes and localized in the intestinal epithelium of adult flukes. It is proposed that, through lysing host cellular components, clonorin could enhance proteolytic digestion in the intestine of C. sinensis.

• Kiyasu T, Asakura A, Nagahashi Y, Hoshino T
• Biotin synthase of Bacillus subtilis shows less reactivity than that of Escherichia coli in in vitro reaction systems.
• Arch Microbiol. 2002; 179: 26-32
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• The biotin synthases of Bacillus subtilis and Escherichia coli were compared in a physiological reduction system using cell-free extracts and in a artificial reduction system using photo-reduced deazariboflavin. The biotin synthase of B. subtilis was less active than that of E. coli in both reaction systems and showed at least ten-fold less biotin-forming activity than that of E. coli in the artificial reduction system. The physiological reduction system using the biotin synthases and cell-free extracts of B. subtilis and E. coli showed species specificity. The results suggest that the activity of the physiological reduction system of B. subtilisis weaker than that of E. coli. Addition of excess dethiobiotin inhibited biotin formation by growing cells of B. subtilis, but not by E. coli.

• Yamamoto I, Kimoto H, Taketo Y, Taketo A
• Mutational and comparative analysis of streptolysin O, an oxygen-labile streptococcal hemolysin.
• Biosci Biotechnol Biochem. 2001; 65: 2682-9
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• The structural gene of streptolysin O was cloned from Streptococcus pyogenes strain Sa and S. equisimilis H46A, and the nucleotide sequences were compared with those of strain Richards. To obtain the minimal active fragment of the toxin and to elucidate structure-function relationships in hemolytic function, streptolysin O mutants deleted in N- and C-terminal regions were constructed. Internal amino acid residues were also replaced by introduction of point mutations. Analyses of these mutants showed that considerable activity was retained even after deletion of the N-terminal 107 residues, but genetic removal of the ultimate C-terminal residue resulted in a marked decrease in hemolytic function. By removal in succession, hemolytic activity declined exponentially, and only 0.002% of the activity remained after deletion of the C-terminal four residues. Nucleotide replacement experiments indicated pivotal roles of I202, V217, D324-L325, V339, and H469 residues in hemolysis.

• Haas M, Beyer D, Gahlmann R, Freiberg C
• YkrB is the main peptide deformylase in Bacillus subtilis, a eubacterium containing two functional peptide deformylases.
• Microbiology. 2001; 147: 1783-91
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• Peptide deformylation is an essential process in eubacteria. The peptide deformylase Def has been suggested to be an attractive target for antibacterial drug discovery. Some eubacteria including medically important pathogens possess two def-like genes. Until now, the functionality of both genes has been tested only in Staphylococcus aureus with the result that one gene copy was functional. Here, expression of two functional def-like gene products in Bacillus subtilis is demonstrated. Besides the def gene, which is chromosomally located close to the formyltransferase gene fmt and which was overexpressed and biochemically tested previously, B. subtilis possesses a second def-like gene, called ykrB. The encoded protein is 32% identical to the def gene product. It was shown that either def or ykrB had to be present for growth of B. subtilis in rich medium (each was individually dispensable). Studies with a def/ykrB double deletion strain with xylose-inducible ykrB copy demonstrated that, besides def, the gene ykrB is a second cellular target of deformylase inhibitors such as the antibiotic actinonin. The gene products exhibited similar enzymic properties, exemplified by similar inhibition efficacy of actinonin in biochemical assays. Antibiotic susceptibility tests with different deletion strains and Northern analyses indicated that YkrB is probably the predominant deformylase in B. subtilis. It was shown that duplication of the deformylase function does not lead to an increased actinonin-resistance frequency in comparison to B. subtilis mutants carrying only one deformylase gene.

• Jones LJ, Carballido-Lopez R, Errington J
• Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis.
• Cell. 2001; 104: 913-22
• Display abstract

• In the absence of an overt cytoskeleton, the external cell wall of bacteria has traditionally been assumed to be the primary determinant of cell shape. In the Gram-positive bacterium Bacillus subtilis, two related genes, mreB and mbl, were shown to be required for different aspects of cell morphogenesis. Subcellular localization of the MreB and Mbl proteins revealed that each forms a distinct kind of filamentous helical structure lying close to the cell surface. The distribution of the proteins in different species of bacteria, and the similarity of their sequence to eukaryotic actins, suggest that the MreB-like proteins have a cytoskeletal, actin-like role in bacterial cell morphogenesis.

• Votava M, Skalka B, Ondrovcik P, Ruzicka F, Svoboda J, Woznicova V
• [A diagnostic medium for Arcanobacterium haemolyticum and other bacterial species reacting with hemolytic synergism to the equi-factor of Rhodococcus equi].
• Epidemiol Mikrobiol Imunol. 2000; 49: 123-9
• Display abstract

• Colonies of Arcanobacterium haemolyticum on common blood agar can be easily overlooked. Therefore a diagnostic medium was developed, on which A. haemolyticum colonies produce a conspicuous zone of complete hemolysis. The medium under question is blood agar prepared from the Columbia Blood Agar Base and 5% washed sheep erythrocytes sensitised with equi factor (EF) of Rhodococcus equi. Optimally, 10 activity units (AU) of EF per 1 ml were used. EF was titrated on a non-nutrient medium consisting of agar Purified (Difco) and 5% washed sheep erythrocytes sensitized with beta-haemolysin (BL) of Staphylococcus aureus, 10 AU/ml. On the same medium, staphylococcal BL was titrated on the basis of its direct haemolytic effect. Despite the very distinct haemolytic reaction of the control strains evident on the diagnostic medium with EF, A. haemolyticum failed to grow from 2.597 throat swabs examined especially for the particular microbe during the a period of two years. However, A. haemolyticum was isolated on this medium twice from 223 swabs from wounds and skin lesions. The proposed medium makes also the rapid diagnosis of species Corynebacterium ulcerans, Dermatophilus congolensis and Listeria monocytogenes on the basis of haemolytic synergism possible.

• Niu L, Lane BG, Ofengand J
• Cloning and characterization of the 23S RNA pseudouridine 2633 synthase from bacillus subtilis
• Biochemistry. 1999; 38: 14117-14117

• Schwartz EN, Schwartz CA, Sebben A
• Occurrence of hemolytic activity in the skin secretion of the caecilian siphonops paulensis.
• Nat Toxins. 1998; 6: 179-82
• Display abstract

• The skin secretion of the caecilian Siphonops paulensis (SpSS) induces a time-and dose-dependent hemolytic response on red blood cells (RBC). When RBC from various animals species were subjected to the action of SpSS, a range of sensitivities was evident, sheep erythrocytes being the most susceptible, human, mouse and rabbit having moderate susceptibility, cow, snake and toad erythrocytes being more resistant, while S. paulensis RBC were entirely resistant. The hemolytic activity of SpSS was inhibited at temperatures higher than 60 degrees C. Both trypsin- and chymotrypsin-treated SpSS were ineffective in inducing RBC lysis. The treatment of SpSS with sheep RBC ghosts reduced its activity. There is no phospholipase activity in the SpSS.

• Gusarov II et al.
• [Primary structure and functional activity of the Bacillus subtilis ribC gene].
• Mol Biol (Mosk). 1997; 31: 820-5

• Amoako KK, Goto Y, Misawa N, Xu DL, Shinjo T
• Interactions between Fusobacterium necrophorum hemolysin, erythrocytes and erythrocyte membranes.
• FEMS Microbiol Lett. 1997; 150: 101-6
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• The interactions between the hemolysin of Fusobacterium necrophorum subsp. necrophorum, erythrocytes and erythrocyte membranes were studied as an attempt to determine the initial characteristics leading to hemolysis. The spectrum of erythrocyte sensitivity indicated that horse, dog and mouse erythrocytes were highly sensitive whereas those of cattle, sheep, goat and chicken were insensitive to the hemolysin. Binding of hemolysin to horse and dog erythrocytes or their ghosts was more pronounced than to those of cattle and sheep as detected by a decrease of hemolytic activity from hemolysin preparations. The kinetics of hemolysis revealed that lysis is preceded by a prelytic phase characterized by binding of hemolysin to erythrocytes. Treatment of horse erythrocytes with hemolysin at various temperatures prior to incubation at 37 degrees C also revealed that this binding prelytic phase is temperature independent. This was followed by a temperature dependent lytic stage since erythrocytes pretreated with hemolysin and incubated at 4 degrees C showed no hemolysis. An inverse relation was found between erythrocyte concentration and hemolytic activity suggesting a multiple-hit mechanism of hemolysis.

• Billington SJ, Jost BH, Cuevas WA, Bright KR, Songer JG
• The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family.
• J Bacteriol. 1997; 179: 6100-6
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• Arcanobacterium (Actinomyces) pyogenes, an animal pathogen, produces a hemolytic exotoxin, pyolysin (PLO). The gene encoding PLO was cloned, and sequence analysis revealed an open reading frame of 1,605 bp encoding a protein of 57.9 kDa. PLO has 30 to 40% identity with the thiol-activated cytolysins (TACYs) of a number of gram-positive bacteria. The activity of PLO was found to be very similar to those of other TACYs, except that it was not thiol activated. The highly conserved TACY undecapeptide is divergent in PLO; in particular, the cysteine residue required for thiol activation has been replaced with alanine. However, mutagenesis of the alanine residue to cysteine did not confer thiol activation on PLO, suggesting a conformational difference in the undecapeptide region of this toxin. Specific antibodies against purified, recombinant PLO completely neutralized the hemolytic activity of A. pyogenes, suggesting that this organism produces a single hemolysin. Furthermore, these antibodies could passively protect mice against lethal challenge with A. pyogenes, suggesting that like other TACYs PLO is an important virulence factor in the pathogenesis of this organism.

• Baida GE, Kuzmin NP
• Mechanism of action of hemolysin III from Bacillus cereus.
• Biochim Biophys Acta. 1996; 1284: 122-4
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• Bacillus cereus hemolysin III activity was tested in crude extracts, from Escherichia coli carrying the hly-III gene. It was concluded that hemolysin III is a pore-forming hemolysin with functional pore diameter of about 3-3.5 nm. Hemolysis occurs in at least three steps: (i) the temperature-dependent binding of the Hly-III monomers to the erythrocyte membrane; (ii) the temperature-dependent formation of the transmembrane oligomeric pore; (iii) the temperature-independent erythrocyte lysis.

• Barnard JP, Stinson MW
• The alpha-hemolysin of Streptococcus gordonii is hydrogen peroxide.
• Infect Immun. 1996; 64: 3853-7
• Display abstract

• The alpha-hemolysin of viridans group streptococci, which causes greening of intact erythrocytes, is a potential virulence factor as well as an important criterion for the laboratory identification of these bacteria; however, it has never been purified and characterized. The alpha-hemolysin of Streptococcus gordonii CH1 caused characteristic shifts in the A403, A430, A578, and A630 of sheep hemoglobin. A spectrophotometric assay was developed and used to monitor purification of alpha-hemolysin during extraction in organic solvents and separation by reverse-phase high-performance liquid chromatography (HPLC). The alpha-hemolysin was identical to hydrogen peroxide with respect to its effects on erythrocyte hemoglobin, oxygen-dependent synthesis by streptococci, insensitivity to proteases, inactivation by catalase, differential solubility, failure to adsorb to ion-exchange chromatography resins, and retention time on a reverse-phase HPLC column. The amount of hydrogen peroxide present in HPLC-fractionated spent culture medium was sufficient to account for all alpha-hemolytic activity observed.

• Kimizuka R, Miura T, Okuda K
• Characterization of Actinobacillus actinomycetemcomitans hemolysin.
• Microbiol Immunol. 1996; 40: 717-23
• Display abstract

• Twenty out of 33 Actinobacillus actinomycetemcomitans strains formed hemolytic colonies on horse blood agar plates under anaerobic conditions. The hemolytic activity found in A. actinomycetemcomitans strain 137HE was examined. This activity was detected in the late exponential to early stationary phases of growth. Human erythrocytes were the most susceptible, followed by rabbit, sheep, horse and swine red blood cells. The majority of activity was detected in the cell-associated vesicle fraction. Zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extract from whole cells was semipurified by ammonium sulfate precipitation, preparative isoelectric focusing (IEF) and gel-filtration chromatography to yield a major band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 12 kDa. Heating at 80 C for 30 min and treatment with proteinase K or trypsin resulted in complete disappearance of the hemolytic activity. Sulphydryl reagents enhanced activity and small amounts of cholesterol inhibited it. In summary, we demonstrated the presence of hemolysin in A. actinomycetemcomitans, and examined and characterized it.

• Platt MW
• In vivo hemolytic activity of group B streptococcus is dependent on erythrocyte-bacteria contact and independent of a carrier molecule.
• Curr Microbiol. 1995; 31: 5-9
• Display abstract

• Experiments were performed to determine the interaction between the hemolysin of group B streptococcus (GBS) and sheep erythrocytes. Growing GBS were shown to possess a potent hemolysin at a very early stage of the growth cycle. After separation of the cells from the growth medium, all the hemolytic activity remained with the bacterial cells, and no activity could be detected in the growth medium. When fetal calf serum was added to the media, some "soluble" activity was detected. This activity, however was completely removed by ultracentrifugation, the hemolytic activity being found solely in the pellet. After the hemolysin had formed, no new protein synthesis was needed to cause hemolysis because the addition of chloramphenicol to cells caused no difference in their hemolytic potential. For proof that no short-lived, soluble factors are produced by the bacteria, bacteria and sheep erythrocytes were incubated in contiguous media, separated by a 0.22-microns membrane. No hemolytic activity was detected on the erythrocyte side of the membrane, although high amounts of hemolysin could be extracted from the bacteria. Only when a detergent was added to the growth medium was hemolysis detected from the erythrocytes, showing that extracted hemolysin could indeed pass through the membrane. These results suggest that the hemolysin is attached to the surface of the cell and that contact is needed between the bacteria and erythrocyte to cause lysis. Where soluble activity was detected, it was connected to bacterial fragments.

• Akamatsu T, Moriyama H, Kaneda T
• Genetic mapping of bfmA mutation causing fatty acid deficiency in Bacillus subtilis.
• Can J Microbiol. 1993; 39: 629-33
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• Bacillus subtilis strain 626, defective in the bfmA gene, is a derivative derivatives of B. subtilis strain 168 and requires branched short-chain carboxylic acids for growth. Branched-chain 2-keto acid decarboxylase activity and fatty acid synthesis in B. subtilis strain 626 were 14 and 7%, respectively, of their levels in strain 626-2R, a spontaneously reverted strain. These results indicate that the bfmA mutation is in either the branched-chain 2-keto acid decarboxylase gene itself or its controlling gene. Thus, primer synthesis from the 2-keto acid substrate in strain 626 is defective, causing a deficiency in fatty acid synthesis. A bfmA mutation was transferred to a suitable genetic background and analysed. The bfmA strain, CAC1, was competent and motile, and required 50 microM CaCl2 or 5 microM FeCl3 for growth on glucose minimal agar plates. The bfmA gene was mapped between glyC (320 degrees) and ctrA (325 degrees) and estimated to be at 320 degrees by protoplast fusion and PBS1 phage transduction.

• Liu J, Li Z, Pan N, Chen Z
• Purification and partial characterization of an antibacterial protein LCIII.
• Chin J Biotechnol. 1992; 8: 187-93
• Display abstract

• Total proteins were precipitated by (NH4)2SO4 from the overnight culture supernatant of antagonistic bacterium Bacillus subtilis A014, applied to CM52 column and separated into three main peaks. The preparation of peak III showed to inhibit specifically the growth of rice bacterial blight pathogen Xanthomonas compestris pv. oryzea was further purified with Mono S column on FPLC and named antibacterial protein LCIII. The molecular weight of this protein is 26915Da, pI = 9.12. Analysis of amino acid composition was revealed to be rich in glycine, threonine and serine, and devoid of proline. Twenty-eight amino acids of N-terminal were sequenced by the Edman degradation and computer analysis of this partial sequence showed that antibacterial protein LCIII is a novel one.

• Beecher DJ, Macmillan JD
• Characterization of the components of hemolysin BL from Bacillus cereus.
• Infect Immun. 1991; 59: 1778-84
• Display abstract

• Previously we described the partial purification of a novel hemolysin from Bacillus cereus and showed that hemolytic activity required the combined action of at least two components, called B and L to signify their cell-binding and cell-lytic roles in this activity. On further purification, as described in the present article, a combination of anion-exchange chromatography and polyacrylamide gel electrophoresis separated three proteins, B, L1, and L2 (35, 36, and 45 kDa, respectively). Individually, these proteins were inactive in hemolytic and vascular permeability assays, and combinations of B and L1 or B and L2 were either inactive or slightly active. Combinations of all three moieties produced the unique ring-shaped zone of hemolysis, a previously described characteristic of hemolysin BL, as well as edema and bluing in the vascular permeability assay. Since the vascular permeability assay is known to correlate with enterotoxicity, these results suggest that hemolysin BL is enterotoxigenic. Furthermore, the molecular weights and isoelectric point values of the hemolysin BL components are consistent with those described by others for the multicomponent diarrheal enterotoxin of B. cereus. Immunofluorescent staining of B-treated erythrocytes confirmed that B binds to cells as an initial step required before the L components can act to cause cell lysis.

• Geoffroy C, Alouf JE
• Molecular cloning and characterization of Bacillus alvei thiol-dependent cytolytic toxin expressed in Escherichia coli.
• J Gen Microbiol. 1988; 134: 1961-70
• Display abstract

• A chromosomal DNA fragment from Bacillus alvei, encoding a thiol-dependent haemolytic product known as alveolysin (Mr 60,000, pI 5.0) was cloned in Escherichia coli SK1592, using pBR322 as the vector plasmid. Only a single haemolysin-positive clone was identified, by testing for haemolysis on blood agar plates. The haemolytic material was associated with the host bacterial cell. It was released by ultrasonic disruption and purified 267-fold. A 64 kDa polypeptide of pI 8.2 cofractionated with haemolytic activity during gel filtration chromatography and isoelectric focusing. It behaved identically to alveolysin in its activation by thiols, inactivation by thiol group reagents, inhibition by cholesterol, and neutralization, immunoprecipitation and immunoblotting by immune sera raised against alveolysin and streptolysin O.

• Lammler C, Blobel H
• [Synergistic and antagonistic hemolytic reactions of bacterial proteins].
• Berl Munch Tierarztl Wochenschr. 1987; 100: 95-9

• Zaremba M, Slojewska-Zareba E
• [Hemolytic properties of Yersinia. I. Use of solid media].
• Med Dosw Mikrobiol. 1984; 36: 113-20

• Kreft J, Berger H, Hartlein M, Muller B, Weidinger G, Goebel W
• Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus.
• J Bacteriol. 1983; 155: 681-9
• Display abstract

• From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertion was recloned as a PstI fragment into pJKK3-1, a shuttle vector which replicates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no external or internal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at levels comparable to those of the B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin O-producing strain of Streptococcus pyogenes or from listeriolysin-producing strains of Listeria monocytogenes, no positive hybridization signals were obtained. These data suggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.

• Zwadyk P Jr, Snyder IS
• Purification and kinetic studies of the hemolysin from Escherichia coli.
• Can J Microbiol. 1971; 17: 741-5

• Gestetner B, Shany S, Assa Y
• Medicagenic acid, the factor responsible for hemolytic activity of lucerne saponins.
• Experientia. 1971; 27: 40-1

• Fievet CJ, Gigandet MP, Ansel HC
• Hemolysis of erythrocytes by primary pharmacologic agents.
• Am J Hosp Pharm. 1971; 28: 961-6

• Bernheimer AW, Avigad LS
• Nature and properties of a cytolytic agent produced by Bacillus subtilis.
• J Gen Microbiol. 1970; 61: 361-9

• Chil-Akopian LA, Afrikian EK
• [Hemolytic properties of aerobic sporogenous bacteria].
• Zh Eksp Klin Med. 1967; 7: 22-6

• Reman FC, van Deenen LL
• The action of some synthetic lysolecithins and lecithins on erythrocytes and lipid bilayers.
• Biochim Biophys Acta. 1967; 137: 592-4

• Wiseman GM, Caird JD
• The nature of staphylococcal beta hemolysin. I. Mode of action.
• Can J Microbiol. 1967; 13: 369-76

• MEDVINSKAIA LIu, KOLCHINSKAIA ID, TINIANOVA NZ
• [EFFECT OF SALT COMPONENTS OF THE MEDIUM ON THE MORPHOLOGY, GROWTH AND PROTEASE ACTIVITY OF B. SUBTILIS AND B. MESENTERICUS].
• Mikrobiol Zh. 1964; 26: 14-8

• IMPERATO S
• [IMMUNOELECTROPHORETIC ANALYSIS OF B. SUBTILIS].
• Nuovi Ann Ig Microbiol. 1964; 15: 48-52

• MOTLIKOVA M, WAGNER V, KOUBA K
• [HEMOLYSIN TESTS WITH CATTLE ERYTHROCYTES IN THE DIAGNOSIS OF INFECTIOUS MONONUCLEOSIS].
• Cesk Pediatr. 1964; 19: 41-6

• ADACHI T, KAMIYA H, KOSUGE T
• [STUDIES ON THE METABOLIC PRODUCTS OF BACILLUS SUBTILIS. IV. DETERMINATION AND MECHANISM OF FORMATION OF TETRAMETHYLPYRAZINE].
• Yakugaku Zasshi. 1964; 84: 545-8

• TAUBMAN SB, SO AG, YOUNG FE, DAVIE EW, CORCORAN JW
• EFFECT OF ERYTHROMYCIN ON PROTEIN BIOSYNTHESIS IN BACILLUS SUBTILIS.
• Antimicrob Agents Chemother (Bethesda). 1963; 161: 395-401

• GUSDON JP
• A bactericidin for Bacillus subtilis in pregnancy.
• J Immunol. 1962; 88: 494-9

• BERK RS
• Production and characterization of Pseudomon as aeruginosa hemolysin.
• J Bacteriol. 1962; 84: 1041-8
• Display abstract

• Berk, Richard S. (Wayne State University, Detroit, Mich.). Production and characterization of Pseudomonas aeruginosa hemolysin. J. Bacteriol. 84:1041-1048. 1962.-Hemolysin from Pseudomonas aeruginosa was obtained by either extraction of blood agar media after bacterial growth or by saline washing of cellophane plate-grown cells, hemolytic activity being greatest in the latter preparations. Except for variations in heat stability, both types of preparations appeared to be similar if not identical. Complete hemolysis of red blood cells was observed over a wide pH range, although the optima appeared to be below 6.0. Acidification of agar extracts resulted in a coarse precipitate which appeared to be a hemolysin-agar complex. Partial purification of hemolysin present in cellophane washings was obtained by fractionation with ammonium sulfate or by gel filtration. Further characterization of hemolysin is described.

• ROMIG WR
• Infection of Bacillus subtilis with phenol-extracted bacteriophages.
• Virology. 1962; 16: 452-9

• POLLOCK MR
• The measurement of the liberation of penicillinase from Bacillus subtilis.
• J Gen Microbiol. 1961; 26: 239-53

• ARMSTRONG JJ, BADDILEY J, BUCHANAN JG
• Structure of the ribitol teichoic acid from the walls of Bacillus subtilis.
• Biochem J. 1960; 76: 610-21

• NISHIMURA S
• Extracellular ribonucleases from Bacillus subtilis. I. Crystallization and specificity.
• Biochim Biophys Acta. 1960; 45: 15-27

• TALIAFERRO WH, JAROSLOW BN
• The restoration of hemolysin formation in x-rayed rabbits by nucleic acid derivatives and antagonists of nucleic acid synthesis.
• J Infect Dis. 1960; 107: 341-50

• DEDONDER R
• [Levan-sucrase in Bacillus subtilis].
• Bull Soc Chim Biol (Paris). 1960; 42: 1745-61

• KOYAMA J, NASHIMOTO H
• Immunochemical studies on rabbit hemolysin to sheep red cells. I. The electrophoretic separation of rabbit antisera to sheep red cells.
• Jpn J Exp Med. 1959; 29: 551-9

• NANI S, GIOLITTI G
• [Research on the behavior of the amino acids in Bacillus subtilis grown in a medium containing high concentrations of sodium chloride. II. Free amino acids].
• Boll Ist Sieroter Milan. 1959; 38: 124-7

• DEDONDER R, SLIZEWICZ P
• [Study of the levans produced by Bacillus subtilis. II. Physical properties and molecular weight of native levans and of the products produced on mild acid hydrolysis].
• Bull Soc Chim Biol (Paris). 1958; 40: 873-86

• WEINRACH RS, LAI M, TALMAGE DW
• The relation between hemolysin concentration and hemolytic rate as measured with chromium 51 labeled cells.
• J Infect Dis. 1958; 102: 60-73

• BARDAROV S
• [Research on penicillinase biosynthesis].
• Arch Mikrobiol. 1958; 29: 143-53

• DEDONDER R
• [Study of the levans produced by Bacillus subtilis. I. Mild acid hydrolysis of native levans].
• Bull Soc Chim Biol (Paris). 1958; 40: 863-71

• CHIBNALL AC, REES MW, RICHARDS FM
• Structure of the polyglutamic acid from Bacillus subtilis.
• Biochem J. 1958; 68: 129-35

• WILLIAMS GR
• Haemolytic material from aerobic sporing bacilli.
• J Gen Microbiol. 1957; 16: 16-21

• SOCIAS A, PEREZ SILVA J
• [Bacillus subtilis as a normal flora of paprika].
• Microbiol Esp. 1956; 9: 383-95

• HOSODA J, NOMURA M
• Nature of the primary action of the autolysin of Bacillus subtilis.
• J Bacteriol. 1956; 72: 573-81

• LULLA BS
• Antibiotic activity as shown by a highly amylolytic strain of Bacillus subtilis.
• Nature. 1949; 163: 489-489

• HASSALL CH
• Subtilin C; an antibiotic concentrate from Bacillus subtilis.
• Nature. 1948; 161: 317-317

• PONDER E
• A method for determining the form of the distribution of red cell resistances to simple hemolysin.
• Blood. 1948; 3: 556-65

• FEENEY RE, GARIBALDI JA, HUMPHREYS EM
• Nutritional studies on subtilin formation by Bacillus subtilis.
• Arch Biochem. 1948; 17: 435-45

• Blank IH, Kersten H
• The Inhibition of Growth of Bacillus subtilis on a Modified Extract Agar by X-radiation of the Medium.
• J Bacteriol. 1935; 30: 21-32

• Nicholls EE
• THE INCIDENCE AND BIOLOGICAL CHARACTERISTICS OF THE HEMOLYTIC BACILLUS COLI IN THE STOOLS OF HEALTHY INDIVIDUALS.
• J Clin Invest. 1934; 13: 479-94

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