SMART MODE:

NORMAL GENOMIC

• Nolan LM et al.
• Extragenic suppressor mutations that restore twitching motility to fimL mutants of Pseudomonas aeruginosa are associated with elevated intracellular cyclic AMP levels.
• Microbiologyopen. 2012; 1: 490-501
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• Cyclic AMP (cAMP) is a signaling molecule that is involved in the regulation of multiple virulence systems of the opportunistic pathogen Pseudomonas aeruginosa. The intracellular concentration of cAMP in P. aeruginosa cells is tightly controlled at the levels of cAMP synthesis and degradation through regulation of the activity and/or expression of the adenylate cyclases CyaA and CyaB or the cAMP phosphodiesterase CpdA. Interestingly, mutants of fimL, which usually demonstrate defective twitching motility, frequently revert to a wild-type twitching-motility phenotype presumably via the acquisition of an extragenic suppressor mutation(s). In this study, we have characterized five independent fimL twitching-motility revertants and have determined that all have increased intracellular cAMP levels compared with the parent fimL mutant. Whole-genome sequencing revealed that only one of these fimL revertants has acquired a loss-of-function mutation in cpdA that accounts for the elevated levels of intracellular cAMP. As mutation of cpdA did not account for the restoration of twitching motility observed in the other four fimL revertants, these observations suggest that there is at least another, as yet unidentified, site of extragenic suppressor mutation that can cause phenotypic reversion in fimL mutants and modulation of intracellular cAMP levels of P. aeruginosa.

• Harvey H, Kus JV, Tessier L, Kelly J, Burrows LL
• Pseudomonas aeruginosa D-arabinofuranose biosynthetic pathway and its role in type IV pilus assembly.
• J Biol Chem. 2011; 286: 28128-37
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• Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with alpha1,5-linked D-arabinofuranose (d-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of alpha1,5-D-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv3791, required for synthesis of a polyprenyl-linked d-ribose precursor and its epimerization to D-Araf. Pa5196 mutants lacking the orthologues of those genes had non-arabinosylated pilins, poor twitching motility, and significantly fewer surface pili than the wild type even in a retraction-deficient (pilT) background. The Pa5196 pilus system assembled heterologous non-glycosylated pilins efficiently, demonstrating that it does not require post-translationally modified subunits. Together the data suggest that pilins of group IV strains need to be glycosylated for productive subunit-subunit interactions. A recombinant P. aeruginosa PAO1 strain co-expressing the genes for d-Araf biosynthesis, the pilin modification enzyme TfpW, and the acceptor PilA(IV) produced arabinosylated pili, confirming that the Pa5196 genes identified are both necessary and sufficient. A P. aeruginosa epimerase knock-out could be complemented with the corresponding Mycobacterium smegmatis gene, demonstrating conservation between the systems of the Corynebacterineae and Pseudomonas. This work describes a novel Gram-negative pathway for biosynthesis of d-Araf, a key therapeutic target in Corynebacterineae.

• Heiniger RW, Winther-Larsen HC, Pickles RJ, Koomey M, Wolfgang MC
• Infection of human mucosal tissue by Pseudomonas aeruginosa requires sequential and mutually dependent virulence factors and a novel pilus-associated adhesin.
• Cell Microbiol. 2010; 12: 1158-73
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• Tissue damage predisposes humans to life-threatening disseminating infection by the opportunistic pathogen Pseudomonas aeruginosa. Bacterial adherence to host tissue is a critical first step in this infection process. It is well established that P. aeruginosa attachment to host cells involves type IV pili (TFP), which are retractile surface fibres. The molecular details of attachment and the identity of the bacterial adhesin and host receptor remain controversial. Using a mucosal epithelium model system derived from primary human tissue, we show that the pilus-associated protein PilY1 is required for bacterial adherence. We establish that P. aeruginosa preferentially binds to exposed basolateral host cell surfaces, providing a mechanistic explanation for opportunistic infection of damaged tissue. Further, we demonstrate that invasion and fulminant infection of intact host tissue requires the coordinated and mutually dependent action of multiple bacterial factors, including pilus fibre retraction and the host cell intoxication system, termed type III secretion. Our findings offer new and important insights into the complex interactions between a pathogen and its human host and provide compelling evidence that PilY1 serves as the principal P. aeruginosa adhesin for human tissue and that it specifically recognizes a host receptor localized or enriched on basolateral epithelial cell surfaces.

• Forslund AL et al.
• The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis.
• BMC Microbiol. 2010; 10: 227-227
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• BACKGROUND: All four Francisella tularensis subspecies possess gene clusters with potential to express type IV pili (Tfp). These clusters include putative pilin genes, as well as pilB, pilC and pilQ, required for secretion and assembly of Tfp. A hallmark of Tfp is the ability to retract the pilus upon surface contact, a property mediated by the ATPase PilT. Interestingly, out of the two major human pathogenic subspecies only the highly virulent type A strains have a functional pilT gene. RESULTS: In a previous study, we were able to show that one pilin gene, pilA, was essential for virulence of a type B strain in a mouse infection model. In this work we have examined the role of several Tfp genes in the virulence of the pathogenic type A strain SCHU S4. pilA, pilC, pilQ, and pilT were mutated by in-frame deletion mutagenesis. Interestingly, when mice were infected with a mixture of each mutant strain and the wild-type strain, the pilA, pilC and pilQ mutants were out-competed, while the pilT mutant was equally competitive as the wild-type. CONCLUSIONS: This suggests that expression and surface localisation of PilA contribute to virulence in the highly virulent type A strain, while PilT was dispensable for virulence in the mouse infection model.

• Harvey H, Habash M, Aidoo F, Burrows LL
• Single-residue changes in the C-terminal disulfide-bonded loop of the Pseudomonas aeruginosa type IV pilin influence pilus assembly and twitching motility.
• J Bacteriol. 2009; 191: 6513-24
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• PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.

• Chiang P, Sampaleanu LM, Ayers M, Pahuta M, Howell PL, Burrows LL
• Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU.
• Microbiology. 2008; 154: 114-26
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• Type IV pili are retractable protein fibres used by many bacterial pathogens for adherence, twitching motility, biofilm development and host colonization. In Pseudomonas aeruginosa, PilB and PilT are bipolar proteins belonging to the secretion NTPase superfamily, and power pilus extension and retraction, respectively, while the unipolar PilT paralogue PilU supports pilus retraction in an unknown manner. Assay of purified 6xHis-tagged PilB, PilT and PilU from P. aeruginosa showed that all three proteins have ATPase activities in vitro. Conserved residues in the Walker A (WA), Walker B (WB), Asp Box and His Box motifs characteristic of secretion NTPases were mutated, and complementation of twitching motility was tested. Mutation of conserved WA or WB residues in any of the three ATPases abrogated twitching motility, and for the WA mutant of PilT caused loss of polar localization. The requirement for three invariant acidic residues in the Asp Box motif, and for two invariant His residues in the His Box motif varied, with PilB being the least tolerant of changes. In all three proteins, the third acidic residue in the Asp Box and the second His of the His Box were crucial for function; mutation of these residues caused loss of PilT ATPase activity in vitro. Modelling of the effects of these mutations on the crystal structures of Aquifex aeolicus PilT and Vibrio cholerae EpsE (a PilB homologue) showed that the critical Asp Box and His Box residues contribute to a catalytic pocket that surrounds the ligand. These results provide experimental evidence differentiating widely conserved Asp and His Box residues that are essential for function from those whose roles are modulated by specific local environments.

• Horzempa J et al.
• Immunization with a Pseudomonas aeruginosa 1244 pilin provides O-antigen-specific protection.
• Clin Vaccine Immunol. 2008; 15: 590-7
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• The O antigen is both a major structural outer membrane component and the dominant epitope of most gram-negative bacteria. Pseudomonas aeruginosa 1244 produces a type IV pilus and covalently links an O-antigen repeating unit to each pilin monomer. Here we show that immunization of mice with pure pilin from strain 1244 by use of either the mouse respiratory model or the thermal injury model resulted in protection from challenge with a pilus-null O-antigen-producing 1244 mutant. These results provide evidence that the pilin glycan stimulates a protective response that targets the O antigen, suggesting that this system could be used as the basis for the development of a variety of bioconjugate vaccines protective against gram-negative bacteria.

• Han X et al.
• Twitching motility is essential for virulence in Dichelobacter nodosus.
• J Bacteriol. 2008; 190: 3323-35
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• Type IV fimbriae are essential virulence factors of Dichelobacter nodosus, the principal causative agent of ovine foot rot. The fimA fimbrial subunit gene is required for virulence, but fimA mutants exhibit several phenotypic changes and it is not certain if the effects on virulence result from the loss of type IV fimbria-mediated twitching motility, cell adherence, or reduced protease secretion. We showed that mutation of either the pilT or pilU gene eliminated the ability to carry out twitching motility. However, the pilT mutants displayed decreased adhesion to epithelial cells and reduced protease secretion, whereas the pilU mutants had wild-type levels of extracellular protease secretion and adherence. These data provided evidence that PilT is required for the type IV fimbria-dependent protease secretion pathway in D. nodosus. It was postulated that sufficient fimbrial retraction must occur in the pilU mutants to allow protease secretion, but not twitching motility, to take place. Although no cell movement was detected in a pilU mutant of D. nodosus, aberrant motion was detected in an equivalent mutant of Pseudomonas aeruginosa. These observations explain how in D. nodosus protease secretion can occur in a pilU mutant but not in a pilT mutant. In addition, virulence studies with sheep showed that both the pilT and pilU mutants were avirulent, providing evidence that mutation of the type IV fimbrial system affects virulence by eliminating twitching motility, not by altering cell adherence or protease secretion.

• Yao H, Zhang L, Shan Z, Li Y, Xu H, Qiao M
• [Impact of Pseudomonas aeruginosa gene PA1550 on its swimming and twitching motility].
• Wei Sheng Wu Xue Bao. 2008; 48: 959-62
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• OBJECTIVE: Studying the genes involved in swimming and twitching motility of Pseudomonas aeruginosa. METHODS: We used Mu transposition technique, gene cloning, nucleotide sequencing and the trans-complementation experiment to study the genes involved in twitching motility and swimming motility of P. aeruginosa strain PA68 isolated from a patient with bronchiectasis. RESULTS: A mutant deficient in both swimming and twitching motility was isolated out of about 2000 mini-Mu insertion mutants. The result of GenBank BLAST showed that the mini-Mu transposon had inserted into the gene PA1550 with unknown function. Analyses on the operon of PA550 and the polar effect revealed that Mu transposon had no effect on the transcription of the downstream genes of PA1550. CONCLUSION: PA1550 is involved in the swimming and twitching motility of Pseudomonas aeruginosa.

• Cui Z, Maruyama Y, Mikami B, Hashimoto W, Murata K
• Crystal structure of glycoside hydrolase family 78 alpha-L-Rhamnosidase from Bacillus sp. GL1.
• J Mol Biol. 2007; 374: 384-98
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• alpha-L-Rhamnosidase (EC 3.2.1.40) catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides. Bacillus sp. GL1 alpha-L-rhamnosidase (RhaB), a member of glycoside hydrolase (GH) family 78, is responsible for degrading the bacterial biofilm gellan, and also functions as a debittering agent for citrus fruit in the food and beverage industries through the release of rhamnose from plant glycoside, naringin. The X-ray crystal structure of RhaB was determined by single-wavelength anomalous diffraction using a selenomethionine derivative and refined at 1.9 A resolution with a final R-factor of 18.2%. As is seen in the homodimeric form of the active enzyme, the structure of RhaB in crystal packing is a homodimer containing 1908 amino acids (residues 3-956), 43 glycerol molecules, four calcium ions, and 1755 water molecules. The overall structure consists of five domains, four of which are beta-sandwich structures designated as domains N, D1, D2, and C, and an (alpha/alpha)(6)-barrel structure designated as domain A. Structural comparison by DALI showed that RhaB shares its highest level of structural similarity with chitobiose phosphorylase (Z score of 25.3). The structure of RhaB in complex with the reaction product rhamnose (inhibitor constant, K(i)=1.8 mM) was also determined and refined at 2.1 A with a final R-factor of 19.5%. Rhamnose is bound to the deep cleft of the (alpha/alpha)(6)-barrel domain, as is seen in the clan-L GHs. Several negatively charged residues, such as Asp567, Glu572, Asp579, and Glu841, conserved in GH family 78 enzymes, interact with rhamnose, and RhaB mutants of these residues have drastically reduced enzyme activity, indicating that the residues are crucial for enzyme catalysis and/or substrate binding. To our knowledge, this is the first report on the determination of the crystal structure of alpha-L-rhamnosidase and identification of its clan-L (alpha/alpha)(6)-barrel as a catalytic domain.

• May M et al.
• Structural and functional analysis of two glutamate racemase isozymes from Bacillus anthracis and implications for inhibitor design.
• J Mol Biol. 2007; 371: 1219-37
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• Glutamate racemase (RacE) is responsible for converting l-glutamate to d-glutamate, which is an essential component of peptidoglycan biosynthesis, and the primary constituent of the poly-gamma-d-glutamate capsule of the pathogen Bacillus anthracis. RacE enzymes are essential for bacterial growth and lack a human homolog, making them attractive targets for the design and development of antibacterial therapeutics. We have cloned, expressed and purified the two glutamate racemase isozymes, RacE1 and RacE2, from the B. anthracis genome. Through a series of steady-state kinetic studies, and based upon the ability of both RacE1 and RacE2 to catalyze the rapid formation of d-glutamate, we have determined that RacE1 and RacE2 are bona fide isozymes. The X-ray structures of B. anthracis RacE1 and RacE2, in complex with d-glutamate, were determined to resolutions of 1.75 A and 2.0 A. Both enzymes are dimers with monomers arranged in a "tail-to-tail" orientation, similar to the B. subtilis RacE structure, but differing substantially from the Aquifex pyrophilus RacE structure. The differences in quaternary structures produce differences in the active sites of racemases among the various species, which has important implications for structure-based, inhibitor design efforts within this class of enzymes. We found a Val to Ala variance at the entrance of the active site between RacE1 and RacE2, which results in the active site entrance being less sterically hindered for RacE1. Using a series of inhibitors, we show that this variance results in differences in the inhibitory activity against the two isozymes and suggest a strategy for structure-based inhibitor design to obtain broad-spectrum inhibitors for glutamate racemases.

• Francetic O, Buddelmeijer N, Lewenza S, Kumamoto CA, Pugsley AP
• Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system.
• J Bacteriol. 2007; 189: 1783-93
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• The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

• Baynham PJ, Ramsey DM, Gvozdyev BV, Cordonnier EM, Wozniak DJ
• The Pseudomonas aeruginosa ribbon-helix-helix DNA-binding protein AlgZ (AmrZ) controls twitching motility and biogenesis of type IV pili.
• J Bacteriol. 2006; 188: 132-40
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• Pseudomonas aeruginosa is an opportunistic pathogen that is commonly found in water and soil. In order to colonize surfaces with low water content, P. aeruginosa utilizes a flagellum-independent form of locomotion called twitching motility, which is dependent upon the extension and retraction of type IV pili. This study demonstrates that AlgZ, previously identified as a DNA-binding protein absolutely required for transcription of the alginate biosynthetic operon, is required for twitching motility. AlgZ may be required for the biogenesis or function of type IV pili in twitching motility. Transmission electron microscopy analysis of an algZ deletion in nonmucoid PAO1 failed to detect surface pili. To examine expression and localization of PilA (the major pilin subunit), whole-cell extracts and cell surface pilin preparations were analyzed by Western blotting. While the PilA levels present in whole-cell extracts were similar for wild-type P. aeruginosa and P. aeruginosa with the algZ deletion, the amount of PilA on the surface of the cells was drastically reduced in the algZ mutant. Analysis of algZ and algD mutants indicates that the DNA-binding activity of AlgZ is essential for the regulation of twitching motility and that this is independent of the role of AlgZ in alginate expression. These data show that AlgZ DNA-binding activity is required for twitching motility independently of its role in alginate production and that this involves the surface localization of type IV pili. Given this new role in twitching motility, we propose that algZ (PA3385) be designated amrZ (alginate and motility regulator Z).

• Brux C et al.
• The structure of an inverting GH43 beta-xylosidase from Geobacillus stearothermophilus with its substrate reveals the role of the three catalytic residues.
• J Mol Biol. 2006; 359: 97-109
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• beta-D-Xylosidases are glycoside hydrolases that catalyze the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicellulose. Here we describe the enzyme-substrate crystal structure of an inverting family 43 beta-xylosidase, from Geobacillus stearothermophilus T-6 (XynB3). Each XynB3 monomeric subunit is organized in two domains: an N-terminal five-bladed beta-propeller catalytic domain, and a beta-sandwich domain. The active site possesses a pocket topology, which is mainly constructed from the beta-propeller domain residues, and is closed on one side by a loop that originates from the beta-sandwich domain. This loop restricts the length of xylose units that can enter the active site, consistent with the exo mode of action of the enzyme. Structures of the enzyme-substrate (xylobiose) complex provide insights into the role of the three catalytic residues. The xylose moiety at the -1 subsite is held by a large number of hydrogen bonds, whereas only one hydroxyl of the xylose unit at the +1 subsite can create hydrogen bonds with the enzyme. The general base, Asp15, is located on the alpha-side of the -1 xylose sugar ring, 5.2 Angstroms from the anomeric carbon. This location enables it to activate a water molecule for a single-displacement attack on the anomeric carbon, resulting in inversion of the anomeric configuration. Glu187, the general acid, is 2.4 Angstroms from the glycosidic oxygen atom and can protonate the leaving aglycon. The third catalytic carboxylic acid, Asp128, is 4 Angstroms from the general acid; modulating its pK(a) and keeping it in the correct orientation relative to the substrate. In addition, Asp128 plays an important role in substrate binding via the 2-O of the glycon, which is important for the transition-state stabilization. Taken together, these key roles explain why Asp128 is an invariant among all five-bladed beta-propeller glycoside hydrolases.

• Popovic B, Tang X, Chirgadze DY, Huang F, Blundell TL, Spencer JB
• Crystal structures of the PLP- and PMP-bound forms of BtrR, a dual functional aminotransferase involved in butirosin biosynthesis.
• Proteins. 2006; 65: 220-30
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• The aminotransferase (BtrR), which is involved in the biosynthesis of butirosin, a 2-deoxystreptamine (2-DOS)-containing aminoglycoside antibiotic produced by Bacillus circulans, catalyses the pyridoxal phosphate (PLP)-dependent transamination reaction both of 2-deoxy-scyllo-inosose to 2-deoxy-scyllo-inosamine and of amino-dideoxy-scyllo-inosose to 2-DOS. The high-resolution crystal structures of the PLP- and PMP-bound forms of BtrR aminotransferase from B. circulans were solved at resolutions of 2.1 A and 1.7 A with R(factor)/R(free) values of 17.4/20.6 and 19.9/21.9, respectively. BtrR has a fold characteristic of the aspartate aminotransferase family, and sequence and structure analysis categorises it as a member of SMAT (secondary metabolite aminotransferases) subfamily. It exists as a homodimer with two active sites per dimer. The active site of the BtrR protomer is located in a cleft between an alpha helical N-terminus, a central alphabetaalpha sandwich domain and an alphabeta C-terminal domain. The structures of the PLP- and PMP-bound enzymes are very similar; however BtrR-PMP lacks the covalent bond to Lys192. Furthermore, the two forms differ in the side-chain conformations of Trp92, Asp163, and Tyr342 that are likely to be important in substrate selectivity and substrate binding. This is the first three-dimensional structure of an enzyme from the butirosin biosynthesis gene cluster.

• Calvio C et al.
• Swarming differentiation and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon.
• J Bacteriol. 2005; 187: 5356-66
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• The number and disposition of flagella harbored by eubacteria are regulated by a specific trait successfully maintained over generations. The genes governing the number of flagella in Bacillus subtilis have never been identified, although the ifm locus has long been recognized to influence the motility phenotype of this microorganism. The characterization of a spontaneous ifm mutant of B. subtilis, displaying diverse degrees of cell flagellation in both liquid and solid media, raised the question of how the ifm locus governs the number and assembly of functional flagella. The major finding of this investigation is the characterization of a newly identified dicistronic operon, named swrA, that controls both swimming motility and swarming differentiation in B. subtilis. Functional analysis of the swrA operon allowed swrAA (previously named swrA [D. B. Kearns, F. Chu, R. Rudner, and R. Losick, Mol. Microbiol. 52:357-369, 2004]) to be the first gene identified in B. subtilis that controls the number of flagella in liquid environments and the assembly of flagella in response to cell contact with solid surfaces. Evidence is given that the second gene of the operon, swrAB, is essential for enabling the surface-adhering cells to undergo swarming differentiation. Preliminary data point to a molecular interaction between the two gene products.

• Camberg JL, Sandkvist M
• Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE.
• J Bacteriol. 2005; 187: 249-56
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• The type II secretion system is a macromolecular assembly that facilitates the extracellular translocation of folded proteins in gram-negative bacteria. EpsE, a member of this secretion system in Vibrio cholerae, contains a nucleotide-binding motif composed of Walker A and B boxes that are thought to participate in binding and hydrolysis of ATP and displays structural homology to other transport ATPases. Here we demonstrate that purified EpsE is an Mg2+-dependent ATPase and define optimal conditions for the hydrolysis reaction. EpsE displays concentration-dependent activity, which may suggest that the active form is oligomeric. Size exclusion chromatography showed that the majority of purified EpsE is monomeric; however, detailed analyses of specific activities obtained following gel filtration revealed the presence of a small population of active oligomers. We further report that EpsE binds zinc through a tetracysteine motif near its carboxyl terminus, yet metal displacement assays suggest that zinc is not required for catalysis. Previous studies describing interactions between EpsE and other components of the type II secretion pathway together with these data further support the hypothesis that EpsE functions to couple energy to the type II apparatus, thus enabling secretion.

• Whitchurch CB et al.
• Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa.
• Mol Microbiol. 2004; 52: 873-93
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• Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine- and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its C-terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.

• Klockgether J, Reva O, Larbig K, Tummler B
• Sequence analysis of the mobile genome island pKLC102 of Pseudomonas aeruginosa C.
• J Bacteriol. 2004; 186: 518-34
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• The Pseudomonas aeruginosa plasmid pKLC102 coexists as a plasmid and a genome island in clone C strains. Whereas the related plasmid pKLK106 reversibly recombines with P. aeruginosa clone K chromosomes at one of the two tRNA(Lys) genes, pKLC102 is incorporated into the tRNA(Lys) gene only close to the pilA locus. Targeting of the other tRNA(Lys) copy in the chromosome is blocked by a 23,395-bp mosaic of truncated PAO open reading frames, transposons, and pKLC102 homologs. Annotation and phylogenetic analysis of the large 103,532-bp pKLC102 sequence revealed that pKLC102 is a hybrid of plasmid and phage origin. The plasmid lineage conferred oriV and genes for replication, partitioning, and conjugation, including a pil cluster encoding type IV thin sex pili and an 8,524-bp chvB glucan synthetase gene that is known to be a major determinant for host tropism and virulence. The phage lineage conferred integrase, att, and a syntenic set of conserved hypothetical genes also observed in the tRNA(Gly)-associated genome islands of P. aeruginosa clone C chromosomes. In subgroup C isolates from patients with cystic fibrosis, pKLC102 was irreversibly fixed into the chromosome by the insertion of the large 23,061-bp class I transposon TNCP23, which is a composite of plasmid, integron, and IS6100 elements. Intramolecular transposition of a copy of IS6100 led to chromosomal inversions and disruption of plasmid synteny. The case of pKLC102 in P. aeruginosa clone C documents the intraclonal evolution of a genome island from a mobile ancestor via a reversibly integrated state to irreversible incorporation and dissipation in the chromosome.

• Shan Z et al.
• Identification of two new genes involved in twitching motility in Pseudomonas aeruginosa.
• Microbiology. 2004; 150: 2653-61
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• Mu transposition complexes were used for transposon mutagenesis of Pseudomonas aeruginosa strain PA68. Mu DNA transposition complexes were assembled with MuA transposase and an artificial mini-Mu transposon in vitro, and introduced into Pseudomonas aeruginosa by electroporation. Eight mutants deficient in twitching motility were isolated. Southern blotting confirmed that the insertions had occurred as single events. DNA sequencing of the region flanking the insertion in the twitching-motility mutants revealed that the mini-Mu transposon had inserted into six different genes, PAO171, PA1822, PAO413, PA4959, PA4551 and PA5040. Four of these have previously been proven to be needed for twitching motility, whereas the PA1822 and PA0171 genes have not previously been shown to be required for twitching motility. The twitching-motility defect in the PA1822 mutant was partially complemented by providing the PA1822 gene in trans, and the defect in the PA0171 mutant was fully complemented when PA0171 was provided. A PA0171 mutant and a PA1822 mutant were constructed by gene replacement in the P. aeruginosa PAO1 strain. These mutants were deficient in twitching motility, showing that both the PA1822 and the PA0171 gene are involved in twitching motility.

• Stevenson A, Macdonald J, Roberts M
• Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae.
• Vet Microbiol. 2003; 92: 121-34
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• Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumoniae. Little is known about the mechanisms by which A. pleuropneumoniae colonises the respiratory tract. Fimbriae are common mediators of bacterial adherence to mucosal epithelia and have been observed on the surface of A. pleuropneumoniae cells. Here we report the identification and characterisation of the type 4 fimbrial structural gene (apfA) from A. pleuropneumoniae. In addition a number of open reading frames were identified in A. pleuropneumoniae that have significant homology to type 4 fimbrial biogenesis genes from other species, including a putative leader specific peptidase (apfD). A. pleuropneumoniae apfA codes for a predicted polypeptide of approximately 16kDa, removal of the leader sequence at the predicted cleavage site would yield a 14.5kDa polypeptide. The first 30 residues of the mature polypeptide are well conserved with other members of the group A type 4 fimbriae family. The signal sequence of ApfA is 13 amino acids in length and, unusually, the residue that precedes the cleavage site is alanine rather than glycine which is found in most other type 4 fimbriae. The C-terminus of ApfA possesses cysteine residues that are conserved in type 4 fimbriae of many species. In other type 4 fimbriae the distal C-terminal cysteines form a disulphide bond that produces a loop, which is important for the function of fimbriae and also comprises a major antigenic determinant. A motif within the predicted loop in ApfA was found to be highly conserved in type 4 fimbriae of other HAP organisms (Haemophilus, Actinobacillus, Pasteurella). The A. pleuropneumoniae type 4 fimbrial biogenesis genes showed the strongest homology to putative type 4 fimbrial genes of Haemophilus ducreyi. A. pleuropneumoniae apfA gene was shown to be present and highly conserved in different serotypes of A. pleuropneumoniae. Recombinant ApfA was produced and used to raise anti-ApfA antisera.

• Chiang P, Burrows LL
• Biofilm formation by hyperpiliated mutants of Pseudomonas aeruginosa.
• J Bacteriol. 2003; 185: 2374-8
• Display abstract

• Under static growth conditions, hyperpiliated, nontwitching pilT and pilU mutants of Pseudomonas aeruginosa formed dense biofilms, showing that adhesion, not twitching motility, is necessary for biofilm initiation. Under flow conditions, the pilT mutant formed mushroom-like structures larger than those of the wild type but the pilU mutant was defective in biofilm formation. Therefore, twitching motility affects the development of biofilm structure, possibly through modulation of detachment.

• Antonyuk SV, Eady RR, Strange RW, Hasnain SS
• The structure of glyceraldehyde 3-phosphate dehydrogenase from Alcaligenes xylosoxidans at 1.7 A resolution.
• Acta Crystallogr D Biol Crystallogr. 2003; 59: 835-42
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• The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the Gram-negative denitrifying bacterial species Alcaligenes xylosoxidans was purified and crystallized as a contaminant protein during purification of nitrous oxide reductase. This is the first structure of a GAPDH from a denitrifying species. The crystal structure was solved at 1.7 A resolution by molecular replacement using the structure of GAPDH from Bacillus stearothermophilus as a starting model. The quality of the structure enabled the amino-acid sequence of the A. xylosoxidans GAPDH to be assigned. The structure is that of the apo-enzyme, lacking the NAD+ cofactor and with the active-site residue Cys154 oxidized. The global structure of the enzyme has a homotetrameric quaternary structure similar to that observed for its bacterial and eukaryotic counterparts. The essential role of Cys154 in the enzyme activity has been confirmed. In monomer O two half-occupancy sulfate ions were found at the active site, which are analogous to the substrate and the "attacking" phosphate seen in B. stearothermophilus. One half-occupancy sulfate ion is also located in the substrate-binding site of monomer P.

• Hondoh H, Kuriki T, Matsuura Y
• Three-dimensional structure and substrate binding of Bacillus stearothermophilus neopullulanase.
• J Mol Biol. 2003; 326: 177-88
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• Crystal structures of Bacillus stearothermophilus TRS40 neopullulanase and its complexes with panose, maltotetraose and isopanose were determined at resolutions of 1.9, 2.4, 2.8 and 3.2A, respectively. Since the latter two carbohydrates are substrates of this enzyme, a deactivated mutant at the catalytic residue Glu357-->Gln was used for complex crystallization. The structures were refined at accuracies with r.m.s. deviations of bond lengths and bond angles ranging from 0.005A to 0.008A and 1.3 degrees to 1.4 degrees, respectively. The active enzyme forms a dimer in the crystalline state and in solution. The monomer enzyme is composed of four domains, N, A, B and C, and has a (beta/alpha)(8)-barrel in domain A. The active site lies between domain A and domain N from the other monomer. The results show that dimer formation makes the active-site cleft narrower than those of ordinary alpha-amylases, which may contribute to the unique substrate specificity of this enzyme toward both alpha-1,4 and alpha-1,6-glucosidic linkages. This specificity may be influenced by the subsite structure. Only subsites -1 and -2 are commonly occupied by the product and substrates, suggesting that equivocal recognition occurs at the other subsites, which contributes to the wide substrate specificity of this enzyme.

• Strych U, Benedik MJ
• Mutant analysis shows that alanine racemases from Pseudomonas aeruginosa and Escherichia coli are dimeric.
• J Bacteriol. 2002; 184: 4321-5
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• Alanine racemases are ubiquitous prokaryotic enzymes providing the essential peptidoglycan precursor D-alanine. We present evidence that the enzymes from Pseudomonas aeruginosa and Escherichia coli function exclusively as homodimers. Moreover, we demonstrate that expression of a K35A Y235A double mutation of dadX in E. coli suppresses bacterial growth in a dominant negative fashion.

• Watnick PI, Lauriano CM, Klose KE, Croal L, Kolter R
• The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139.
• Mol Microbiol. 2001; 39: 223-35
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• Throughout most of history, epidemic and pandemic cholera was caused by Vibrio cholerae of the serogroup O1. In 1992, however, a V. cholerae strain of the serogroup O139 emerged as a new agent of epidemic cholera. Interestingly, V. cholerae O139 forms biofilms on abiotic surfaces more rapidly than V. cholerae O1 biotype El Tor, perhaps because regulation of exopolysaccharide synthesis in V. cholerae O139 differs from that in O1 El Tor. Here, we show that all flagellar mutants of V. cholerae O139 have a rugose colony morphology that is dependent on the vps genes. This suggests that the absence of the flagellar structure constitutes a signal to increase exopolysaccharide synthesis. Furthermore, although exopolysaccharide production is required for the development of a three-dimensional biofilm, inappropriate exopolysaccharide production leads to inefficient colonization of the infant mouse intestinal epithelium by flagellar mutants. Thus, precise regulation of exopolysaccharide synthesis is an important factor in the survival of V. cholerae O139 in both aquatic environments and the mammalian intestine.

• Schmidt A et al.
• Three-dimensional structure of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli complexed with a PLP-substrate intermediate: inferred reaction mechanism.
• Biochemistry. 2001; 40: 5151-60
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• 2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phosphate (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine. It acts in concert with threonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of cofactor CoA, to glycine and acetyl-CoA. The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escherichia coli and human enzymes. We present the three-dimensional structure of E. coli KBL determined at 2.0 A resolution. KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspartate aminotransferase. Its closest structural homologue is E. coli 8-amino-7-oxononanoate synthase. Like many other members of the alpha family, the functional form of KBL is a dimer, and one such dimer is found in the asymmetric unit in the crystal. There are two active sites per dimer, located at the dimer interface. Both monomers contribute side chains to each active/substrate binding site. Electron density maps indicated the presence in the crystal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an external aldimine, which remained bound to KBL throughout the protein purification procedure. The observed interactions between the aldimine and the side chains in the substrate binding site explain the specificity for the substrate and provide the basis for a detailed proposal of the reaction mechanism of KBL. A putative binding site of the CoA cofactor was assigned, and implications for the cooperation with threonine dehydrogenase were considered.

• Morais MC, Zhang W, Baker AS, Zhang G, Dunaway-Mariano D, Allen KN
• The crystal structure of bacillus cereus phosphonoacetaldehyde hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily.
• Biochemistry. 2000; 39: 10385-96
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• Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor. The reaction proceeds via a novel bicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group from the Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this study, the X-ray crystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product) analogue tungstate (K(i) = 50 microM) and the Mg(II) cofactor was determined to 3.0 A resolution with an R(cryst) = 0.248 and R(free) = 0.284. Each monomer is made up of an alpha/beta core domain consisting of a centrally located six-stranded parallel beta-sheet surrounded by six alpha-helices. Two flexible, solvated linkers connect to a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two alpha8 helices from the respective core domains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interface of each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group of Arg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-line attack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domain residues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution. The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conserved active-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residues at these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.

• Wolfgang M, van Putten JP, Hayes SF, Dorward D, Koomey M
• Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili.
• EMBO J. 2000; 19: 6408-18
• Display abstract

• Type IV pili (Tfp) are a unique class of multifunctional surface organelles in Gram-negative bacteria, which play important roles in prokaryotic cell biology. Although components of the Tfp biogenesis machinery have been characterized, it is not clear how they function or interact. Using Neisseria gonorrhoeae as a model system, we report here that organelle biogenesis can be resolved into two discrete steps: fiber formation and translocation of the fiber to the cell surface. This conclusion is based on the capturing of an intermediate state in which the organelle is retained within the cell owing to the simultaneous absence of the secretin family member and biogenesis component PilQ and the twitching motility/pilus retraction protein PilT. This finding is the first demonstration of a specific translocation defect associated with loss of secretin function, and additionally confirms the role of PilT as a conditional antagonist of stable pilus fiber formation. These findings have important implications for Tfp structure and function and are pertinent to other membrane translocation systems that utilize a highly related set of components.

• Pintar A, Guez V, Castagne C, Bedouelle H, Delepierre M
• Secondary structure of the C-terminal domain of the tyrosyl-transfer RNA synthetase from Bacillus stearothermophilus: a novel type of anticodon binding domain?
• FEBS Lett. 1999; 446: 81-5
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• The tyrosyl-tRNA synthetase catalyzes the activation of tyrosine and its coupling to the cognate tRNA. The enzyme is made of two domains: an N-terminal catalytic domain and a C-terminal domain that is necessary for tRNA binding and for which it was not possible to determine the structure by X-ray crystallography. We determined the secondary structure of the C-terminal domain of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus by nuclear magnetic resonance methods and found that it is of the alpha+beta type. Its arrangement differs from those of the other anticodon binding domains whose structure is known. We also found that the isolated C-terminal domain behaves as a folded globular protein, and we suggest the presence of a flexible linker between the two domains.

• Pujol C, Eugene E, Marceau M, Nassif X
• The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion.
• Proc Natl Acad Sci U S A. 1999; 96: 4017-22
• Display abstract

• The ability of Neisseria meningitidis (MC) to interact with cellular barriers is essential to its pathogenesis. With epithelial cells, this process has been modeled in two steps. The initial stage of localized adherence is mediated by bacterial pili. After this phase, MC disperse and lose piliation, thus leading to a diffuse adherence. At this stage, microvilli have disappeared, and MC interact intimately with cells and are, in places, located on pedestals of actin, thus realizing attaching and effacing (AE) lesions. The bacterial attributes responsible for these latter phenotypes remain unidentified. Considering that bacteria are nonpiliated at this stage, pili cannot be directly responsible for this effect. However, the initial phase of pilus-mediated localized adherence is required for the occurrence of diffuse adherence, loss of microvilli, and intimate attachment, because nonpiliated bacteria are not capable of such a cellular interaction. In this work, we engineered a mutation in the cytoplasmic nucleotide-binding protein PilT and showed that this mutation increased piliation and abolished the dispersal phase of bacterial clumps as well as the loss of piliation. Furthermore, no intimate attachment nor AE lesions were observed. On the other hand, PilT- MC remained adherent as piliated clumps at all times. Taken together these data demonstrate that the induction of diffuse adherence, intimate attachment, and AE lesions after pilus-mediated adhesion requires the cytoplasmic PilT protein.

• Denessiouk KA, Denesyuk AI, Lehtonen JV, Korpela T, Johnson MS
• Common structural elements in the architecture of the cofactor-binding domains in unrelated families of pyridoxal phosphate-dependent enzymes.
• Proteins. 1999; 35: 250-61
• Display abstract

• A detailed comparison of the structures of aspartate aminotransferase, alanine race-mase, the beta subunit of tryptophan synthase, D-amino acid aminotransferase and glycogen phosphorylase has revealed more extensive structural similarities among pyridoxal phosphate (PLP)-binding domains in these enzymes than was observed previously. These similarities consist of seven common structural segments of the polypeptide chain, which form an extensive common structural organization of the backbone chain responsible for the appropriate disposition of key residues, some from the aligned fragments and some from variable loops joined to these fragments, interacting with PLPs in these enzymes. This common structural organization contains an analogous hydrophobic minicore formed from four amino acid side chains present in the two most conserved structural elements. In addition, equivalent alpha-beta-alpha-beta supersecondary structures are formed by these seven fragments in three of the five structures: alanine racemase, tryptophan synthase and glycogen phosphorylase. Despite these similarities, it is generally accepted that these proteins do not share a common heritage, but have arisen on five separate occasions. The common and contiguous alpha-beta-alpha-beta structure accounts for only 28 residues and all five enzymes differ greatly in both the orientation of the PLP pyridoxal rings and their contacts with residues close to the common structural elements.

• Anantha RP, Stone KD, Donnenberg MS
• Role of BfpF, a member of the PilT family of putative nucleotide-binding proteins, in type IV pilus biogenesis and in interactions between enteropathogenic Escherichia coli and host cells.
• Infect Immun. 1998; 66: 122-31
• Display abstract

• Adherence of enteropathogenic Escherichia coli (EPEC) to epithelial cells is dependent on a type IV fimbria, termed the bundle-forming pilus (BFP). A cluster of 14 genes is required for expression of BFP. The eighth gene in the cluster, bfpF, encodes a putative nucleotide-binding protein which resembles the PilT protein of Pseudomonas aeruginosa. It has been proposed that PilT is required for the retraction of the P. aeruginosa pilus, which results in twitching motility. To test the role of BfpF in BFP function and EPEC pathogenesis, two different mutations were constructed in the bfpF gene, one in the cloned gene cluster in a laboratory E. coli strain and one in wild-type EPEC. Neither mutation affected prepilin synthesis, leader sequence processing, or pilus biogenesis. However, both mutations resulted in increased localized adherence. In addition, the EPEC bfpF mutant displayed increased aggregation. The EPEC bfpF mutant was not deficient in attaching and effacing activity or invasion capacity. These results suggest that BfpF decreases aggregation and adherence by EPEC but that subsequent steps in EPEC pathogenesis do not require this protein.

• Takatsu H, Sakurai M, Shin HW, Murakami K, Nakayama K
• Identification and characterization of novel clathrin adaptor-related proteins.
• J Biol Chem. 1998; 273: 24693-700
• Display abstract

• We have identified a human approximately 87-kDa protein, designated as gamma2-adaptin, that is similar to gamma-adaptin (called gamma1-adaptin in this paper), a large chain of the AP-1 clathrin-associated adaptor complex, not only in the primary structure (60% amino acid identity) but also in the domain organization. Northern blot analysis has shown that its mRNA is expressed in a variety of tissues. Analysis using a yeast two-hybrid system has revealed that, similarly to gamma1-adaptin, gamma2-adaptin is capable of interacting not only with the sigma1 chain (called as sigma1A in this paper), the small chain of the AP-1 complex, but also with a novel sigma1-like protein, designated as sigma1B, which shows an 87% amino acid identity to sigma1A; and that, unlike gamma1-adaptin, it is unable to interact with beta1-adaptin, another large chain of the AP-1 complex. Immunofluorescence microscopy analysis has revealed that gamma2-adaptin is localized to paranuclear vesicular structures that are not superimposed on structures containing gamma1-adaptin. Furthermore, unlike gamma1-adaptin, gamma2-adaptin is recruited onto membranes in the presence of a fungal antibiotic, brefeldin A. These data suggest that gamma2-adaptin constitute a novel adaptor-related complex that participates in a transport step different from that of AP-1.

• Bussiere DE et al.
• Structure of the E2 DNA-binding domain from human papillomavirus serotype 31 at 2.4 A.
• Acta Crystallogr D Biol Crystallogr. 1998; 54: 1367-76
• Display abstract

• The papillomaviruses are a family of small double-stranded DNA viruses which exclusively infect epithelial cells and stimulate the proliferation of those cells. A key protein within the papillomavirus life-cycle is known as the E2 (Early 2) protein and is responsible for regulating viral transcription from all viral promoters as well as for replication of the papillomavirus genome in tandem with another protein known as E1. The E2 protein itself consists of three functional domains: an N-terminal trans-activation domain, a proline-rich linker, and a C-terminal DNA-binding domain. The first crystal structure of the human papillomavirus, serotype 31 (HPV-31), E2 DNA-binding domain has been determined at 2.4 A resolution. The HPV DNA-binding domain monomer consists of two beta-alpha-beta repeats of approximately equal length and is arranged as to have an anti-parallel beta-sheet flanked by the two alpha-helices. The monomers form the functional in vivo dimer by association of the beta-sheets of each monomer so as to form an eight-stranded anti-parallel beta-barrel at the center of the dimer, with the alpha-helices lining the outside of the barrel. The overall structure of HVP-31 E2 DNA-binding domain is similar to both the bovine papillomavirus E2-binding domain and the Epstein-Barr nuclear antigen-1 DNA-binding domain.

• Kasukabe T, Okabe-Kado J, Honma Y
• TRA1, a novel mRNA highly expressed in leukemogenic mouse monocytic sublines but not in nonleukemogenic sublines.
• Blood. 1997; 89: 2975-85
• Display abstract

• Mouse monocytic Mm-A, Mm-P, Mm-S1, and Mm-S2 cells are sublines of mouse monocytic and immortalized Mm-1 cells derived from spontaneously differentiated, mouse myeloblastic M1 cells. Although these subline cells retain their monocytic characteristics in vitro, Mm-A and Mm-P cells are highly leukemogenic to syngeneic SL mice and athymic nude mice, whereas Mm-S1 and Mm-S2 cells are not or are only slightly leukemogenic. To better understand the molecular mechanisms of these levels of leukemogenicity, we investigated putative leukemogenesis-associated genes or oncogenes involved in the maintenance of growth, especially in vivo, by means of differential mRNA display. We isolated a fragment clone (15T01) from Mm-P cells. The mRNA probed with 15T01 was expressed at high levels in leukemogenic Mm-P and Mm-A cells but not in nonleukemogenic Mm-S1 and Mm-S2 cells. The gene corresponding to 15T01, named TRA1, was isolated from an Mm-P cDNA library. The longest open reading frame of the TRA1 clone predicts a peptide containing 204 amino acids with a calculated molecular weight of 23,049 D. The predicted TRA1 protein is cysteine-rich and contains multiple cysteine doublets. A putative normal counterpart gene, named NOR1, was also isolated from a normal mouse kidney cDNA library and sequenced. NOR1 cDNA predicts a peptide containing 234 amino acids. The sequence of 201 amino acids from the C-terminal NOR1 was completely identical to that of TRA1, whereas the remaining N-terminal amino acids (33 amino acids) were longer than that (3 amino acids) of TRA1 and the N-terminus of NOR1 protein contained proline-rich sequence. A similarity search against current nucleotide and protein sequence databases indicated that the NOR1/TRA1 gene(s) is conserved in a wide range of eukaryotes, because apparently homologous genes were identified in Caenorhabditis elegans and Saccharomyces cerevisiae genomes. Northern blotting using TRA1-specific and NOR1-specific probes indicated that TRA1 mRNA is exclusively expressed in leukemogenic but not in nonleukemogenic Mm sublines and normal tissues and also indicated that NOR1 mRNA is expressed in normal tissues, especially in kidney, lung, liver, and bone marrow cells but not in any Mm sublines. After leukemogenic Mm-P cells were induced to differentiate into normal macrophages by sodium butyrate, the normal counterpart, NOR1, was expressed, whereas the TRA1 level decreased. Furthermore, transfection of TRA1 converted nonleukemogenic Mm-S1 cells into leukemogenic cells. These results indicate that the TRA1 gene is associated at least in part with the leukemogenesis of monocytic Mm sublines.

• Eide LG, Sander C, Prydz H
• Sequencing and analysis of a 35.4 kb region on the right [corrected] arm of chromosome IV from Saccharomyces cerevisiae reveal 23 open reading frames.
• Yeast. 1996; 12: 1085-90
• Display abstract

• The complete DNA sequence of cosmid clone 31A5 containing a 35 452 bp segment from the right [corrected] arm of chromosome IV from Saccharomyces cerevisiae, was determined from an ordered set of subclones in combination with primer walking on the cosmid. The sequence contains 23 open reading frames (ORFs) of more than 100 amino acid residues and the tRNA-Va12a gene. Five ORFs corresponded to the known yeast genes SNQ2, SES1, GCV1, RPL2B and RPS18A. The DNA sequence for RPS18A is interrupted by an intron. One ORF corresponded to a part of the yeast gene HEX2 at the end of the cosmid insert. Four ORFs encoded putative proteins which showed strong homologies to other previously known proteins, three of yeast origin and one of non-yeast origin. Two ORFs were classified as having borderline homologies: one had similarity to two protein families and another to two protein products of unknown function from other species. The remaining 11 ORFs bore no significant similarity to any published protein.

• Alm RA, Hallinan JP, Watson AA, Mattick JS
• Fimbrial biogenesis genes of Pseudomonas aeruginosa: pilW and pilX increase the similarity of type 4 fimbriae to the GSP protein-secretion systems and pilY1 encodes a gonococcal PilC homologue.
• Mol Microbiol. 1996; 22: 161-73
• Display abstract

• Type 4 fimbriae of Pseudomonas aeruginosa are surface filaments involved in host colonization. They mediate both attachment to host epithelial cells and flagelia-independent twitching motility. Four additional genes, pilW, pilX, pilY1 and pilY2, are located on Spel fragment E in the 5 kb intergenic region between the previously characterized genes pilV and pilE, which encode prepilin-like proteins involved in type 4 fimbrial biogenesis. The phenotypes of a transposon insertion and other mutations constructed by allelic exchange show that these genes are involved in the assembly of type 4 fimbriae. The PilW and PilX proteins are membrane located, possess the hydrophobic N-terminus characteristic of prepilin-like proteins, and appear to belong to the GspJ and GspK group of proteins that are required for protein secretion in a wide range of Gram-negative bacteria. These findings increase the similarities between the fimbrial biogenesis and the Gsp-based protein-secretion supersystems. PilY1 is a large protein with C-terminal homology to the PilC2 protein of Neisseria gonorrhoeae, thought to be a fimbrial tip-associated adhesin, and which, like PilY1, is involved in fimbrial assembly. PilY1 appears to be located in both the membrane and the external fimbrial fractions. PilY2 is a small protein that appears to play a subtle role in fimbrial biogenesis and represents a new class of protein.

• Sohel I, Puente JL, Ramer SW, Bieber D, Wu CY, Schoolnik GK
• Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis.
• J Bacteriol. 1996; 178: 2613-28
• Display abstract

• Sequence flanking the bfpA locus on the enteroadherent factor plasmid of the enteropathogenic Escherichia coli (EPEC) strain B171-8 (O111:NM) was obtained to identify genes that might be required for bundle-forming pilus (BFP) biosynthesis. Deletion experiments led to the identification of a contiguous cluster of at least 12 open reading frames, including bfpA, that could direct the synthesis of a morphologically normal BFP filament. Within the bfp gene cluster, we identified open reading frames that share homology with other type IV pilus accessory genes and with genes required for transformation competence and protein secretion. Immediately upstream of the bfp gene cluster, we identified a potential replication origin including genes that are predicted to encode proteins homologous with replicase and resolvase. Restriction fragment length polymorphism analysis of DNA from six additional EPEC serotypes showed that the organization of the bfp gene cluster and its juxtaposition with a potential plasmid origin of replication are highly conserved features of the EPEC biotype.

• Chiang PW et al.
• Isolation and characterization of the human and mouse homologues (SUPT4H and Supt4h) of the yeast SPT4 gene.
• Genomics. 1996; 34: 368-75
• Display abstract

• To study gene regulation mediated by chromatin in mammals, we isolated the human (SUPT4H) and murine (Supt4h) counterparts of the yeast gene encoding SPT4; the product of this gene presumably interacts with the products of the mammalian homologues (which we have also cloned) of yeast SPT5 and SPT6, thereby modulating chromatin formation and activity. We isolated two different sized human SUPT4H cDNA clones (1464 and 728 nt) and one murine Supt4h (688 nt) cDNA clone; all three encode the same 117-amino-acid protein with conservation of the zinc finger motif found in SPT4. Conservation of this zinc finger motif from yeast to mouse and human implies functional importance. Although the overall sequence homology at the DNA level between the human 728-nt transcript and the murine 688-nt transcript is only 78.4%, the DNA sequence homology is 97.7% within the coding region. At the protein level, the amino acid sequences of the translated murine Supt4h and the human SUPT4H gene products are identical. The likely functional copy of SUPT4H, which has at least two introns, maps to human chromosome 17, with candidate intronless pseudogenes on chromosomes 2, 12, and 20. Buttressing the hypothesis that this is a gene required constitutively, both the human SUPT4H transcripts and the murine Supt4h transcript are expressed widely, although not at equal levels (e.g., such as most histones), in all fetal and adult tissues that we examined.

• Escribano V, Eraso P, Portillo F, Mazon MJ
• Sequence analysis of a 14.6 kb DNA fragment of Saccharomyces cerevisiae chromosome VII reveals SEC27, SSM1b, a putative S-adenosylmethionine-dependent enzyme and six new open reading frames.
• Yeast. 1996; 12: 887-92
• Display abstract

• The nucleotide sequence of a fragment from the left arm of Saccharomyces cerevisiae chromosome VII has been determined. Analysis of the 14,607 bp DNA segment reveals nine open reading frames (ORFs) longer than 300 bp. G2827 is the SEC 7 gene, an essential coatomer complex subunit. G2834 encodes SSM1b, a ribosomal protein. The G2838 product shows homology to hypothetical yeast proteins, YIF0 and YE09, of unknown function. The G2830 product shows homology with the cell division protein FtsJ from Escherichia coli, with two hypothetical proteins from yeast, YCF4 and YBR1, and with R74.7, a hypothetical protein from Caenorhabditis elegans. Two of the ORFs are completely internal to longer ones and a third is partially embedded in G2850. The remaining ORFs give no significant homology with proteins in the databases.

• Parle-McDermott AG, Hand NJ, Goulding SE, Wolfe KH
• Sequence of 29 kb around the PDR10 locus on the right arm of Saccharomyces cerevisiae chromosome XV: similarity to part of chromosome I.
• Yeast. 1996; 12: 999-1004
• Display abstract

• We report a 29,445 bp sequence from the right arm of yeast chromosome XV. It contains the genes MYO2, SNC2, PDR10, SCD5 (also called FTB1), MIP1, VMA4, MRS2, ALA1, KRE5, TEA1, and a homologue of YAL034c. Several discrepancies with previously published sequences were found. PDR10 encodes a protein highly similar to the pleiotropic drug resistance protein Pdr5p. This sequence contig forms part of a region of extended similarity to part of the left arm of chromosome I, which is a relic of an ancient duplicated chromosomal region.

• Dijkstra AJ, Keck W
• Peptidoglycan as a barrier to transenvelope transport.
• J Bacteriol. 1996; 178: 5555-62

• Toikkanen J et al.
• Yeast protein translocation complex: isolation of two genes SEB1 and SEB2 encoding proteins homologous to the Sec61 beta subunit.
• Yeast. 1996; 12: 425-38
• Display abstract

• A yeast gene (cDNA clone) was isolated in a screen for suppressors of secretion-defective sec15-1 mutation. This gene encodes a protein homologous to the beta subunit of the mammalian Sec61 protein complex functioning in protein translocation into the endoplasmic reticulum (ER). The predicted protein, Seb1p, consists of 82 amino acids and contains one potential membrane-spanning region at the C-terminus but no N-terminal signal sequence. Seb1p shows 30% identity to the mammalian Sec61 beta subunit and 34% identity to the Arabidopsis thaliana Sec61 beta subunit. Overexpression of SEB1 from a multicopy plasmid suppressed the temperature sensitivity of sec61-2 and sec61-3 mutants. Immunofluorescence and immunoelectron microscopy indicated that Seb1p resides in the ER membranes with the hydrophilic N-terminus exposed to the cytoplasm. The in vitro translated Seb1p was post-translationally inserted into microsomal membranes. As the chromosomal disruption of the SEB1 gene was not lethal, potential homologous genes were screened by heterologous hybridization. The SEB1 homologue thus isolated, SEB2, encodes a protein 53% identical to Seb1p. Disruption of the chromosomal SEB2 was not lethal whereas the double disruption of SEB1 and SEB2 resulted in a temperature-sensitive phenotype. This study further emphasizes the evolutionary conservation of the ER protein translocation apparatus and provides novel genetic tools for its functional analysis.

• de Kievit TR, Dasgupta T, Schweizer H, Lam JS
• Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype O5).
• Mol Microbiol. 1995; 16: 565-74
• Display abstract

• Previous work from our laboratory has shown that cosmid clone pFV100, containing a 26 kb insert, is able to restore O-antigen synthesis in serotype O5 rough mutants of Pseudomonas aeruginosa. Mobilization of pFV100 into two P. aeruginosa semi-rough (SR) mutants, AK14O1 and rd7513, resulted in O-antigen expression, indicating that pFV100 may contain an O-polymerase (rfc) gene. pFV.TK6, a subclone of pFV100 that contains a 5.6 kb chromosomal insert, was able to complement O-antigen expression in these SR mutants. Mutagenesis of pFV.TK6 using Tn1000 exposed a 1.5 kb region that was essential for complementing O-antigen expression in AK14O1. A 2.0 kb XhoI-HindIII fragment, containing this region, was cloned into vector pUCP26 and the resulting plasmid called pFV.TK8. In Southern analysis of the 20 P. aeruginosa serotypes using a probe generated from the 1.5 kb XhoI fragment of pFV.TK8, the rfc probe hybridized to a common fragment of the cross-reactive O2-O5-O16-O18-O20 serogroup, suggesting that these serotypes may share a common O-polymerase gene. In functional studies of the rfc gene, the PAO1 (serotype O5) chromosomal rfc was mutated using a gene-replacement strategy. These knockout mutants expressed the SR lipopolysaccharide (LPS) phenotype, which indicated that they were no longer producing a functional O-polymerase enzyme. Nucleotide sequence analysis of the insert DNA of pFV.TK8 revealed one open reading frame (ORF), designated ORF48.9, which could code for a 48.9 kDa protein. In comparisons of the P. aeruginosa rfc nucleotide and amino acid sequences with DNA and protein databases, no significant homology was found. However, the deduced structure of the P. aeruginosa Rfc protein indicated that it is very hydrophobic and contains 11 putative membrane-spanning domains. Therefore, the predicted structure is similar to that of other reported Rfc proteins. Furthermore, comparison of the amino acid composition and codon usage of the P. aeruginosa Rfc with other Rfc proteins revealed significant similarity between them.

• Van Dyck L, Pascual-Ahuir A, Purnelle B, Goffeau A
• An 8.2 kb DNA segment from chromosome XIV carries the RPD3 and PAS8 genes as well as the Saccharomyces cerevisiae homologue of the thiamine-repressed nmt1 gene and a chromosome III-duplicated gene for a putative aryl-alcohol dehydrogenase.
• Yeast. 1995; 11: 987-91
• Display abstract

• A 8.2 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome XIV (GenBank/EMBL accession number: X83226) encompasses four open reading frames (ORFs) longer than 100 residues. The ORF N0295 is highly similar to the Aspergillus parasiticus and Schizosaccharomyces pombe nmt1 gene products, which are involved in thiamine biosynthesis and are strongly repressed by thiamine. N0300 is 76% identical to YCR107w, a hypothetical protein of yeast chromosome III, and 55% identical to a ligninolytic aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. In addition, this fragment encodes Rpd3, a pleiotropic transcription factor (Vidal and Gaber, 1991), and part of Pas8, a protein essential for the biogenesis of peroxisomes (Voorn-Brouwer et al., 1993).

• Verhasselt P, Aert R, Voet M, Volckaert G
• Nucleotide sequence analysis of an 8887 bp region of the left arm of yeast chromosome XIV, encompassing the centromere sequence.
• Yeast. 1994; 10: 945-51
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• The nucleotide sequencing of 8887 bp of the left arm of chromosome XIV is described. The sequence includes the centromeric region. Both strands were sequenced with an average redundancy of 5.09 per base pair. The overall G+C content is 37.3% (39.2% for putative coding regions versus 32.5% for non-coding regions). Six open reading frames (ORFs) greater than 100 amino acids were detected, all of which are completely confined to the 8.9 kbp region. Codon frequencies of the six ORFs agree with codon usage in Saccharomyces cerevisiae and all show the characteristics of low-level expressed genes. Comparison of the translated sequences with protein sequences in data bases suggests the presence of two ORFs (N2014 and N2007) encoding ribosomal proteins, the latter of which is the previously sequenced MRP7 gene. Another ORF (N2012) could encode a membrane-associated protein since it contains secretory signal sequence and two presumed transmembrane helices. This protein might be involved in mitochondrial energy transfer. ORF N2016 is immediately adjacent to the centromere, suggesting that it corresponds to the SPO1 gene, which is very tightly linked to the centromere at the left arm side of chromosome XIV (Mortimer et al., 1989).

• Garcia-Cantalejo J et al.
• The complete sequence of an 18,002 bp segment of Saccharomyces cerevisiae chromosome XI contains the HBS1, MRP-L20 and PRP16 genes, and six new open reading frames.
• Yeast. 1994; 10: 231-45
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• We report the sequence of an 18,002 bp DNA fragment from the right arm of Saccharomyces cerevisiae chromosome XI. This segment contains nine complete open reading frames (ORFs), YKR401 to YKR409, and part of another ORF, YKR400, covering altogether 87.2% of the entire sequence. One of them, YKR400, encodes an NAD-dependent 5,10-methylene-tetrahydrofolate dehydrogenase. YKR404, YKR405 and YKR406 correspond to the previously characterized HBS1, MRP-L20 and PRP16 genes, coding for a translation elongation factor, a mitochondrial ribosomal protein and an ATP-binding protein, respectively. The putative product of YKR407 contains the zinc-binding region signature of neutral zinc metallopeptidases. The five other ORFs do not show significant homology to any known protein.

• Mai B, Lipp M
• Cloning and chromosomal organization of a gene encoding a putative amino-acid permease from Saccharomyces cerevisiae.
• Gene. 1994; 143: 129-33
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• A new member of the yeast amino acid (aa) permease gene family has been cloned, mapped and sequenced. The sequence of the PAP1 (putative aa permease 1) gene contains an open reading frame of 566 aa corresponding to a polypeptide with a calculated M(r) of 62,704. Its hydropathy profile suggests the presence of 13 membrane-spanning regions and a charged N-terminal domain. It does not resemble hydrophobic signal sequences found in secreted proteins. Hence, PAP1 encodes a protein with characteristics typical of integral membrane proteins translocating ligands across cellular membranes. Sequence comparisons indicate strong homology to the five known aa permeases of Saccharomyces cerevisiae and to an aa transporter in Trichoderma harzianum. Primer extension analysis revealed one major and one minor transcription start point located 121 and 125 nucleotides upstream from the ATG start codon, corresponding to a 2.1-kb transcript. PAP1 was mapped in a contig of three known (DBF4, TPI and HEM12), but so far unlinked, genes on chromosome IV.

• Zumstein E, Griffin H, Schweizer M
• Sequence of a 10.27 kb segment on the left arm of chromosome XV from Saccharomyces cerevisiae includes part of the IRA2 gene and a putative new gene.
• Yeast. 1994; 10: 1383-7
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• A 10,270 bp fragment from the left arm of chromosome XV of Saccharomyces cerevisiae was sequenced and analysed. The sequence reveals the presence of two open reading frames (ORFs), one of them is the larger part of the previously sequenced gene IRA2 (YOL0951). The other ORF, YOL0950, has a length of 1245 nucleotides and exhibits no significant homology with any known gene, although there is some similarity of its upstream region to the corresponding region of the Schizosaccharomyces pombe cdr1/nim1 gene which is involved in the control of mitotic cell size.

• James CM, Gent ME, Indge KJ, Oliver SG
• Sequence analysis of a 10 kb fragment of yeast chromosome XI identifies the SMY1 locus and reveals sequences related to a pre-mRNA splicing factor and vacuolar ATPase subunit C plus a number of unidentified open reading frames.
• Yeast. 1994; 10: 247-55
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• We report the DNA sequence analysis of a region on the left arm of chromosome XI of Saccharomyces cerevisiae extending over 10 kb. The region contains five open reading frames (ORFs) of greater than 100 amino acids which do not show significant overlap with other ORFs. YKL408 contains a sequence with strong similarity to the RNA helicase pre-mRNA splicing factors PRP2, PRP16 and PRP22 (Burgess et al., 1990; Company et al., 1991; Ruby et al., 1991). YKL409 corresponds to the gene SMY1, the sequence of which was previously reported by Lillie and Brown (1992). YKL410 is identical to ATPase subunit C (Beltran et al., 1992) except for an N-terminal extension. YKL406 and YKL407 show no significant identity with any sequences in the databases searched.

• Lalo D, Stettler S, Mariotte S, Gendreau E, Thuriaux P
• Organization of the centromeric region of chromosome XIV in Saccharomyces cerevisiae.
• Yeast. 1994; 10: 523-33
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• A 15.1 kb fragment of the yeast genome was allocated to the centromeric region of chromosome XIV by genetic mapping. It contained six bona fide genes, RPC34, FUN34, CIT1 (Suissa et al., 1984), RLP7, PET8 and MRP7 (Fearon and Mason, 1988) and two large open reading frames, DOM34 and TOM34. RPC34 and RLP7 define strictly essential functions, whereas CIT1, PET8 and MRP7 encode mitochondrial proteins. The PET8 product belongs to a family of mitochondrial carrier proteins. FUN34 encodes a putative transmembraneous protein that is non-essential as judged from the normal growth of the fun34-::LUK18(URA3) allele even on respirable substrates. TOM34 codes for a putative RNA binding protein, and DOM34 defines a hypothetical polypeptide of 35 kDa, with no significant homology to known proteins. The region under study also contains two divergently transcribed tDNAs, separated only by a chimeric transposable element. This tight tDNA linkage pattern is commonly encountered in yeast, and a general hypothesis is proposed for its emergence on the Saccharomyces cerevisiae genome. RPC34, RLP7, PET8 and MRP7 are unique on the yeast genome, but the remaining genes belong to an extant centromeric duplication between chromosome III and XIV.

• Boyd JM, Koga T, Lory S
• Identification and characterization of PilS, an essential regulator of pilin expression in Pseudomonas aeruginosa.
• Mol Gen Genet. 1994; 243: 565-74
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• Expression of the pilin gene, pilA, of Pseudomonas aeruginosa requires the alternative sigma factor, sigma 54, and also two other transcriptional regulators encoded by the pilS and pilR genes. These two linked genes, which have been identified by transposon insertion mutagenesis, share significant amino acid sequence homology with members of the two-component family of regulators. The transcriptional regulator, PilR, has been described previously. PilS, a 37,285 Dalton protein, shares significant homology with the protein kinase sensors of the two-component regulatory family. PilS, however, has no hydrophobic domains which might be membrane-spanning alpha-helices, suggesting that PilS is a cytoplasmic protein. Characterization of the pilS gene revealed that when overexpressed in Escherichia coli by the bacteriophage T7 promoter it specifies a protein of approximately 40,000 daltons, corresponding to the molecular weight of PilS predicted from the deduced amino acid sequence. Deletion analysis of the pilS promoter fused to a promoterless lacZ gene further showed that a significant region upstream of pilS is essential for expression of pilS and pilR, suggesting a need for transcriptional activation. The pilA promoter can be activated in E. coli but only when PilR and sigma 54 are present. This work suggests that the PilS activation signal is received in the bacterial cytoplasm, and that the mechanism of PilS/PilR-mediated signal transduction resulting in activation of the pilin gene promoter is likely to be similar to that of other two-component systems.

• Roemer T, Fortin N, Bussey H
• DNA sequence analysis of a 10.4 kbp region on the right arm of yeast chromosome XVI positions GPH1 and SGV1 adjacent to KRE6, and identifies two novel tRNA genes.
• Yeast. 1994; 10: 1527-30
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• Determination of DNA sequence in the KRE6 region of the Saccharomyces cerevisiae genome completes a 10.4 kbp section on the extreme right arm of chromosome XVI. This segment contains two additional genes, GHP1 and SGV1 (Hwang et al., 1989; Irei et al., 1991) previously assigned physically to chromosome XVI, as well as a new tRNA(Gly) gene, and a novel tRNA(Ala) gene which is flanked by a sigma element.

• Van Dyck L, Jonniaux JL, de Melo Barreiros T, Kleine K, Goffeau A
• Analysis of a 17.4 kb DNA segment of yeast chromosome II encompassing the ribosomal protein L19 as well as proteins with homologies to components of the hnRNP and snRNP complexes and to the human proliferation-associated p120 antigen.
• Yeast. 1994; 10: 1663-73
• Display abstract

• We report the nucleotide sequence of a 17.4 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome II. This sequence contains 12 open reading frames (ORFs) longer than 300 bp and a putative autonomously replicating sequence (ARS). The ORF YBL0418 contains the KH motif present in several nucleic acid-binding proteins and shares homologies with the mouse X protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complexes involved in pre-mRNA processing. YBL0424 is the yeast member of the ribosomal protein L19 (YL14) family. YBL0425 is related to the D1 core polypeptide of the small nuclear ribonucleoprotein (snRNP) particles involved in the splicing of introns. YBL0437 is a putative homologue of the human protein p120, one of the major antigens associated with malignant tumours. Mcm2, a protein important for ARS activity, as well as Aac2, one of the three isoforms of the mitochondrial ATP/ADP carrier, were previously described (Yan et al., 1991; Lawson and Douglas, 1988). Four ORFs show no homology or particular features that could help to assess their functions. The last ORFs are not likely to be expressed for they are localized on the complementary strand of longer ORFs.

• Martin PR, Hobbs M, Free PD, Jeske Y, Mattick JS
• Characterization of pilQ, a new gene required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa.
• Mol Microbiol. 1993; 9: 857-68
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• Type 4 fimbriae are produced by a variety of pathogens, in which they appear to function in adhesion to epithelial cells, and in a form of surface translocation called twitching motility. Using transposon mutagenesis of Pseudomonas aeruginosa, we have identified a new locus required for fimbrial assembly. This locus contains the gene pilQ which encodes a 77 kDa protein with an N-terminal hydrophobic signal sequence characteristic of secretory proteins. pilQ mutants lack the spreading colony morphology characteristic of twitching motility, are devoid of fimbriae, and are resistant to the fimbrial-specific bacteriophage PO4. The pilQ gene was mapped to Spel fragment 2, which is located at 0-5 minutes on the P. aeruginosa PAO1 chromosome, and thus it is not closely linked to the previously characterized pilA-D, pilS,R or pilT genes. The pilQ region also contains ponA, aroK and aroB-like genes in an organization very similar to that of corresponding genes in Escherichia coli and Haemophilus influenzae. The predicted amino acid sequence of PilQ shows homology to the PulD protein of Klebsiella oxytoca and related outer membrane proteins which have been found in association with diverse functions in other species including protein secretion, DNA uptake and assembly of filamentous phage. PilQ had the highest overall homology to an outer membrane antigen from Neisseria gonorrhoeae, encoded by omc, that may fulfil the same role in type 4 fimbrial assembly in this species.

• Purnelle B, Tettelin H, Van Dyck L, Skala J, Goffeau A
• The sequence of a 17.5 kb DNA fragment on the left arm of yeast chromosome XI identifies the protein kinase gene ELM1, the DNA primase gene PRI2, a new gene encoding a putative histone and seven new open reading frames.
• Yeast. 1993; 9: 1379-84
• Display abstract

• A 17.5 kb DNA fragment of chromosome XI, located between the genetic loci mif2 and mak11 was sequenced and analysed. Ten open reading frames were identified. Two of them are the previously sequenced genes ELM1 and PRI2, two (YKL253 and YKL256) show homologies to proteins from other organisms and one (YKL262) to yeast and mouse histone.

• Nunn DN, Lory S
• Cleavage, methylation, and localization of the Pseudomonas aeruginosa export proteins XcpT, -U, -V, and -W.
• J Bacteriol. 1993; 175: 4375-82
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• Four components of the apparatus of extracellular protein secretion of Pseudomonas aeruginosa, Xcpt, -U, -V, and -W (XcpT-W), are synthesized as precursors with short N-terminal leader peptides that share sequence similarity with the pilin subunit of this organism. A specialized leader peptidase/methylase, product of the pilD gene, has been shown to cleave the leader peptide from prepilin and to methylate the N-terminal phenylalanine of the mature pilin. Antibodies were prepared against XcpT-W and used to purify each of these proteins. Sequence analysis of XcpT-W has shown that these proteins, like mature pilin, contain N-methylphenylalanine as the N-terminal amino acid. Analysis of cellular fractions from wild-type and pilD mutant strains of P. aeruginosa showed that the precursor forms of XcpT-W are located predominantly in the bacterial inner membrane, and their localization is not altered after PilD-mediated removal of the leader sequence. These studies demonstrate that the biogenesis of the apparatus of extracellular protein secretion and that of type IV pili share a requirement for PilD. This bifunctional enzyme, acting in the inner membrane, cleaves the leader peptides from precursors of pilins and XcpT-W and subsequently methylates the amino group of the N-terminal phenylalanine of each of its substrates.

• Turner LR, Lara JC, Nunn DN, Lory S
• Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa.
• J Bacteriol. 1993; 175: 4962-9
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• The process of extracellular secretion in Pseudomonas aeruginosa requires specialized machinery which is widely distributed among bacteria that actively secrete proteins to the extracellular medium. One of the components of this machinery is the product of the xcpR gene, which is homologous to pilB, a gene encoding a protein essential for the biogenesis of type IV pili. Both XcpR and PilB are characterized by the presence of a conserved ATP-binding motif (Walker sequence). The codons of highly conserved glycine residues within the Walker sequences of xcpR and pilB were altered to encode a serine, and the effects of these substitutions were examined. Bacteria expressing mutant XcpR or PilB were unable to secrete exotoxin A or assemble pili, respectively. In addition, high-level expression of mutant XcpR in wild-type P. aeruginosa led to a pleiotropic extracellular secretion defect, resulting in the periplasmic accumulation of enzymes that are normally secreted from the cell. These studies show that the putative ATP-binding sites of XcpR and PilB are essential for their functions in protein secretion and assembly of pili, respectively. Moreover, the observed dominant negative phenotype of mutant XcpR suggests that this protein functions as a multimer or, alternatively, interacts with another essential component of the extracellular protein secretion machinery.

• Friedrich MJ, Kinsey NE, Vila J, Kadner RJ
• Nucleotide sequence of a 13.9 kb segment of the 90 kb virulence plasmid of Salmonella typhimurium: the presence of fimbrial biosynthetic genes.
• Mol Microbiol. 1993; 8: 543-58
• Display abstract

• The 90kb plasmid resident in Salmonella typhimurium confers increased virulence in mice by promoting the spread of infection after invasion of the intestinal epithelium. The nucleotide sequence of a 13.9kb segment of this plasmid known to encode an outer membrane protein related in sequence to components of fimbrial biosynthesis in enteric bacteria was determined. This cloned segment between the repB and repC replicon regions programmed expression of abundant surface fimbriae in Escherichia coli and S. typhimurium cells. A 7kb region contained seven open reading frames, the protein products of five of which were related in sequence to regulatory, structural, and assembly proteins of adherence fimbriae/pili, such as the P and K88 pili. These five genes and two adjacent ones which were not markedly related to proteins in the data bases comprise the pef (plasmid-encoded fimbriae) locus. Transposon TnphoA insertions in four genes in the pef locus (pefA, pefC, orf5 and orf6) resulted in active PhoA fusions and blocked or reduced the surface presentation of fimbriae, indicating that the proteins encoded by these four genes are translocated at least across the cytoplasmic membrane and contribute to formation of the fimbrial structure. The differences in genetic organization and protein sequence relatedness from other fimbrial gene clusters suggest that the pef locus might encode a novel type of fimbria. Between the pef and the repB loci, there were five open reading frames, one of which (orf8) gave rise to active PhoA fusions but was not necessary for fimbrial expression. Two of the other proteins were homologous to transcription regulatory proteins and a third was the rck gene, which encodes an outer membrane protein that confers complement resistance to serum-sensitive hosts.

• Purnelle B, Skala J, Goffeau A
• The product of the YCR105 gene located on the chromosome III from Saccharomyces cerevisiae presents homologies to ATP-dependent permeases.
• Yeast. 1991; 7: 867-72
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• During the systematic sequencing of chromosome III from Saccharomyces cerevisiae, carried out by a network of laboratories sponsored by the Commission of the European Community, we have identified the open reading frame YCR105 located on fragment J11D from the circular derivative of chromosome III in strain XJ24-24a (Palzkill et al., 1986). YCR105 is immediately centromere proximal to the PGK gene (opposite strand) on the right arm of chromosome III about 20 kb from the centromere.

• Toyama H, Esaki N, Yoshimura T, Tanizawa K, Soda K
• Thermostable alanine racemase of Bacillus stearothermophilus: subunit dissociation and unfolding.
• J Biochem. 1991; 110: 279-83
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• The guanidine hydrochloride-induced subunit dissociation and unfolding of thermostable alanine racemase from Bacillus stearothermophilus have been studied by circular dichroism, fluorescence and absorption spectroscopies, and gel filtration. The overall process was found to be reversible: more than 75% of the original activity was recovered upon reduction of the denaturant concentration. In the range of 0.6 to 1.5 M guanidine hydrochloride, the dimeric enzyme was dissociated into a monomeric form, which was catalytically inactive. The monomeric enzyme appeared to bind the cofactor pyridoxal phosphate by a non-covalent linkage, although the native dimeric enzyme binds the cofactor through an aldimine Schiff base linkage. The monomer was mostly unfolded, with the transition occurring in the range of 1.8 to 2.2 M guanidine hydrochloride.

• Holm L, Koivula AK, Lehtovaara PM, Hemminki A, Knowles JK
• Random mutagenesis used to probe the structure and function of Bacillus stearothermophilus alpha-amylase.
• Protein Eng. 1990; 3: 181-91
• Display abstract

• Mutations that cover the sequence of Bacillus stearothermophilus alpha-amylase were produced by an efficient in vitro enzymatic random mutagenesis method and the mutant alpha-amylases were expressed in Escherichia coli, which also secreted the product. Ninety-eight mutants were identified by sequencing and their enzyme activities were classified into three classes: wild-type, reduced or null. A molecular model of the enzyme was constructed using the coordinates of Takaamylase A and a consensus alignment of mammalian, plant, and bacterial alpha-amylases. The location of mutant amino acids on the model indicate that mutations which destroy or decrease the catalytic activity are particularly clustered: (i) around the active site and along the substrate-binding groove and (ii) in the interface between the central alpha/beta barrel and the C-terminal domain. Exposed loops are typically tolerant towards mutations.

• Lindqvist Y, Branden CI
• Structure of glycolate oxidase from spinach.
• Proc Natl Acad Sci U S A. 1985; 82: 6855-9
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• A high-resolution structure determination of glycolate oxidase from spinach is reported. X-ray data were collected on films at the synchrotron radiation source in Daresbury, England. The structure was solved by using two heavy-atom derivatives and a solvent-flattening procedure developed by B.-C. Wang. The subunit structure is essentially a structure of the eight-stranded alpha/beta-barrel type first described for triosephosphate isomerase. In addition, there are 70 residues at the NH(2) terminus and 45 residues between strand four and helix four of the barrel, which are arranged in a helical domain outside the COOH end of the parallel strands of the barrel. The active site is in a cleft between these domains with the coenzyme FMN essentially bound to the barrel and a substrate analogue, thioglycolate, bound to the helical domain. The molecule is octameric with 422 symmetry and has a 15- to 20-A-wide hole in the middle.

• van Die I, van Geffen B, Hoekstra W, Bergmans H
• Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene.
• Gene. 1985; 34: 187-96
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• The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.

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