NORMAL GENOMIC

• Alcaraz LD, Moreno-Hagelsieb G, Eguiarte LE, Souza V, Herrera-Estrella L, Olmedo G
• Understanding the evolutionary relationships and major traits of Bacillusthrough comparative genomics.
• BMC Genomics. 2010; 11: 332-332
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• BACKGROUND: The presence of Bacillus in very diverse environments reflectsthe versatile metabolic capabilities of a widely distributed genus.Traditional phylogenetic analysis based on limited gene sampling is notadequate for resolving the genus evolutionary relationships. Bydistinguishing between core and pan-genome, we determined the evolutionaryand functional relationships of known Bacillus. RESULTS: Our analysis isbased upon twenty complete and draft Bacillus genomes, including a newlysequenced Bacillus isolate from an aquatic environment that we report forthe first time here. Using a core genome, we were able to determine thephylogeny of known Bacilli, including aquatic strains whose position inthe phylogenetic tree could not be unambiguously determined in the past.Using the pan-genome from the sequenced Bacillus, we identified functionaldifferences, such as carbohydrate utilization and genes involved in signaltransduction, which distinguished the taxonomic groups. We also assessedthe genetic architecture of the defining traits of Bacillus, such assporulation and competence, and showed that less than one third of the B.subtilis genes are conserved across other Bacilli. Most variation wasshown to occur in genes that are needed to respond to environmental cues,suggesting that Bacilli have genetically specialized to allow for theoccupation of diverse habitats and niches. CONCLUSIONS: The aquaticBacilli are defined here for the first time as a group through thephylogenetic analysis of 814 genes that comprise the core genome. Our datadistinguished between genomic components, especially core vs. pan-genometo provide insight into phylogeny and function that would otherwise bedifficult to achieve. A phylogeny may mask the diversity of functions,which we tried to uncover in our approach. The diversity of sporulationand competence genes across the Bacilli was unexpected based on previousstudies of the B. subtilis model alone. The challenge of uncovering thenovelties and variations among genes of the non-subtilis groups stillremains. This task will be best accomplished by directing efforts towardunderstanding phylogenetic groups with similar ecological niches.

• Paredes-Sabja D, Setlow P, Sarker MR
• SleC is essential for cortex peptidoglycan hydrolysis during germinationof spores of the pathogenic bacterium Clostridium perfringens.
• J Bacteriol. 2009; 191: 2711-20
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• Clostridial spore germination requires degradation of the spore'speptidoglycan (PG) cortex by cortex-lytic enzymes (CLEs), and twoClostridium perfringens CLEs, SleC and SleM, degrade cortex PG in vitro.We now find that only SleC is essential for cortex hydrolysis andviability of C. perfringens spores. C. perfringens sleC spores did notgerminate completely with nutrients, KCl, or a 1:1 chelate of Ca(2+) anddipicolinic acid (Ca-DPA), and the colony-forming efficiency of sleCspores was 10(3)-fold lower than that of wild-type spores. However, sleCspores incubated with various germinants released most of their DPA,although slower than wild-type or sleM spores, and DPA release from sleCsleM spores was very slow. In contrast, germination and viability of sleMspores were similar to that of wild-type spores, although sleC sleM sporeshad 10(5)-fold-lower viability. These results allow the followingconclusions about C. perfringens spore germination: (i) SleC is essentialfor cortex hydrolysis; (ii) although SleM can degrade cortex PG in vitro,this enzyme is not essential; (iii) action of SleC alone or with SleM canaccelerate DPA release; and (iv) Ca-DPA does not trigger spore germinationby activation of CLEs.

• Sebaihia M et al.
• Genome sequence of a proteolytic (Group I) Clostridium botulinum strainHall A and comparative analysis of the clostridial genomes.
• Genome Res. 2007; 17: 1082-92
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• Clostridium botulinum is a heterogeneous Gram-positive species thatcomprises four genetically and physiologically distinct groups of bacteriathat share the ability to produce botulinum neurotoxin, the most poisonoustoxin known to man, and the causative agent of botulism, a severe diseaseof humans and animals. We report here the complete genome sequence of arepresentative of Group I (proteolytic) C. botulinum (strain Hall A, ATCC3502). The genome consists of a chromosome (3,886,916 bp) and a plasmid(16,344 bp), which carry 3650 and 19 predicted genes, respectively.Consistent with the proteolytic phenotype of this strain, the genomeharbors a large number of genes encoding secreted proteases and enzymesinvolved in uptake and metabolism of amino acids. The genome also revealsa hitherto unknown ability of C. botulinum to degrade chitin. There is asignificant lack of recently acquired DNA, indicating a stable genomiccontent, in strong contrast to the fluid genome of Clostridium difficile,which can form longer-term relationships with its host. Overall, thegenome indicates that C. botulinum is adapted to a saprophytic lifestyleboth in soil and aquatic environments. This pathogen relies on its toxinto rapidly kill a wide range of prey species, and to gain access tonutrient sources, it releases a large number of extracellular enzymes tosoften and destroy rotting or decayed tissues.

• Omelchenko MV, Makarova KS, Wolf YI, Rogozin IB, Koonin EV
• Evolution of mosaic operons by horizontal gene transfer and genedisplacement in situ.
• Genome Biol. 2003; 4: 55-55
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• BACKGROUND: Shuffling and disruption of operons and horizontal genetransfer are major contributions to the new, dynamic view of prokaryoticevolution. Under the 'selfish operon' hypothesis, operons are viewed asmobile genetic entities that are constantly disseminated via horizontalgene transfer, although their retention could be favored by the advantageof coregulation of functionally linked genes. Here we apply comparativegenomics and phylogenetic analysis to examine horizontal transfer ofentire operons versus displacement of individual genes within operons byhorizontally acquired orthologs and independent assembly of the same orsimilar operons from genes with different phylogenetic affinities.RESULTS: Since a substantial number of operons have been identifiedexperimentally in only a few model bacteria, evolutionarily conserved genestrings were analyzed as surrogates of operons. The phylogeneticaffinities within these predicted operons were assessed first by sequencesimilarity analysis and then by phylogenetic analysis, includingstatistical tests of tree topology. Numerous cases of apparent horizontaltransfer of entire operons were detected. However, it was shown thatapparent horizontal transfer of individual genes or arrays of genes withinoperons is not uncommon either and results in xenologous gene displacementin situ, that is, displacement of an ancestral gene by a horizontallytransferred ortholog from a taxonomically distant organism without changeof the local gene organization. On rarer occasions, operons might haveevolved via independent assembly, in part from horizontally acquiredgenes. CONCLUSIONS: The discovery of in situ gene displacement shows thatcombination of rampant horizontal gene transfer with selection forpreservation of operon structure provides for events in prokaryoticevolution that, a priori, seem improbable. These findings also emphasizethat not all aspects of operon evolution are selfish, with operonintegrity maintained by purifying selection at the organism level.

• Lopez-Contreras AM et al.
• Production by Clostridium acetobutylicum ATCC 824 of CelG, a cellulosomalglycoside hydrolase belonging to family 9.
• Appl Environ Microbiol. 2003; 69: 869-77
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• The genome sequence of Clostridium acetobutylicum ATCC 824, anoncellulolytic solvent-producing strain, predicts the production ofvarious proteins with domains typical for cellulosomal subunits. Most ofthe genes coding for these proteins are grouped in a cluster similar tothat found in cellulolytic clostridial species, such as Clostridiumcellulovorans. CAC0916, one of the open reading frames present in theputative cellulosome gene cluster, codes for CelG, a putativeendoglucanase belonging to family 9, and it was cloned and overexpressedin Escherichia coli. The overproduced CelG protein was purified by makinguse of its high affinity for cellulose and was characterized. Thebiochemical properties of the purified CelG were comparable to those ofother known enzymes belonging to the same family. Expression of CelG by C.acetobutylicum grown on different substrates was studied by Westernblotting by using antibodies raised against the purified E. coli-producedprotein. Whereas the antibodies cross-reacted with CelG-like proteinssecreted by cellobiose- or cellulose-grown C. cellulovorans cultures, CelGwas not detectable in extracellular medium from C. acetobutylicum grown oncellobiose or glucose. However, notably, when lichenan-grown cultures wereused, several bands corresponding to CelG or CelG-like proteins werepresent, and there was significantly increased extracellular endoglucanaseactivity.

• Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS
• Microbial cellulose utilization: fundamentals and biotechnology.
• Microbiol Mol Biol Rev. 2002; 66: 506-77
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• Fundamental features of microbial cellulose utilization are examined atsuccessively higher levels of aggregation encompassing the structure andcomposition of cellulosic biomass, taxonomic diversity, cellulase enzymesystems, molecular biology of cellulase enzymes, physiology ofcellulolytic microorganisms, ecological aspects of cellulase-degradingcommunities, and rate-limiting factors in nature. The methodological basisfor studying microbial cellulose utilization is considered relative toquantification of cells and enzymes in the presence of solid substrates aswell as apparatus and analysis for cellulose-grown continuous cultures.Quantitative description of cellulose hydrolysis is addressed with respectto adsorption of cellulase enzymes, rates of enzymatic hydrolysis,bioenergetics of microbial cellulose utilization, kinetics of microbialcellulose utilization, and contrasting features compared to solublesubstrate kinetics. A biological perspective on processing cellulosicbiomass is presented, including features of pretreated substrates andalternative process configurations. Organism development is considered for"consolidated bioprocessing" (CBP), in which the production ofcellulolytic enzymes, hydrolysis of biomass, and fermentation of resultingsugars to desired products occur in one step. Two organism developmentstrategies for CBP are examined: (i) improve product yield and tolerancein microorganisms able to utilize cellulose, or (ii) express aheterologous system for cellulose hydrolysis and utilization inmicroorganisms that exhibit high product yield and tolerance. A concludingdiscussion identifies unresolved issues pertaining to microbial celluloseutilization, suggests approaches by which such issues might be resolved,and contrasts a microbially oriented cellulose hydrolysis paradigm to themore conventional enzymatically oriented paradigm in both fundamental andapplied contexts.

• Grishin NV, Wolf YI, Koonin EV
• From complete genomes to measures of substitution rate variability withinand between proteins.
• Genome Res. 2000; 10: 991-1000
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• Accumulation of complete genome sequences of diverse organisms creates newpossibilities for evolutionary inferences from whole-genome comparisons.In the present study, we analyze the distributions of substitution ratesamong proteins encoded in 19 complete genomes (the interprotein ratedistribution). To estimate these rates, it is necessary to employ anotherfundamental distribution, that of the substitution rates among sites inproteins (the intraprotein distribution). Using two independentapproaches, we show that intraprotein substitution rate variabilityappears to be significantly greater than generally accepted. This yieldsmore realistic estimates of evolutionary distances from amino-acidsequences, which is critical for evolutionary-tree construction. Wedemonstrate that the interprotein rate distributions inferred from thegenome-to-genome comparisons are similar to each other and can beapproximated by a single distribution with a long exponential shoulder.This suggests that a generalized version of the molecular clock hypothesismay be valid on genome scale. We also use the scaling parameter of theobtained interprotein rate distribution to construct a rooted whole-genomephylogeny. The topology of the resulting tree is largely compatible withthose of global rRNA-based trees and trees produced by other approaches togenome-wide comparison.

• Fiegler H, Bassias J, Jankovic I, Bruckner R
• Identification of a gene in Staphylococcus xylosus encoding a novelglucose uptake protein.
• J Bacteriol. 1999; 181: 4929-36
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• By transposon Tn917 mutagenesis, two mutants of Staphylococcus xylosuswere isolated that showed higher levels of beta-galactosidase activity inthe presence of glucose than the wild type. Both transposons integrated ina gene, designated glcU, encoding a protein involved in glucose uptake inS. xylosus, which is followed by a glucose dehydrogenase gene (gdh).Glucose-mediated repression of beta-galactosidase, alpha-glucosidase, andbeta-glucuronidase activities was partially relieved in the mutantstrains, while repression by sucrose or fructose remained as strong as inthe wild type. In addition to the pleiotropic regulatory effect,integration of the transposons into glcU reduced glucose dehydrogenaseactivity, suggesting cotranscription of glcU and gdh. Insertionalinactivation of the gdh gene and deletion of the glcU gene withoutaffecting gdh expression showed that loss of GlcU function is exclusivelyresponsible for the regulatory defect. Reduced glucose repression is mostlikely the consequence of impaired glucose uptake in the glcU mutantstrains. With cloned glcU, an Escherichia coli mutant deficient in glucosetransport could grow with glucose as sole carbon source, provided afunctional glucose kinase was present. Therefore, glucose is internalizedby glcU in nonphosphorylated form. A gene from Bacillus subtilis, ycxE,that is homologous to glcU, could substitute for glcU in the E. coliglucose growth experiments and restored glucose repression in the S.xylosus glcU mutants. Three more proteins with high levels of similarityto GlcU and YcxE are currently in the databases. It appears that theseproteins constitute a novel family whose members are involved in bacterialtransport processes. GlcU and YcxE are the first examples whosespecificity, glucose, has been determined.

• Koonin EV, Mushegian AR, Galperin MY, Walker DR
• Comparison of archaeal and bacterial genomes: computer analysis of proteinsequences predicts novel functions and suggests a chimeric origin for thearchaea.
• Mol Microbiol. 1997; 25: 619-37
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• Protein sequences encoded in three complete bacterial genomes, those ofHaemophilus influenzae, Mycoplasma genitalium and Synechocystis sp., andthe first available archaeal genome sequence, that of Methanococcusjannaschii, were analysed using the BLAST2 algorithm and methods for aminoacid motif detection. Between 75% and 90% of the predicted proteinsencoded in each of the bacterial genomes and 73% of the M. jannaschiiproteins showed significant sequence similarity to proteins from otherspecies. The fraction of bacterial and archaeal proteins containingregions conserved over long phylogenetic distances is nearly the same andclose to 70%. Functions of 70-85% of the bacterial proteins and about 70%of the archaeal proteins were predicted with varying precision. Thiscontrasts with the previous report that more than half of the archaealproteins have no homologues and shows that, with more sensitive methodsand detailed analysis of conserved motifs, archaeal genomes become asamenable to meaningful interpretation by computer as bacterial genomes.The analysis of conserved motifs resulted in the prediction of a number ofpreviously undetected functions of bacterial and archaeal proteins and inthe identification of novel protein families. In spite of the generallyhigh conservation of protein sequences, orthologues of 25% or less of theM. jannaschii genes were detected in each individual completely sequencedgenome, supporting the uniqueness of archaea as a distinct domain of life.About 53% of the M. jannaschii proteins belong to families of paralogues,a fraction similar to that in bacteria with larger genomes, such asSynechocystis sp. and Escherichia coli, but higher than that in H.influenzae, which has approximately the same number of genes as M.jannaschii. Certain groups of proteins, e.g. molecular chaperones and DNArepair enzymes, thought to be ubiquitous and represented in the minimalgene set derived by bacterial genome comparison, are missing in M.jannaschii, indicating massive non-orthologous displacement of genesresponsible for essential functions. An unexpectedly large fraction of theM. jannaschii gene products, 44%, shows significantly higher similarity tobacterial than to eukaryotic proteins, compared with 13% that haveeukaryotic proteins as their closest homologues (the rest of the proteinsshow approximately the same level of similarity to bacterial andeukaryotic homologues or have no homologues). Proteins involved intranslation, transcription, replication and protein secretion are mostclosely related to eukaryotic proteins, whereas metabolic enzymes,metabolite uptake systems, enzymes for cell wall biosynthesis and manyuncharacterized proteins appear to be 'bacterial'. A similar prevalence ofproteins of apparent bacterial origin was observed among the currentlyavailable sequences from the distantly related archaeal genus, Sulfolobus.It is likely that the evolution of archaea included at least one majormerger between ancestral cells from the bacterial lineage and the lineageleading to the eukaryotic nucleocytoplasm.

• Cornillot E, Croux C, Soucaille P
• Physical and genetic map of the Clostridium acetobutylicum ATCC 824chromosome.
• J Bacteriol. 1997; 179: 7426-34
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• A physical and genetic map of the Clostridium acetobutylicum ATCC 824chromosome was constructed. The macrorestriction map for CeuI, EagI, andSstII was created by ordering the 38 restriction sites by one- andtwo-dimensional pulsed-field gel electrophoresis (PFGE) and by using anoriginal strategy based on the CeuI enzyme and indirect end labelling byhybridization on both sides of the CeuI sites with rrs (16S RNA) and 3'rrl (23S RNA) probes. The circular chromosome was estimated to be 4.15 Mbin size, and the average resolution of the physical map is 110 kb. Thechromosome contains 11 rrn loci, which are localized on 44% of thechromosome in a divergent transcriptional orientation regarding thepresumed location of the replication origin. In addition to these 11 rrnoperons, a total of 40 identified genes were mapped by hybridizationexperiments with genes from C. acetobutylicum and from various otherclostridia as probes. The genetic map of C. acetobutylicum was compared tothat of the three other endospore-forming bacteria characterized so far:Bacillus subtilis, Clostridium beijerinckii, and Clostridium perfringens.Parodoxically, the chromosomal backbone of C. acetobutylicum showed moresimilarity to that of B. subtilis than to those of the clostridia.

• Gerischer U, Durre P
• mRNA analysis of the adc gene region of Clostridium acetobutylicum duringthe shift to solventogenesis.
• J Bacteriol. 1992; 174: 426-33
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• By using primer extension analysis, we located the transcription startpoint of the acetoacetate decarboxylase (adc) gene of Clostridiumacetobutylicum 90 nucleotides upstream from the initiation codon with A asthe first transcribed nucleotide. From this site the promoter structureTTTACT(18 bp)TATAAT was identified; it shows high homology to theconsensus sequences of gram-positive bacteria and Escherichia coli.Northern blot experiments revealed a length of 850 bases for thetranscript of the adc gene. It thus represents a monocistronic operon.Transcription of adc was induced by conditions necessary for the onset ofsolvent formation. Induction occurred long before the respectivefermentation product (acetone) could be detected in the medium.Transcription of the operon containing the genes for acetoacetyl coenzymeA:acetate/butyrate:coenzyme A transferase (designated ctf) downstream ofthe adc gene but divergently transcribed is also induced by conditionsnecessary for the onset of solvent formation. The length of the respectiveRNA transcript, 4.1 kb, indicates additional coding capacity, since thegenes for the two subunits of the coenzyme A transferase cover onlyapproximately 1.5 kb. No distinct transcripts for the other open readingframes of the adc gene region, ORF1 and ORF2, could be detected. Computeranalysis indicated that ORF1, which showed significant similarity to thealpha-amylase gene of Bacillus subtilis (U. Gerischer and P. Durre, J.Bacteriol. 172:6907-6918, 1990), probably is indeed a coding region. ORF2,however, does not seem to have a coding function.

• TCHAPEROFF IC
• Determination of the effect of irradiation by x-ray on p-aminobenzoicacid, using Clostridium acetobutylicum for assay.
• Can J Res. 1946; 24: 49-54