Subtilis Genome Research Articles

Novel phosphotransferase system genes revealed by genome analysis - the complete complement of PTS proteins encoded within the genome of Bacillus subtilis.

Bacillus subtilis can utilize several sugars as single sources of carbon and energy. Many of these sugars are transported and concomitantly phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In addition to its role in sugar uptake, the PTS is one of the major signal transduction systems in B. subtilis. In this study, an analysis of the complete set of PTS proteins encoded within the B. subtilis genome is presented. Fifteen sugar-specific PTS permeases were found to be present and the functions of novel PTS permeases were studied based on homology to previously characterized permeases, analysis of the structure of the gene clusters in which the permease encoding genes are located and biochemical analysis of relevant mutants. Members of the glucose, sucrose, lactose, mannose and fructose/mannitol families of PTS permeases were identified. Interestingly, nine pairs of IIB and IIC domains belonging to the glucose and sucrose permease families are present in B. subtilis; by contrast only five Enzyme IIA(Glc)-like proteins or domains are encoded within the B. subtilis genome. Consequently, some of the EIIA(Glc)-like proteins must function in phosphoryl transfer to more than one IIB domain of the glucose and sucrose families. In addition, 13 PTS-associated proteins are encoded within the B. subtilis genome. These proteins include metabolic enzymes, a bifunctional protein kinase/phosphatase, a transcriptional cofactor and transcriptional regulators that are involved in PTS-dependent signal transduction. The PTS proteins and the auxiliary PTS proteins represent a highly integrated network that catalyses and simultaneously modulates carbohydrate utilization in this bacterium.

Estimation of P-to-O ratio in Bacillus subtilis and its influence on maximum riboflavin yield.

Simultaneous growth and riboflavin overproduction were investigated using a previously developed stoichiometric model of Bacillus subtilis metabolism. A fit of model predictions to experimental data was used to obtain estimates of fundamental energetic parameters of B. subtilis. Although multiple solutions describe the experimental data, evidence for a P-to-O ratio of about 1(1/3) mole of ATP produced per atom of oxygen consumed in oxidative phosphorylation was provided by genomic analysis of electron transport components, because no homologue of the proton-translocating NADH dehydrogenase I was found in the B. subtilis genome database. These results allow us to devise a rational metabolic engineering strategy to improve riboflavin production. The potential influence of increased energy coupling in oxidative phosphorylation on riboflavin yield is discussed. Higher coupling is most significant under carbon-limiting conditions in slow-growing cells, that is, in fed-batch processes of industrial interest.

Bacillus subtilis Contains Two Small c-Type Cytochromes with Homologous Heme Domains but Different Types of Membrane Anchors

We demonstrate that the cccB gene, identified in the Bacillus subtilis genome sequence project, is the structural gene for a 10-kDa membrane-bound cytochrome c(551) lipoprotein described for the first time in B. subtilis. Apparently, CccB corresponds to cytochrome c(551) of the thermophilic bacterium Bacillus PS3. The heme domain of B. subtilis cytochrome c(551) is very similar to that of cytochrome c(550), a protein encoded by the cccA gene and anchored to the membrane by a single transmembrane polypeptide segment. Thus, B. subtilis contains two small, very similar, c-type cytochromes with different types of membrane anchors. The cccB gene is cotranscribed with the yvjA gene, and transcription is repressed by glucose. Mutants deleted for cccB or yvjA-cccB show no apparent growth, sporulation, or germination defect. YvjA is not required for the synthesis of cytochrome c(551), and its function remains unknown.

Transcription of genes near the sspE locus of the Bacillus subtilis genome.

The yfhP, yfhQ (mutY homologue), yfhS and yfhR (oxidoreductase homologue) genes, which are located upstream of the sspE locus, have been identified in the Bacillus subtilis genome. Transcriptional analysis showed that yfhP, yfhQ and yfhR are transcribed during the exponential growth phase, and sspE is monocistronically transcribed in the late sporulation phase and co-transcribed with yfhQ and/or yfhR during exponential growth. However, SspE was not translated during this period. Northern blot and primer extension analyses indicated that yfhS is transcribed by E sigma E during sporulation. No significant difference between wild-type and yfhS mutant strains was found in the rate of sporulation or germination, the heat tolerance of spores or the transcription of the sspE locus during sporulation. The transcription of the yfhP and yfhQ-yfhR-sspE loci increased 2.5- and 5.3-fold in a yfhP-deficient strain compared to the wild-type strain at t-2 (2 h before initiation of sporulation). In addition, transcription corresponding to the yfhR-sspE loci increased more than twofold with maximum values observed at t-1.5. These results suggest that YfhP may act as a negative regulator for the transcription of yfhQ, yfhR, sspE and yfhP.

ClpE, a novel type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis.

Clp ATPases, which include the ubiquitous HSP100 family, are classified according to their structural features and sequence similarities. During the course of the Bacillus subtilis genome sequencing project, we identified a gene encoding a new member of the HSP100 family. We designated this protein ClpE, as it is the prototype of a novel subfamily among the Clp ATPases, and have identified homologues in several bacteria, including Listeria monocytogenes, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, Lactobacillus sakei and Clostridium acetobutylicum. A unique feature of these Hsp100-type Clp ATPases is their amino-terminal zinc finger motif. Unlike the other class III genes of B. subtilis (clpC and clpP ), clpE does not appear to be required for stress tolerance. Transcriptional analysis revealed two sigmaA-type promoters, expression from which was shown to be inducible by heat shock and puromycin treatment. Investigation of the regulatory mechanism controlling clpE expression indicates that this gene is controlled by CtsR and is thus a member of the class III heat shock genes of B. subtilis. CtsR negatively regulates clpE expression by binding to the promoter region, in which five CtsR binding sites were identified through DNase I footprinting and sequence analysis.

Fate of unstable Bacillus subtilis subgenome: re-integration and amplification in the main genome

The plastic Bacillus subtilis genome was dissected into two physically separate genomes, the 3.9 Mb main genome and the 0.3 Mb subgenome. DNA replication of the main genome was initiated from the normal replication origin ( oriC) and that of the subgenome was from a 7.2 kb oriN-containing fragment artificially inserted. When the 7.2 kb fragment was shortened to a 1.5 kb fragment that contains oriN but lacks the segregational function, the subgenome became unstable and was rapidly lost from the cell, producing inviable cells due to the loss of essential genes carried by the subgenome. Stable survivors were isolated in which the subgenome had re-integrated and multiplied in the main genome. These results suggest that a reduced genetic stability of the subgenome induces size variation of the B. subtilis genome.

Inventory, assembly and analysis of Bacillus subtilis ABC transport systems

We have undertaken the inventory and assembly of the ATP binding cassette (ABC) transporter systems in the complete genome of Bacillus subtilis. We combined the identification of the three protein partners that compose an ABC transporter (nucleotide-binding domain, NBD; membrane spanning domain, MSD; and solute-binding protein, SBP) with constraints on the genetic organization. This strategy allowed the identification of 86 NBDs in 78 proteins, 103 MSD proteins and 37 SBPs. The analysis of transcriptional units allows the reconstruction of 59 ABC transporters, which include at least one NBD and one MSD. A particular class of five dimeric ATPases was not associated to MSD partners and is assumed to be involved either in macrolide resistance or regulation of translation elongation. In addition, we have detected five genes encoding ATPases without any gene coding for MSD protein in their neighborhood and 11 operons that encode only the membrane and solute-binding proteins. On the bases of similarities, three ATP-binding proteins are proposed to energize ten incomplete systems, suggesting that one ATPase may be recruited by more than one transporter. Finally, we estimate that the B. subtilis genome encodes for at least 78 ABC transporters that have been split in 38 importers and 40 extruders. The ABC systems have been further classified into 11 sub-families according to the tree obtained from the NBDs and the clustering of the MSDs and the SBPs. Comparisons with Escherichia coli show that the extruders are over-represented in B. subtilis, corresponding to an expansion of the sub-families of antibiotic and drug resistance systems.

Molecular phylogeny of phi29-like phages and their evolutionary relatedness to other protein-primed replicating phages and other phages hosted by gram-positive bacteria.

The phi29-like phage genus of Podoviridae family contains phages B103, BS32, GA-1, M2, Nf, phi15, phi29, and PZA that all infect Bacillus subtilis. They have very similar morphology and their genomes consist of linear double-stranded DNA of approximately 20 kb. The nucleotide sequences of individual genomes or their parts determined thus far show that these phages evolved from a common ancestor. A terminal protein (TP) that is covalently bound to the DNA 5'-end primes DNA replication of these phages. The same mechanism of DNA replication is used by the Cp-1 related phages (also members of the Podoviridae family) and by the phage PRD1 (member of the Tectoviridae family). Based on the complete or partial genomic sequence data of these phages it was possible to analyze the evolutionary relationship within the phi29-like phage genus as well as to other protein-primed replicating phages. Noncoding regions containing origins of replication were used in the analysis, as well as amino acid sequences of DNA polymerases, and with the phi29-like phages also amino acid sequences of the terminal proteins and of the gene 17 protein product, an accessory component of bacteriophage DNA replicating machinery. Included in the analysis are also results of a comparison of these phage DNAs with the prophages present in the Bacillus subtilis genome. Based on this complex analysis we define and describe in more detail the evolutionary branches of phi29-like phages, one branch consisting of phages BS32, phi15, phi29, and PZA, the second branch composed of phages B103, M2, and Nf, and the third branch having phage GA-1 as its sole member. In addition, amino acid sequences of holins, proteins involved in phage lysis were used to extend the evolutionary study to other phages infecting Gram-positive bacteria. The analysis based on the amino acid sequences of holins showed several weak points in present bacteriophage classification.

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125.

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15-20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a lambda phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis.

Effective Cloning of Unmarked DNA Fragments in the Bacillus subtilis 168 Genome.

The Bacillus subtilis 168 genome is being used to clone DNA segments, particularly unmarked DNA segments between the two available clones. To facilitate this cloning, a counter selection method was developed in which loss of a cI repressor gene rendered the host strain resistant to neomycin. This method is promoted to use the B. subtilis genome as a general cloning vehicle.

Characterization of yrpC gene product of Bacillus subtilis IFO 3336 as glutamate racemase isozyme.

Glr, the glutamate racemase of Bacillus subtilis (formerly Bacillus natto) IFO 3336 encoded by the glr gene, and YrpC, a protein encoded by the yrpC gene, which is located at a different locus from that of the glr gene in the B. subtilis genome, share a high sequence similarity. The yrpC gene complemented the D-glutamate auxotrophy of Escherichia coli WM335 cells defective in the glutamate racemase gene. Glutamate racemase activity was found in the extracts of E. coli WM335 clone cells harboring a plasmid, pYRPC1, carrying its gene. Thus, the yrpC gene encodes an isozyme of glutamate racemase of B. subtilis IFO 3336. YrpC is mostly found in an inactive inclusion body in E. coli JM109/pYRPC1 cells. YrpC was solubilized readily, but glutamate racemase activity was only slightly restored. We purified YrpC from the extracts of E. coli JM109/pYRPC2 cells using a Glutathione S-transferase Gene Fusion System to characterize it. YrpC is a monomeric protein and contains no cofactors, like Glr. Enzymological properties of YrpC, such as the substrate specificity and optimum pH, are also similar to those of Glr. The thermostability of YrpC, however, is considerably lower than that of Glr. In addition, YrpC showed higher affinity and lower catalytic efficiency for L-glutamate than Glr. This is the first example showing the occurrence and properties of a glutamate racemase isozyme.

The Bacillus subtilis genome sequence: the molecular blueprint of a soil bacterium

The rate at which entire microbial genomes are being sequenced has accelerated rapidly over the past two years, promising to revolutionise our understanding of microbial molecular biology and genetics. The Bacillus subtilis genome sequence is the first complete genome of a free-living soil and rhizosphere bacterium. Data derived from the genome sequence and the systematic functional analysis programme, together with the wealth of knowledge already available for this organism, open up new opportunities to study the behaviour and ecology of this soil and plant growth-promoting rhizobacterium at the molecular level. In this review we examine the Bacillus subtilis 168 genome sequence in the light of clues it might provide for the role of this species in natural environments and discuss suitable methods for applying the available data and resources to the study of this and related organisms in natural systems.

Analyses of a Bacillus subtilis homologue of the Na +/H + antiporter gene which is important for pH homeostasis of alkaliphilic Bacillus sp. C-125

Bacillus subtilis was revealed to have a homologous region to the DNA fragment responsible for alkaliphily of alkaliphilic Bacillus sp. C-125 on the genome, as reported previously [1]. The yufT gene on the B. subtilis genome showed a significant similarity with ORF1 of Bacillus sp. C-125, which is related to membrane potential ( ΔΨ)-driven Na +/H + antiport activity and is important for pH homeostasis in an alkaline condition. Disruption of the yufT gene resulted in the decrease of Na +/H + antiport activity, and the growth of the yufT disrupted strain was impaired with an increase in the external Na + concentration. We conclude that the yufT gene encodes a Na +/H + antiporter, which has a dominant role in the extrusion of cytotoxic Na +.

Purification and characterization of a novel glycine oxidase from Bacillus subtilis

The open reading frame yjbR which had been sequenced as a part of the Bacillus subtilis genome project encodes a putative 40.9-kDa protein. The yjbR-coding sequence was slightly similar to those of bacterial sarcosine oxidases and possibly compatible with the tertiary structure of the porcine kidney d-amino acid oxidase. The yjbR gene product was overproduced in Escherichia coli, purified to homogeneity from the recombinant strain, and characterized. This protein effectively catalyzed the oxidation of sarcosine ( N-methylglycine), N-ethylglycine and glycine. Lower activities on d-alanine, d-valine, and d-proline were detected although no activities were shown on l-amino acids and other d-amino acids. Since glycine is a product and not a substrate for sarcosine oxidase, this protein is not a type of demethylating enzymes but a novel deaminating oxidase, named glycine oxidase as a common name. Several enzymatic properties of the B. subtilis glycine oxidase were also investigated.

The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in gram-positive bacteria.

The molecular mechanisms for regulation of the genes involved in the biosynthesis of methionine and cysteine are poorly characterized in Bacillus subtilis. Analyses of the recently completed B. subtilis genome revealed 11 copies of a highly conserved motif. In all cases, this motif was located in the leader region of putative transcriptional units, upstream of coding sequences that included genes involved in methionine or cysteine biosynthesis. Additional copies were identified in Clostridium acetobutylicum and Staphylococcus aureus, indicating conservation in other Gram-positive genera. The motif includes an element resembling an intrinsic transcriptional terminator, suggesting that regulation might be controlled at the level of premature termination of transcription. The 5' portion of all of the leaders could fold into a conserved complex structure. Analysis of the yitJ gene, which is homologous to Escherichia coli metH and metF, revealed that expression was induced by starvation for methionine and that induction was independent of the promoter and dependent on the leader region terminator. Mutation of conserved primary sequence and structural elements supported a model in which the 5' portion of the leader forms an anti-antiterminator structure, which sequesters sequences required for the formation of an antiterminator, which, in turn, sequesters sequences required for the formation of the terminator; the anti-antiterminator is postulated to be stabilized by the binding of some unknown factor when methionine is available. This set of genes is proposed to form a new regulon controlled by a global termination control system, which we designate the S box system, as most of the genes are involved in sulphur metabolism and biosynthesis of methionine and cysteine.

A five-nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis.

Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme. This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme. From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E. coli. Using protection of linear double-stranded DNA as a criterion, we identified the five-nucleotide sequence 5'-AGCGG-3', or its complement 5'-CCGCT-3', as being sufficient for AddAB nuclease attenuation. We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme. Finally, inspection of the complete B. subtilis genome revealed that this five-nucleotide sequence is over-represented and is, in a majority of cases, co-oriented with DNA replication. Based on these observations, we propose that 5'-AGCGG-3', or its complement, is the B. subtilis analogue of the E. coli chi sequence.

Combining diverse evidence for gene recognition in completely sequenced bacterial genomes

Analysis of a newly sequenced bacterial genome starts with identification of protein-coding genes. Functional assignment of proteins requires the exact knowledge of protein N-termini. We present a new program ORPHEUS that identifies candidate genes and accurately predicts gene starts. The analysis starts with a database similarity search and identification of reliable gene fragments. The latter are used to derive statistical characteristics of protein-coding regions and ribosome-binding sites and to predict the complete set of genes in the analyzed genome. In a test on Bacillus subtilis and Escherichia coli genomes, the program correctly identified 93.3% (resp. 96.3%) of experimentally annotated genes longer than 100 codons described in the PIR-International database, and for these genes 96.3% (83.9%) of starts were predicted exactly. Furthermore, 98.9% (99.1%) of genes longer than 100 codons annotated in GenBank were found, and 92.9% (75.7%) of predicted starts coincided with the feature table description. Finally, for the complete gene complements of B.subtilis and E.coli, including genes shorter than 100 codons, gene prediction accuracy was 88.9 and 87.1%, respectively, with 94.2 and 76.7% starts coinciding with the existing annotation.

Combining diverse evidence for gene recognition in completely sequenced bacterial genomes.

Analysis of a newly sequenced bacterial genome starts with identification of protein-coding genes. Functional assignment of proteins requires the exact knowledge of protein N-termini. We present a new program ORPHEUS that identifies candidate genes and accurately predicts gene starts. The analysis starts with a database similarity search and identification of reliable gene fragments. The latter are used to derive statistical characteristics of protein-coding regions and ribosome-binding sites and to predict the complete set of genes in the analyzed genome. In a test on Bacillus subtilis and Escherichia coli genomes, the program correctly identified 93.3% (resp. 96.3%) of experimentally annotated genes longer than 100 codons described in the PIR-International database, and for these genes 96.3% (83.9%) of starts were predicted exactly. Furthermore, 98.9% (99.1%) of genes longer than 100 codons annotated in GenBank were found, and 92.9% (75.7%) of predicted starts coincided with the feature table description. Finally, for the complete gene complements of B.subtilis and E.coli , including genes shorter than 100 codons, gene prediction accuracy was 88.9 and 87.1%, respectively, with 94.2 and 76.7% starts coinciding with the existing annotation.

The competence transcription factor of Bacillus subtilis recognizes short A/T-rich sequences arranged in a unique, flexible pattern along the DNA helix.

The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which leads to the synthesis of the competence transcription factor (CTF). Previous studies suggested that CTF is encoded by comK. ComK is required for the transcription of comK itself, as well as of the late competence genes encoding the DNA uptake machinery and of genes required for homologous recombination. Here, we used purified ComK to study its role in transcription and to determine the DNA recognition sequence for ComK. In vitro transcription from the comG promoter, which depends on ComK in vivo, was observed on the addition of purified ComK together with Bacillus subtilis RNA polymerase, proving that ComK is CTF. To determine the DNA sequences involved in ComK recognition, footprinting analysis was performed with promoter fragments of the CTF-dependent genes: comC, comE, comF, comG, comK, and addAB. The ComK binding sites determined by DNase I protection experiments were unusually long, with average lengths of approximately 65 bp, and displayed only weak sequence similarities. Hydroxy-radical footprinting, performed with the addAB promoter, revealed a unique arrangement of four short A/T-rich sequences. Gel retardation experiments indicated that four molecules of ComK bound the addAB promoter and the dyad symmetrical arrangement of the four A/T-rich sequences implied that ComK functions as a tetramer composed of two dimers each recognizing the motif AAAAN5TTTT. Comparable A/T-rich sequences were identified in all six DNase I footprints and could be used to predict ComK targets in the B. subtilis genome. On the basis of the variability in distance between the ComK-dimer binding sites, ComK-regulated promoters could be divided into three classes, demonstrating a remarkable flexibility in the binding of ComK. The pattern of hydroxy-radical protections suggested that ComK binds at one face of the DNA helix through the minor groove. This inference was strengthened by the observation that minor groove binding drugs inhibited the binding of ComK.

The 172 kb prkA-addAB region from 83 degrees to 97 degrees of the Bacillus subtilis chromosome contains several dysfunctional genes, the glyB marker, many genes encoding transporter proteins, and the ubiquitous hit gene.

A 171812 bp nucleotide sequence between prkA and addAB (83 degrees to 97 degrees) on the genetic map of the Bacillus subtilis 168 chromosome was determined and analysed. An accurate physical/genetic map of this previously poorly described chromosomal region was constructed. One hundred and seventy open reading frames (ORFs) were identified on the DNA fragment. These include the previously described genes cspB, glpPFKD, spoVR, phoAIV, papQ, citRA, sspB, prsA, hpr, pbpF, hemEHY, aprE, comK and addAB. ORF yhaF in this region corresponds to the glyB marker. Among the striking features of this region are: an abundance of genes encoding (putative) transporter proteins, several dysfunctional genes, the ubiquitous hit gene, and five multidrug-resistance-like genes. These analyses have also revealed the existence of numerous paralogues of ORFs in this region: about two-thirds of the putative genes seem to have at least one paralogue in the B. subtilis genome.