Subtilis Genome Research Articles

Purification and characterization of NfrA1, a Bacillus subtilis nitro/flavin reductase capable of interacting with the bacterial luciferase.

ipa-43d is a hypothetical gene identified by the Bacillus subtilis genome project (Mol. Microbiol. 10, 371-384 1993; Nature 390, 249-256 1997). The ipa-43d protein overexpressed in E. coli was purified to homogeneity and its properties were analyzed biochemically. The ipa-43d protein was found to be tightly associated with FMN and to be capable of reducing both nitrofurazone and FMN effectively. Although the ipa-43d protein catalysis obeys the ping-pong Bi-Bi mechanism, catalysis mode was changed to the sequential mechanism upon coupling with the bioluminescent reaction. Database search showed that B. subtilis possessed four genes (ipa-44d, ytmO, yddN, and yvbT), encoding proteins similar in amino acid sequence to LuxA and LuxB of Photobacterium fischeri, and, in particular, ipa-44d is immediately adjacent to the ipa-43d gene on the chromosome.

An 8 kb nucleotide sequence at the 3' flanking region of the sspC gene (184 degrees) on the Bacillus subtilis 168 chromosome containing an intein and an intron.

As part of the Bacillus subtilis genome sequencing project, we determined the complete nucleotide sequence of an 8000-bp fragment downstream of the sspC gene (184 degrees) of the B. subtilis 168 chromosome. The sequence analysis shows that the sspC gene is located inside of the SP beta region, which differs from the current genetic map of B. subtilis 168. This region contains 12 putative ORFs (yojQ through yojZ and sspC). A homology search for the deduced products of the ORFs shows significant similarities to enzymes involved in deoxyribonucleotide metabolism: ribonucleotide reductase (Nrd) E, NrdF, thioredoxin and dUTPase. Interestingly, this DNA fragment includes two split genes, yojP containing conserved motifs of an intein and yojQ and yojS with an 808-bp intervening sequence for a putative intron structure. In addition, the yojR gene includes a putative new DNA replication terminator.

SigX of Bacillus subtilis replaces the ECF sigma factor fecI of Escherichia coli and is inhibited by RsiX.

Analysis of the Bacillus subtilis genome sequence revealed two open reading frames, designated sigX and ypuN (now termed rsiX), that are homologous to fecI and fecR, respectively, of Escherichia coli. fecI encodes a sigma 70-type factor that is necessary for transcription of the ferric citrate transport genes fecABCDE. fecR encodes a cytoplasmic transmembrane protein that is required for the induction of fec transport gene transcription by ferric citrate binding to the FecA outer membrane receptor protein. Investigation of the SigX and RsiX activities disclosed that they are not involved in ferric citrate utilization--since ferric citrate did not serve as an iron source for B. subtilis SG64--or in the regulation of any other ferric siderophore transport system tested. Strains deleted for sigX or rsiX displayed no phenotype under aerobic or anoxic conditions. However, cloned sigX complemented an E. coli fecI mutant, and the Fur box upstream of sigX responded to the E. coli iron regulatory protein Fur. The purified SigX protein was required for in vitro transcription of a sigX-containing DNA fragment by the E. coli RNA polymerase core enzyme. Autoregulation of sigX was also found in vivo using a sigX'-lacZ gene fusion. RsiX inhibited SigX activity in vivo and in vitro and stabilized the SigX protein. RsiX was localized in the membrane fraction. When RsiX is present, SigX is found in the membrane fraction; in the absence of RsiX, some SigX is detectable in the cytoplasm. We conclude that SigX is a sigma factor that belongs to the ECF (extracytoplasmic function) sigma 70-factor family. It is not known which promoters are recognized by SigX in B. subtilis. SigX may be involved in the regulation of iron metabolism, as evidenced by its activity in E. coli.

Characterization of an lrp-like (IrpC ) gene from Bacillus subtilis

In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4 kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilis lrp-like-gene is a bona fide Lrp protein-the first one to be detected in gram-positive bacteria. When expressed in E. coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B subtilis Lrp-like protein plays a role in the growth phase transition.

Cloning and sequencing of a 35.7 kb in the 70°–73° region of the Bacillus subtilis genome reveal genes for a new two-component system, three spore germination proteins, an iron uptake system and a general stress response protein

In the framework of the international project aimed at sequencing the Bacillus subtilis ( Bs) genome, a 35.7-kb chromosome segment around the pel locus has been cloned and sequenced. This region (35 745 bp; 70°–73° of the genetic map) contains two partial and 38 complete orfs. A homology search for the products deduced from the 39 orfs revealed that 26 of them exhibit significant similarity to known proteins, e.g. germination proteins, sodium-alanine symporter, PTS system, methionine amino peptidase, 2-oxoglutarate/malate translocater, pectate lyase, general stress response protein, RNA helicase, iron uptake and two-component systems.

Organization and transcription of the myo-inositol operon, iol, of Bacillus subtilis.

Previous determination of the nucleotide sequence of the iol region of the Bacillus subtilis genome allowed us to predict the structure of the iol operon for myo-inositol catabolism, consisting of 10 iol genes (iolA to iouJ); iolG corresponds to idh, encoding myo-inositol 2-dehydrogenase (Idh). Primer extension analysis suggested that an inositol-inducible promoter for the iol operon (iol promoter) might be a promoter-like sequence in the 5' region of iolA, which is probably recognized by sigmaA. S1 nuclease analysis implied that a rho-independent terminator-like structure in the 3' region of iolJ might be a terminator for iol transcription. Disruption of the iol promoter prevented synthesis of the iol transcript as well as that of Idh, implying that the iol operon is most probably transcribed as an 11.5-kb mRNA containing the 10 iol genes. Immediately upstream of the iol operon, two genes (iolR and iolS) with divergent orientations to the iol operon were found. Disruption of iolR (but not iolS) caused constitutive synthesis of the iol transcript and Idh, indicating that the iolR gene encodes a transcription-negative regulator (presumably a repressor) for the iol operon. Northern and S1 nuclease analyses revealed that the iolRS genes were cotranscribed from another inositol-inducible promoter, which is probably recognized by sigmaA. The promoter assignments of the iol and iolRS operons were confirmed in vivo with a lacZ fusion integrated into the amyE locus.

Identification and characterization of pbpA encoding Bacillus subtilis penicillin-binding protein 2A.

Amino acid sequence analysis of tryptic peptides derived from purified penicillin-binding protein PBP2a of Bacillus subtilis identified the coding gene (now termed pbpA) as yqgF, which had been sequenced as part of the B. subtilis genome project; pbpA encodes a 716-residue protein with sequence similarity to class B high-molecular-weight PBPs. Use of a pbpA-lacZ fusion showed that pbpA was expressed predominantly during vegetative growth, and the transcription start site was mapped using primer extension analysis. Insertional mutagenesis of pbpA resulted in no changes in the growth rate or morphology of vegetative cells, in the ability to produce heat-resistant spores, or in the ability to trigger spore germination when compared to the wild type. However, pbpA spores were unable to efficiently elongate into cylindrical cells and were delayed significantly in spore outgrowth. This provides evidence that PBP2a is involved in the synthesis of peptidoglycan associated with cell wall elongation in B. subtilis.

A 23.4 kb segment at the 69 degrees-70 degrees region of the Bacillus subtilis genome.

Within the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, a 23.4 kb chromosome segment has been cloned and sequenced. This region (23 433 bp; 69 degrees-70 degrees of the genetic map) contains 17 complete ORFs and a partial one. A homology search for the products deduced from the 18 ORFs revealed that twelve of them had significant similarity to known proteins, including the quinolone-resistance protein, ABC transporter, aldehyde dehydrogenase, amino acid transporter, fosmidomycin-resistance protein, CDP-glucose 4,6-dehydratase, glucose-1-phosphate cytidyltransferase and cytochrome P450/NADPH-cytochrome P450 reductase.

A 23911 bp region of the Bacillus subtilis genome comprising genes located upstream and downstream of the lev operon.

Within the framework of the European project to sequence the whole Bacillus subtilis 168 genome, a 23911 bp long chromosomal DNA fragment located around 233 degrees on the B. subtilis genetic map was cloned and sequenced. From the generated sequencing data and the results of the homology search, the primary structure of this region was determined. In addition to the whole lev operon, the region contains putative genes for an amino acid permease, two different alcohol dehydrogenases, a chitosanase, a protein belonging to the LysR family of transcriptional regulators, a protein related to the MerR transcriptional regulator, up to four proteins related to the product of the spoF gene, and genes coding for nine more inferred proteins of unknown function.

An internal FK506-binding domain is the catalytic core of the prolyl isomerase activity associated with the Bacillus subtilis trigger factor.

Two major families of peptidylprolyl cis-trans-isomerases, the cyclophilins and the structurally unrelated FK506-binding proteins (FKBPs), have been identified as cellular factors involved in protein folding in vitro. Here we report on the biochemical characterization of a second prolyl isomerase of Bacillus subtilis that was purified from a cyclophilin-negative (ppiB null) mutant and was shown to be the trigger factor (TigBS). N-terminal sequencing of 27 amino acid residues of the purified protein revealed 100% identity to the deduced sequence encoded by the tig gene, sequenced as a part of the B. subtilis genome project. The tigBS gene, located at 246 degrees on the genetic map upstream of the clpX and lonA,B genes, encodes an acidic protein (pI 4.3) of 47.5 kDa. Purified and recombinant TigBS-His proteins share the same substrate specificity and catalytic activity (Kcat/K(m) of 1.5 microM-1 s-1); both are inhibited by the macrolide FK506 with IC50 the range of 500 nM. We also demonstrate that the prolyl isomerase activity of TigBS is mediated by an internal domain of about 13 kDa (homologous to FKPB12) that represents the catalytic core of the trigger factor.

Sequence analysis of the 36-kb region between gntZ and trnY genes of Bacillus subtilis genome.

Within the framework of an international Bacillus subtilis genome sequencing project, we have determined a 36-kb sequence covering the region between the gntZ and trnY genes. In addition to five genes sequenced and characterized previously, 27 putative protein coding sequences (open reading frame; ORF) were identified. A homology search for the newly identified ORFs revealed that six of them had similarities to known proteins. It is notable that new ORFs belonging to response-regulator aspartate phosphatase (Rap) and its regulator (Phr) families, and response regulator and sensory kinase families of two-component signal transduction systems have been identified. Furthermore, we found that some 180-bp non-coding sequence, that might be an remnant of an ancient IS element, is preserved in at least five loci of the B. subtilis genome.

Predicted and Unsuspected Alterations of the Genomes Structure of Genetically DefinedBacillus subtilis168 Strains

We scrutinized the genome structures of Bacillus subtilis 168 derivative strains the isolation procedure of which was well described. NotI and SfiI restriction fragment analyses indicated that strains constructed by transformation using non-168 Bacillus strains as donors had replacement of DNA segments in the predicted region. Interesting examples were found in a lineage including strains 168trpC2-YSl 1-RM125-MI112, the last two of which have a restriction-deficiency mutation (hsdM) introduced from a non-168 strain DNA. As expected, the loss of the hsdM gene was a result of replacement of the hsdM region by non-168 DNA fragments. Moreover, RM125 acquired a previously unnoticed NotI site that gave no obvious phenotypes. Among 168 derivative strains investigated, the most extreme case had changes at six different regions of the genome, probably introduced by repeated transformations. Unsuspected DNA alterations were found even in isogenic strains constructed by phage PBS1-mediated transduction. These acquired structural variations of the B. subtilis genome were distinct from the spontaneous DNA deletions previously characterized [M. Itaya, J. Bacteriol., 175, 741–749 (1993)]. Caution should therefore be taken for nominally isogenic strains that are thought to have identical genome structure except for the gene(s) of interest. We propose the term isogenomic strains for strains that shows the same macrorestriction fragment pattern.

Sequence analysis of a 25-kb segment in the 17 degrees-19 degrees region of the Bacillus subtilis chromosome containing ada locus.

As a part of the Bacillus subtilis genome sequencing project, we have determined a 25-kb sequence covering the 17 degrees-19 degrees region. This region contains 26 complete open reading frames (ORFs) including the alkA and adaA/B operon, which encode genes for adaptive response to DNA alkylation. A homology search for the newly identified 21 ORFs revealed that 4 of them exhibit a significant similarity to known proteins, e.g., methicillin-resistant Staphylococcus aureus (MRSA) protein homolog, proteins involved in chloramphenicol resistance, glucosamine synthase and an ABC transporter protein. The remaining 17 ORFs did not show any significant sequence similarities to known gene products in the database.

Sequence analysis of a 45-kb segment in the 19 degrees-23 degrees region of the Bacillus subtilis chromosome containing glpT and mpr loci.

The nucleotide sequence of a 45,137-bp segment covering the 19 degrees and 23 degrees region in the 360 degrees map of the Bacillus subtilis genome was determined. This region contained 45 open reading frames (ORFs) including 7 which corresponded to the products of genes with known functions that had been previously sequenced. The known genes were: glpT and glpQ for glycerol utilization pathway; purT for a part of the purine synthesis pathway; mpr for an extracellular metalloprotease; pss and psd for the parts of the phospholipid synthesis pathway; and gltP for a glutamine transporter. Deduced amino acid sequences of the 22 newly identified ORFs showed significant homologies to known gene products in the database such as a Methicillin-resistant Staphylococcus aureus (MRSA) gene which is related to drug resistance, a two-component response regulator, a series of amino acid permeases, transcriptional regulators, beta-lactamase, the phosphotransferase system (PTS) enzyme II for sugar uptake, and the eukaryotic ECA39 gene which is associated with cancer and apoptosis, etc. The remaining 16 ORFs did not show any significant sequence similarities to known gene products in the database.

Micado--a network-oriented database for microbial genomes.

We created Micado, a database for managing genomic information, as part of the Bacillus subtilis genome programs. Its content will be progressively extended to the whole microbial world. A relational schema is defined for selective queries. It links eubacterial and archaeal sequences, genetic maps for Bacillus subtilis and Escherichia coli, and information on mutants. The latter comes from a new functional analysis project of unknown genes in B subtilis, and the database allows the community to curate information. To help queries from users, a graphical interface is built on SQL access to the database and provided through the WWW. We have automated imports of microbial sequences, and E. coli genetic map, by programming parsers of flat file distributions. These ensure smooth updates from molecular biology repositories on the Internet. Hyperlinks are created as a complement, to reference other general and specialized related information resources.

Sequence analysis of the groESL-cotA region of the Bacillus subtilis genome, containing the restriction/modification system genes.

We have determined a 35-kb sequence of the groESL-gutR-cotA (45 degrees-52 degrees) region of the Bacillus subtilis genome. In addition to the groESL, gutRB and cotA genes reported previously, we have newly identified 24 ORFs including gutA and fruC genes, encoding glucitol permease and fructokinase, respectively. The inherent restriction/modification system genes, hsdMR and hsdMM, were mapped between groESL and gutRB, and we have identified two open reading frames (ORFs) encoding 5-methylcytosine forming DNA methyl transferase and an operon probably encoding a restriction enzyme complex. The unusual genome structure of few ORFs and lower GC content around the restriction/modification genes strongly suggests that the region originated from a bacteriophage integrated during evolution.

Systematic sequencing of the 283 kb 210 degrees-232 degrees region of the Bacillus subtilis genome containing the skin element and many sporulation genes.

As part of the Bacillus subtilis genome sequencing project, we have determined a 283 kb contiguous sequence from 210 degrees to 232 degrees of the B. subtilis genome. This region contains the 48 kb skin element which is excised during sporulation by a site-specific recombinase. In this region, 310 complete ORFs and one tRNA gene were identified: 66 ORFs have been sequenced and characterized previously by other workers, e.g. acc, ans, bfm, blt, bmr, comE, comG, dnaK, rpoD and sin operons; cwiA, gpr and lysA genes; many sporulation genes and operons, spo0A, spoIIA, spoIIM, spoiiP, spoIIIA, spoIIIC, spoIVB, spoIVCA, spoIVCB and spoVA, etc. The products of 84 ORFs were found to display significant similarity to proteins with known function in data banks, e.g., proteins involved in nucleotide metabolism, lipid biosynthesis, amino acid transport (ABC transporter), phosphate-specific transport, the glycine cleavage system, the two-component regulatory system, cell wall autolysis, ferric uptake and sporulation. However, the functions of more than half of the ORFs (52%, 160 ORFs) are still unknown. In the skin element containing 60 ORFs, 32 ORFs (53%) encode proteins which have significant homology to gene products of the B. subtilis temperate phage phi 105 and/or the defective phage PBSX.

The systematic sequencing of the Bacillus subtilis genome in Japan

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Sequencing of a 65 kb region of the Bacillus subtilis genome containing the lic and cel loci, and creation of a 177 kb contig covering the gnt-sacXY region.

Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, this paper communicates the sequencing of a chromosome region containing the lic and cel loci (65 kb), which creates a 177 kb contig covering the region from gnt to sacXY. This 65 kb region contains 64 ORFs (62 complete and two partial genes). The 14th, 15th and 17th genes correspond to licT, licS and katE, encoding the antiterminator for licS transcription, beta-glucanase (lichenase) and catalase 2, respectively. The 11th, 30th, 36th, 39th, 41st, 45th-48th, 51st and 58th genes are designated deaD, pepT, galE, aldY, msmX, cydABCD, sigY and katX because their products probably encode ATP-dependent RNA helicase, tripeptidase, UDP-glucose 4-epimerase, aldehyde dehydrogenase, multiple sugar-binding transport ATP-binding protein, the respective components of cytochrome d ubiquinol oxidase and ATP-binding cassette transporter, sigma-factor of RNA polymerase and catalase, respectively. The 60th-64th genes are celRABCD, which are probably involved in cellobiose utilization. Gene organization and gene features in the gnt-sacXY region are discussed.

Cloning and sequencing of a 40.6 kb segment in the 73 degrees-76 degrees region of the Bacillus subtilis chromosome containing genes for trehalose metabolism and acetoin utilization.

In the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, a 40.6 kb chromosome segment, which contains the tre locus, has been cloned and sequenced. This region (40 601 bp; 73 degrees-76 degrees on the genetic map) contains 38 complete ORFs and one partial one. Three ORFs, the closest to the hsdC locus, correspond to the treP, treA and treR genes encoding enzyme IITre, trehalose-6-phosphate hydrolase and the repressor of the tre operon, respectively. A homology search for the products deduced from the 39 ORFs revealed that 23 exhibit significant similarity to known proteins, e.g. proteins involved in acetoin utilization, deoxyribonuclease, methyladenine glycosidase, hydroxyisobutyrate dehydrogenase, multidrug resistance proteins, protein phosphatase, cyclic-nucleotide phosphodiesterase, 5'-nucleotidase and NADP(H)-flavin oxidoreductase. Based on the gene organization and the results of the homology search, it is predicted that YfjG, YfjH, YfjI, YfjJ and YfjK form an acetoin dehydrogenase system (acetoin regulatory protein, and acetoin dehydrogenase components/subunits E3, E2, E1 beta and E1 alpha respectively). yfkN, an extremely large ORF comprising 4386 nucleotides, seems to correspond to the fusion of the genes for 2',3'-cyclic-nucleotide 2'-phosphodiesterase and 5'-nucleotidase precursor.