- Volume 142, Issue 11, 1996

A continuous 75627 bp segment of the Mycobacterium leprae chromosome spanning the oriC region was sequenced. The gene order at this locus was similar to that found in the replication origin region of many other prokaryotes, particularly Mycobacterium tuberculosis and Streptomyces coelicolor. As in the case of several Gram-positive bacteria, essential genes involved in basic cellular functions, such as DNA or RNA metabolism (dnaA, dnaB, dnaN, gyrB, gyrA, pcnB, recF, rnpA, ssb), cell wall synthesis (ponA, pbpA) and probably cell division (gidB, rodA) were found. Strikingly, the gidA gene was absent from this part of the genome and there was no rRNA operon near oriC. The gyrA gene harbours an intein coding sequence indicating that protein splicing is required to produce the mature A subunit of DNA gyrase. Among the many other noteworthy features were ORFs encoding putative serine/threonine protein kinases and a protein phosphatase, three tRNA genes, one M. leprae-specific repetitive element and a glnQ pseudogene.

We have cloned and sequenced the nrd (nucleotide reductase) locus of Bacillus subtilis. The locus seems to be organized in an operon comprising four ORFs. The first three encode polypeptides highly similar to the product of the coding sequences characterizing the nrdEF operons of Enterobacteriaceae. The sequencing of the conditional lethal mutation ts-A13, localized in the nrdE cistron, and the lethality of insertional mutations targeted in the internal region of nrdE and nrdF, demonstrated the essential role of this locus. The fourth ORF, ymaB, part of the putative operon, which is not similar to any known protein, is also essential. The regulation of expression of the operon, monitored by lacZ transcriptional fusions, is similar to the regulation of the functionally relevant nrdAB operon of Escherichia coli. The operon was induced by thymidine starvation and its expression was directly or indirectly affected by RecA function. Genetic and functional analysis strongly indicates that in B. subtilis the class I ribonucleotide reductase encoded by this nrd operon is evolutionary distant from the homologous class I enzyme of Enterobacteria.

Within the Bacillus subtilis genome sequencing project, the region between lysA and ilvA was assigned to our laboratory. In this report we present the sequence of the last 36 kb of this region, between the kdg operon and the attachment site of the SPβ prophage. A two-step strategy was used for the sequencing. In the first step, total chromosomal DNA was cloned in phage M13-based vectors and the clones carrying inserts from the target region were identified by hybridization with a cognate yeast artificial chromosome (YAC) from our collection. Sequencing of the clones allowed us to establish a number of contigs. In the second step the contigs were mapped by Long Accurate (LA) PCR and the remaining gaps closed by sequencing of the PCR products. The level of sequence inaccuracy due to LA PCR errors appeared to be about 1 in 10000, which does not affect significantly the final sequence quality. This two-step strategy is efficient and we suggest that it can be applied to sequencing of longer chromosomal regions. The 36 kb sequence contains 38 coding sequences (CDSs), 19 of which encode unknown proteins. Seven genetic loci already mapped in this region, xpt, metB, ilvA, ilvD, thyB, dfrA and degR were identified. Eleven CDSs were found to display significant similarities to known proteins from the data banks, suggesting possible functions for some of the novel genes: cspD may encode a cold shock protein; bcsA, the first bacterial homologue of chalcone synthase; exol, a 5′ to 3′ exonuclease, similar to that of DNA polymerase I of Escherichia coli; and bsaA, a stress-response-associated protein. The protein encoded by ypIP has homology with the transcriptional NifA-like regulators. The arrangement of the genes relative to possible promoters and terminators suggests 19 potential transcription units.

We constructed a PCR map of the 150 kb spoIIIC-pheA region of the Bacillus subtilis chromosome. It was established using known sequences of the spoIIIC, blt, aadK, sacC, spoVB and pheA loci and eight random sequence tags. The tags were generated using PFGE-purified DNA of yeast artificial chromosome (YAC) 11–17 from the yeast clone which carries the major part of this region. The ends of two other YACs were positioned on the map using total DNA extracted from yeast cells carrying them. The procedure allowed the placement of precisely known and new (putative) genes on the physical chromosome map and the generation of sufficient amounts of DNA for sequencing this region. Apart from allowing correction of the genetic map in this region, these results demonstrate how a collection of long segments of bacterial chromosome and Long Accurate PCR can be used for reliable high-resolution physical mapping of an extended chromosome area.

A 21808 bp nucleotide sequence at 75° on the genetic map of the Bacillus subtilis chromosome was determined. The sequence of this region is adjacent to the glpPFKD operon involved in glycerol utilization. Twenty-six ORFs were identified, one of which corresponds to the cspB gene, encoding a cold-shock protein. Seventeen of the deduced protein sequences of these ORFs displayed significant homology to known proteins in the data banks. One putative operon was identified, consisting of five ORFs, that is probably involved in the uptake and processing of copper. The location of cspB in this sequence does not confirm the genetic mapping data, indicating that the gene is closely linked to comK, which is located at 80° on the B. subtilis chromosome.

Within the framework of the international project for sequencing the entire Bacillus subtilis genome, we have determined the complete sequence of the segment flanking the purE-D gene cluster (55°) as far as cotA (52°). This segment (34960 bp) contains, as well as 12 genes already identified as part of the pur operon, 17 putative ORFs and one partial one. Two of them (gabP and guaA) are known B. subtilis genes. The gene product of cotA (formerly pig) shows significant similarity to oxidoreductases (phenoxazine synthase and bilirubin oxidase). The putative products of ORFs yeaB (Czd protein), yeaC (MoxR), yebA (CNG-channel and cGMP-channel proteins from eukaryotes), yebB (hypothetical 32·9 kDa protein of Escherichia coli), yecA (amino acid permease) and yecB (adenine deaminase) were similar to proteins in data banks.

A stretch of DNA approximately 27 kb in length, adjacent to the nprE gene of Bacillus subtilis, has been sequenced. The sequenced fragment carries a total of 23 ORFs. Of these, 15 could be assigned probable functions based on homologies to characterized genes either in B. subtilis or in other organisms. The sequencing of this region has also allowed us to assign to this area adeC and strB, previously located on the other side of nprE, between nprE and the pyr operon.

The 49630 bp spoOH-rrnH region of the Bacillus subtilis genome has been fully sequenced. The sequence contains one partial and 62 complete ORFs, one partial and three complete rRNA genes and a cluster of six tRNA genes. The direction of the transcription and translation of 61 ORFs is the same as that of the movement of the replication fork. A homology search of 40 ORFs in newly determined sequence revealed that 27 of them had significant similarity to known proteins such as elongation factor G, elongation factor Tu, pseudouridine synthase I and ribosomal proteins. Two adjacent genes, ybaD and ybaE, appeared to encode proteins belonging to the ATP-binding cassette (ABC) family.

We determined a 146 kb contiguous sequence at the 25°–36° region of the Bacillus subtilis chromosome containing the amyE-srfA segment. Among the 113 ORFs identified, 33 are already known. Functions were assigned to 38 ORFs by a search of non-redundant protein sequence data banks and those of 16 ORFs were suggested through significant similarity with reported sequences. The amino acid sequences of 13 of the ORFs were similar to proteins of unknown function of Escherichia coli, Haemophilus influenzae and other species. We did not find similarities for 29 ORFs to any known proteins. The 146 kb region is rich in enzymes (35 ORFs) related to the metabolism of low molecular mass compounds and five genes for surfactin production occupy about 26 kb of the region.

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 (40601 bp; 73°–76° 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, E1β and E1α, 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.

Within the framework of the international programme to sequence the genome of Bacillus subtilis strain 168, we were allocated the region between dnaB (256°) and pheA (240°). The sequencing of this region is now complete and we report our primary analysis of the 114 kb region containing 114 ORFs. In addition to previously characterized genes, we have identified genes involved in the utilization of plant cell wall polysaccharides, stress responses and the metabolism of amino acids, cell walls, DNA and fatty acids. We also discuss various structural and physical features, including the orientation of genes with respect to replication, putative start and stop codons, ribosome binding sites and ρ-independent transcription terminators.

The nucleotide sequence of the Bacillus subtilis 168 chromosomal segment located between yvhJ (307°) and secA (305°) was determined. This 20·3 kb region encompasses 23 ORFs, 17 of which have been sequenced previously. Comparison of sequences obtained here with the previously obtained ones revealed seven discrepancies. The products of the sequenced genes are involved in the regulation of degradative enzymes, competence, flagellar motility and protein secretion. Putative functions of newly identified genes are based on sequence homologies.

A sequence strategy which combines a low redundancy shotgun approach and directed sequencing has been elaborated. Essentially, the sequences, as well as the size of the fragments utilized for a low coverage shotgun approach, were exploited for the construction of a physical map of the region to be sequenced. The latter considerably simplified the subsequent directed sequencing steps. We report the physical mapping of a 115 kb segment which covers nearly 100 kb of the hisA-cysB region of the Bacillus subtilis chromosome and contains previously sequenced genes sigL and sacB. Sequencing and analysis of a 21305 bp segment, which includes the sigL locus, revealed 21 ORFs, apparently belonging to at least seven transcription units. This segment has a G+C content greater than 47%, compared to 43% characteristic of the flanking regions, and mainly consists of genes whose products seem to be involved in the synthesis of an exopolysaccharide. These observations leave open the possibility that the analysed fragment has been acquired through horizontal transfer.

A DNA contig of 26·2 kb covering the 170° region of the Bacillus subtilis strain 168 genome was isolated and sequenced. For DNA isolation, suitable restriction sites at the end of previously known genes were chosen to amplify adjacent unknown DNA regions by inverse PCR. On the basis of the DNA sequence, 26 ORFs were identified of which egIS and ccdA, as well as parts of citB and tkt have been described previously. Here we report the complete sequences of the aconitase (citB) and transketolase (tkt) genes. Of the other proteins encoded on the 26·2 kb fragment, eight revealed similarities to previously described proteins. These included a pair of newly identified DNA gyrase subunits A (grIA) and B (grIB), a sodium/proton-dependent alanine carrier (alsT), a member of the thioredoxin family (tlpA), an endo-1,4-β-xylanase (xynD) and a response regulator protein. Comparison of the physical and the genetic maps revealed several differences. According to its flanking sequences the lexA (dinR) gene which was previously mapped at 162° was found to be adjacent to yneA localized at 170°. Genes citB and egIS were located the opposite way round and closer together than expected from the genetic map (citB at 173° and egIS at 170°). The prkA gene, which was mapped at 169°, was not present on the respective fragment. Sequence comparison actually showed that prkA is located close to 70° on the B. subtilis genome.

As part of the Bacillus subtilis genome sequencing project, we have determined a 283 kb contiguous sequence from 210° to 232° 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, spoOA, 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. subtilistemperate phage ø105 and/or the defective phage PBSX.

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, β-glucanase (lichenase) and catalase 2, respectively. The 11th, 30th, 36th, 39th, 41st, 45th–48th, 51st and 58th genes are designated deaD, pepT, gaIE, 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, σ-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.