A 10.3 kbp Segment from nprB to argJ at the 102 Region of the Bacillus Subtilis Chromosome

Abstract

The approximately 10 kbp region encompassing nprB and argJ at 102" on the Bacillus subtilis chromosome was sequenced, revealing 12 ORFs, four known genes (argJ, argC, ipi and nprB) and two genes, yitV and yitS, whose products respectively display significant homology with L-gulono-y-lactone oxidase of rat and dihydrofolate reductase of Staphy/ococcus aureus. The data also indicated that nprB mapped to a different position than previously published.Keywords : Bacillus subtilis, genome sequencing, 102' region, argJ, nprB Within the European programme for sequencing the Bacillus subtilis genome, a 60 kbp DNA fragment located between nprB (95") and arg] (102") was originally assigned to our group. Since, as described in this paper, nprB was, in fact, found to be located approximately 8 kb from arg], the assignment was redefined and the region to be sequenced was extended from arg] (102") to addAB (89"). In this first communication, we describe our cloning and sequencing strategy and we report the sequencing and gene features of the 10336 bp region from nprB to arg].For this study, we employed a chromosome walking strategy using the integrative plasmid pDIA5304 (Glaser et al., 1993), into which we inserted a 1 kb fragment from arg] obtained from pUL710. This allowed us, after digestion of the chromosomal DNA with an appropriate restriction enzyme ( S a d ) , to pick up a fragment of more than 10 kbp adjacent to argJ. The recombinant plasmid, pOMG3001, was propagated in Escherichia coli TP611, a mutant strain (pcnB) which facilitates cloning of B. subtilis DNA by reducing the copy number of ColEl derivatives (Xu et al., 1993). The integrity of the clone was verified by Southern hybridization. For shotgun analysis, pOMG3001 was subjected to a nebulization procedure and 1-1-5 kb fragments were subcloned into pUCl8. After identification and selection by Southern hybridization, the fragments corresponding to the insert were sequenced. The sequencing reactions were performed using the Taq Dye Primer Cycle Sequencing kit and the Taq Dye Deoxy Terminator Cycle Sequencing kit, followed by analysis with a 373A DNA sequencer Abbreviation: SD, Shine-Dalgarno.The EMBL accession number for the nucleotide sequence reported in this paper is 279580.from Applied Biosystems. The sequences were compiled using the Autoassembler program (Perkin Elmer) and analysed for possible ORFs (GENEMARK). The predicted amino acid sequence of the products of each ORF was used to search for similarity to sequences reported previously in databases using BLAST/FASTA.From the sequence data, all six possible reading frames were screened with the DNA Strider program. Based on starting codons ATG, GTG or TTG preceded by a putative Shine-Dalgarno (SD) sequence, 12 putative ORFs were detected. The direction of transcription and translation of all except orf2 ( yitS) and orf. ( yitU), was, like the majority of genes on the B. subtilis chromosome, 0

Sequencing of regions downstream of addA (98°) and citG (28°) in Bacillus subtilis

“…The reported sequences for these genes were found to be identical to our sequence, except for one position in sbcD (an additional G at position 1332 in our sequence) and two positions in wprA (T at position 11012 and C at position 11026 in our sequence instead of C and A, respectively). From these results and our previous data (Levine et al , 1997; Roche et al , 1997) we deduced that addA was located 53·5 kbp from argj. Consequently, in agreement with these results, its position on the B. subtilis genetic map has been remapped at 98° (Biaudet et al , 1996) instead of 86° indicated previously (Anagnostopoulos et al , 1993).…”

“…This completes our sequencing of the 54 kbp addA-argJ chromosomal region which proved to be much shorter than originally estimated. The sequencing strategy mainly involved Long Range PCR between addA and yisP (Roche et al , 1997), shotgun analysis after nebulization and subcloning in pUC18, as described previously (Levine et al , 1997). The gaps were filled by normal PCR.…”

The identification of response regulators ofBranhamella catarrhalisusing PCR

“…Over 180 bp, there was 63.3% homology to the pho P gene of Bacillus subtilis [11]. Across 170 bp of the third clone there was 65.4% identity to the ntr A gene which codes for a Pseudomonas putida sigma factor [5], while the fourth clone had 58.6% identity over 140 bp to a B. subtilis npr B gene [6]. The final two clones had significant homology to the dtx R gene which regulates toxin production in Corynebacterium diphtheriae [1].…”

The identification of response regulators of Branhamella catarrhalis using PCR

“…Over 180 bp, there was 63.3% homology to the phoP gene of Bacillus subtilis [11]. Across 170 bp of the third clone there was 65.4% identity to the ntrA gene which codes for a Pseudomonas putida sigma factor [5], while the fourth clone had 58.6% identity over 140 bp to a B. subtilis nprB gene [6]. The ¢nal two clones had signi¢cant homology to the dtxR gene which regulates toxin production in Corynebacterium diphtheriae [1].…”