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 λ 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. Received: April 17, 1998 / Accepted: June 23, 1998     

News & Notes: Molecular Characterization of the xerC Gene of Lactobacillus leichmannii Encoding a Site-Specific Recombinase and Two Adjacent Heat Shock Genes

Sequencing of four overlapping DNA fragments comprising 3.527 kb isolated from a L. leichmannii genomic library revealed three complete open reading frames (ORFs) and one that was truncated. The deduced amino acid sequences of the complete ORFs showed considerable similarities with the already known sequences of the xerC, hslV, and hslU gene products of Escherichia coli: the site-specific XerC recombinase, a member of the lambda integrase family, and the HtpI resp. HtpO heat shock proteins. The deduced amino acid sequence of the fourth, incomplete ORF upstream the xerC gene showed strong homology with the gidA gene product of B. subtilis.     

Complete nucleotide sequence of pGS18, a 62.8-kb plasmid from <Emphasis Type="Italic">Geobacillus stearothermophilus</Emphasis> strain 18

The complete nucleotide sequence (62.8 kb) of pGS18, the largest sequenced plasmid to date from the species Geobacillus stearothermophilus, was determined. Computational analysis of sequence data revealed 65 putative open reading frames (ORFs); 38 were carried on one strand and 27 were carried on the other. These ORFs comprised 84.1% of the pGS18 sequence. Twenty-five ORFs (38.4%) were assigned to putative functions; four ORFs (6.2%) were annotated as pseudogenes. The amino acid sequences obtained from 29 ORFs (44.6%) had the highest similarity to hypothetical proteins of the other microorganisms, and seven (10.8%) had no significant similarity to any genes present in the current open databases. Plasmid replication region, strongly resembling that of the theta-type replicon, and genes encoding three different plasmid maintenance systems were identified, and a putative discontinuous transfer region was localized. In addition, we also found several mobile genetic elements and genes, responsible for DNA repair, distributed along the whole sequence of pGS18. The alignment of pGS18 with two other large indigenous plasmids of the genus Geobacillus highlighted the presence of well-conserved segments and has provided a framework that can be exploited to formulate hypotheses concerning the molecular evolution of these three plasmids.     

Sequence analysis and functional studies of a chromosomal region of alkaliphilic Bacillus firmus OF4 encoding an ABC-type transporter with similarity of sequence and Na+ exclusion capacity to the Bacillus subtilis NatAB transporter

A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the alkaliphile natCAB catalyzes Na⁺ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na⁺ extrusion. The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na⁺/H⁺ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs. Received: August 30, 1998 / Accepted: November 13, 1998     

Sequence Analysis of the 36-kb Region Between gntZ and trnY Genes of Bacillus Subtilis Genome*

Within the framework of an international Bacillus subtilis genomesequencing project, we have determined a 36-kb sequence coveringthe region between the gntZ and trnY genes. In addition to fivegenes sequenced and characterized previously, 27 putative proteincoding sequences (open reading frame; ORF) were identified.A homology search for the newly identified ORFs revealed thatsix of them had similarities to known proteins. It is notablethat new ORFs belonging to response-regulator aspartate phosphatase(Rap) and its regulator (Phr) families, and response regulatorand sensory kinase families of two-component signal transductionsystems have been identified. Furthermore, we found that some180-bp non-coding sequence, that might be an remnant of an ancientIS element, is preserved in at least five loci of the B. subtilisgenome.     

Sequence Analysis of the Bacillus subtilis 168 Chromosome Region between the sspC and odhA Loci (184{degrees}-180{degrees})

The nucleotide sequence of 45,389 bp in the 184°-;180°region of the Bacillus subtilis chromosome, containing the cgecluster, which is controlled by the sporulation regulatory proteinGerE, was determined. Fifty-four putative ORFs with putativeribosome-binding sites were recognized. Seven of them correspondto previously characterized genes: cgeB, cgeA, cgeC, cgeD, cgeE,ctpA, and odhA. The deduced products of 25 ORFs were found todisplay significant similarities to proteins in the data banks.We have identified genes involved in detoxification, cell walls,and in the metabolism of biotins, purines, fatty acids, carbohydratesand amino acids. The remaining 22 ORFs showed no similarityto known proteins. Both an attachment site of the SPßprophage and 2 new putative DNA replication terminators wereidentified in this region.     

Microbial genes homologous to the peptidyl-tRNA hydrolase-encoding gene of Escherichia coli

We have cloned and determined the nucleotide (nt) sequences of the genes encoding peptidyl-tRNA hydrolase (Pth) homologues of Salmonella typhi (St) and the Lyme disease spirochaete, Borrelia burgdorferi (Bb). We also completed the nt sequence of a pth homologous gene contained in a Chlamydia trachomatis (Ct) clone identified in the databanks. The open reading frames (ORFs) of the Pth homologues encode putative polypeptides of 194 (St), 188 (Bb) and 194 (Ct) amino acids exhibiting significant identity with Escherichia coli (Ec) Pth. Together with the products of two previously unidentified ORFs from Bacillus subtilis and Saccharomyces cerevisiae, and the recently recognized Haemophilus influenzae and Mycoplasma genitalium pth genes, these seven putative polypeptides and the Ec Pth form a group of homologous basic proteins spanning eubacteria and eukaryota which can be defined by at least three conserved regions. Previously known Ec pth mutations were located in highly conserved residues     

Organization and Sequence of Histidine Biosynthesis Genes hisH, -A, -F, and -IE in Thermoanaerobacter ethanolicus

Nucleotide sequence analysis of a 3.5-kb chromosomal fragment from the low G + C Gram-positive bacterium Thermoanaerobacter ethanolicus revealed a cluster of five contiguous open reading frames (ORFs) designated hisH, hisA, hisF, hisIE, and ORF5. The first four ORFs showed homology to genes of the histidine biosynthesis pathway, and ORF5 encoded a product with no significant similarities to polypeptides presently known. The hisH ORF was partial (truncated by cloning) and ORF5 was adjacent to xylF, which codes for a xylose-binding periplasmic protein. The five genes encoded putative proteins of >104, 237, 254, 216, and 169 amino acids, respectively. Amino acid sequence comparison of the four his gene products indicated closely related homologs in prokaryotes, varying from low G + C Gram-positive bacteria to archaea. This is the first report of his anabolic genes in a thermophilic anaerobic bacterium. Received: 8 July 1999 / Accepted: 23 August 1999     

Cloning,Sequencing, and Characterization of the Genetic Region Relevant to Biosynthesis of the Lipopeptides Iturin A and Surfactin in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis B3 was found to produce lipopeptides iturins and fengycin that have activity against several plant pathogens such as Fusarium graminearum, Rhizoctonia solani, Rhizoctonia cerealis, and Pyricularia grisea. A 3642-bp genomic region of B. subtilis B3 comprising srfDB3, aspB3, lpaB3, and yczEB3 genes that resulted in biosynthesis of surfactin in B. subtilis 168 was cloned, sequenced, and characterized. Among them, the srfDB3 gene encodes thioesterase, which is required for biosynthesis of surfactin in B. subtilis; the aspB3 gene encodes a putative aspartate aminotransferase-like protein; the lpaB3 encodes phosphopantetheinyl transferase, which shows high identity to the product of lpa-14 gene regulating the biosynthesis of iturin A and surfactin in B. subtilis RB14; the yczEB3 encodes a YczE-like protein with significant similarities in signal peptide and part of the ABC transport system. The genetic regions between the srfD gene and lpa gene from B. subtilis B3 and B. subtilis A13, which produces iturin A, contain an approximate 1-kb nucleotide fragment encoding an aspartate aminotransferase-like protein; however, the relevant regions from B. subtilis 168 and B. subtilis ATCC21332 producing surfactin comprise an approximately 4-kb nucleotide fragment encoding four unknown proteins. There is 73% identity between the Lpa family and the Sfp family, although both are highly conserved.Received: 29 October 2002 / Accepted: 6 December 2002     

Molecular Cloning of the Transglutaminase Gene from Bacillus subtilis and Its Expression in Escherichia coli

We cloned and characterized a gene, tgl, encoding transglutaminase in Bacillus subtilis. The tgl gene contained a open reading frame 735-nucleotides long that encoded a 245-residue protein with the molecular weight of 28,300. The deduced amino acid sequence had little sequence similarity with sequences of other transglutaminases from a Streptoverticillium sp. or from mammals. The -10 and -35 regions of a putative promoter resembled the consensus sequence for the σ^K-dependent promoter. In addition, a sequence similar to the consensus sequence for the GerE binding site was found upstream from this region. These findings suggested that tgl was transcribed in the mother cells during a late stage of sporulation. Evidence for this suggestion was that transglutaminase activity was detected in sporulating cells during the same stage. Transglutaminase activity was detected in Escherichia coli cells transformed with a plasmid for expression of the tgl gene.     

Comparative Genomics Reveals Long, Evolutionarily Conserved, Low-Complexity Islands in Yeast Proteins

Eukaryotic proteomes abound in low-complexity sequences, including tandem repeats and regions with significantly biased amino acid compositions. We assessed the functional importance of compositionally biased sequences in the yeast proteome using an evolutionary analysis of 2838 orthologous open reading frame (ORF) families from three Saccharomyces species (S. cerevisiae, S. bayanus, and S. paradoxus). Sequence conservation was measured by the amino acid sequence variability and by the ratio of nonsynonymous-to-synonymous nucleotide substitutions (K _a /K _s ) between pairs of orthologous ORFs. A total of 1033 ORF families contained one or more long (at least 45 residues), low-complexity islands as defined by a measure based on the Shannon information index. Low-complexity islands were generally less conserved than ORFs as a whole; on average they were 50% more variable in amino acid sequences and 50% higher in K _a /K _s ratios. Fast-evolving low-complexity sequences outnumbered conserved low-complexity sequences by a ratio of 10 to 1. Sequence differences between orthologous ORFs fit well to a selectively neutral Poisson model of sequence divergence. We therefore used the Poisson model to identify conserved low-complexity sequences. ORFs containing the 33 most conserved low-complexity sequences were overrepresented by those encoding nucleic acid binding proteins, cytoskeleton components, and intracellular transporters. While a few conserved low-complexity islands were known functional domains (e.g., DNA/RNA-binding domains), most were uncharacterized. We discuss how comparative genomics of closely related species can be employed further to distinguish functionally important, shorter, low-complexity sequences from the vast majority of such sequences likely maintained by neutral processes. [Reviewing Editor: Dr. Stuart Newfeld]     

DNA Sequence of Bacillus subtilis (natto) NR-1 γ-Glutamyltranspeptidase Gene,ggt

The ggt encoding γ-glutamyltranspeptidase (GGT) from Bacillus subtilis (natto) was cloned and sequenced. The DNA sequence contains a single open reading frame of 1761 bp that might be translated to a protein of 587 amino acid residues, and indicates that B. subtilis (natto) GGT is synthesized as prepro-GGT and processed later into large and small subunits. The putative catabolite responsive element (CRE) was located upstream of the ggt coding region.     

A novel ICE in the genome of Shewanella putrefaciens W3-18-1: comparison with the SXT/R391 ICE-like elements

A novel R391-like ICE (integrating conjugative element) has been detected in the 4.2 MB genome of Shewanella putrefaciens W3-18-1 located on three different contigs. Assembly of the ICE encoding contigs based on similarity with R391 revealed a mosaic element of plasmid, phage and transposon-like sequences typical of SXT/R391 ICE-like elements. The element, which is 110 057 bp in length, was highly similar to R391 sequences, with most related ORFs showing >96% amino acid sequence identity. The element, designated ICESpuPO1, contained a number of inserts determining resistance to copper and other heavy metals and a broad-spectrum RND efflux pump similar to antibiotic efflux systems. The element was integrated into the Shewanella prfC gene in a manner similar to related ICE-like elements. The chromosomal element junctions contained a 17-bp SXT/R391-like attL and attR site and an unannotated ORF between attL and the ICE integrase encoding a putative recombinational directional factor necessary for excision, with 100% amino acid identity to the R391 ORF4 product.     

Sequence Analysis of a 32-kb Region Including the Major Ribosomal Protein Gene Clusters from Alkaliphilic Bacillus sp. Strain C-125

Forty-one open reading frames (ORFs) were identified in a 32-kb DNA fragment of alkaliphilic Bacillus sp. C-125. A similarity search using the BSORF database found 37 ORFs with significant sequence similarity to B. subtilis RNA polymerase subunits, elongation factor G, elongation factor Tu, and ribosomal proteins. Each ORF product showed more than 70% identity to those of B. subtilis. Gene organization in the region of str, S10, spc, and the α cluster was highly conserved among three strains, C-125, B. subtilis, and B. stearothermophilus.     

Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae

Open reading frames in the genome of Saccharomyces cerevisiae were screened for potential glycosylphosphatidylinositol (GPI)-attached proteins. The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a signal sequence for secretion and a serine- or threonine-rich sequence. In all, 53 ORFs met these three criteria and 38 were further analyzed as follows. The sequence encoding the 40 C-terminal amino acids of each was fused with the structural gene for a reporter protein consisting of a secretion signal, α-galactosidase and a hemagglutinin (HA) epitope, and examined for the ability to become incorporated into the cell wall. On this basis, 14 of fusion proteins were classified as GPI-dependent cell wall proteins because cells expressing these fusion proteins: (i) had high levels of α-galactosidase activity on their surface; (ii) released significant amounts of the fusion proteins from the membrane on treatment with phosphatidylinositol-specific phospholipase C (PI-PLC); and (iii) released fusion proteins from the cell wall following treatment with laminarinase. Of the 14 identified putative GPI-dependent cell wall proteins, 12 had novel ORFs adjacent to their GPI-attachment signal sequence. Amino acid sequence alignment of the C-terminal sequences of the 12 ORFs, together with those of known cell wall proteins, reveals some sequence similarities among them. Received: 1 September 1997 / Accepted: 20 November 1997     

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°–52°) 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. Theinherent restriction/modification system genes, hsdMR and hsdMM,were mapped between groESL and gutRB, and we have identifiedtwo open reading frames (ORFs) encoding 5-methylcytosine formingDNA methyl transferase and an operon probably encoding a restrictionenzyme complex. The unusual genome structure of few ORFs andlower GC content around the restriction/modification genes stronglysuggests that the region originated from a bacteriophage integratedduring evolution.