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Analysis of the Bacillus subtilis S10 ribosomal protein gene cluster identifies two promoters that may be responsible for transcription of the entire 15-kilobase S10-spc-alpha cluster.

ABSTRACT: We have sequenced a previously uncharacterized region of the Bacillus subtilis S10 ribosomal protein gene cluster. The new segment includes genes for S10, L3, L4, L23, L2, S19, L22, S3, and part of L16. These B. subtilis genes map in the same order as the genes in the Escherichia coli S10 ribosomal protein operon. Two potential promoter sequences were identified, one approximately 200 bases and the other approximately 140 bases upstream of the S10 gene. The activities of the two promoters were demonstrated by primer extension analysis, in vitro transcription experiments, and in vivo promoter fusion plasmid studies. In agreement with previous reports, our Northern analysis of exponentially growing cells failed to identify terminators or other active promoters within the S10-spc-alpha region. Our observations suggest that the two S10 promoters reported here are responsible for transcribing a 15-kb-long transcript for all of the genes in the B. subtilis S10, spc, and alpha clusters.

SUBMITTER: Li X

PROVIDER: S-EPMC179646 | biostudies-other | 1997 Nov

REPOSITORIES: biostudies-other

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Analysis of the Bacillus subtilis S10 ribosomal protein gene cluster identifies two promoters that may be responsible for transcription of the entire 15-kilobase S10-spc-alpha cluster.

Li X X Lindahl L L Sha Y Y Zengel J M JM

Journal of bacteriology 19971101 22

We have sequenced a previously uncharacterized region of the Bacillus subtilis S10 ribosomal protein gene cluster. The new segment includes genes for S10, L3, L4, L23, L2, S19, L22, S3, and part of L16. These B. subtilis genes map in the same order as the genes in the Escherichia coli S10 ribosomal protein operon. Two potential promoter sequences were identified, one approximately 200 bases and the other approximately 140 bases upstream of the S10 gene. The activities of the two promoters were d ...[more]

PMID: 9371452

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Project description:S10-spc-alpha is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-alpha locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-alpha locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-alpha locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.

Project description:BACKGROUND:?-Mannanase catalyzes the cleavage of ?-1,4-linked internal linkages of mannan backbone randomly to produce new chain ends. Alkaline and thermostable ?-mannanases provide obvious advantages for many applications in biobleaching of pulp and paper, detergent industry, oil grilling operation and enzymatic production of mannooligosaccharides. However, only a few of them are commercially exploited as wild or recombinant enzymes, and none heterologous and secretory expression of alkaline ?-mannanase in Bacillus subtilis expression system was reported. Alkaliphilic Bacillus clausii S10 showed high ?-mannanase activity at alkaline condition. In this study, this ?-mannanase was cloned, purified and characterized. The high-level secretory expression in B. subtilis was also studied. RESULTS:A thermo-alkaline ?-mannanase (BcManA) gene encoding a 317-amino acid protein from alkaliphilic Bacillus clausii strain was cloned and expressed in Escherichia coli. The purified mature BcManA exhibited maximum activity at pH 9.5 and 75 °C with good stability at pH 7.0-11.5 and below 80 °C. BcManA demonstrated high cleavage capability on polysaccharides containing ?-1,4-mannosidic linkages, such as konjac glucomannan, locust bean gum, guar gum and sesbania gum. The highest specific activity of 2366.2 U mg-1 was observed on konjac glucomannan with the Km and kcat value of 0.62 g l-1 and 1238.9 s-1, respectively. The hydrolysis products were mainly oligosaccharides with a higher degree of polymerization than biose. BcManA also cleaved manno-oligosaccharides with polymerization degree more than 3 without transglycosylation. Furthermore, six signal peptides and two strong promoters were used for efficiently secreted expression optimization in B. subtilis WB600 and the highest extracellular activity of 2374 U ml-1 with secretory rate of 98.5% was obtained using SPlipA and P43 after 72 h cultivation in 2?×?SR medium. By medium optimization using cheap nitrogen and carbon source of peanut meal and glucose, the extracellular activity reached 6041 U ml-1 after 72 h cultivation with 6% inoculum size by shake flask fermentation. CONCLUSIONS:The thermo-alkaline ?-mannanase BcManA showed good thermal and pH stability and high catalytic efficiency towards konjac glucomannan and locust bean gum, which distinguished from other reported ?-mannanases and was a promising thermo-alkaline ?-mannanase for potential industrial application. The extracellular BcManA yield of 6041 U ml-1, which was to date the highest reported yield by flask shake, was obtained in B. subtilis with constitutive expression vector. This is the first report for secretory expression of alkaline ?-mannanase in B. subtilis protein expression system, which would significantly cut down the production cost of this enzyme. Also this research would be helpful for secretory expression of other ?-mannanases in B. subtilis.

Dataset Information

Analysis of the Bacillus subtilis S10 ribosomal protein gene cluster identifies two promoters that may be responsible for transcription of the entire 15-kilobase S10-spc-alpha cluster.

Publications

Analysis of the Bacillus subtilis S10 ribosomal protein gene cluster identifies two promoters that may be responsible for transcription of the entire 15-kilobase S10-spc-alpha cluster.

Similar Datasets

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