Browsing by Subject "Bacillus subtilis"
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Item A Study of the Antagonistic Activity of Bacillus subtilis Strain T1 Against Shrimp Pathogen Vibrio parahaemolyticus Strain D4(2018-01-01) Avery, Sarah; Schreier, Harold J; Biological Sciences; Biological SciencesAcute hepatopancreatic necrosis disease (AHPND), also known as early mortality syndrome (EMS), is caused by strains of Vibrio parahaemolyticus containing the PirAB toxin and has been devastating to shrimp aquaculture globally. Current methods for prevention rely on the use of antibiotics that leads to the development of antibioticresistant bacteria. In vitro, Bacillus subtilis strain T1 was found to possess antagonistic activity against EMS-causing V. parahaemolyticus strain D4 and is a candidate for use as a probiotic in the aquaculture industry. Competitive growth experiments examined the effect of T1 on D4 growth. Using qPCR to assess T1 and D4 growth, these studies showed that T1 was capable of inhibiting D4 growth in a density-dependent manner, with complete inhibition occurring when the T1 starting density was 104 -fold higher than D4. Using a mariner-based transposon system, T1 mutants were generated to identify genes involved in D4-inhibitory activity. Of over 3,000 colonies screened using an overlaybased assay, 17 were identified as having either complete or partial loss of activity. Eleven mutants contained insertions within an ~30-kb DNA cluster that included lipopeptide and polyketide biosyntheses genes. One of these mutants, A3-41, which contained an insertion within a non-ribosomal peptide synthetase gene utilized for lipopeptide biosyntheses, was found to have lost the ability to inhibit D4 growth in coculture experiments. Two mutants were found to contain insertions within stationary phase regulators, spo0A (sporulation gene regulator) and oppA (first gene of the oligopeptide transporter system operon) suggesting that D4 inhibitory activity is associated with a stationary phase product. Consistent with the mutagenesis results, supernatant fractions prepared from stationary phase cultures of T1 were found to inhibit D4 growth in a dose-dependent manner while culture supernatants prepared from mutant A3-41 were not inhibitory. These results indicate that T1 produces and excretes a stationary phase metabolite(s) that has inhibitory activity against D4 and has implications for the use of T1 as a probiotic in shrimp aquaculture.Item 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(American Society for Microbiology, 1997-11) Li, X.; Lindahl, L.; Sha, Y.; Zengel, J. M.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.Item Regulation of expression from the glnA promoter of Bacillus subtilis requires the glnA gene product(American Society for Microbiology, 1985-05) Schreier, Harold J.; Fisher, Susan H.; Sonenshein, Abraham L.Expression of the cloned glnA gene [coding for glutamine synthetase (EC 6.3.1.2)] of Bacillus subtilis was 10-fold higher in an Escherichia coli strain grown under nitrogen-limiting conditions than in the same strain under nitrogen-excess conditions. Mutations in the E. coli glnA, glnB, glnD, glnE, glnF, glnG, and glnL genes had no effect on the observed regulation. To test whether sequences within the B. subtilis DNA (3.2 kilobase pairs) were responsible for the observed regulation, a plasmid carrying a transcriptional fusion of the B. subtilis glnA promoter with E. coli lacZ was constructed. beta-Galactosidase levels coded for by this plasmid were found to be negatively regulated in trans by a plasmid carrying the entire B. subtilis glnA gene. Analysis of various deletion plasmids showed that the 1.4-kilobase-pair region encoding glutamine synthetase was necessary for the observed regulation of beta-galactosidase. Plasmids coding for 67% or more of the glutamine synthetase polypeptide gave at least partial repression, but a plasmid carrying 30% of the structural gene, as well as a plasmid carrying a deletion internal to glnA, gave no repression. DNA downstream from glnA (to within 130 base pairs of the end of the gene) was not required for the observed regulation. These results suggest that the glnA gene of B. subtilis is autoregulated, supporting the model for glnA control proposed by Dean et al. [Dean, D. R., Hoch, J. A. & Aronson, A. I. (1977) J. Bacteriol. 131, 981-987].