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Physical and functional characterization of the Bacillus subtilis spoIIM gene.

ABSTRACT: The spoIIM locus of Bacillus subtilis is the most recently discovered of six genetic loci in which mutations can prevent the synthesis of a normal asymmetric septum or prevent migration of the septal structure to engulf the forespore compartment of the sporangium. Ultrastructure studies of a spoIIM mutant confirmed a block prior to the completion of engulfment. Introduction of a spoIIM mutation into a panel of strains containing lacZ fusions belonging to different regulatory classes allowed us to determine that the spoIIM gene product is required for the efficient expression of genes transcribed by sigma G-associated RNA polymerase but is not required for the expression of sigma F-controlled genes, including spoIIIG, which encodes sigma G. The results of complementation studies, gene disruption analysis, and DNA sequencing revealed that the spoIIM locus contains a single sporulation-essential gene encoding a polypeptide with a predicted molecular mass of 24,850 Da. The predicted spoIIM gene product is highly hydrophobic and very basic, and it does not exhibit significant homology to sequence files in several major data bases.

SUBMITTER: Smith K

PROVIDER: S-EPMC204762 | biostudies-other | 1993 Jun

REPOSITORIES: biostudies-other

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Physical and functional characterization of the Bacillus subtilis spoIIM gene.

Smith K K Bayer M E ME Youngman P P

Journal of bacteriology 19930601 11

The spoIIM locus of Bacillus subtilis is the most recently discovered of six genetic loci in which mutations can prevent the synthesis of a normal asymmetric septum or prevent migration of the septal structure to engulf the forespore compartment of the sporangium. Ultrastructure studies of a spoIIM mutant confirmed a block prior to the completion of engulfment. Introduction of a spoIIM mutation into a panel of strains containing lacZ fusions belonging to different regulatory classes allowed us t ...[more]

PMID: 8501064

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Project description:BACKGROUND: Microbial gene expression is strongly influenced by environmental growth conditions. Comparison of gene expression under different conditions is frequently used for functional analysis and to unravel regulatory networks, however, gene expression responses to co-cultivation with other microorganisms, a common occurrence in nature, is rarely studied under laboratory conditions. To explore cellular responses of the antibiotic-producing fungus Penicillium chrysogenum to prokaryotes, the present study investigates its transcriptional responses during co-cultivation with Bacillus subtilis. RESULTS: Steady-state glucose-limited chemostats of P. chrysogenum grown under penillicin-non-producing conditions were inoculated with B. subtilis. Physiological and transcriptional responses of P. chrysogenum in the resulting mixed culture were monitored over 72 h. Under these conditions, B. subtilis outcompeted P. chrysogenum, as reflected by a three-fold increase of the B. subtilis population size and a two-fold reduction of the P. chrysogenum biomass concentration. Genes involved in the penicillin pathway and in synthesis of the penicillin precursors and side-chain were unresponsive to the presence of B. subtilis. Moreover, Penicillium polyketide synthase and nonribosomal peptide synthase genes were either not expressed or down-regulated. Among the highly responsive genes, two putative ?-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold, respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production. Mutanase activity was neither observed in pure cultures of P. chrysogenum or B. subtilis, nor during exposure of P. chrysogenum to B. subtilis culture supernatants or heat-inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum exposed to filter-sterilized supernatants of mixed cultures of P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that Pc12g07500 encoded an active ?-1,3 endoglucanase. CONCLUSION: Time-course transcriptional profiling of P. chrysogenum revealed differentially expressed genes during co-cultivation with B. subtilis. Penicillin production was not induced under these conditions. However, induction of a newly characterized P. chrysogenum gene encoding ?-1,3 endoglucanase may enhance the efficacy of fungal antibiotics by degrading bacterial exopolysaccharides.

Project description:Background: Microbial gene expression is to a large extend determined by environmental growth conditions. Differential gene expression analysis between two conditions has been frequently used to reveal regulatory networks and to assign physiological function to unknown genes. In nature, microorganisms cohabit however these interactions have been rarely studied and reproduced in laboratory set-up. Thus to quantitatively explore the genome-wide responses of microbial interaction, we co-cultivated Penicillium chrysogenum and Bacillus subtilis in chemostat culture. Results: Time course expression analysis of P. chrysogenum to co-cultivation with B. subtilis was carried out to understand the natural responses of P. chrysogenum to prokaryotes. Steady state chemostats of P. chrysogenum in non-B-lactam producing conditions was pulsed with B. subtilis and co-cultivation was followed for 72 hours. The dynamic physiological and transcriptional responses of P. chrysogenum in mixed culture were monitored. B. subtilis outcompeted growth of P. chrysogenum resulting in an increased B. subtilis biomass by more than three fold of its original size and a reduction in P. chrysogenum biomass to half of its original size after 72 h of mixed culture. Genes of the penicillin pathway, synthesis of the side-chain and precursors were overall unresponsive to the presence of B. subtilis. Moreover Penicillium polyketide synthase and nonribosomal peptide synthetase genes either remained silent or down-regulated, whereas genes responsible for protein synthesis, metabolism, energy conservation, respiration and transport were upregulated in the presence of B. subtilis. Among highly responsive genes, two putative B-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production in P. chrysogenum. Mutanase activity was not observed in pure cultures of P. chrysogenum and B. subtilis or when P. chrysogenum was co-cultured with B. subtilis supernatant or heat inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum pulsed with filter sterilized supernatants from mixed cultures P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that at least Pc12g07500 encoded an B-1,3 endoglucanase. Conclusion: Time course transcriptional profiling of P. chrysogenum revealed several differentially expressed genes during mixed culture, potentially reflecting interactions between the eukaryotic and the prokaryotic systems. -1,3 endoglucanase produced by P. chrysogenum against B. subtilis signals may have application in improving the efficacy of antibiotics by degrading exopolysacchride biofilms of pathogenic bacteria.

Dataset Information

Physical and functional characterization of the Bacillus subtilis spoIIM gene.

Publications

Physical and functional characterization of the Bacillus subtilis spoIIM gene.

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