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Inactivation of ?E and ?G in Clostridium acetobutylicum illuminates their roles in clostridial-cell-form biogenesis, granulose synthesis, solventogenesis, and spore morphogenesis.

ABSTRACT: Central to all clostridia is the orchestration of endospore formation (i.e., sporulation) and, specifically, the roles of differentiation-associated sigma factors. Moreover, there is considerable applied interest in understanding the roles of these sigma factors in other stationary-phase phenomena, such as solvent production (i.e., solventogenesis). Here we separately inactivated by gene disruption the major sporulation-specific sigma factors, ?(E) and ?(G), and performed an initial analysis to elucidate their roles in sporulation-related morphogenesis and solventogenesis in Clostridium acetobutylicum. The terminal differentiation phenotype for the sigE inactivation mutant stalled in sporulation prior to asymmetric septum formation, appeared vegetative-like often with an accumulation of DNA at both poles, frequently exhibited two longitudinal internal membranes, and did not synthesize granulose. The sigE inactivation mutant did produce the characteristic solvents (i.e., butanol and acetone), but the extent of solventogenesis was dependent on the physiological state of the inoculum. The sigG inactivation mutant stalled in sporulation during endospore maturation, exhibiting engulfment and partial cortex and spore coat formation. Lastly, the sigG inactivation mutant did produce granulose and exhibited wild-type-like solventogenesis.

SUBMITTER: Tracy BP

PROVIDER: S-EPMC3067626 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Inactivation of σE and σG in Clostridium acetobutylicum illuminates their roles in clostridial-cell-form biogenesis, granulose synthesis, solventogenesis, and spore morphogenesis.

Tracy Bryan P BP Jones Shawn W SW Papoutsakis Eleftherios T ET

Journal of bacteriology 20110107 6

Central to all clostridia is the orchestration of endospore formation (i.e., sporulation) and, specifically, the roles of differentiation-associated sigma factors. Moreover, there is considerable applied interest in understanding the roles of these sigma factors in other stationary-phase phenomena, such as solvent production (i.e., solventogenesis). Here we separately inactivated by gene disruption the major sporulation-specific sigma factors, σ(E) and σ(G), and performed an initial analysis to ...[more]

PMID: 21217008

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Project description:The recently revived Clostridium acetobutylicum-based acetone-butanol-ethanol (ABE) fermentation is widely celebrated and studied for its impact on industrial biotechnology. C. acetobutylicum has been studied and engineered extensively, yet critical areas of the molecular basis for how solvent formation is regulated remain unresolved. The core solventogenic genes (adhE1/aad, ctfA, ctfB, and adc) are carried on the sol locus of the pSOL1 megaplasmid, whose loss leads to asporogenous, "degenerate" cells. The sol locus includes a noncoding small RNA (sRNA), SolB, whose role is presumed to be critical for solventogenesis but has eluded resolution. In the present study, SolB overexpression downregulated the sol-locus genes at the transcript level, resulting in attenuated protein expression and a solvent-deficient phenotype, thus suggesting that SolB affects expression of all sol-locus transcripts and seemingly validating its hypothesized role as a repressor. However, deletion of solB resulted in a total loss of acetone production and severe attenuation of butanol formation, with complex effects on sol-locus genes and proteins: it had a small impact on adc mRNA or its corresponding protein (acetoacetate decarboxylase) expression level, somewhat reduced adhE1 and ctfA-ctfB mRNA levels, and abolished the ctfA-ctfB-encoded coenzyme A transferase (CoAT) activity. Computational predictions support a model whereby SolB expressed at low levels enables the stabilization and translation of sol-locus transcripts to facilitate tuning of the production of various solvents depending on the prevailing culture conditions. A key predicted SolB target is the ribosome binding site (RBS) of the ctfA transcript, and this was verified by expressing variants of the ctfA-ctfB genes to demonstrate the importance of SolB for acetone formation.IMPORTANCE Small noncoding RNAs regulate many important metabolic and developmental programs in prokaryotes, but their role in anaerobes has been explored minimally. Regulation of solvent formation in the important industrial organism C. acetobutylicum remains incompletely understood. While the genes for solvent formation and their promoters are known, the means by which this organism tunes the ratios of key solvents, notably the butanol/acetone ratio to balance its electron resources, remains unknown. Significantly, the roles of several coding and noncoding genes in the sol locus in tuning the solvent formation ratios have not been explored. Here we show that the small RNA SolB fine-tunes the expression of solvents, with acetone formation being a key target, by regulating the translation of the acetone formation rate-limiting enzyme, the coenzyme A transferase (CoAT). It is notable that SolB expressed at very low levels enables CoAT translation, while at high, nonphysiological expression levels, it leads to degradation of the corresponding transcript.

Dataset Information

Inactivation of ?E and ?G in Clostridium acetobutylicum illuminates their roles in clostridial-cell-form biogenesis, granulose synthesis, solventogenesis, and spore morphogenesis.

Publications

Inactivation of σE and σG in Clostridium acetobutylicum illuminates their roles in clostridial-cell-form biogenesis, granulose synthesis, solventogenesis, and spore morphogenesis.

Similar Datasets

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