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An agr quorum sensing system that regulates granulose formation and sporulation in Clostridium acetobutylicum.

ABSTRACT: The Gram-positive, anaerobic, endospore-forming bacterium Clostridium acetobutylicum has considerable biotechnological potential due to its ability to produce solvents as fermentation products, in particular the biofuel butanol. Its genome contains a putative agr locus, agrBDCA, known in staphylococci to constitute a cyclic peptide-based quorum sensing system. In staphylococci, agrBD is required for the generation of a peptide signal that, upon extracellular accumulation, is sensed by an agrCA-encoded two-component system. Using ClosTron technology, agrB, agrC, and agrA mutants of C. acetobutylicum ATCC 824 were generated and phenotypically characterized. Mutants and wild type displayed similar growth kinetics and no apparent differences in solvent formation under the conditions tested. However, the number of heat-resistant endospores formed by the mutants in liquid culture was reduced by about one order of magnitude. On agar-solidified medium, spore formation was more strongly affected, particularly in agrA and agrC mutants. Similarly, accumulation of the starch-like storage compound granulose was almost undetectable in colonies of agrB, agrA, and agrC mutants. Importantly, these defects could be genetically complemented, demonstrating that they were directly linked to agr inactivation. A diffusible factor produced by agrBD-expressing strains was found to restore granulose and spore formation in the agrB mutant. Furthermore, a synthetic cyclic peptide, designed on the basis of the C. acetobutylicum AgrD sequence, was also capable of complementing the defects of the agrB mutant when added exogenously to the culture. Together, these findings support the hypothesis that agr-dependent quorum sensing is involved in the regulation of sporulation and granulose formation in C. acetobutylicum.

SUBMITTER: Steiner E

PROVIDER: S-EPMC3273008 | biostudies-other | 2012 Feb

REPOSITORIES: biostudies-other

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An agr quorum sensing system that regulates granulose formation and sporulation in Clostridium acetobutylicum.

Steiner Elisabeth E Scott Jamie J Minton Nigel P NP Winzer Klaus K

Applied and environmental microbiology 20111216 4

The Gram-positive, anaerobic, endospore-forming bacterium Clostridium acetobutylicum has considerable biotechnological potential due to its ability to produce solvents as fermentation products, in particular the biofuel butanol. Its genome contains a putative agr locus, agrBDCA, known in staphylococci to constitute a cyclic peptide-based quorum sensing system. In staphylococci, agrBD is required for the generation of a peptide signal that, upon extracellular accumulation, is sensed by an agrCA-e ...[more]

PMID: 22179241

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Project description:Since both the Agr (accessory gene regulator)-like quorum sensing (QS) system and VirS/VirR (VirS/R) two-component regulatory system of Clostridium perfringens positively regulate production of several toxins, including C. perfringens beta toxin (CPB), it has been hypothesized the VirS membrane sensor protein is an Agr-like QS signaling peptide (SP) receptor. To begin evaluating whether VirS is an SP receptor, this study sequenced the virS gene in C. perfringens strains CN3685 and CN1795 because it was reported that agrB mutants of both strains increase CPB production in response to the pentapeptide 5R, likely the natural SP, but only the CN3685 agrB mutant responds to 8R, which is 5R plus a 3-amino-acid tail. This sequencing identified differences between the predicted VirS extracellular loop 2 (ECL2) of CN3685 versus that of CN1795. To explore if those ECL2 differences explain strain-related variations in SP sensitivity and support VirS as an SP receptor, virS agrB double-null mutants of each strain were complemented to swap which VirS protein they produce. CPB Western blotting showed that this complementation changed the natural responsiveness of each strain to 8R. A pulldown experiment using biotin-5R demonstrated that VirS can bind SP. To further support VirS:SP binding and to identify a VirS binding site for SP, a 14-mer peptide corresponding to VirS ECL2 was synthesized. This ECL2 peptide inhibited 5R signaling to agrB mutant and wild-type strains. This inhibition was specific, since a single N to D substitution in the ECL2 peptide abrogated these effects. Collectively, these results support VirS as an important SP receptor and may assist development of therapeutics.IMPORTANCEC. perfringens beta toxin (CPB) is essential for the virulence of type C strains, a common cause of fatal necrotizing enteritis and enterotoxemia in humans and domestic animals. Production of CPB, as well as several other C. perfringens toxins, is positively regulated by both the Agr-like QS system and the VirS/R two-component regulatory system. This study presents evidence that the VirS membrane sensor protein is a receptor for the AgrD-derived SP and that the second extracellular loop of VirS is important for SP binding. Understanding interactions between SP and VirS improves knowledge of C. perfringens pathogenicity and may provide insights for designing novel strategies to reduce C. perfringens toxin production during infections.

Dataset Information

An agr quorum sensing system that regulates granulose formation and sporulation in Clostridium acetobutylicum.

Publications

An agr quorum sensing system that regulates granulose formation and sporulation in Clostridium acetobutylicum.

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