AnagrQuorum 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 agrCAencoded 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.

The putative influence of the agr operon upon survival mechanisms used by Clostridium acetobutylicum

“…Variations to the parameter set allow sporulation to be triggered, with cells still remaining in acidogenic phase; additional signals are capable of restoring the solventogenic response. The converse was not possible, matching the experimental results of [14], while demonstrating that the agr system could still be involved in solventogenesis in other clostridial bacteria.…”

“…Experimental data used to generate wild-type results for [14] were used to fit specific parameters and conditions. Because the methods used do not calculate vegetative and spore cells directly, some data conversion was required to make them suitable for use with this model.…”

“…The agr quorum-sensing system has recently been shown to form part of the regulation of sporulation in C. acetobutylicum [14]. Quorum sensing is a cell signalling mechanism whereby individual cells secrete signal molecules into the external environment.…”

Energia da biomassa de cana-de-açúcar sob influência de hidrogel, cobertura vegetal e profundidade de plantio

“…Alguns pesquisadores acreditam que até a próxima ela será denominada "Era da bioenergia" (Mintom, 2009) oriunda principalmente de material vegetal transformado por micro-organismos (Steiner et al, 2011). O aproveitamento da energia da biomassa desponta ecológica, financeira e tecnologicamente adequado.…”

ClosTron-Mediated Engineering of Clostridium

“…By way of example, it has been used to establish the role of bacterial factors (toxins, flagella and adhesins) in infection, 23,[26][27][28][29] to garner a greater understanding of the role and activities of enzymes in primary metabolism, [30][31][32][33] to begin to elucidate mechanistic details of the developmental process of spore formation/ germination 21,34,35 and provide fundamental information on regulatory processes important in virulence factor and metabolite production. [36][37][38][39] Its availability has provided the research community with a sequence of the re-targeted region used to generate the insertion should be given. So, for example, when creating a toxin A mutant in C. difficile strain R20291, the implementation of the Perutka algorithm 13 at www.clostron.com suggests a possible insertion site in the sense orientation after nucleotide 1584.…”