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Agr Quorum Sensing influences the Wood-Ljungdahl pathway in Clostridium autoethanogenum.

ABSTRACT: Acetogenic bacteria are capable of fermenting CO₂ and carbon monoxide containing waste-gases into a range of platform chemicals and fuels. Despite major advances in genetic engineering and improving these biocatalysts, several important physiological functions remain elusive. Among these is quorum sensing, a bacterial communication mechanism known to coordinate gene expression in response to cell population density. Two putative agr systems have been identified in the genome of Clostridium autoethanogenum suggesting bacterial communication via autoinducing signal molecules. Signal molecule-encoding agrD1 and agrD2 genes were targeted for in-frame deletion. During heterotrophic growth on fructose as a carbon and energy source, single deletions of either gene did not produce an observable phenotype. However, when both genes were simultaneously inactivated, final product concentrations in the double mutant shifted to a 1.5:1 ratio of ethanol:acetate, compared to a 0.2:1 ratio observed in the wild type control, making ethanol the dominant fermentation product. Moreover, CO₂ re-assimilation was also notably reduced in both hetero- and autotrophic growth conditions. These findings were supported through comparative proteomics, which showed lower expression of carbon monoxide dehydrogenase, formate dehydrogenase A and hydrogenases in the ∆agrD1∆agrD2 double mutant, but higher levels of putative alcohol and aldehyde dehydrogenases and bacterial micro-compartment proteins. These findings suggest that Agr quorum sensing, and by inference, cell density play a role in carbon resource management and use of the Wood-Ljungdahl pathway as an electron sink.

SUBMITTER: Piatek P

PROVIDER: S-EPMC8748961 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:Korarchaeota, due to its rarity in common environments, is one of the archaeal phyla that has received the least attention from researchers. It was previously thought to consist solely of strict thermophiles. However, our study provides genetic evidence for the presence of korarchaeal members in temperate subsurface seawater. Furthermore, a systematic reclassification of the Korarchaeota based on 16S rRNA genes and genomes has revealed three novel marine groups (Kor-6 to Kor-8) at the root of the Korarchaeota branch. Kor-6 contains microbes that are present in moderate temperatures. All three novel marine phyla possess genes for the Wood-Ljungdahl pathway, and Kor-7 and Kor-8 possess fewer genes encoding oxygen resistance traits than other korarchaeal groups, suggesting a distinct lifestyle for these novel phyla. Our results, together with estimations of Korarchaeota divergence times, suggest that oxygen availability may be one of the important factors that have influenced the evolution of Korarchaeota. IMPORTANCE Korarchaeota were previously thought to inhabit exclusively high-temperature environments. However, our study provides genetic evidence for their unexpected presence in temperate marine waters. Through analysis of publicly available korarchaeal reference data, we have systematically reclassified Korarchaeota and identified the existence of three previously unknown marine groups (Kor-6, Kor-7, and Kor-8) at the root of the Korarchaeota branch. Comparative analysis of their gene content revealed that these novel groups exhibit a lifestyle distinct from other Korarchaeota. Specifically, they have the ability to fix carbon exclusively via the Wood-Ljungdahl (WL) pathway, and the genomes within Kor-7 and Kor-8 contain few genes encoding antioxidant enzymes, indicating their strictly anaerobic lifestyle. Further studies suggest that the genes related to methane metabolism and the WL pathway may have been inherited from a common ancestor of the Korarchaeota and that oxygen availability may be one of the important evolutionary factors that shaped the diversification of this archaeal phylum.

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Agr Quorum Sensing influences the Wood-Ljungdahl pathway in Clostridium autoethanogenum.

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