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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:UNLABELLED:The future sustainable production of chemicals and fuels from nonpetrochemical resources and reduction of greenhouse gas emissions are two of the greatest societal challenges. Gas fermentation, which utilizes the ability of acetogenic bacteria such as Clostridium autoethanogenum to grow and convert CO2 and CO into low-carbon fuels and chemicals, could potentially provide solutions to both. Acetogens fix these single-carbon gases via the Wood-Ljungdahl pathway. Two enzyme activities are predicted to be essential to the pathway: carbon monoxide dehydrogenase (CODH), which catalyzes the reversible oxidation of CO to CO2, and acetyl coenzyme A (acetyl-CoA) synthase (ACS), which combines with CODH to form a CODH/ACS complex for acetyl-CoA fixation. Despite their pivotal role in carbon fixation, their functions have not been confirmed in vivo By genetically manipulating all three CODH isogenes (acsA, cooS1, and cooS2) of C. autoethanogenum, we highlighted the functional redundancies of CODH by demonstrating that cooS1 and cooS2 are dispensable for autotrophy. Unexpectedly, the cooS1 inactivation strain showed a significantly reduced lag phase and a higher growth rate than the wild type on H2 and CO2 During heterotrophic growth on fructose, the acsA inactivation strain exhibited 61% reduced biomass and the abolishment of acetate production (a hallmark of acetogens), in favor of ethanol, lactate, and 2,3-butanediol production. A translational readthrough event was discovered in the uniquely truncated (compared to those of other acetogens) C. autoethanogenum acsA gene. Insights gained from studying the function of CODH enhance the overall understanding of autotrophy and can be used for optimization of biotechnological production of ethanol and other commodities via gas fermentation. IMPORTANCE:Gas fermentation is an emerging technology that converts the greenhouse gases CO2 and CO in industrial waste gases and gasified biomass into fuels and chemical commodities. Acetogenic bacteria such as Clostridium autoethanogenum are central to this bioprocess, but the molecular and genetic characterization of this microorganism is currently lacking. By targeting all three of the isogenes encoding carbon monoxide dehydrogenase (CODH) in C. autoethanogenum, we identified the most important CODH isogene for carbon fixation and demonstrated that genetic inactivation of CODH could improve autotrophic growth. This study shows that disabling of the Wood-Ljungdahl pathway via the inactivation of acsA (encodes CODH) significantly impairs heterotrophic growth and alters the product profile by abolishing acetate production. Moreover, we discovered a previously undescribed mechanism for controlling the production of this enzyme. This study provides valuable insights into the acetogenic pathway and can be used for the development of more efficient and productive strains for gas fermentation.

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

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