Lactiplantibacillus plantarum is a beneficial lactic acid bacterium that is commonly found in the digestive tract and fermented foods. This probiotic bacterium enhances the gut microbiota composition and the immune system, which could contribute to a lowered risk of chronic illnesses. It's a commensal gram-positive bacterium communicating through quorum-sensing, a type of cellular signaling that allows the bacteria to respond to cell population density, resulting in the adjustment of gene expression accordingly. LamD peptide is hypothesized to be the cyclic peptide with five amino acids (CVGIW) regulating this quorum-sensing circuit. It is assumed to be natively cyclized in the form of a thiolactone (LamD-S). LamD-S can proceed to a lactam via an S-to-N acyl shift under mildly basic conditions to form a cyclic amide (LamD-N). We don't know if one or both forms of LamD are biologically relevant. To explore this, synthetic versions of LamD were produced using the Solid-Phase Peptide Synthesis (SPPS) method and incorporated into bioassays. Quorum sensing was evaluated using a reporter strain carrying the GusA gene in L. plantarum, with PNPG cleavage and yellow color production as indicators, but the results were inconclusive due to the self-quorum-sensing nature of wild-type L. plantarum. To address this issue, a specialized reporter strain was developed to investigate this density-dependent chemical communication mechanism within the context of lamBDCA total knockout L. plantarum. This new reporter strain is generated from the pSIP403 plasmid, which contains the GusA gene and ERY resistance gene. The lamC and lamA genes, which respectively encode histidine kinase and response regulator, were incorporated downstream of a constitutive promoter. We hypothesize that this lamCA reporter strain would exclusively activate in the lamBDCA total knockout bacteria when synthetic LamD peptides were introduced. This activation would serve as an indicator of an up-regulated quorum-sensing process. Understanding which peptide controls this quorum sensing process is crucial in manipulating the communication and coordination mechanisms within L. plantarum. This knowledge can provide valuable insights into how this beneficial bacterium regulates its activities and interacts with its surroundings.

Michael Bertucci

Lafayette College

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