Exploration of the transcriptomes and functional contributions of Listeria monocytogenes plasmids during food production-associated stress conditions

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Anast, Justin Michael
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Schmitz-Esser, Stephan Brehm-Stecher, Byron Dickson, James Mellata, Melha Shaw, Angela
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Listeria monocytogenes is the causative agent of the foodborne disease listeriosis, which is often fatal in susceptible individuals. The persistence of L. monocytogenes in the food production environment (FPE), defined as the repeated isolation of the same strain over several months, leads to the reoccurrence of food product recalls and listeriosis outbreaks. Recently, it was revealed that the plasmids of L. monocytogenes are highly conserved and distributed among persistent strains in the FPE and that some plasmids enhanced survival when cells were exposed to FPE-associated stress conditions. Much is known of the chromosomally-encoded molecular mechanisms of L. monocytogenes that increase tolerance to FPE-associated stress conditions. However, our understanding of the stress response roles of L. monocytogenes plasmids is limited. A more comprehensive understanding of the L. monocytogenes survival mechanisms that enable its persistence in food and FPEs might ultimately improve regulations to mitigate L. monocytogenes contamination of food products. Transcriptome sequencing is a standard method used to identify genes that are important for the response to stresses relevant to food production. We conducted differential gene expression analysis of plasmid-harboring L. monocytogenes strains in response to diverse FPE-relevant stress conditions. We show significant shifts in plasmid gene expression and that many plasmid genes, mainly those with no predicted putative function, are differentially expressed in diverse conditions. We also reveal that plasmid-harbored non-coding RNAs are highly prevalent in Listeria plasmids and are significantly induced during stress response. Our transcriptome data will serve as a foundation for future studies by highlighting which plasmid genes may be involved in stress response and could be used for intervention targets to alleviate the problematic persistence of L. monocytogenes. Ultraviolet (UV) light is used in food production to mitigate microbial contamination. Plasmid-encoded UV tolerance mechanisms have been described in bacteria other than L. monocytogenes. We compared CFUs of wildtype L. monocytogenes strains against plasmid-cured strains post UV exposure and revealed that L. monocytogenes plasmids significantly increased survival. From sequence analysis, we identified two candidate genes that may be involved in UV stress response and can be used in future functional characterization studies. Finally, we cloned and expressed two L. monocytogenes plasmid genes that were significantly induced during lactic acid stress, the clpL of pLM6179 (previously shown to have a role in heat stress), and the mco gene of pLMR479a. Previously, our group found that the plasmids pLM6179 and pLMR479a significantly increased survival during lactic acid and hydrogen peroxide stress exposure. Thus, we investigated whether the clpL and mco genes are important in response to lactic acid and hydrogen peroxide stress on a single gene level. Our data show that the clpL gene of L. monocytogenes plasmids enhanced growth in media containing hydrogen peroxide, demonstrating that the clpL gene has an important role in other FPE-associated stress conditions in addition to heat stress. We were not able to elucidate a potential role for the mco gene in the lactic acid and hydrogen peroxide stress response. However, we provide several suggestions for improving the experimental design and encourage future research to reveal if clpL and mco genes are suitable candidates for targets to combat L. monocytogenes contamination in the FPE. The mco and clpL genes are conserved among persistent L. monocytogenes strains’ plasmids. Thus, work pertaining to their function is relevant for multiple L. monocytogenes strains that colonize the FPE.
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