Identification and functional characterization of genes involved in the Listeria monocytogenes stress response

Abstract

Listeria monocytogenes is a gram-positive, facultatively anaerobic foodborne pathogen. Systemic infection with L. monocytogenes is associated with high mortality, and it is therefore imperative to prevent the organism from contaminating foods. However, L. monocytogenes can survive in food production environments despite measures designed to mitigate microbial contamination. This dissertation focuses on elucidating genetic mechanisms involved in the response of L. monocytogenes to stressors encountered in food processing. Transcriptomic analysis was conducted to identify genes involved in the L. monocytogenes response to the preservative lactic acid. Indeed, lactic acid exposure resulted in a major shift in gene expression. Two genes, rli47 and lmo2230, demonstrated particularly high upregulation in response to lactic acid and were chosen for further characterization. Rli47 is a noncoding RNA that is known to suppress isoleucine biosynthesis. Interestingly, survival assays revealed that an rli47 deletion mutant was more resistant to lactic acid stress than the wild type L. monocytogenes strain. Subsequent analysis indicated that this was likely because the L. monocytogenes membrane was more resistant to lactic acid stress in the absence of rli47. The gene lmo2230 is upregulated under a variety of stressors and encodes a putative arsenate reductase. However, sequence analysis revealed that Lmo2230 lacks the amino acid residues essential for arsenate reduction in functionally characterized arsenate reductases. This was supported by heterologous expression in Escherichia coli which demonstrated that Lmo2230 confers less resistance to arsenate stress than a characterized arsenate reductase from Bacillus subtilis. In silico modeling revealed a putative DNA-binding domain in Lmo2230, indicating that it may function in L. monocytogenes as a regulator of stress response. Thus, the work presented here furthers the knowledge of the genetic mechanisms underlying the L. monocytogenes stress response.