Abstract

In the genus Listeria, the receptor for antimicrobial peptides known as class IIa bacteriocins is the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) permease EII_t^Man. EII_t^Man is the principal glucose transporter in Listeria and plays a central role in carbon catabolite repression. Thus, carbohydrate metabolism and bacteriocin action are interconnected in this genus.

In the first part of the research, genes that are important for class IIa bacteriocin interaction and resistance in Listeria species were identified by screening transposon Tn917 knockout libraries constructed in Listeria innocua strain Lin11 for mutants that are resistant to pediocin AcH. A highly resistant mutant G7, in which the transposon integrated into the putative promoter of the lin0142 gene, was isolated. lin0142 is located immediately upstream of the mpt operon that encodes the EII_t^Man permease. The transcription of the mpt operon is known to be positively controlled by σ ⁵⁴ factor and ManR (a σ⁵⁴-associated activator). Transcripts for lin0142 and mpt were undetectable in the G7 mutant, based on quantitative real-time reverse transcriptase (RT) PCR analysis. When the wild-type lin0142 gene was expressed at a 7.9-fold-elevated level in the mutant, the level of mpt mRNA became 70% higher than that in the wild-type strain. In addition, the complementation strain reverted back to the pediocin AcH-susceptible phenotype. The combined results show that the lin0142 gene encodes a putative transcription regulator of the mpt operon.

In the second part of the research, the basic mechanism of mpt operon regulation by the ManR protein was examined in L. innocua. ManR contains conserved histidine residues within two PTS regulation domains (PRDs) that have been shown to be modified by phosphorylation in related regulatory proteins. Site-directed mutagenesis experiments demonstrated that His-871 in ManR PRD-II is needed for mpt repression in glucose-free media, and His-506 in PRD-I is needed for mpt induction by glucose. The results are consistent with a model in which ManR is inactivated in media lacking glucose by phosphorylation at both sites, and activated in the presence of glucose by phosphorylation at His-506.

In the last part of the research, the function of the 1,2-propanediol (1,2-PDL) utilization (pdu) operon was analyzed. Research performed with the pediocin AcH-resistant D2 Tn917 mutant suggests that pdu and mpt operon expression are somehow linked. It was determined that the pdu operon is induced by 1,2-PDL and required for degradation of 1,2-PDL to the intermediates propanol and propionate in L. innocua. Additional research will be needed to clarify the relationship between pdu and mpt operon expression.