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Strains of the food-borne pathogenListeria monocytogenes, showing either intermediate or high-level resistance to class IIa bacteriocins, were investigated to determine characteristics that correlated with their sensitivity levels. Two intermediate and one highly resistant spontaneous mutant ofL. monocytogenesB73, a highly resistant mutant ofL. monocytogenes412, and a highly resistant, defined (mptA) mutant ofL. monocytogenesEGDe were compared with their respective wild-type strains in order to investigate the contribution of different factors to resistance. Decreased mannose-specific phosphotransferase system gene expression (mptA, EIIABMancomponent) was implicated in all levels of resistance, confirming previous studies by the authors' group. However, a clear correlation betweend-alanine content in teichoic acid (TA), in particular the alanine : phosphorus ratio, and a more positive cell surface, as determined by cytochromecbinding, were found for the highly resistant strains. Furthermore, two of the three highly resistant strains showed a significant increase in sensitivity towardsd-cycloserine (DCS). However, real-time PCR of thedltA(d-alanine esterification), anddalandddlAgenes (peptidoglycan biosynthesis) showed no change in transcriptional levels. The link between DCS sensitivity and increasedd-alanine esterification of TA may be that DCS competes with alanine for transport via the alanine transporter. A possible tendency towards increased lysinylation of membrane phospholipid in the highly resistant strains was also found. A previous study reported that cell membranes of all the resistant strains, including the intermediate resistant strains, contained more unsaturated phosphatidylglycerol, which is an indication of a more fluid cell membrane. The results of that study correlate with the possible lysinylation, decreasedmptAexpression,d-alanine esterification of TA and more positive cell surface charge found in this study for resistant strains. The authors' findings strongly indicate that all these factors could contribute to class IIa bacteriocin resistance and that the combination and contribution of each of these factors determine the level of bacteriocin resistance.

The role of the alternativeσ54factor, encoded by therpoNgene, was investigated inListeria monocytogenesby comparing the global gene expression of the wild-type EGDe strain and anrpoNmutant. Gene expression, using whole-genome macroarrays, and protein content, using two-dimensional gel electrophoresis, were analysed. Seventy-seven genes and nine proteins, whose expression was modulated in therpoNmutant as compared to the wild-type strain, were identified. Most of the modifications were related to carbohydrate metabolism and in particular to pyruvate metabolism. However, under the conditions studied, only themptACDoperon was shown to be directly controlled byσ54. Therefore, the remaining modifications seem to be due to indirect effects. In parallel, anin silicoanalysis suggests thatσ54may directly control the expression of four different phosphotransferase system (PTS) operons, includingmptACD. PTS activity is known to have a direct effect on the pyruvate pool and on catabolite regulation. These results suggest thatσ54is mainly involved in the control of carbohydrate metabolism inL. monocytogenesvia direct regulation of PTS activity, alteration of the pyruvate pool and modulation of carbon catabolite regulation.

Summary: Because of their potential usefulness as natural food preservatives, increased interest has focused on bacteriocins from lactic acid bacteria. Mesentericin Y105 is a small non-lantibiotic bacteriocin (class II) encoded within a 35 kb plasmid from Leuconostoc mesenteroides Y105 and it is active against Listeria monocytogenes. Using reverse genetic methodologies, an 8 kb Drall fragment has been cloned that contains the mesentericin Y105 structural gene, mesY, which encodes a precursor of the bacteriocin with a 24 amino acid N-terminal extension ending with a Gly-Gly motif upstream of the cleavage site, which is typical of class II bacteriocins. Four other putative genes are associated with mesY within two divergent putative operons. In addition to mesY, the first putative operon is predicted to encode a protein, similar to that encoded by ORF2 in the leucocin A operon, whose function remains to be elucidated. The second putative operon contains three ORFs, two of which, mesD and mesE, encode proteins that resemble ATP-dependent transporters and accessory factors, respectively. For three other class II bacteriocin systems (lactococcin A, pediocin PA-1, colicin V), these proteins have been shown to be involved in bacteriocin secretion independently of the general sec-dependent secretion pathway. The last putative gene (mesC) does not resemble any previously characterized gene. Results concerning the heterologous expression of the cloned mesY in Lactobacillus johnsonii NCK64 suggest that the maturation and secretion functions dedicated to lactacin F (another class II bacteriocin) are efficient for mesentericin Y105 as well. This characteristic may be of great interest in the development of industrial fermentation starters producing multiple bactericidal activities.