Class IIa bacteriocins may be used as natural food preservatives, yet resistance development in the target organisms is still poorly understood. In this study, the understanding of class IIa resistance development in Listeria monocytogenes is extended, linking the seemingly diverging results previously reported. Eight resistant mutants having a high resistance level (at least a 10 3 -fold increase in MIC), originating from five wild-type listerial strains, were independently isolated following exposure to four different class IIa bacteriocin-producing lactic acid bacteria (including pediocin PA-1 and leucocin A producers). Two of the mutants were isolated from food model systems (a saveloy-type sausage at 10 SC, and salmon juice at 5 SC). Northern blot analysis showed that the eight mutants all had increased expression of EII Bgl and a phospho-β-glucosidase homologue, both originating from putative β-glucosidespecific phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). However, disruption of these genes in a resistant mutant did not confer pediocin sensitivity. Comparative two-dimensional gel analysis of proteins isolated from mutant and wild-type strains showed that one spot was consistently missing in the gels from mutant strains. This spot corresponded to the MptA subunit of the mannose-specific PTS, EII Man t , found only in the gels of wild-type strains. The mptACD operon was recently shown to be regulated by the σ 54 transcription factor in conjunction with the activator ManR. Class IIa bacteriocin-resistant mutants having defined mutations in mpt or manR also exhibited the two diverging PTS expression changes. It is suggested here that high-level class IIa resistance in L. monocytogenes and at least some other Gram-positive bacteria is developed by one prevalent mechanism, irrespective of wild-type strain, class IIa bacteriocin, or the tested environmental conditions. The changes in expression of the β-glucoside-specific and the mannose-specific PTS are both influenced by this mechanism. The current understanding of the actual cause of class IIa resistance is discussed.

Leucocin A is a class IIa bacteriocin produced by Leuconostoc spp. that has previously been shown to inhibit the growth of Listeria monocytogenes. A spontaneous resistant mutant of L. monocytogenes was isolated and found to be resistant to leucocin A at levels in excess of 2 mg/ml. The mutant showed no significant cross-resistance to nontype IIa bacteriocins including nisaplin and ESF1-7GR. However, there were no inhibition zones found on a lawn of the mutant when challenged with an extract containing 51,200 AU of pediocin PA-2 per ml as determined by a simultaneous assay on the sensitive wild-type strain. DNA and protein analysis of the resistant and susceptible strains were carried out using silver-stained amplified fragment length polymorphism (ssAFLP) and one-and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Two-dimensional SDS-PAGE clearly showed a 35-kDa protein which was present in the sensitive but absent from the resistant strain. The N-terminal end of the 35-kDa protein was sequenced and found to have an 83% homology to the mannose-specific phosphotransferase system enzyme IIAB of Streptococcus salivarius.Bacteriocins are proteinaceous antimicrobial peptides synthesized by bacteria and are usually active against strains closely related to the producing organism (13, 21). There is increasing interest in the use of bacteriocins from lactic acid bacteria in foods (20,22). For example, the class I bacteriocin nisin was approved for use in 1969 and has been applied to several foods (6). Class II bacteriocins are characteristically small, heat-stable peptides (15), some of which contain the consensus motif YGNGV and include leucocin A and pediocin PA-2. These were isolated from food-related lactic acid bacteria and have potential as food preservatives. With the addition of nisin (and other bacteriocins) into the environment, there is a concomitant interest in resistance to these antimicrobial compounds (4,5,10,18,19,33). The mechanism of resistance has not been fully established, but has been attributed to cell membrane and S-layer changes.Leucocin A is produced by several Leuconostoc spp. It inhibits the growth of the important potential food-borne pathogen Listeria monocytogenes (11,24). Resistance to class IIa bacteriocins has been reported to be a stable phenomenon (8,30). Transposon-mediated inactivation of 54 in L. monocytogenes has rendered it resistant to mesentericin Y105 (a class IIa bacteriocin) (31). There is, however, very little known about the molecular basis of resistance in naturally isolated strains.In an attempt to discover resistance-associated phenomena at both the DNA and proteiomic levels, amplified fragment length polymorphism (AFLP) and two-dimensional (2-D) gel electrophoresis were employed, respectively. AFLP is a genome fingerprinting technique based on the selective amplification of a subset of DNA fragments generated by restriction enzyme digestion (34). 2-D gel electrophoresis is a powerful tool for the analysis of complex p...

It was previously shown that enhanced nisin resistance in some mutants was associated with increased expression of three genes, pbp2229, hpk1021, and lmo2487, encoding a penicillin-binding protein, a histidine kinase, and a protein of unknown function, respectively. In the present work, we determined the direct role of the three genes in nisin resistance. Interruption of pbp2229 and hpk1021 eliminated the nisin resistance phenotype. Interruption of hpk1021 additionally abolished the increase in pbp2229 expression. The results indicate that this nisin resistance mechanism is caused directly by the increase in pbp2229 expression, which in turn is brought about by the increase in hpk1021 expression. We also found a degree of cross-protection between nisin and class IIa bacteriocins and investigated possible mechanisms. The expression of virulence genes in one nisin-resistant mutant and two class IIa bacteriocin-resistant mutants of the same wild-type strain was analyzed, and each mutant consistently showed either an increase or a decrease in the expression of virulence genes (prfA-regulated as well as prfA-independent genes). Although the changes mostly were moderate, the consistency indicates that a mutant-specific change in virulence may occur concomitantly with bacteriocin resistance development.Nisin and the class IIa bacteriocins (also called pediocin-like bacteriocins) are antimicrobial peptides that are produced by lactic acid bacteria and that have the greatest potential as biopreservatives for food. One of the main target organisms in this context is Listeria monocytogenes, a food-borne pathogen that causes severe human illness as well as economic losses for the food industry.Nisin exerts its antimicrobial action by forming pores in the cytoplasmic membrane through an interaction with the peptidoglycan precursor lipid II (for a recent review, see reference 17). Enhanced nisin resistance in L. monocytogenes generally constitutes less than a 10-fold increase in the MIC. Nisin resistance in several, but not all, spontaneous mutants of L.

Sensitivity to class IIa bacteriocins from lactic acid bacteria was recently associated with the mannose phosphotransferase system (PTS) permease, EII Man t , in Listeria monocytogenes. To assess the involvement of this protein complex in class IIa bacteriocin activity, the mptACD operon, encoding EII Man t , was heterologously expressed in an insensitive species, namely Lactococcus lactis, using the NICE double plasmid system. Upon induction of the cloned operon, the recombinant Lc. lactis became sensitive to leucocin A. Pediocin PA-1 and enterocin A also showed inhibitory activity against Lc. lactis cultures expressing mptACD. Furthermore, the role of the three genes of the mptACD operon was investigated. Derivative plasmids containing various combinations of these three genes were made from the parental mptACD plasmid by divergent PCR. The results showed that expression of mptC alone is sufficient to confer sensitivity to class IIa bacteriocins in Lc. lactis.