Leucocin A-UAL 187 is a bacteriocin produced by Leuconostoc gelidum UAL 187, a lactic acid bacterium isolated from vacuum-packaged meat. The bacteriocin was purified by ammonium sulfate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration, and reversed-phase high-performance liquid chromatography with a yield of 58% of the original activity. Leucocin A is stable at low pH and heat resistant, and the activity of the pure form is enhanced by the addition of bovine serum albumin. It is inactivated by a range of proteolytic enzymes. The molecular weight was determined by mass spectrometry to be 3,930.3 0.4. Leucocin A-UAL 187 contains 37 amino acids with a calculated molecular weight of 3,932.3. A mixed oligonucleotide (24-mer) homologous to the sequence of the already known N terminus of the bacteriocin hybridized to a 2.9-kb HpaII fragment of a 7.6-MDa plasmid from the producer strain. The fragment was cloned into pUC118 and then subcloned into a lactococcal shuttle vector, pNZ19. DNA sequencing revealed an operon consisting of a putative upstream promoter, a downstream terminator, and two open reading frames flanked by a putative upstream promoter and a downstream terminator. The first open reading frame downstream of the promoter contains 61 amino acids and is identified as the leucocin structural gene, consisting of a 37-amino-acid bacteriocin and a 24-residue N-terminal extension. No phenotypic expression of the bacteriocin was evident in several lactic acid bacteria that were electrotransformed with pNZ19 containing the 2.9-kb cloned fragment of the leucocin A plasmid.Bacteriocins are antimicrobial peptides or proteins formed by bacteria. The potential applications for bacteriocin-producing lactic acid bacteria in food preservation has stimulated interest in the characterization of these substances. However, maintaining activity during isolation and purification has proved difficult, and the full or partial amino acid sequences of only five such bacteriocins have been reported. The most extensively studied is nisin A (2, 3, 7, 34), a posttranslationally modified bacteriocin from Lactococcus lactis subsp. lactis. Nisin has been approved for use as a preservative in foods in over 45 countries (9). It is ribosomally synthesized (6, 10) and is one of a group of lantibiotics that possess lanthionine-or methyllanthionine-containing rings resulting from the attack of cysteine sulfhydryl groups on dehydroalanine or dehydrobutyrine residues (derived from serine or threonine). Partial characterization of lactacin 481 from L. lactis shows that it also contains lanthionine rings, but the full sequence has not yet been published (31). In contrast, lactocin S from Lactobacillus sake (27)
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...
Strains of the food-borne pathogen Listeria 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 of L. monocytogenes B73, a highly resistant mutant of L. monocytogenes 412, and a highly resistant, defined (mptA) mutant of L. monocytogenes EGDe 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, EIIAB Man component) was implicated in all levels of resistance, confirming previous studies by the authors' group. However, a clear correlation between D-alanine content in teichoic acid (TA), in particular the alanine : phosphorus ratio, and a more positive cell surface, as determined by cytochrome c binding, were found for the highly resistant strains. Furthermore, two of the three highly resistant strains showed a significant increase in sensitivity towards D-cycloserine (DCS). However, real-time PCR of the dltA (D-alanine esterification), and dal and ddlA genes (peptidoglycan biosynthesis) showed no change in transcriptional levels. The link between DCS sensitivity and increased D-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, decreased mptA expression, 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.
A major concern in the use of class IIa bacteriocins as food preservatives is the well-documented resistance development in target Listeria strains. We studied the relationship between leucocin A, a class IIa bacteriocin, and the composition of the major phospholipid, phosphatidylglycerol (PG), in membranes of both sensitive and resistant L. monocytogenes strains. Two wild-type strains, L. monocytogenes B73 and 412, two spontaneous mutants of L. monocytogenes B73 with intermediate resistance to leucocin A (؎2.4 and ؎4 times the 50% inhibitory concentrations [IC 50 ] for sensitive strains), and two highly resistant mutants of each of the wild-type strains (>500 times the IC 50 for sensitive strains) were analyzed. Electrospray mass spectrometry analysis showed an increase in the ratios of unsaturated to saturated and short-to long-acyl-chain species of PG in all the resistant L. monocytogenes strains in our study, although their sensitivities to leucocin A were significantly different. This alteration in membrane phospholipids toward PGs containing shorter, unsaturated acyl chains suggests that resistant strains have cells with a more fluid membrane. The presence of this phenomenon in a strain (L. monocytogenes 412P) which is resistant to both leucocin A and pediocin PA-1 may indicate a link between membrane composition and class IIa bacteriocin resistance in some L. monocytogenes strains. Treatment of strains with sterculic acid methyl ester (SME), a desaturase inhibitor, resulted in significant changes in the leucocin A sensitivity of the intermediate-resistance strains but no changes in the sensitivity of highly resistant strains. There was, however, a decrease in the amount of unsaturated and short-acyl-chain PGs after treatment with SME in one of the intermediate and both of the highly resistant strains, but the opposite effect was observed for the sensitive strains. It appears, therefore, that membrane adaptation may be part of a resistance mechanism but that several resistance mechanisms may contribute to a resistance phenotype and that levels of resistance vary according to the type of mechanisms present.
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