Absence of a Putative Mannose-Specific Phosphotransferase System Enzyme IIAB Component in a Leucocin A-Resistant Strain of Listeria monocytogenes , as Shown by Two-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Abstract

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...

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…For example, the EIIAB component of a mannose class PTS (called Mpt) was found to be absent from several spontaneous leucocin A-resistant L. monocytogenes mutants (693), while overproduction of a ␤-glucoside-specific PTS in the same organism led to pediocin PA-1 resistance (294). The role of the Mpt PTS in class IIa bacteriocin resistance has been studied in more detail.…”

Novel Activator of Mannose-Specific Phosphotransferase System Permease Expression in Listeria innocua , Identified by Screening for Pediocin AcH Resistance

“…In Listeria species, low-level resistance (two-to fourfold) to class IIa bacteriocins is caused by alterations in membrane lipid composition (13,49,50). High-level resistance (1,000-fold) in Listeria monocytogenes and Enterococcus faecalis results primarily from the loss of EII t Man , which is encoded by the mpt (mannose permease two) operon (14,26,38). Highlevel resistance in L. monocytogenes is also caused by the loss of 54 factor (encoded by rpoN) (43) and the 54 -associated activator ManR (14), which together positively regulate the mpt operon.…”

“…Initially, these peptides are thought to bind to negatively charged molecules, such as teichoic acids in the cell wall (7). Subsequently, they transfer to the cytoplasmic membrane, where they interact with anionic phospholipids (9) and a phosphoenolpyruvate-dependent phosphotransferase system (PTS) permease of the mannose structural family (EII t Man ) (38). Once they are bound to cell membranes, class IIa bacteriocins fold into a ␤␣ conformation (19,48,51) in which the nonpolar ␣-helical region is inserted into the phospholipid bilayer (32).…”

Influence of Peptide P34 on Gene Expression of Listeria Monocytogenes and Listeria Seelegeri

“…The expression of this gene was highly suppressed after incubation for 240 h at 4 °C, and the treatment with nisin resulted in lower dltA expression as compared with the peptide P34. Previous studies have shown that a 1000-fold increased resistance to class IIa bacteriocins in L. monocytogenes and Enterococcus faecalis resulted from the loss of mptA expression [38,42,43]. The cell wall of L. monocytogenes is composed by a thick peptidoglycan layer containing two types of anionic polymers: teichoic acids (TA), which are covalently linked to the peptidoglycan, and lipoteichoic acids (LTAs), which are poly phosphoglycerols substituted with a Dalanyl ester or a glycosyl residue and anchored in the membrane by their glycolipid moiety [44].…”