1 Department of Biochemistry, University of Stellenbosch, Private Bag X1, 7602 Matieland, South Africa
2 Laboratory of Fundamental and Applied Microbiology, LCEE UMR CNRS 6008, University of Poitiers, 40, avenue du Recteur Pineau, 86022 Poitiers Cedex, France
3 The Royal Veterinary and Agricultural University, Department of Dairy and Food Science, Centre for Advanced Food Studies, LMC, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
Correspondence
Yann Héchard
yann.hechard{at}univ-poitiers.fr
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
These authors contributed equally to this work.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To further evaluate the role of in bacterial sensitivity to class IIa bacteriocins, we heterologously expressed the mptACD operon of Ls. monocytogenes in an insensitive Lactococcus lactis strain. Genetic constructs were made to determine the effect of expressing individual genes and combinations of genes in the operon. Our results show that the expression of mptC induces sensitivity to various class IIa bacteriocins in Lc. lactis.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
RT-PCR experiments.
Lc. lactis was grown to an OD630 of 0·2. Nisin A was added to the relevant cultures and grown for another 2 h to allow mptACD expression. The cells were harvested (8000 g, 10 min at 4 °C), resuspended in a lysis buffer (5 mg lysozyme ml1, 100 g glucose l1, 5 mM Tris-EDTA, pH 8) and incubated for 1 h at 37 °C. Total RNA was isolated with the RNAwiz kit (Ambion), according to the manufacturer's instructions, and treated with 4 units RNase-free DNase (Invitrogen) for 1 h at 37 °C. The quality of the RNA samples was assessed by 1 % formaldehyde agarose gel electrophoresis and the quantity was estimated by spectrophotometry. The expression of each gene from the mptACD operon in the various constructions was assessed by RT-PCR or quantitative RT-PCR (qRT-PCR). Reverse transcription, leading to cDNA synthesis, was performed using 2 µg total RNA, with random hexamers (15 ng ml1) and the Superscript II RNase H kit (Invitrogen), according to the manufacturer's instructions. PCR reactions using specific primers were performed from cDNA to check that each gene of the mptACD operon had been transcribed. qPCR was performed with the TaqMan Universal PCR Master kit (Applied Biosystems) to quantify expression of the mptA operon upon nisin A induction, using specific primers (5'-CAGGACTTAATTTGCCAATGTTG-3' and 5'-CGCGAACACCTTCTTGAGCT-3') and probe (5'-Fam-AGCGCACACGAAATCGCAGCAA-Tamra-3'). The PCR reactions were performed and analysed on an ABI Prism 7700 sequence detector (Applied Biosystems) under the following conditions: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 1 min. The control templates corresponded to different dilutions of genomic DNA. All the reactions were performed in triplicate. The Ct value was defined as the cycle number at which a significant increase in amplification product occurs. For each sample, a mean Ct was calculated from triplicate reactions. The Ct value is inversely correlated to the cDNA quantity. The relative expression was calculated as 2Ct, according to the manufacturer's instructions.
Bacteriocin preparation.
Nisin A was purchased as a 2·5 % (w/w) powder (Sigma) and reconstituted in water. Leucocin A was synthesized as described previously (Ramnath et al., 2000) and resuspended in 50 % acetonitrile to a final concentration of 2 mg ml1. Pediocin PA-1 (Henderson et al., 1992
) and enterocin A (Aymerich et al., 1996
) were produced by Pediococcus acidilactici NRRL B5627 and Enterococcus faecium 336, respectively, and purified as described by Guyonnet et al. (2000)
. All bacteriocin stocks were stored at 20 °C until used.
Sensitivity assays.
The sensitivity assays were performed in microtitre plates where an overnight culture of Lc. lactis was added as a 1 % inoculum and growth was monitored at OD630. When an OD630 of 0·05 was reached, nisin A was added to a final concentration of 2·5 ng ml1 and the cultures were grown for a further 1·5 h before the addition of bacteriocin extracts or gramicidin S (Sigma).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
mptACD expression leads to Lc. lactis sensitivity
Fig. 2. shows the effect of the addition of leucocin A to nisin-induced and uninduced cultures of Lc. lactis MG-ACD as well as MG-Con. Growth of the induced MG-ACD cultures was arrested immediately following addition of leucocin in the range of 20 µg ml1 to 156 ng ml1 (Fig. 2a
). In contrast, growth of the control strain MG-Con was unaffected by the added concentrations of nisin A and leucocin A (Fig. 2b
). An additional experiment with Lc. lactis MG-Con showed that growth was not influenced by a concentration of leucocin A as high as 400 µg ml1.
|
The effect of two other class IIa bacteriocins on nisin-induced Lc. lactis MG-ACD cultures was compared to leucocin treatment (Fig. 3). Addition of pediocin PA-1 or enterocin A resulted in a complete inhibition of growth.
|
The sensitivity of the nisin-induced strains to leucocin was determined (Fig. 4). In the presence of nisin A, all five strains showed a reduction in growth that was similar to Lc. lactis MG-ACD. The growth of Lc. lactis MG-CD, MG-AC and MG-C was rapidly inhibited by leucocin A to the same extent as the parental MG-ACD strain. On the contrary, the addition of leucocin A had no effect on the growth of both Lc. lactis MG-AD and Lc. lactis MG-D. Thus, mptC was the only gene present in all the sensitive strains, but not in the insensitive ones.
|
No difference in sensitivity was observed between uninduced and induced cultures to gramicidin S (no growth above 5 µg ml1). This molecule acts non-specifically on the cytoplasmic membrane (Prenner et al., 1999), indicating that the sensitivity to the class IIa bacteriocins was not due to a membrane perturbation by the induced proteins.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We subsequently analysed the role of the three subunits of the mptACD operon in class IIa sensitivity. The results showed that expression of mptC alone is sufficient to confer sensitivity to class IIa bacteriocins in Lc. lactis. A BLAST search showed that MptC has very high identity (88 %) to the Enterococcus faecalis mannose IIC subunit, which belongs to the mannose permease associated to class IIa bacteriocin sensitivity of this organism (Héchard et al., 2001). This high level of homology was expected in proteins that may be similarly involved in the class IIa mode of action. However, the region required for sensitivity may not be extensive as the search also showed that MptC has 59 % identity to the mannose IIC subunit (PtnC) of the class IIa-insensitive Lc. lactis.
The IIC subunit of a mannose permease has been shown to be specifically involved in the transfer of bacteriophage DNA across the inner membrane in Escherichia coli (Esquinas-Rychen & Erni, 2001
).
The involvement of the IIC subunit in the sensitivity to class IIa bacteriocins was unexpected because a partial mptD deletion experiment had previously suggested that the MptD subunit was specifically involved in the sensitivity of Ls. monocytogenes (Dalet et al., 2001). However, we subsequently observed that MptA was not expressed in the mptD deletion mutant, suggesting that the entire operon is switched off in this mutant (Gravesen et al., 2002
). This observation may possibly be a regulatory consequence of defective permease activity, as it is known that PTS permease activity affects its own expression (Deutscher et al., 2002
). In the present work, mptACD was under the control of the inducible nis promoter to avoid possible feedback regulation of the permease and to tightly control the expression.
In summary, the results of this study show that expression of mptC renders Lc. lactis sensitive to IIa bacteriocins, and indicate that these bacteriocins could possibly exert their antimicrobial activity through interaction with the MptC subunit in the cytoplasmic membrane.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aymerich, T., Holo, H., Havarstein, L. S., Hugas, M., Garriga, M. & Nes, I. F. (1996). Biochemical and genetic characterization of enterocin A from Enterococcus faecium, a new antilisterial bacteriocin in the pediocin family of bacteriocins. Appl Environ Microbiol 62, 16761682.[Abstract]
Chikindas, M. L., Garcia-Garcera, M. J., Driessen, A. J., Ledeboer, A. M., Nissen-Meyer, J., Nes, I. F., Abee, T., Konings, W. N. & Venema, G. (1993). Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells. Appl Environ Microbiol 59, 35773584.[Abstract]
Dalet, K., Cenatiempo, Y., Cossart, P. & Héchard, Y. (2001). A sigma(54)-dependent PTS permease of the mannose family is responsible for sensitivity of Listeria monocytogenes to mesentericin Y105. Microbiology 147, 32633269.[Medline]
de Ruyter, P. G., Kuipers, O. P. & de Vos, W. M. (1996). Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62, 36623667.
Deutscher, J., Galinier, A. & Martin-Verstraete, I. (2002). Carbohydrate uptake and metabolism. In Bacillus subtilis and its Closest Relatives: from Genes to Cells. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC: American Society for Microbiology.
Eichenbaum, Z., Federle, M. J., Marra, D., de Vos, W. M., Kuipers, O. P., Kleerebezem, M. & Scott, J. R. (1998). Use of the lactococcal nisA promoter to regulate gene expression in gram-positive bacteria: comparison of induction level and promoter strength. Appl Environ Microbiol 64, 27632769.
Eijsink, V. G., Skeie, M., Middelhoven, P. H., Brurberg, M. B. & Nes, I. F. (1998). Comparative studies of class IIa bacteriocins of lactic acid bacteria. Appl Environ Microbiol 64, 32753281.
Ennahar, S., Deschamps, N. & Richard, J. (2000a). Natural variation in susceptibility of Listeria strains to class IIa bacteriocins. Curr Microbiol 41, 14.[CrossRef][Medline]
Ennahar, S., Sashihara, T., Sonomoto, K. & Ishizaki, A. (2000b). Class IIa bacteriocins: biosynthesis, structure and activity. FEMS Microbiol Rev 24, 85106.[CrossRef][Medline]
Esquinas-Rychen, M. & Erni, B. (2001). Facilitation of bacteriophage lambda DNA injection by inner membrane proteins of the bacterial phosphoenol-pyruvate : carbohydrate phosphotransferase system (PTS). J Mol Microbiol Biotechnol 3, 361370.[Medline]
Glaser, P., Frangeul, L., Buchrieser, C. & 52 other authors (2001). Comparative genomics of Listeria species. Science 294, 849852.
Gravesen, A., Ramnath, M., Rechinger, K. B., Andersen, N., Jansch, L., Héchard, Y., Hastings, J. W. & Knöchel, S. (2002). High-level resistance to class IIa bacteriocins is associated with one general mechanism in Listeria monocytogenes. Microbiology 148, 23612369.[Medline]
Guyonnet, D., Fremaux, C., Cenatiempo, Y. & Berjeaud, J. M. (2000). Method for rapid purification of class IIa bacteriocins and comparison of their activities. Appl Environ Microbiol 66, 17441748.
Héchard, Y. & Sahl, H. G. (2002). Mode of action of modified and unmodified bacteriocins from Gram-positive bacteria. Biochimie 84, 545557.[CrossRef][Medline]
Héchard, Y., Pelletier, C., Cenatiempo, Y. & Frère, J. (2001). Analysis of 54-dependent genes in Enterococcus faecalis: a mannose PTS permease (EIIMan) is involved in sensitivity to a bacteriocin, mesentericin Y105. Microbiology 147, 15751580.[Medline]
Henderson, J. T., Chopko, A. L. & van Wassenaar, P. D. (1992). Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC-1.0. Arch Biochem Biophys 295, 512.[Medline]
Kleerebezem, M., Beerthuyzen, M. M., Vaughan, E. E., de Vos, W. M. & Kuipers, O. P. (1997). Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl Environ Microbiol 63, 45814584.[Abstract]
Mengaud, J., Geoffroy, C. & Cossart, P. (1991). Identification of a new operon involved in Listeria monocytogenes virulence: its first gene encodes a protein homologous to bacterial metalloproteases. Infect Immun 59, 10431049.[Medline]
Montville, T. J. & Chen, Y. (1998). Mechanistic action of pediocin and nisin: recent progress and unresolved questions. Appl Microbiol Biotechnol 50, 511519.[CrossRef][Medline]
Prenner, E. J., Lewis, R. N. A. H. & McElhaney, R. N. (1999). The interaction of the antimicrobial peptide gramicidin S with lipid bilayer model and biological membranes. Biochim Biophys Acta 1462, 201221.[Medline]
Ramnath, M., Beukes, M., Tamura, K. & Hastings, J. W. (2000). 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. Appl Environ Microbiol 66, 30983101.
Rasch, M. & Knöchel, S. (1998). Variations in tolerance of Listeria monocytogenes to nisin, pediocin PA-1 and bavaricin A. Lett Appl Microbiol 27, 275278.[CrossRef][Medline]
Sambrook, J. & Russell, D. (2000). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Vadyvaloo, V., Hastings, J. W., van der Merwe, M. J. & Rautenbach, M. (2002). Membranes of class IIa bacteriocin-resistant Listeria monocytogenes cells contain increased levels of desaturated and short-acyl-chain phosphatidylglycerols. Appl Environ Microbiol 68, 52235230.
Yan, L. Z., Gibbs, A. C., Stiles, M. E., Wishart, D. S. & Vederas, J. C. (2000). Analogues of bacteriocins: antimicrobial specificity and interactions of leucocin A with its enantiomer, carnobacteriocin B2, and truncated derivatives. J Med Chem 43, 45794581.[CrossRef][Medline]
Received 23 December 2003;
revised 9 April 2004;
accepted 28 April 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |