Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Apdo 85, 33300-Villaviciosa, Asturias, Spain1
Departamento de Biología Funcional, Area de Microbiología, Universidad de Oviedo, 33006-Oviedo, Spain2
Author for correspondence: Ana Rodríguez. Tel: +34 98 589 21 31. Fax: +34 98 589 22 33. e-mail: iplacsic{at}greencom.net
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: lactococcin 972, Lactococcus lactis, bacteriocin, plasmid-encoded genes
Abbreviations: LAB, lactic acid bacteria
The GenBank accession number for the sequence reported in this paper is AJ002203.
a Present address: TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, PO Box 360, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacteriocins have been defined as proteins or protein complexes which are bactericidal against closely related bacteria (Tagg et al., 1976 ). The concept was widened to include peptides of a fairly broad inhibitory spectrum (Klaenhammer, 1993
). LAB bacteriocins have been recently classified into three classes (Nes et al., 1996
): I, lantibiotics; II, small heat-stable unmodified peptides; and III, large heat-labile proteins. Classes I and II comprise the most abundant and best studied bacteriocins. The diversity of class II bacteriocins has motivated its division into three sub-groups: IIa, bacteriocins with antilisterial effect; IIb, two-peptide bacteriocins; and IIc, sec-dependent secreted bacteriocins.
The potential application of LAB bacteriocins as natural food preservatives, as well as the approval of nisin for that purpose, has encouraged interest in them. Most are plasmid-encoded (Davey, 1984 ; Dufour et al., 1991
) and even a single plasmid has been found to encode three different bacteriocins (van Belkum et al., 1992
). However, nisin is encoded by a 70 kb conjugative transposon (Rauch & de Vos, 1992
). The structural genes are usually clustered with accessory genes that encode proteins associated with immunity and/or maturation (Kolter & Moreno, 1992
). Finally, bacteriocin synthesis appears to be a cell-density-regulated process, dependent on interaction of peptide pheromones with devoted two-component signal transduction systems (Diep et al., 1995
; Kuipers et al., 1995
).
In this paper we report the cloning and characterization of the genetic determinant for lactococcin 972, a bacteriocin produced by Lactococcus lactis subsp. lactis IPLA 972, that is active on all lactococci tested to date (Martínez et al., 1995 ). This would protect the producing strain from contamination by other lactococci when used as a unique starter in cheese fermentation. Additionally, the bacteriocin would be used as a potential accelerator of maturation through lysis of the starters. Lactococcin 972 is composed of two copies of a single 7500-Da peptide whose amino-terminal sequence is E-G-T-W-Q-X-G-Y-G-V-, where the X stands for an undetermined residue. The isolated monomers do not show any antimicrobial activity (Martínez et al., 1996
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Determination of lactococcin 972 activity.
Production of the bacteriocin was tested in filter-sterilized culture supernatants throughout the growth cycle of L. lactis IPLA 972. The inhibitory activity of lactococcin 972 was quantified using the agar diffusion test of twofold serial lactococcin 972 dilutions (Reddish, 1929 ), using L. lactis MG 1614 as an indicator. The highest dilution that produced a clear zone of growth inhibition was defined as one arbitrary unit of activity (AU) ml-1. Concentration and partial purification of lactococcin 972 was performed from supernatants of late-exponential-phase cultures of L. lactis IPLA 972 which were precipitated with 5 vols acetone at -20 °C for 30 min. After centrifugation at 4 °C, the pellet was dried under vacuum and resuspended in 50 mM sodium phosphate buffer, pH 7 (Martínez et al., 1996
). The bacteriocin was visualized through Tricine/SDS-PAGE (Schägger & von Jagow, 1987
).
DNA techniques.
Lactococcal plasmids were extracted by the method of OSullivan & Klaenhammer (1993) . L. lactis MG 1614 cells were electroporated by the method of Leenhouts et al. (1990)
. Selection of the transformants was made by including serial dilutions of the transformed cultures in top-agar overlays (GM17, 0·7% agar) containing streptomycin (500 µg ml-1) and partially purified lactococcin 972 (250 AU ml-1). Electroporation of E. coli was performed as described by Dower et al. (1988)
. Standard DNA manipulations such as E. coli plasmid isolation, restriction endonuclease analysis, ligations and gel electrophoresis were performed according to Sambrook et al. (1989)
. To localize the genetic determinant of lactococcin 972, a degenerate 17-mer oligonucleotide (5'-TGGCARTGYGGNTAYGG-3', R=A or G, Y=C or T, N=A, T, C or G) was designed from its amino-terminal sequence (EGTWQXGYGV) (Martínez et al., 1996
), assuming that X corresponded to cysteine. The oligonucleotide was labelled with [
-32P]ATP using T4 polynucleotide kinase (Boehringer Mannheim). Southern hybridization was carried out in Rapid-Hyb buffer (Amersham) at 37 °C for 16 h. Washes were performed under low stringency conditions [2x SSC (0·3 M sodium chloride, 0·03 M sodium citrate)/0·1% SDS, at 37 °C]. PCR amplifications were done with the proofreading Pwo polymerase (Boehringer Mannheim). Nucleotide sequencing was performed according to the dideoxy chain-termination method (Sanger et al., 1977
) using sequenase 2.0 (Amersham). Sequences were analysed using the GCG software package (Devereux et al., 1984
).
RNA isolation, Northern hybridization and primer extension analysis.
RNA extraction was done by a modification of the method of Chomczynski & Sacchi (1987) . In brief, samples of L. lactis IPLA 972 grown in M17 broth were harvested at different time intervals, resuspended in denaturing solution and disrupted with 4·55·5 mm glass balls by vigorous vortexing. After several phenol/chloroform extractions, nucleic acids were treated with DNase (0·05 U µl-1, Boehringer Mannheim) and stored as ethanol precipitates. For Northern analysis, 5 µg RNA was fractionated on agarose gels (Sambrook et al., 1989
) and transferred to Hybond-N membrane (Amersham). After addition of the probe, hybridization was performed at 42 °C in Rapid-Hyb buffer and the membrane was washed according to the manufacturers instructions. Double-stranded DNA probes were labelled with [
-32P]dCTP using the Rediprime labelling kit (Amersham).
Primer extension experiments were performed with a 15-mer oligonucleotide (5'-ATAATTTGAATATGC-3') complementary to the coding sequence in the region of the amino terminus of the mature bacteriocin, end-labelled with [-32P]ATP (Amersham) using T4 polynucleotide kinase. The primer (10 pmol) was annealed to 20 µg DNase-treated total RNA and cDNA synthesis was performed by using a cDNA synthesis kit (Promega) following the manufacturers protocol. The product was analysed on a 6% polyacrylamide/urea sequencing gel next to standard sequencing reactions as size markers.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Identification and sequencing of lclA
The oligonucleotide mix corresponding to the amino-terminal end of lactococcin 972 was used to localize lclA in the plasmid map (Fig. 1). Sequencing of that region revealed two ORFs (Fig. 1
). The first ORF corresponds to lclA and encodes a predicted polypeptide of 91 aa that presents a potential ribosome-binding site (RBS; GGAGGA) 6 bp upstream of the translation start codon ATG (Fig. 3
). The previously determined N-terminal amino acid sequence of lactococcin 972 starts at Glu-26. This indicates that lclA encodes a pre-bacteriocin with a 25-residue N-terminal extension and a 66-aa C-terminal portion that would yield the mature lactococcin 972. The calculated molecular mass of this second part (7381 Da) fits with the estimation obtained by Tricine/SDS-PAGE of the pure bacteriocin (Martínez et al., 1996
). The presumptive leader has characteristics typical of signal peptides, such as a positively charged amino end, a hydrophobic central region and a sec-dependent processing signal, Ala-Gln-Ala, that conforms to the -3,-1 rule of von Heijne (1983)
. Curiously, this amino-terminal stretch also shows features that resembles those of double glycine leaders, which are frequently found in Class II bacteriocins, namely, the dimers G-G and L-S at positions 1920 and 1011, respectively (Havarstein et al., 1994
). However, in the purification experiments of lactococcin 972 we never observed a second peak of antimicrobial activity, nor did we find a double signal during sequencing of the bacteriocin. Of course this does not rule out the possibility that some processing of lactococcin 972 occurs at the double glycine motif but, in that case, the resulting polypeptide would not have any appreciable growth-inhibitory activity. Three nucleotides downstream of the stop codon (TAA) of lclA a 16 bp inverted repeat sequence was found, which could act as a
-independent terminator with a
G value of -35 kcal mol-1. A search of protein sequence databases revealed no polypeptides with homology to lactococcin 972. In contrast with most bacteriocins, the hydrophobicity plot of lactococcin 972 indicates that it is a hydrophilic peptide that presents up to 53% polar amino acids. It is also quite basic with a predicted pI of 10·57.
|
Downstream of lclA a second ORF, corresponding to lclB and with coding capacity for a polypeptide of 563 aa, was found. They are separated by a stretch of 57 nt that includes the putative -independent terminator described above. Analysis of the primary sequence of this second polypeptide (LclB) revealed seven hydrophobic zones, each composed of 1420 aa, indicating that the putative protein might span the plasma membrane. Five of the seven hydrophobic domains were clustered in the central region of the polypeptide and were well separated from the other two that occupied the extreme amino and carboxy ends of the protein. A potential RBS (GAGGGA) was found 6 nt before the start codon (ATG). An inverted repeat with a
G of -20 kcal mol-1 was located 152 nt after the stop codon (TGA). No homologies were found between LclB and proteins stored in databases.
Production of lactococcin 972 depends on expression of the operon formed by lclA-lclB
Lactococcin 972 is produced by L. lactis IPLA 972 during the exponential phase of growth that lasts about 6 h under standard conditions. The concentration of the bacteriocin in culture supernatants remains constant during early stationary phase and is abruptly lowered after 10 h cultivation (Martínez et al., 1996 ; Fig. 4a
). To determine whether lclA and lclB were independently transcribed or not and if the kinetics of accumulation of lactococcin 972 was dependent on the rate of transcription of lclA, total RNA was extracted from L. lactis IPLA 972 cultures periodically through its life cycle (Fig. 4b
) and hybridized to (i) a PCR-amplified segment spanning the putative RBS of lclA to the end of this gene and (ii) a 600 bp PstI segment internal to lclB. In the first case two bands were observed (Fig. 4c
). One of them, of about 2·5 kb, was only present in exponential-phase cultures, while the other, of about 400 bp, showed maximum levels in exponentially growing cells but was evident even in late-stationary-phase cultures. When the lclB internal segment was used as probe, only the 2·5 kb transcript became evident (Fig. 4d
). The size of this band corresponds to the length of the segment that comprises lclA plus lclB which, together with the hybridization data with the probes specific for each gene, indicates that both form a single bicistronic operon. Furthermore, the presence of the largest transcript only in exponential-phase cultures may be a consequence of a non-absolute stop of transcription at the level of the terminator that lies between lclA and lclB. Since the accumulation of specific RNA and, presumably, its synthesis is maximal in exponential-phase cultures, it may well be that the stop signal was missed more frequently at this time of the cycle, producing a detectable 2·5 kb band.
|
The abundance of the transcripts paralleled the accumulation rate of lactococcin 972 in culture supernatants (Fig. 4). This was high throughout exponential phase, coinciding with a maximum in the concentration of both RNA species. The stationary phase was accompanied by a plateau in the concentration of bacteriocin and by a decrease in the abundance of specific transcripts. Finally, the concentration of active lactococcin 972 decreased, possibly as a consequence of the resolution of the peptide dimers that form the active bacteriocin, to reach a new plateau, which might represent an equilibrium between synthesis the transcript was evident even in 24-h-old cultures and degradation upon export.
Therefore, it appears that lclA and lclB form a single transcriptional unit. The first gene would encode the pre-bacteriocin which, upon export, probably through the general sec-secretion system of the cell, would reach the external medium, being processed at the same time to give the final 66 aa polypeptide. This forms a dimer to produce the active lactococcin 972. The product of lclB might be the immunity protein that protects the producing cell from suicide. This hypothesis is based (i) on its location, immediately downstream of the structural bacteriocin gene, (ii) on the fact that the two genes form an operon and (iii) on the possible location of LclB spanning the plasma membrane. All these properties are typical of bacteriocin immunity systems (Nes et al., 1996 ). Furthermore, all the attempts to disrupt lclB have failed, possibly indicating that its product is necessary to protect the cell from the potential toxicity of lactococcin 972 (B. Martínez, A. Rodríguez & J. E. Suárez, unpublished data). However, lclB would encode a 563-aa polypeptide while the usual Class II bacteriocin immunity proteins are fairly small, in all cases less than 200 aa and in some cases as small as around 50 aa.
To date, four other bacteriocins have been reported to have signal peptides matching the requirements of the sec-export system, but none were isolated from lactococci: acidocin B and divergicin A from Lactobacillus acidophilus and Carnobacterium divergens, respectively (Leer et al., 1995 ; Worobo et al., 1995
), and two pediocin-like peptides isolated from enterococci (Cintas et al., 1997
; Tomita et al., 1996
). In all cases production was dependent on the expression of a single putative operon that comprised the structural and putative immunity genes, without the need of dedicated secretion or maturation proteins. It is tempting to speculate that production of active lactococcin 972 will also be achieved by the expression of lclA and lclB. In any case, the information needed is probably encoded by plasmid pBL1, since its transformants produced the bacteriocin and were immune to it.
Lactococcin 972 presents a series of features that make it difficult to include it in any of the existing bacteriocin classes. It was previously shown that its active form is a homodimer and that it does not affect plasma membrane integrity (Martínez et al., 1996 ). The data reported here further confirm these two peculiar findings. First, there is only one structural gene; all two-component bacteriocins described so far are encoded by two adjacent ORFs, each encoding one of the polypeptides that constitute the active antimicrobial compound. Second, the monomer of lactococcin 972 is hydrophilic, which confirms that the membrane would not be its primary target. In fact, preliminary data seem to indicate that lactococcin 972 inhibits the synthesis of the cell wall (B. Martínez, A. Rodríguez & J. E. Suárez, unpublished data). This might justify the large size of the putative immunity protein LclB and its location, with several domains facing the outside of the plasma membrane. It is becoming clear that lactococcin 972 may be considered to be a hybrid between Classes IIb and IIc, although it presents some peculiarities that may force the definition of a new class of bacteriocins. Further studies, some of which are under way, will help in its definitive classification among LAB antimicrobial peptides.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Víctor Ladero for helping us with the artwork.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
van Belkum, M. J., Kok, J. & Venema, G. (1992). Cloning, sequencing and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6. Appl Environ Microbiol 58, 572-577.[Abstract]
Chang, A. C. & Cohen, S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15 cryptic miniplasmid. J Bacteriol 134, 1141-1156.[Medline]
Chomczynski, P. & Sacchi, N. (1987). Single step method of RNA isolation by acid guanidinum thiocyanate-phenol-chlorophorm extraction. Anal Biochem 162, 156-159.[Medline]
Cintas, L. M., Casaus, P., Havarstein, L. S., Hernández, P. E. & Nes, I. F. (1997). Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl Environ Microbiol 63, 4321-4330.[Abstract]
Daeschel, M. A. (1989). Antimicrobial substances from lactic acid bacteria for use as food preservatives. Food Technol 43, 164-167.
Davey, G. P. (1984). Plasmid associated with diplococcin production in Streptococcus cremoris. Appl Environ Microbiol 48, 895-896.[Medline]
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395.[Abstract]
Diep, D. B., Havarstein, L. S. & Nes, I. F. (1995). A bacteriocin-like peptide induces bacteriocin synthesis in Lactobacillus plantarum C11. Mol Microbiol 18, 631-639.[Medline]
Dower, W. J., Miller, J. F. & Ragsdale, C. W. (1988). High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16, 6127-6145.[Abstract]
Dufour, A., Thuault, D., Boulliou, A., Bourgeois, C. M. & Le Pennec, J. P. (1991). Plasmid-encoded determinants for bacteriocin production and immunity in a Lactococcus lactis strain and purification of the inhibitory peptide. J Gen Microbiol 137, 2423-2429.[Medline]
Gasson, M. J. (1983). Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154, 1-9.[Medline]
Havarstein, L. S., Holo, H. & Nes, I. F. (1994). The leader peptide of colicin V shares consensus sequences with leader peptides that are common among peptide bacteriocins produced by Gram-positive bacteria. Microbiology 140, 2383-2389.[Abstract]
von Heijne, G. (1983). Patterns of amino acids near signal-sequence cleavage sites. Eur J Biochem 133, 17-21.[Abstract]
Klaenhammer, T. R. (1993). Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol Rev 12, 39-86.[Medline]
Kolter, R. & Moreno, F. (1992). Genetics of ribosomally synthesized peptide antibiotics. Annu Rev Microbiol 46, 141-163.[Medline]
Kuipers, O. P., Rollema, H. S., Beerthuyezen, M. M., Siezen, R. J. & de Vos, W. M. (1995). Protein engineering and biosynthesis of nisin and regulation of the transcription of the structural nisA gene. Int Dairy J 5, 785-795.
Leenhouts, K. J., Kok, J. & Venema, G. (1990). Stability of integrated plasmids in the chromosome of Lactococcus lactis. Appl Environ Microbiol 56, 2726-2735.
Leer, R. J., van der Vossen, J. M. B. M., van Giezen, M., van Noort, J. M. & Pouwels, P. (1995). Genetic analysis of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus. Microbiology 141, 1629-1635.[Abstract]
Martínez, B., Suárez, J. E. & Rodríguez, A. (1995). Antagonistic activities of wild lactococcal strains isolated from homemade cheeses. J Food Prot 58, 1118-1123.
Martínez, B., Suárez, J. E. & Rodríguez, A. (1996). Lactococcin 972, a homodimeric lactococcal bacteriocin whose primary target is not the plasma membrane. Microbiology 142, 2393-2398.
Nes, I. F., Diep, D. B., Havarstein, L. S., Brurberg, M. B., Eisink, V. & Holo, H. (1996). Biosynthesis of bacteriocins in lactic acid bacteria. Antonie Leeuwenhoek 70, 113-128.
OSullivan, D. J. & Klaenhammer, T. R. (1993). Rapid mini-prep isolation of high-quality plasmid DNA from Lactococcus and Lactobacillus spp. Appl Environ Microbiol 59, 2730-2733.[Abstract]
Ra, S. R., Qiao, M., Immonen, T., Pujana, I. & Saris, P. E. J. (1996). Genes responsible for nisin synthesis, regulation and immunity form a regulon of two operons and are induced by nisin in Lactococcus lactis N8. Microbiology 142, 1281-1288.[Abstract]
Rauch, P. J. G. & de Vos, W. M. (1992). Characterization of the novel nisin-sucrose conjugative transposon Tn5276 and its insertion in Lactococcus lactis. J Bacteriol 174, 1280-1287.[Abstract]
Reddish, G. F. (1929). Methods for testing antiseptics. J Lab Clin Med 14, 649-658.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[Abstract]
Schägger, H. & von Jagow, G. (1987). Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368-379.[Medline]
Tagg, J. R., Dajani, A. S. & Wannamaker, L. W. (1976). Bacteriocins of Gram positive bacteria. Bacteriol Rev 40, 722-756.[Medline]
Tomita, H., Fujimoto, S., Tanimoto, K. & Ike, Y. (1996). Cloning and genetic organization of the bacteriocin 31 determinant encoded on the Enterococcus faecalis pheromone responsive conjugative plasmid pYI17. J Bacteriol 178, 3585-3593.[Abstract]
Worobo, R. W., van Belkum, M. J., Sailer, M., Roy, K. L., Vederas, J. C. & Stiles, M. E. (1995). A signal peptide secretion-dependent bacteriocin from Carnobacterium divergens. J Bacteriol 177, 3143-3149.[Abstract]
Vanderbergh, P. A. (1993). Lactic acid bacteria, their metabolic products and interference with microbial growth. FEMS Microbiol Rev 12, 221-238.
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119.[Medline]
Received 16 February 1999;
revised 21 July 1999;
accepted 23 July 1999.