Departments of Microbiology, and Food Science and Technology, and National Food Biotechnology Centre, National University of Ireland, Cork, Ireland1
Laboratoire de Recherche sur la Viande2 and Laboratoire de Génétique Microbienne3, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France
Author for correspondence: Douwe van Sinderen. Tel: +353 21 902811. Fax: +353 21 903101. e-mail: douwe{at}ucc.ie
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ABSTRACT |
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Keywords: Lactococcus lactis, signal transduction, response regulator, two-component regulatory systems, histidine protein kinase
Abbreviations: 2CS, two-component system; HPK, histidine protein kinase; RR, response regulator
The GenBank accession numbers for the sequences of the six 2CSs and surrounding ORFs determined in this work are AF172649, AF176556, AF178425, AF172650, AF176557 and AF176809 for systems AF, respectively.
a Present address: Laboratoire de Reproduction et Développement des Plantes, Ecole Normale Supérieure, 46 allée dItalie, 69364 Lyon Cedex 07, France.
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INTRODUCTION |
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The HPK and RR proteins are composed of distinct domains or modules which can be arranged in different configurations to build signalling circuits (for reviews, see Stock et al., 1989 ; Parkinson & Kofoid, 1992
; Parkinson, 1993
; Alex & Simon, 1994
; Egger et al., 1997
). In the simplest circuit, the HPK is composed of a C-terminal transmitter module attached to an N-terminal signal-input domain, whilst the RR consists of an N-terminal receiver module connected to a C-terminal signal-output domain. Another phosphotransfer signalling device, referred to as a histidine phosphotransfer protein (HPT) (Ishige et al., 1994
) consists of approximately 120 amino acids and contains a short consensus motif of approximately 25 amino acids including an invariant histidine residue, which can acquire a phosphoryl group from a cognate HPK and/or RR and transmits this phosphoryl group to the receiver domain of the appropriate RR. Hybrid or unorthodox HPK proteins have also been described (Parkinson & Kofoid, 1992
), some of which harbour the signal-input and output domains, as well as the transmitter and receiver modules within one protein.
To date, members of the HPK and RR families have been described for over fifty different bacterial species and for several eukaryotic organisms, including Neurospora crassa, Saccharomyces cerevisiae, Arabidopsis thaliana and Dictyostelium discoideum (Alex & Simon 1994 ; Chang & Meyerowitz, 1994
; Appleby et al., 1996
; Pao & Saier, 1997
; Loomis et al., 1997
) and it can be inferred that such systems may be ubiquitous in many living organisms. An apparent exception to this rule is the methanogenic archaeon, Methanococcus jannaschii (Bult et al., 1996
).
In Gram-positive bacteria, several processes are regulated by 2CSs. Analysis of the genome sequence of Bacillus subtilis identified the presence of 36 HPKs and 34 RRs. Several 2CSs in B. subtilis have been extensively studied, these include those involved in chemotaxis (CheA-CheY), phosphate metabolism (PhoR-PhoP), anaerobic growth (ResE-ResD), competence development (ComP-ComA) and degradative enzyme production (DegS-DegU) (Fabret et al., 1999 ). Quorum sensing mechanisms in Gram-positive bacteria are also regulated by 2CSs but in a cell-density-dependent manner. These include the development of genetic competence in B. subtilis (Dubnau, 1991
) and Streptococcus pneumoniae (Pestova et al., 1996
), virulence responses in Staphylococcus aureus (Morfeldt et al., 1996
) and the production of antimicrobial peptides by lactic acid bacteria (Diep et al., 1994
, 1996
; Kuipers et al., 1993
, 1995
; Quadri et al., 1997
; Van der Meer et al., 1993
; OKeeffe et al., 1999
). In each case, a secreted peptide functions as the input signal for a specific sensor of a 2CS. The genes which encode the precursor of the peptide and genes encoding the proteins involved in the 2CS and/or those involved in the secretion of the peptide pheromone are transcriptionally linked and the synthesis of the peptide pheromones appears to be an autoregulatory process. Another common feature of these systems is the involvement of an ATP-binding cassette (ABC) exporter in the secretion of the peptide pheromone. A plasmid-borne 2CS which regulates copper resistance in Lactococcus lactis has also been described (Khunajakr et al., 1999
).
Previously, we reported the identification of five HPKs from L. lactis MG1363 by complementation of HPK-deficient Escherichia coli mutants (OConnell-Motherway et al., 1997 ). This study reports on the identification and sequence of the five cognate RR genes and identification of a novel 2CS from L. lactis by a PCR approach, as well as preliminary characterization of the six putative 2CSs from L. lactis MG1363.
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METHODS |
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Reagents and molecular cloning procedures.
Restriction enzymes, ligase, reverse transcriptase and T4 DNA polymerase were purchased from commercial sources and were used according to the suppliers recommendations. [-32P]dCTP (300 Ci mmol-1; 11·1 TBq mmol-1) and [
-32P]ATP (5000 Ci mmol-1; 185 TBq mmol-1) were purchased from Amersham.
Chromosomal DNA from L. lactis was extracted as previously described (Leenhouts et al., 1989 , 1990
). Minipreparation of plasmid DNA from E. coli was done either by the alkaline lysis method of Birnboim & Doly (1979)
or (for sequencing purposes) by using the QIAprep Spin Plasmid Miniprep kit (Qiagen). DNA restriction enzyme fragments were isolated from agarose gels using the Qiagen Gel Extraction kit as recommended by the manufacturer. PCR fragments were purified using the Boehringer High Pure PCR Purification kit (Roche Diagnostics).
Electrotransformation of E. coli was accomplished using the Bio-Rad Gene Pulser apparatus according to the manufacturers instructions. Preparation and transformation of competent L. lactis cells were performed as described by De Vos et al. (1989) . Southern transfer and hybridizations were all performed according to standard procedures (Sambrook et al., 1989
) using the ECL kit (Amersham).
PCR and inverse PCR.
PCR reagents (Taq polymerase, Expand DNA polymerase and dNTPs) were purchased from Roche Diagnostics and used according to the manufacturers instructions, employing a Perkin Elmer Gene Amp PCR 2400 system. Synthetic single-stranded DNA oligonucleotide primers used in this study were synthesized on a Beckman Oligo 1000M DNA synthesizer (Beckman Instruments). The degenerate primers (DD and K primers) used in this study were described previously (Morel-Deville et al., 1997 ) and are based on two highly conserved regions found within the receiver modules of published RR proteins, i.e. the DD and the K boxes (Parkinson, 1993
).
The oligonucleotide primers used for amplification of RR-encoding genes by inverse PCR, transcription start site mapping, amplification of putative promoter regions and construction of insertion mutants were designed based on the nucleotide sequences of the RR- and HPK-encoding genes or surrounding DNA sequences.
Template DNA for inverse PCR was prepared essentially as described by Ochman et al. (1990) with the following modifications. DNA restriction fragments were ligated under conditions that favoured the formation of monomeric circles (DNA concentrations ranging from 0·1 µg to 0·5 µg in a total volume of 50 µl were ligated for 16 h at 15 °C). The ligated DNA mixture was phenol-extracted, precipitated and subsequently used as template for inverse PCR, which was performed using a Boehringer ExpandTM Long Template PCR system as recommended by the manufacturer.
Nucleotide sequence analysis.
Nucleotide sequence determination was performed on an ABI 373A automatic sequencer using the Dye Terminator Sequencing kit (Applied Biosystems). Sequence data assembly and analysis was performed using DNASTAR software. Sequence comparisons and database searches were performed using non-redundant sequences present at the NCBI internet site (http://www.ncbi.nlm.nih.gov/) using TBLASTN and BLASTP programs (Altschul et al., 1997 ). Sequence alignments were performed using the CLUSTAL method of the MEGALIGN program of the DNASTAR software package. Protein analysis was performed by KyteDoolittle hydrophilicity plots of the PROTEAN program of the DNASTAR software package.
Construction of promoter fusions.
DNA fragments containing presumptive promoters for the 2CSs were amplified by PCR using L. lactis MG1363 chromosomal DNA as template, ligated to the BglII and PstI restriction sites of the promoter probe vector pAK80 (Israelsen et al., 1995 ) and introduced into E. coli XL-1 Blue by electrotransformation. The expected structure of the recombinant plasmids was confirmed by restriction mapping, PCR and sequencing of the cloned fragment, prior to their introduction into L. lactis MG1363 by electrotransformation and subsequent selection on M17 glucose agar plates containing erythromycin (5 µg ml-1) and X-Gal (40 µg ml-1).
Northern blot and primer extension analysis.
Total RNA was isolated from L. lactis by the Macaloid method described by Kuipers et al. (1993) . For Northern blot analysis, RNA was glyoxylated, separated by agarose gel (1%) electrophoresis, blotted and hybridized as described previously (Van Rooijen & de Vos, 1990
). Hybridization probes derived from each 2CS [internal 300 bp fragments of each RR-encoding gene cloned in pBluescript SK(-)] were radiolabelled with [
-32P]dCTP by random priming (Roche Diagnostics). Primer extension was performed by annealing 10 pmol 5'-end
-32P-labelled synthetic 18-mer oligonucleotides to 50 µg of RNA as described by Pujic et al. (1998)
. Sequence ladders were produced using the same primers as the primer extension product with the aid of the Sequenase sequencing kit (version 2.0) (US Biochemical).
Construction of RR and HPK mutants.
Internal 500 and 800 bp fragments of the six identified RR and HPK genes, respectively, were amplified by PCR using L. lactis MG1363 chromosomal DNA as template. DNA fragments to be amplified were chosen so that after insertion into the chromosome the HPK-and RR-encoding genes would be interrupted, and in the protein products the conserved histidine/aspartate residues, which are the sites of phosphorylation, would be separated from their respective C-terminal transmitter/effector domains.
The amplified fragments were cloned in the integration insertion vector pRV300 (Leloup et al., 1997 ) and introduced into competent L. lactis MG1363 cells by electrotransformation, with subsequent selection on M17 glucose agar plates containing erythromycin (5 µg ml-1). The colonies arising on this plate were suspected to have integrated pRV300 derivatives into their chromosome by a Campbell-like mechanism as plasmid pRV300 is incapable of replication in L. lactis (Leloup et al., 1997
). This assumption was confirmed by PCR mapping and Southern hybridization analyses using chromosomal DNA prepared from these transformants.
Sugar metabolism profiles.
Sugar metabolism profiles were determined using API 20 Strep as recommended by the manufacturer (BioMérieux). The resulting fermentation patterns were inspected following incubation at 30 °C for 4 and 24 h. Fermentation of carbohydrates was detected by acid production and a change in colour of the pH indicator.
Determination of acid stress resistance.
Overnight cultures were diluted 100-fold in TYG broth and grown at 30 °C to early-exponential-growth phase (OD600 ~0·3). These cells were used for acid stress resistance experiments using two distinct procedures: (1) cells were spread-plated on modified GM17 agar (ß-glycerophosphate absent and pH adjusted to a value of 5·5 with acetic acid after sterilization) and incubated at 30 °C overnight, after which colony numbers were evaluated; and (2) (according to OSullivan, 1996 ) cells were harvested, resuspended in TYG broth, which had been adjusted to pH 4·0 with acetic acid, incubated at 30 °C and spread-plated on TYG agar at hourly intervals for 3 h. The plates were incubated at 30 °C overnight, after which the number of colony-forming units was determined.
Determination of osmotic and oxidative stress resistance.
Overnight cultures were diluted 100-fold in TYG broth and grown at 30 °C to early-exponential-growth phase (OD600 ~0·3). They were then harvested and resuspended in TYG broth containing 20% NaCl or 4 mM H2O2 to assess osmotic or oxidative stress resistance, respectively. Cultures were subsequently incubated at 30 °C for 8 h and 20 min, respectively, and serial dilutions spread-plated at appropriate time intervals. Plates were incubated at 30 °C overnight, after which the stress resistances were determined by colony-counting (OSullivan, 1996 ).
Phosphatase activity and ß-galactosidase assays.
Qualitative phosphatase activity was determined by streaking cultures on GM17 agar containing X-P (40 µg ml-1) and incubating overnight at 30 °C. A blue colour indicated a phosphatase-positive culture. ß-Galactosidase activity was determined as described by Israelsen et al. (1995) .
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RESULTS |
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The HPK-encoding genes were used as probes to determine the restriction map of their surrounding regions by Southern hybridization (data not shown). Based on the five HPK nucleotide sequences, primers were designed to amplify DNA regions neighbouring each HPK gene by inverse PCR. Sequence analysis of the PCR products allowed the identification of a putative RR-specifying gene for each of the putative HPK-encoding genes. These genes were designated llrAE (where the capital letter is identical to that of the corresponding HPK-encoding genes llkinAE) and were located either upstream (in the case of llkinA, llkinB, llkinC and llkinE) or downstream (in the case of llkinD) of the putative HPK-encoding gene (Fig. 1.
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This strategy was applied to isolate (internal fragments of) RR-encoding genes from L. lactis MG1363. A unique and intense band of the expected size (approx. 290 bp) was generated (data not shown). The assumed mixture of DNA fragments was purified, cloned into the unique EcoRI/XbaI sites of the pBluescript II SK(-) vector and sequenced on both strands. Analysis of the nucleotide sequence of 48 independent clones revealed that they corresponded to four different DNA sequences. Each of the four sequences of the coding strand contained one uninterrupted ORF. Further inspection of the four DNA sequences revealed that three fragments were internal segments of the putative RR-encoding genes designated llrA, llrC and llrE described above. Analysis of the deduced amino acid sequence of the ORF contained within the fourth segment also showed significant similarity to RR receiver modules in the sequence databases (data not shown). These results provided strong evidence that this DNA segment amplified by the primer set originated from a RR gene of L. lactis MG1363. Extensive screening of 600 independent clones by colony hybridization with the four cloned DNA segments as probes did not allow identification of other putative RR-encoding genes in MG1363. This may be due to the primer design, which may have to be adapted for L. lactis (Morel-Deville et al., 1997 ), or to the possible paucity of 2CSs in this bacterium.
The entire sequence of the newly identified putative RR-encoding gene, designated llrF, was determined by cloning a 4·2 kb XbaI chromosomal DNA fragment from L. lactis MG1363 which hybridized with the fourth DNA fragment described above in pBluescript II SK(+) to produce plasmid pLlrF. This clone contained the complete coding region (llrF) of this putative RR protein as well as two upstream ORFs. Inverse PCR allowed amplification of DNA downstream of llrF. Subsequent chromosome walking and DNA sequence analysis revealed the presence of a putative HPK-encoding gene, designated llkinF, immediately downstream of llrF. The deduced protein product of llkinF is significantly similar to llkinA, llkinB, llkinC and llkinEorf1 (OConnell-Motherway et al., 1997 ) and to the Env/PhoR family of HPKs, possessing the four conserved histidine kinase motifs: the autophosphorylation site (H box), at which a conserved histidine is phosphorylated upon signal detection; the phenylalanine/aspartate (F/D) and glycine (G) boxes, which are thought to be involved in nucleotide binding; and the asparagine (N) box, which is of unknown function. Two hydrophobic membrane-spanning segments were identified, suggesting that this protein is composed of a non-conserved N-terminal membrane-associated signal input (sensor) domain and a conserved C-terminal intracytoplasmic domain required for subsequent signal transmission to the regulatory partner through phosphorylation (Stock et al., 1995
) (Fig. 2
).
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Identification of genes surrounding each putative 2CS by chromosome walking
Sequencing of the DNA regions adjacent to the six 2CSs allowed the identification of a number of neighbouring ORFs. These are summarized in Table 2 together with their similarities to known protein products. Their positions and orientations are schematically depicted in Fig. 1
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Growth profiles were determined in GM17, TYG broth and minimal medium at 30 °C and ß-galactosidase activity was measured as a function of growth. In all media MGpAK80-B and MGpAK80-F exhibited a sharp increase in ß-galactosidase activity which started at the mid-exponential phase of growth and continued to increase into stationary phase (Fig. 5). MGpAK80-D and MGpAK80-E also exhibited inducible ß-galactosidase activities but in these two cases the induction level appeared to be much lower. Strain MGpAK80-E displayed a ß-galactosidase activity profile similar to that of MGpAK80-B and MGpAK80-F, whilst in the case of MGpAK80-D activity started to increase in late-exponential phase and this increase continued into stationary phase (Fig. 5
).
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API profiles
API 20 Strep profiles were performed to determine if the mutants had been altered with respect to sugar metabolism. No differences in sugar metabolism were observed; however, it was observed that MGKinA and MGRrA were arginine-deaminase-negative whilst the parent strain, MG1363, and the other mutant strains gave a positive reaction. This observation was confirmed by growing MGKinA and MGRrA in Nivens arginine broth and testing for the production of ammonia from arginine using Nesslers reagent. Upon addition of Nesslers reagent, a brown precipitate was observed for MG1363, indicating a positive arginine deaminase reaction, but was not observed for MGKinA and was observed to a lesser extent for MGRrA. This result indicates that system A is involved in regulation of arginine metabolism in the lactococcal cell.
Acid stress
Two methods were used to assess the effect of acid stress on the MG1363 parent and the HPK and RR mutant strains (see Methods). Using the first procedure, it was found that MGKinA and MGRrA were 10-fold more acid-resistant, whilst MGKinB and MGRrB were 100-fold more acid-resistant, compared to the parent strain, MG1363. However, it should be noted that the colonies of MGKinB and MGRrB strains were pinpoint-sized and only became visible after 72 h incubation when cultures were plated on pH-adjusted, unbuffered GM17 agar. This acid resistance was not observed using the second procedure whereby parent and mutant cultures grown in TYG broth to early-exponential phase were harvested and resuspended in TYG broth which had been adjusted to pH 4·0 with acetic acid and incubated at 30 °C. Serial dilutions were plated at time zero and subsequently at hourly intervals to obtain plate counts (Table 3). After 3 h incubation, 0·4% of the original MG1363 cells were viable. MGKinB and MGRrB displayed slight acid resistance, having 1% survival after 3 h. An acid-sensitive phenotype was also observed for MGKinC and MGRrC, with only 0·008 and 0·000002% survival, respectively, after 3 h at pH 4·0. Strains MGKinA and MGRrA exhibited a more acid-sensitive phenotype, with only 0·002 and <0·1%, respectively, surviving the 3 h incubation at pH 4·0. The acid-sensitive phenotype observed for mutants of system A combined with the arginine deaminase-negative phenotype described above suggests that this 2CS may be involved in modulating the internal pH of the lactococcal cell and counteracting acid stress through arginine metabolism and its concomitant production of ammonia.
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Oxidative stress
Early-exponential-phase cultures grown in TYG broth were harvested and resuspended in TYG broth containing 4 mM H2O2 and incubated at 30 °C. Samples were taken at time zero and after 10 and 20 min and serial dilutions plated. After 20 min incubation, 70% of the MG1363 cells remained viable. Of the mutant strains tested, only MGRrF exhibited greater H2O2 sensitivity as compared to the parent culture MG1363 with just 9% of MGRrF cells remaining viable after 20 min (data not shown). This observation suggests that system F may be involved in the response of L. lactis to oxidative stress.
Phosphatase activity
Qualitative phosphatase assays of MG1363 and the insertion mutant strains were established by streaking overnight cultures on GM17 agar containing an indicator of phosphatase activity, X-P, and incubating at 30 °C overnight. Phosphatase activity was observed for MG1363, indicated by blue-coloured colonies. All mutant colonies displayed a blue colony morphology similar to the wild-type, MG1363, except MGKinE, which produced colonies remaining completely white indicating an absence of phosphatase activity, and MGRrE, which formed pale blue colonies, thus exhibiting very weak phosphatase activity.
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DISCUSSION |
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The genes encoding corresponding HPK and RR proteins of a specific 2CS are frequently organized in an operon. This appears to be valid for all six 2CSs described here. Putative RR genes (designated llrAD) were identified upstream of llkinA, llkinB and llkinC, and downstream of llkinD. One RR-encoding gene, llrE, was identified upstream of llkinE but separated by a putative ORF of 513 bp. This putative ORF, designated lltcsEorf2, bears no significant homology to any known sequence in the databases, but the deduced amino acid composition and hydrophilicity profiles suggest that the gene product is a membrane-associated protein. Three of the four RR-encoding fragments identified using a PCR strategy were shown to be internal fragments of llrA, llrC and llrE. The remaining DNA fragment appeared to be part of an as yet unidentified RR-encoding gene, which was designated llrF. The complete DNA sequence of llrF was determined and a putative HPK gene designated llkinF was identified downstream of llrF.
Analysis of the deduced protein products of the six putative lactococcal RR-encoding genes demonstrated that they all possess the conserved N-terminal motifs characteristic of other RRs. They all contain an N-terminal receiver module of approximately 110 amino acids, which harbours the three signature amino acids typical for RRs.
Five of the lactococcal RRs, i.e. LlrA, LlrB, LlrC, LlrE and LlrF, exhibit extensive similarity to the OmpR family of RRs, not only in the receiver domain but also in the effector domain (Fig. 3a). LlrD on the other hand appears to belong to the NarL subfamily of RRs (Fig. 3b
). In both OmpR and NarL subfamilies, the effector domain is a DNA-binding module, allowing the RR to function as a transcription factor. Phosphorylation serves to control the ability of the RR to either bind to a target DNA sequence or to interact with specific components of the transcription machinery.
The deduced protein product of llkinF exhibits strong structural similarity to members of the EnvZ family of orthodox HPKs. As with the other lactococcal HPKs (OConnell-Motherway et al., 1997 ), two hydrophobic membrane-spanning segments were identified in LlkinF, suggesting that this protein is composed of a non-conserved N-terminal membrane-associated signal input (sensor) domain and a conserved C-terminal intracytoplasmic domain required for subsequent signal transmission to the regulatory partner through transphosphorylation. These features suggest that all six identified L. lactis 2CS protein pairs function as signal transduction proteins via a mechanism of phosphorylation and dephosphorylation similar to those described for EnvZ-OmpR, NarX and NarQ-NarL and are likely to be involved in linking environmental awareness to physiological adaptation through transcriptional regulation.
Northern analysis performed using RNA isolated during various stages of growth in GM17 at 30 °C established that only two of the systems, systems A and C, are constitutively expressed. DNA regions containing promoter activity were identified upstream of the other 2CSs. Examination of these regions by ß-galactosidase gene fusions suggests that expression of these 2CSs is under the control of transcriptional regulation and is induced either during mid-exponential phase (systems B, E and F) or at the very end of the exponential phase (system D). The effect of various environmental factors on the mutant strains of the 2CSs was also examined. Mutant strains were constructed by disruption of the HPK- and RR-encoding genes by plasmid insertion and examined under various growth conditions. Polar effects of plasmid insertion on the expression of downstream-located genes cannot be excluded, but since RR- and HPK-encoding genes frequently form operons with genes involved in the same signalling pathway, no function other than that controlled by the 2CS should have been affected. Characterization of the insertion mutants obtained revealed that each had phenotypic differences from the wild-type, L. lactis MG1363, which ranged from acid sensitivity/resistance, altered phosphatase activity, increased salt sensitivity and H2O2 sensitivity.
From this preliminary characterization, it can be inferred that the adjacent gene couples of L. lactis MG1363 identified during this study probably encode members of homologous families of signal transduction proteins. The results obtained also suggest that all six systems are functional since promoter activity could be detected for each by either Northern analysis or constructing ß-galactosidase fusions, whilst in all cases phenotypic differences between the wild-type and mutant strains were observed.
Experiments are in progress to construct RR deletion mutants to verify the phenotypes observed with the insertion mutants and also to find target genes for at least some of the lactococcal 2CSs.
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ACKNOWLEDGEMENTS |
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This work was supported by Enterprise Ireland (contract SC/99/394) and the European Commission Biotechnology programme (19941998) (contract Bio4-CT96-0498), and a long term EMBO fellowship (ALTF 431-1995) to D.v.S.
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Received 22 October 1999;
revised 17 December 1999;
accepted 14 January 2000.