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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Lacticin 3147 is a membrane-active, two-component lantibiotic produced by Lactococcus lactis ssp. lactis DPC3147. In this study, the promoters of the lacticin 3147 gene cluster were mapped to the intergenic region between ltnR and ltnA1 (the genes encoding the regulatory protein LtnR and the first structural gene, LtnA1), and Northern analyses revealed that the biosynthetic and immunity genes are divergently transcribed in two operons, ltnA1A2M1TM2D and ltnRIFE respectively. Although the promoter controlling biosynthesis (Pbac) appears to be constitutive, characterization of a downstream β-galactosidase (β-gal) fusion beyond an intragenic stem–loop structure in ltnM1 confirmed that this putative transcriptional attenuator allows limited readthrough to the downstream biosynthetic genes, thus maintaining the correct stoichiometry between structural peptides and biosynthetic machinery. The promoter of the ltnRIFE operon (Pimm) was shown to be regulated by the transcriptional repressor LtnR. A mutant with a truncated ltnR gene exhibited a hyperimmune phenotype, whereas overexpression of ltnR resulted in cells with increased sensitivity to lacticin 3147. Gel mobility shift analysis indicated that LtnR binds to the Pimm promoter region, and fusion of this promoter to the β-gal gene of pAK80 revealed that expression from Pimm is significantly reduced in the presence of LtnR. Thus, we have demonstrated that lacticin 3147 uses a regulatory mechanism not previously identified in lantibiotic systems.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Lantibiotics are small, membrane-active peptides that contain the thioether amino acids lanthionine and β-methyllanthionine, as well as a number of dehydrated amino acids (Sahl et al., 1995; de Vos et al., 1995; Sahl and Bierbaum, 1998). These unusual residues are introduced by post-translational modification of the lantibiotic precursor peptides, followed by processing of the leader peptide and translocation across the membrane (Schnell et al., 1988). Recently, a number of two-component lantibiotics have been identified, including cytolysin produced by Enterococcus faecalis (Gilmore et al., 1990), staphylococcin C55 produced by Staphylococcus aureus C55 (Navaratna et al., 1998) and lacticin 3147 produced by Lactococcus lactis ssp. lactis DPC3147 (Ryan et al., 1996; 1999). Lacticin 3147 is a broad-spectrum lantibiotic that exerts its primary bactericidal activity by the formation of potential-dependent, ion-selective pores in the cytoplasmic membranes of sensitive organisms (McAuliffe et al., 1998). In addition to the thioether amino acids, a number of d-alanine residues have also been identified in the lacticin 3147 peptides (Ryan et al., 1999).

The lacticin 3147 genetic determinants are found on the 60.2 kb plasmid pMRC01, the sequencing of which has been described previously (Dougherty et al., 1998). Ten genes organized in two divergently transcribed clusters, ltnRIFE and ltnA1A2M1TM2D, have been shown to be involved in the biosynthesis and immunity to lacticin 3147 (McAuliffe et al., 2000a). Of these genes, ltnA1 and ltnA2 encode the lacticin 3147 precursor peptides of 59 and 64 residues, respectively; these precursors are processed to give rise to the mature LtnA1 and LtnA2 peptides (Ryan et al., 1999). Based on homology with genes identified in other lantibiotic gene clusters, ltnM1 and ltnM2 encode enzymes that catalyse the dehydration/thioether-forming reactions involved in post-translational modification. Indeed, we have recently demonstrated that each of the lacticin 3147 prepeptides requires a separate modification enzyme (McAuliffe et al., 2000b). ltnT encodes a putative transport protein of the ABC (ATP-binding cassette) superfamily, whereas the gene product of ltnD shows significant sequence similarity to a number of dehydrogenase enzymes. In the smaller four-gene cluster, LtnR shares homology with a number of transcriptional repressors of the PBSX (Xre) family. The gene product of ltnI has been shown to confer immunity to lacticin 3147 (McAuliffe et al., 2000a), whereas LtnFE comprises putative components of another ABC transporter possibly involved in immunity.

The regulation of biosynthesis and immunity of a number of lantibiotics has been studied in detail. In the case of nisin and subtilin, biosynthesis has been shown to be growth phase dependent and under the control of two-component signal transduction systems (Klein et al., 1993; Engelke et al., 1994). Genes encoding histidine kinases (LanK) and response regulators (LanR) have been identified in the respective gene clusters (Klein et al., 1993; Engelke et al., 1994), the latter most probably acting at the promoter of the lantibiotic structural gene, lanA. Although the nature of the signalling molecule is unknown in the subtilin biosynthesis pathway, it has been demonstrated that nisin autoregulates its own biosynthesis (Kuipers et al., 1995) by acting as a peptide pheromone for quorum sensing involving NisK and NisR (Kleerebezem et al., 1997). Although the promoter controlling the nisFEG genes is also subject to nisin control, the nisRK promoter exhibits nisin-independent expression (de Ruyter et al., 1996a; de Ruyter, 1998). Recently, three regulatory genes have been identified within the mersacidin gene cluster (Altena et al., 2000); mrsR2 and mrsK2 encode a two-component regulatory system that appears to be necessary for the transcription of the immunity gene cluster mrsFGE. In addition, mrsR1 encodes a protein with similarity to response regulators. However, mersacidin does not appear to be the signalling molecule in this system (Altena et al., 2000). Regulation of epidermin biosynthesis is controlled by EpiQ (Peschel et al., 1993); this protein bears significant homology to other response regulators at the C-terminal region. EpiQ activates the expression of the biosynthetic genes by binding to the promoter region in front of the epiABCD transcriptional unit. As a cognate histidine kinase has not been identified in the epidermin gene cluster, it has been proposed that a host-encoded histidine kinase may complete the signal transduction pathway (Peschel et al., 1993). In the lactocin S gene cluster, open reading frame (ORF) 239 has a significant level of identity (22%) to WrbA, which is involved in the regulation of trp operon expression in Escherichia coli (Skaugen et al., 1997). Putative regulators have been identified in the mutacin II (Qi et al., 1999) and cytolysin (Haas and Gilmore, 1999) gene clusters; whether these proteins are involved in regulating the biosynthesis of their respective lantibiotics remains to be verified experimentally.

In this report, the transcriptional analysis of the lacticin 3147 gene cluster is described. The transcription initiation sites and promoters were identified, and Northern analyses established that the lacticin 3147 locus is transcribed as two operons, ltnRIFE and ltnA1A2M1TM2D. Furthermore, we demonstrate that the transcriptional repressor, LtnR, regulates producer immunity to lacticin 3147 by binding to a region that overlaps the Pimm promoter, whereas lacticin 3147 biosynthesis appears to be constitutive.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Transcriptional analysis of the lacticin 3147 biosynthetic and immunity gene clusters

Production of and immunity to lacticin 3147 have been localized to a 12.6 kb region of the 60.2 kb plasmid pMRC01, originally identified in the producer strain L. lactis ssp. lactis DPC3147 (McAuliffe et al., 2000). Ten genes are arranged in two divergent clusters, one of which comprises the genes ltnA1A2M1TM2D and is most probably responsible for lacticin 3147 biosynthesis, whereas the immunity gene cluster contains ltnRIFE. The transcription start sites of the ltnA1 and ltnR promoters were determined by the 5′/3′ rapid amplification of cDNA ends (RACE) method with RNA isolated from L. lactis ssp. lactis DPC3147. Using an ltnA2-specific primer, the start site of the ltnA1 promoter was found to be located at the A residue 23 nucleotides (nt) upstream of the ltnA1 start codon ATG (Fig. 1). This result correlates with the start site identified previously for this promoter (Martinez-Cuesta et al., 2000). The −10 region (TAAAAT) differs from the canonical L. lactis−10 promoter sequence by only one nucleotide (van de Guchte et al., 1992; Jensen and Hammer, 1998), whereas the −35 region (TTGACA) is identical, suggesting that transcription of ltnA1 is likely to be constitutive. For the ltnR promoter, the transcription start site is located at the A residue 17 nucleotides upstream of the TTG ltnR start codon (Fig. 1). In this case, the −10 region (TATAAT) is identical to that of the L. lactis consensus promoter. However, the −35 region (TTTACT) deviates somewhat from the consensus sequence TTGACA, suggesting that Pimm may be a weak promoter or, alternatively, may be regulated.

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Figure 1. Characterization of the lacticin 3147 promoters. Nucleotide sequence of the divergent promoters Pimm and Pbac; their −35 and −10 regions, ribosomal binding sites (RBS) and the start codons for the ltnR and ltnA1 genes are indicated. Transcriptional start sites are also shown as determined by 5′/3′ RACE.

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Transcript size was analysed by Northern hybridization using RNA isolated from four strains: DPC3147, plasmid-free L. lactis MG1363, an MG1363 transconjugant containing pMRC01 and L. lactis MG1363 containing pOM02 (a clone containing the 12.6 kb coding region). All strains were grown in M17 broth containing 0.5% glucose at 30°C. Under these conditions, three distinct transcripts were identified. An ltnA1A2-specific probe hybridized to an abundant mRNA of ≈ 0.75 kb (Fig. 2A); this transcript is larger than the expected 0.4 kb transcript necessary to encompass ltnA1A2. Indeed, the ltnA1A2 transcript extends ≈ 350 bp into the downstream modification gene ltnM1. A triple stem–loop structure, which is likely to serve as a transcription attenuator (ΔG = −15 kcal), can be identified at a location consistent with the size of the abundant transcript (Fig. 2B). The ltnA1A2-specific probe also hybridized to an mRNA transcript of ≈ 9.5 kb, which was detected at extremely low concentrations (Fig. 2A). This transcript was only rarely detected using the ltnA1A2-specific probe and never with a probe specific for the downstream ltnD gene, which suggests that this mRNA is highly unstable. A transcript of this size is large enough to cover all six genes in the lacticin 3147 biosynthetic gene cluster and is evidence of an operon structure. A rho-independent transcription terminator can be identified 9573 nt downstream of the transcription start site (ΔG = −15 kcal; data not shown). In addition, a 2.4 kb transcript corresponding to the immunity gene cluster ltnRIFE was identified using a probe to ltnFE (Fig. 2A), confirming that these genes are also co-transcribed. Once again, a terminator (ΔG = −12 kcal) was identified 2495 nt downstream of the start site. The concentration of ltnRIFE mRNA was also quite low in comparison with the ltnA1A2 transcript. None of the transcripts described was detected in the non-producing, lacticin-sensitive L. lactis MG1363.

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Figure 2. Schematic representation of the lacticin 3147 operons.

A. Northern blot analysis of L. lactis ssp. lactis DPC3147. The 0.75 kb and 9.5 kb transcripts identified using an ltnA1A2-specific probe and the 2.4 kb mRNA detected with an ltnFE-specific probe are shown.

B. Nucleotide sequence of the triple stem–loop structure located within the ltnM1 gene. The free energy (ΔG) of the largest stem–loop was calculated as −15 kcal. Nucleotides are numbered from the transcription start site.

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Role of LtnR in the regulation of lacticin immunity

We have reported previously that the gene product of ltnR shares significant sequence similarity with a number of repressor proteins from the PBSX (Xre) family of transcriptional repressors (McAuliffe et al., 2000a). To confirm that LtnR plays a role in regulation of lacticin immunity (and possibly in biosynthesis), we created a version of the lacticin 3147 operon with a truncated ltnR gene (described in Experimental procedures). After the introduction of the resultant plasmid, pOM34, into L. lactis MG1363, the phenotype of a number of independent clones was examined. The ltnR mutant exhibited significantly more immunity to concentrated lacticin 3147 than the strain containing pOM02. An inhibition zone of 4 mm was observed against MG1363.pOM02 when assaying with a concentrated preparation of lacticin 3147 (160 000 AU ml−1); in contrast, no zone of inhibition was observed against the ltnR mutant, MG1363.pOM34 (Fig. 3). Interestingly, production of lacticin 3147 by the mutant was reduced by ≈ 50% when compared with the levels produced by MG1363.pOM02 (data not shown). This suggests that LtnR represses the ltnRIFE immunity operon, as cells that lack a functional LtnR become more immune to the inhibitory action of lacticin 3147. This is consistent with our previous results, in which we have shown that overproduction of LtnI can lead to a hyperimmunity phenotype (McAuliffe et al., 2000a).

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Figure 3. Inhibitory action of concentrated lacticin 3147 (160 000 AU ml−1) against the indicators, L. lactis MG1363.pOM34 and L. lactis MG1363.pOM02, indicating the hyperimmune phenotype of L. lactis MG1363.pOM34. Heat-treated bacteriocin (50 µl) was added to each well.

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Overexpression of ltnR results in an increase in sensitivity to lacticin 3147

Given that eliminating ltnR led to hyperimmunity, it could be expected that overexpressing the gene would have the opposite effect, i.e. increased sensitivity. To test this hypothesis further, a two-step strategy was devised. First, a DNA fragment corresponding to the ltnR gene was amplified by polymerase chain reaction (PCR) and cloned into the nisin-inducible expression vector, pNZ8048, in order to place LtnR production under the control of the nisin expression system (de Ruyter et al., 1996b). The resultant plasmid, pTOK01, was electroporated into the nisin expression host, L. lactis NZ9800. To complete the strategy, pMRC01 was introduced to L. lactis NZ9800.pTOK01 in a conjugal mating using L. lactis MG1363.pMRC01 as the donor, with both lacticin 3147 and chloramphenicol as selective agents. Putative transconjugants, which contain all the lacticin 3147 machinery and the ltnR gene under nisin control, were confirmed by plasmid isolation. The level of immunity exhibited by the resultant strain, grown in various concentrations of purified nisin, was assessed by means of the agar well diffusion assay. It was observed that, when L. lactis NZ9800.pTOK01.pMRC01 was used as the sensitive indicator, the addition of purified nisin to the wells, at levels below the minimum inhibitory concentration (MIC) for these strains, resulted in zones of inhibition that decreased as the concentration of nisin decreased (Fig. 4A). As these zones were not detected in control assays (i.e. cultures without nisin added or in assays with the control culture L. lactis NZ9800.pNZ8048.pMRC01), we concluded that perhaps nisin-induced overexpression of ltnR resulted in cells adjacent to the wells becoming sensitive to the lacticin 3147 produced by pMRC01. This finding was substantiated by following the growth of these cultures in the presence of nisin; it was found that, at a nisin concentration of 2.5 ng ml−1, the growth of L. lactis NZ9800.pTOK01.pMRC01 was significantly slower than that of the same culture grown in the absence of nisin (Fig. 4B), which grew at the same rate as L. lactis NZ9800.pNZ8048.pMRC01 grown in both the presence and the absence of nisin (data not shown). To ensure that this slow-down effect resulted from an increase in sensitivity to lacticin 3147 and not solely from overexpression of ltnR, L. lactis NZ9800.pTOK01 was included as a control; this strain is not capable of producing lacticin 3147. The growth of this culture was not affected by ltnR overexpression at the same concentration of nisin (data not shown). It was concluded from these results that overexpression of ltnR results in cells that are more sensitive to lacticin 3147. Thus, we can conclude that LtnR functions to limit the amount of R itself but, more importantly, the immunity proteins LtnI, F and E. Furthermore, we also observed that the level of active lacticin 3147 produced was significantly reduced upon overexpression of LtnR, similar to that observed on deletion of ltnR.

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Figure 4. A. Inhibitory action of 1 mg ml−1 purified nisin A (left well) and a fivefold serial dilution of the same preparation (concentration decreasing from left to right) against the indicator L. lactis NZ9800.pTOK01.pMRC01.

B. Growth profile of L. lactis NZ9800.pTOK01.pMRC01 in GM17 (□) and in GM17 containing 2.5 ng ml−1 purified nisin A (◆). At the times indicated, samples were taken to determine the absorbance at OD600. These data are representative of repeated experiments.

The Pimm promoter is repressed by LtnR at the transcriptional level

The promoters of the lacticin 3147 gene clusters, ltnRIFE and ltnA1A2M1TM2D, were characterized by transcriptional fusions to the promoterless β-gal gene (lacLlacM) in pAK80. As the promoters are located within the non-coding region between ltnR and ltnA1 (Fig. 1), a DNA fragment corresponding to this intergenic region was cloned into pAK80 in both orientations. The plasmid containing the ltnA1 promoter (designated Pbac) fused to the β-gal gene was designated pOM52, and that with the ltnR promoter (designated Pimm) was designated pOM56 (Fig. 5). After electroporation into L. lactis MG1363, growth of transformants was accompanied in both cases by the development of a deep blue colour on GM17 plates containing Xgal, indicating β-gal activity and, therefore, the presence of active promoters. To quantify the levels of β-gal activity, Miller assays were performed on liquid cultures of both strains according to the method of Israelsen et al. (1995). The levels of β-gal were in the same range for both cultures, with high levels of expression from both promoters (Fig. 5). Thus, in the absence of any regulatory factors, both promoters are strongly and constitutively expressed.

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Figure 5. β-Gal activities of L. lactis MG1363 cells containing the indicated plasmids.

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A similar experiment was performed to establish the role of ltnR in the control of expression from these promoters. β-Gal fusions were again made with both the Pbac and the Pimm promoters but, on this occasion, the ltnR gene was also included (Fig. 5). Construct pOM53, i.e. Pbac promoter fused to β-gal plus ltnR, produced levels of β-gal activity comparable with those for this promoter in the absence of ltnR (Fig. 5). These results confirm that LtnR does not play a role in the regulation of Pbac and, consequently, does not control transcription of the biosynthetic genes. In contrast, when the Pimm promoter was cloned in pAK80 (pOM55) in the presence of the ltnR gene, the levels of β-gal expressed were reduced to ≈ 10% of that observed for Pimm without ltnR (Fig. 5), i.e. 90 Miller units compared with 805 Miller units. This verifies the previous proposal that LtnR autoregulates its own expression and that of the downstream immunity genes through repression of transcription from the Pimm promoter.

Gel mobility shift experiments

Characterization of the β-gal fusions demonstrated that LtnR negatively regulates the Pimm promoter at the transcriptional level. By analogy with the Xre system, we have proposed previously that a possible operator site for this repressor is the intergenic region between ltnR and ltnA1, where the promoters for these genes have now been identified (Fig. 1). The probe and crude protein extracts were prepared as described in Experimental procedures. The labelled probe representing the intergenic region was then incubated with protein extract from L. lactis NZ9800.pTOK01 induced with nisin to overexpress LtnR and from an uninduced culture. Gel shifts were observed only in the presence of protein extract from the induced strain and not in the presence of the protein extract from the uninduced strain (Fig. 6). When a non-specific DNA probe of approximately the same size was incorporated into the binding reaction mixture as a control, no retardation was observed. This experiment confirms that LtnR binds to this region in a specific manner, possibly at the operator site that overlaps the Pimm promoter (Fig. 7). Significantly, the proposed operator bears similarity at the sequence level to the consensus operator defined for the Xre repressor (Fig. 7).

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Figure 6. Autoradiograph of gel mobility shift analysis of the DNA fragment indicated with protein extracts with and without LtnR. Lane 1, DNA fragment without protein extract; lane 2, protein extract from nisin-induced L. lactis NZ9800.pTOK01; lane 3, protein extract from nisin-induced L. lactis NZ9800.pNZ8048; lane 4, protein extract from uninduced L. lactis NZ9800.pTOK01.

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Figure 7. Nucleotide sequence comparison of the potential LtnR operator site with the Xre consensus operator.

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The transcription attenuator identified in ltnM1 allows limited readthrough to the biosynthetic genes

The transcriptional analysis of the lacticin 3147 gene locus revealed that the ltnA1A2 transcript of ≈ 0.75 kb extends into the ltnM1 gene. To determine whether the triple stem–loop structure located in ltnM1 (Fig. 2B) acts as a stop signal for the termination of this abundant mRNA, a β-gal transcriptional fusion was constructed containing the Pbac promoter and extending into ltnM1 beyond this inverted repeat. The resultant plasmid, pOM57, was transformed into L. lactis MG1363 and gave rise to colonies that were very pale blue on Xgal plates after prolonged storage. When the β-gal activity in liquid cultures was assessed by triplicate Miller assay, it was observed that this fusion gave rise to ≈ 25% of the β-gal levels of the ltnA1 promoter (a representative set of data is presented in Fig. 5), suggesting that this repeat acts as an attenuator, allowing partial readthrough from the Pbac promoter to the downstream biosynthetic genes.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

There appears to be a high level of diversity in the regulation of lantibiotic biosynthesis and immunity. Although genes encoding two-component regulatory systems (LanRK) have been identified in the nisin and subtilin gene clusters (Kuipers et al., 1993; van der Meer et al., 1993; Klein and Entian, 1994), only a response regulator homologue, EpiQ, seems to be required for the regulation of epidermin biosynthesis (Peschel et al., 1993). In the case of nisin, it is mature nisin itself that acts as the extracellular signal in a three-component regulatory pathway (Kuipers et al., 1995). Genes encoding such a signal transduction mechanism have not been identified in the lacticin 3147 gene cluster. The only candidate regulator identified thus far is LtnR, a protein with a high degree of homology to the PBSX (Xre) family of transcriptional repressors (McAuliffe et al., 2000a). It has been reported that, at high concentrations, Xre is known to autoregulate its own production (Wood et al., 1990; McDonnell and McConnell, 1994). By comparison with this system, we proposed previously that LtnR may repress the divergently transcribed biosynthetic and immunity genes of the lacticin 3147 system (McAuliffe et al., 2000a). In this study, we have verified that LtnR is indeed responsible for the repression of its own transcription and that of the downstream immunity genes. Regulation of expression from Pimm is achieved through binding of LtnR to the intergenic region encompassing Pbac and Pimm, where an operator site with similarity to the consensus Xre operator most probably serves as a specific binding site. Thus, once a certain concentration of LtnR and, consequently, LtnIFE has accumulated within the cell, expression of ltnRIFE is repressed to ≈ 10% of the levels observed in the absence of regulation. Subsequently, once LtnR becomes limiting as a result of cell growth and the cell risks becoming sensitive to lacticin 3147 because of low levels of LtnIFE, the repression is lifted, and more LtnRIFE is produced. Thus, a steady-state immunity is maintained by the level of LtnR within the cell. Furthermore, we have established that biosynthesis of lacticin 3147 is not regulated by LtnR. However, although expression from Pbac appears to be constitutive, an intragenic rho-independent attenuator in ltnM1 most probably controls the level of transcription of downstream genes. This transcriptional organization ensures a high level of transcription of the ltnA1A2 mRNA in comparison with the mRNA encoding the biosynthetic genes, resulting in the maintenance of the correct stoichiometry between the structural peptides and the biosynthetic machinery.

Northern blot analyses using probes specific for ltnA1A2 and ltnFE revealed that the lacticin 3147 biosynthesis and immunity locus consists of two operons: the ltnRIFE and the ltnA1A2M1TM2D operons. The mapping of the Pimm promoter and the size of the transcript detected using the ltnFE probe showed that ltnRIFE form one operon of 2.4 kb, confirming that the transcription terminator downstream of ltnE is functional. However, the majority of transcripts initiated at the Pbac promoter presumably terminate at the stem–loop structure within the ltnM1 gene. This structure most probably acts as a rho-independent attenuator that functions as the signal for the internal processing of ltnA1A2M1TM2D transcript. This may also stabilize the ltnA1A2 mRNA by protection against ribonuclease digestion (Belasco and Higgins, 1988; Peterson, 1992) and facilitate the translation of the structural peptides. Characterization of a β-gal fusion beyond this putative attenuator indicated that ≈ 25% of transcripts are not terminated at this stop signal. However, the amount of transcript for the biosynthetic genes detected by Northern analyses seemed to be significantly less; this is presumably because of the instability of such a large transcript. A weak intergenic stem–loop structure between the structural gene (lanA) and the downstream biosynthetic genes is highly conserved among the lantibiotic operons characterized thus far, e.g. nisin (Kuipers et al., 1993), Pep5 (Pag et al., 1999), mutacin II (Qi et al., 1999) and the type B lantibiotic, mersacidin (Altena et al.. 2000). The structure generally acts as a transcriptional terminator for the differential expression of structural gene and accessory gene mRNAs. A stem–loop structure identified in the Pep5 system downstream of pepA was shown to act as a stabilizing element for the pepIpepA transcript (Pag et al., 1999); in the absence of this secondary structure, the mRNA produced is highly unstable. This regulating element also allows partial readthrough to the biosynthetic genes downstream (Pag et al., 1999). To our knowledge, the stem–loop structure identified in ltnM1 of the lacticin 3147 system is the only reported case of an intragenic transcriptional attenuator among lantibiotic operons. It is tempting to speculate that this positioning of the putative attenuator within an essential gene for lacticin 3147 production (McAuliffe et al., 2000b) provides an added ‘built-in’ protection, which ensures that the attenuator structure, and thus the stoichiometry between transcripts, remains unaltered.

Previously, we reported that the inactivation of ltnR in a construct containing ltnRI under the control of the native Pimm promoter (pOM23) did not affect the level of immunity conferred, when compared with the unmutated plasmid (pOM14; McAuliffe et al., 2000a). In contrast, we observed a hyperimmune phenotype on the deletion of ltnR from pOM02; this result makes sense in view of the fact that ltnR represses the expression of the downstream immunity genes. This phenotype may not have been observed previously (McAuliffe et al., 2000a), as the ltnFE genes were missing from the pOM23 construct; it is possible that the ltnFE gene products are involved in immunity, but this remains to be investigated. Furthermore, the hyperimmune phenotype detected on the deletion of ltnR correlated with the observation that overexpression of ltnR in nisin-induced cells resulted in substantial growth rate decreases resulting from a heightened sensitivity to lacticin 3147. The most likely explanation for this phenotype is that, in the presence of excess LtnR, transcription of the ltnRIFE operon is repressed before the cells have accumulated sufficient immunity machinery; as a consequence, the cells cannot protect themselves from the inhibitory action of their own bacteriocin. The role of LtnR in the repression of ltnRIFE is also evident from the β-gal activities demonstrated in L. lactis MG1363 cells harbouring pOM55; gel mobility shift analysis verified that this repression is effected by the interaction between LtnR and the intergenic promoter region. These data clearly demonstrate the involvement of LtnR in transcriptional repression of the Pimm promoter.

An interesting anomaly arising from the experiments outlined here is that, on both deletion and overexpression of ltnR, the levels of lacticin 3147 detected in culture supernatants were significantly reduced. On phenotypic examination of the ltnR deletion mutant, MG1363.pOM34, lacticin 3147 was detected at ≈ 50% of the levels observed in the parent strain, MG1363.pOM02 (data not shown). Although we have no direct evidence to support it, one possibility is that a proportion of the lacticin 3147 produced may bind to the excess LtnI and thus be unavailable to inhibit the target organism. Similarly, on overexpression of ltnR, an obvious reduction in lacticin 3147 production was observed. In this case, however, it is most probably caused by the decreased cell numbers exhibited by the strain in which ltnR was overexpressed as a result of inhibition by lacticin 3147.

Negative regulation of gene expression through a repressor protein has not been reported to date in lantibiotic gene clusters. A protein with sequence similarity to HipB, a repressor involved in peptidoglycan synthesis (Black et al., 1994), has been identified in the region preceding the cytolysin gene cluster (Haas and Gilmore, 1999), but a role for this regulator in cytolysin production and/or immunity has not been demonstrated. We have shown that lacticin 3147 uses a regulatory system that has not been identified previously in lantibiotic systems, in which lacticin 3147 immunity is repressed at the level of transcription by LtnR, whereas the structural peptides are produced constitutively, and the level of biosynthetic machinery is controlled by an intragenic transcription attenuator. In a variety of organisms, back-to-back promoter configurations have been identified (Beck and Warren, 1988), in which a regulatory molecule acts within the divergent transcription unit to control transcription of the structural genes and also often regulates its own synthesis, e.g. the lac operon of L. lactis (van Rooijen and de Vos, 1990; van Rooijen et al., 1992). The divergent promoters identified in the lacticin 3147 system differ somewhat in that, although transcription of ltnR and the downstream genes is regulated by LtnR, expression of the biosynthetic gene cluster is not under LtnR control. The observed organization has the advantage that the cell can control the synthesis of the immunity machinery when sufficient amounts are present, thus conserving energy for lacticin 3147 production.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial strains, plasmids and media

The lacticin 3147-producing strain L. lactis ssp. lactis DPC3147 (Ryan et al., 1996), the indicator organism L. lactis ssp. cremoris HP and derivatives of L. lactis MG1363 (Gasson, 1983) and NZ9800 (de Ruyter et al., 1996b) were routinely grown at 30°C without aeration in M17 broth (Oxoid) supplemented with 0.5% glucose (GM17). E. coli DH5α (Life Technologies) was used as an intermediate host for cloning. This strain was grown at 37°C in LB broth (Sambrook et al., 1989) with vigorous agitation. Plasmids used and created in this study are listed in Table 1. Antibiotics were used, where indicated, at the following concentrations: erythromycin, 200 µg ml−1 for E. coli and 1 µg ml−1 for L. lactis; chloramphenicol, 20 µg ml−1 for E. coli and 5 µg ml−1 for L. lactis. Xgal was used at a concentration of 40 µg ml−1 for E. coli and 160 µg ml−1 for L. lactis. The chemical reagents listed were purchased from Sigma Chemical.

Table 1. Plasmids used in this study.
PlasmidRelevant propertiesaReference
  1. a . Cm R, chloramphenicol resistance; EmR, erythromycin resistance; Lcn+, lacticin 3147 production; Imm+, immunity to lacticin 3147.

pCI372 E. coli/L. lactis shuttle vector, CmR Hayes et al. (1990)
pNZ8048Nisin-inducible expression vector, CmR de Ruyter et al. (1996b)
pAK80Promoter selection vector containing promoterless β-gal gene, EmR Israelsen et al. (1995)
pMRC01Ltn+, Imm+ from L. lactis DPC3147 Ryan et al. (1996)
pOM02Lacticin-coding region in pCI372 McAuliffe et al. (2000a)
pOM34pOM02 ΔltnRThis study
pTOK01pNZ8048 containing ltnRThis study
pOM52pAK80 containing a 400 bp PCR-generated fragment of the Pimm and Pbac promoters with the Pbac promoter fused to the β-gal geneThis study
pOM53pAK80 containing a 700 bp PCR-generated fragment of the Pimm and Pbac promoters + ltnR with the Pbac promoter fused to the β-gal geneThis study
pOM55pAK80 containing the above 700 bp fragment with the Pimm promoter fused to the β-gal geneThis study
pOM56pAK80 containing the above 400 bp fragment with the Pimm promoter fused to the β-gal geneThis study
pOM57pAK80 containing a 1.25 kb fragment of both promoters extending beyond the terminator in ltnM1This study

General methods

Plasmid DNA was isolated from E. coli strains using the Qiagen column purification kit and from L. lactis using the method described by Anderson and McKay (1983). pMRC01 DNA (Ryan et al., 1996), which was used as template DNA in PCR, was purified by equilibrium centrifugation in CsCl–ethidium bromide gradients (Sambrook et al., 1989). L. lactis and E. coli were transformed by electroporation with a Gene-Pulser apparatus (Bio-Rad Laboratories) as described by Wells et al. (1993) and Sambrook et al. (1989) respectively. PCR was performed according to standard procedures using BioTaq DNA polymerase (Bioline). Thirty cycles were performed with the following conditions: 30 s at 90°C, 1 min at 50°C and 1 min at 72°C. The Expand High Fidelity PCR system (Roche Diagnostics) was used to generate PCR products for cloning purposes. Where longer PCR products were sought, the Expand Long Template PCR system from Roche Diagnostics was used according to the manufacturer's instructions. Vent DNA polymerase (New England Biolabs) was used to generate the probe used in gel mobility shift assays. Primers for PCR were synthesized using an Applied Biosystems PCR-MATE DNA synthesizer. Restriction digestion, Klenow treatment and DNA ligations were performed according to standard procedures (Sambrook et al., 1989). Restriction enzymes, the Klenow fragment of the E. coli DNA polymerase I and T4 DNA ligase were purchased from New England Biolabs. Restriction endonuclease-digested DNA was eluted from agarose gels using a Gene-Clean II kit from Bio101.

RNA isolation

Cultures (6 ml) were grown in GM17 broth to the late exponential growth phase and harvested by centrifugation. The cell pellets were resuspended in 500 µl of ice-cold lysis buffer (25 mM sodium acetate, pH 5.2, 1% SDS). Aliquots of 500 µl of 70°C preheated acid phenol–chloroform (1:1) and 200 µl of glass beads (Sigma Chemical) were subsequently added, and the cells were incubated at 70°C for 10 min with repeated vortexing. The samples were centrifuged to separate the aqueous phase, and the extraction was repeated with 500 µl of acid phenol–chloroform. RNA was precipitated with 1 ml of absolute ethanol (−20°C), washed with 70% ethanol, dried and resuspended in 50 µl of diethylpyrocarbonate (DEPC)-treated water containing 10 U of RNase inhibitor (Roche Diagnostics) and RNase-free DNase I (Roche Diagnostics). After 20 min at room temperature, the RNA was re-extracted with phenol–chloroform, precipitated and resuspended in 6 µl of DEPC-treated water.

Northern analysis

For Northern hybridization, ≈ 10 µg of RNA per sample was added to 15 µl of sample buffer, heated to 65°C for 15 min and loaded on a 1.2% agarose gel containing formaldehyde. A 0.24–9.5 kb RNA ladder (Life Technologies) was used to determine the transcript size. After separation at 50 V, the RNA was transferred onto Hybond N+ membrane (Amersham Pharmacia Biotech UK) using 10 mM NaOH, and probe labelling, hybridization and detection were performed with the ECL nucleic acid detection kit (Amersham Pharmacia Biotech UK).

Determination of transcription start sites

The 5′ ends of the lacticin 3147 transcripts were determined with the 5′/3′ RACE kit (Roche Diagnostics) according to the manufacturer's instructions, using the ltnA2-specific primer 5434 and the ltnR-specific primer 5147. The sequences of these primers are presented in Table 2. DNA sequence determination was performed by MWG-Biotech.

Table 2. Primers used in this study.
Primer name/no.aSequence (5′−3′)bTemperature (°C)
  • a

    . F, forward; R, reverse.

  • b

    . Restriction sites in primer sequences are underlined.

5088t t g c t g g t g t t g t t c56
5089t g a a a c a c a a c c a g t52
ltnF (F)a t g t c g a c t t t g a t t c a g t g a t t t a t a58
ltnF (R)a c a a a g c t t a t a t a t t c a t a g c a g t a g a58
ltnD (F)a t g g a t c c a t c a g t a g g t a g a a c t a61
ltnD (R)g c t g t c g a c a a a t t a a c c a c c c a a a62
5434c a g c t a a t t c a a t c a t a t c51
5147c a a c a t c a a a a a a g t42
ltnR (F)t a t a c c a t g g c a t a g a t a g g a g g a a a a t g63
ltnR (R)t a g a c t g c a g a a a a g g t a t t g a t a t t t t58
5423a a g a g c t c t t a t a t a c a g a g t t a c58
5424c t g g t t g t g t g t t t c a a t t t c a t t57
3951(b)a a a g a g c t c t g c g a a t a a c a t c a a g g g a a64
5557a t g g t a c c c t a t a c a c c t t c t t t59
5558a g g t a c c c a g a g t t a c t a a t a g a a59
5146g c t g t c g a c t t a a g t a t a g g g c a a t62
6737a g g a t c c t t a t a t a c a g a g t t a c t a58
6738a g g a t c c a t t t c a t t t t t g t t c a t56
6739a g g a t c c t a a g c c t a a a a g t a t a t57
Bgl2(400)t t t a g a t c t a t t t c a t t t t t g t t c a t53
Pst1(400)t t t a g a t c t t t a t a t a c a g a g t t g c t a57
Pst1(700)a a a c t g c a g t a a g c c t a a a a g t a t a t58
Bgl2(1000)t t t a g a t c t t t a t a t a c a g a g t t g c t a57
Pst1(1000)t t t c t g c a g c a g g a t a t t c a t c t g a61

Plasmid construction and mutagenesis

Plasmid pTOK01 was constructed by the generation of a 297 bp fragment corresponding to ltnR, using the ltnR primer set, followed by ligation to the nisin-inducible expression vector pNZ8048. Also, plasmids pOM52–57 were constructed by PCR amplification of the appropriate fragment followed by ligation to the promoter probe vector pAK80. The plasmid pOM34, i.e. pOM02 ΔltnR, was constructed by amplifying a 2.4 kb fragment using the primer set 3951(b)/5557 and a 10.2 kb fragment using the primer set 5558/5146, which correspond to the lacticin 3147 operon with a deletion in ltnR. These fragments were then sequentially cloned into the E. coli/L. lactis shuttle vector pCI372 to create the plasmid pOM34. Primer sequences are presented in Table 2.

Gel mobility shift analysis

For the preparation of crude protein extracts, cells were grown in 20 ml of GM17 broth. ltnR expression was induced with purified nisin A; 10 ng ml−1 was used for preinduction and 300 ng ml−1 for induction. Cells were resuspended in 500 µl of buffer (20 mM Tris, pH 7.0) and ruptured in a bead beater. Samples were centrifuged at 15 000 g for 5 min in a bench top centrifuge to remove cell debris. Protein concentrations of the sample supernatants were determined using the Bio-Rad protein assay. A 417 bp DNA fragment, corresponding to the non-coding region between ltnR and ltnA1, was amplified by PCR using primers 5423 and 5424 (Table 2). This fragment was digested with SacI, treated with alkaline phosphatase and end-labelled with 32P by a T4 polynucleotide kinase reaction. Appropriate amounts of crude protein extract were incubated with calf thymus DNA and 0.3 ng of labelled DNA in binding buffer [50 mM Tris, pH 8.0, 5 mM MgCl2, 500 mM KCl, 2 mM dithiothreitol, 50 µg ml−1 BSA, 75 µg ml−1 poly-(dI–dC), 10% (v/v) glycerol, 1 mM EDTA] in a volume of 40 µl at room temperature for 10 min. Reactions were stopped on the addition of 5 µl of 50% glycerol. Samples were applied to a 4% polyacrylamide gel containing 2.5% glycerol. Gels were run in TAE buffer (0.04 M Tris-acetate, pH 7.5, 2 mM EDTA) at 120 V for 4 h, dried and exposed overnight at −70°C to X-Omat film (Kodak).

Conjugations

Conjugal matings were set up using L. lactis MG1363 containing pMRC01 (Ltn+, Imm+) as the donor strain and L. lactis NZ9800 containing either pNZ8048 or pTOK01 (Ltn, Imm, CmR) as the recipient. Both strains were grown to mid-log phase (OD600 of 0.5–1). Aliquots (1 ml) of these cultures were harvested, and the resultant pellets were washed once in GM17. The pellets thus obtained were resuspended in 25 µl of GM17, mixed and spotted on a non-selective GM17 agar plate. Donor and recipient controls were prepared in the same manner. After overnight incubation at 30°C, the cultures were resuspended in GM17 broth, diluted and plated on selective agar, i.e. GM17 with 100 AU ml−1 lacticin 3147 and 10 µg ml−1 chloramphenicol. The conjugation frequency was estimated as the number of transconjugants (appearing on selection plates) per number of donor cells. Putative transconjugants were confirmed by plasmid isolation and restriction analysis.

Bacteriocin preparation and assay

Concentrated lacticin 3147 was prepared as described previously (McAuliffe et al., 1999) from the supernatant of L. lactis ssp. lactis DPC3147, and the activity was determined by critical dilution assay (Ryan et al., 1996). Basically, molten agar was cooled to 48°C and seeded with the indicator strain L. lactis ssp. cremoris HP (≈ 2 × 107 fresh overnight-grown cells). The inoculated medium was dispensed into sterile Petri plates, allowed to solidify and dried. Wells (≈ 4.6 mm diameter) were made in the seeded agar plates. Aliquots of a twofold serial dilution of the bacteriocin preparation were dispensed into wells, and the plates were incubated overnight at 30°C. The arbitrary units (AU ml−1) were determined as described by Ryan et al. (1996).

Measurement of β-galactosidase activity in liquid cultures

β-Galactosidase activity was measured as described by Israelsen et al. (1995).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The authors wish to thank Eric Johansen for providing the promoter probe vector pAK80. This work was funded by Bioresearch Ireland and the Non-Commissioned Food Research Programme, operated by the Irish Department of Agriculture, Food and Forestry and supported by national and European funds.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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Footnotes
  1. Present address: Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 0JG, UK.