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Applied and Environmental Microbiology, November 2002, p. 5508-5516, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5508-5516.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Cadmium Resistance in Streptococcus thermophilus Strain 4134: an Example of Lateral Gene Transfer

Jan Schirawski,^1* Werner Hagens,¹ Gerald F. Fitzgerald,^1,2,3 and Douwe van Sinderen^1,2

National Food Biotechnology Centre,¹ Departments of Microbiology,² Food Science, Food Technology and Nutrition, University College Cork, Cork, Ireland³

Received 24 May 2002/ Accepted 22 August 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two genes (cadC_St and cadA_St [subscript St represents Streptococcus thermophilus]), located on the chromosome of S. thermophilus 4134, were shown to constitute a cadmium/zinc resistance cassette. The genes seem to be organized in an operon, and their transcription is cadmium dependent in vivo. The proposed product of the cadA open reading frame (CadA_St) is highly similar to P-type cadmium efflux ATPases, whereas the predicted protein encoded by cadC_St (CadC_St) shows high similarity to ArsR-type regulatory proteins. The observed homologies and G+C content of this cassette and surrounding regions suggest that this DNA was derived from Lactococcus lactis and may have been introduced relatively recently into the S. thermophilus 4134 genome by a lateral gene transfer event. The complete cassette confers cadmium and zinc resistance to both S. thermophilus and L. lactis, but expression of cadA_St alone is sufficient to give resistance. By using electrophoretic mobility shift assays it was shown that the CadC_St protein is a DNA binding protein that binds specifically to its own promoter region, possibly to two copies of an inverted repeat, and that this CadC_St-DNA interaction is lost in the presence of cadmium. Using lacZ fusion constructs it was shown that the cadmium-dependent expression of CadA_St is mediated by the negative regulator CadC_St. A model for the regulation of the expression of cadmium resistance in S. thermophilus is discussed.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lactic acid bacteria (LAB) have been used for centuries in food preservation processes and are used with increasing intensity for specific industrial food fermentation processes. The efforts to create functional foods that have additional beneficial properties for human health, like increased vitamin or amino acid content, necessitate the development of food-grade marker systems as well as the discovery of regulated promoters that can be used for targeted protein expression during food fermentation. Antibiotic resistance has been rejected as a selectable marker for microorganisms destined for use in food production because of the risk of spreading the antibiotic resistance in the human intestinal microflora and ultimately to pathogenic bacteria. Hence, efforts have focused on the identification of alternative selectable markers such as bacteriocin immunity or heavy metal resistance (15, 30).

The heavy metal cadmium is toxic in its ionized form to many organisms, including microbes and humans. It is believed to enter the bacterial cell via transport systems normally used for essential divalent cations and to exert its toxic effect by inhibiting respiration through binding to sulfhydryl groups of essential proteins (39). Bacteria carrying cadmium resistance mechanisms have a selective advantage for survival in the environment (17). Unlike resistance systems to mercury and arsenic, which are highly homologous in all bacteria studied, cadmium resistance seems to have evolved at least three times, having given rise to the ATP-dependent cadmium efflux transporters found in gram-positive bacteria, the unrelated chemiosmotic cation-proton antiporters present in gram-negative bacteria, and the metallothionein system used by cyanobacteria (34).

The cadmium resistance system found in gram-positive bacteria is encoded by two adjacent genes, cadA and cadC, and was first identified in Staphylococcus aureus, the only organism in which the function of the two genes was studied in detail (10, 27, 28). Since then, homologues of these genes have been identified in many other bacterial species, e.g., Bacillus firmus, Listeria monocytogenes, Lactococcus lactis, Listeria innocua, Stenotrophomonas maltophilia, and Geobacillus stearothermophilus (1, 12, 14, 18, 20, 25a). However, only for a few organisms (e.g., S. aureus, L. monocytogenes, and L. lactis) were they actually shown to confer cadmium resistance (18, 20, 28).

The cadmium resistance system of S. aureus is specified by two adjacent genes, cadA and cadC, both of which are necessary for cadmium resistance in vivo (42). The CadA protein has been shown to be a cadmium efflux ATPase of the P type, i.e., a transport ATPase that has a covalent phospho-protein intermediate (38). CadC was identified by in vitro assays as a trans-acting DNA binding negative regulatory protein, whose DNA binding capacity is diminished in vitro in the presence of cadmium (10). The binding properties of CadC from S. aureus on the cadA operator/promoter DNA region were investigated by DNase I footprinting experiments, which showed that the recognition site of CadC contains an inverted repeat sequence (10).

Genes highly homologous to the cadA and cadC genes of S. aureus and L. monocytogenes have been identified on plasmids of L. lactis (20, 30), but gene regulation or the mechanism of their action has never been investigated. This may be of interest in view of recent reports, which showed that the cadmium resistance cassette can be used as a selectable marker in Lactococcus (30) and that it can be transferred into industrially relevant L. lactis strains (37).

Thermophilic starter strains are used for the production of certain fermented milk products. Streptococcus thermophilus is an important species of LAB in the dairy industry, mainly used as a starter culture component in yogurt fermentation and cheese making (11). It is also present as a major microbial component in combination with other LAB species in natural thermophilic starters used for the production of certain cheeses (3). This paper reports the identification of a cadmium resistance cassette on the genome of an industrial S. thermophilus strain. We demonstrate the functionality of this cassette in two LAB, S. thermophilus and L. lactis, and present a model for the regulation of the expression of cadmium resistance in LAB. Throughout the text the expression "cadmium" or "zinc" refers to cadmium or zinc in its ionized form (Cd²⁺ or Zn²⁺).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, and growth conditions.
S. thermophilus strains, which are or have been used in industrial fermentations, were taken from the University College Cork collection, Cork, Ireland (strains 4035, 4038, 4039, 4044, 4116, 4134, 4141, 90461, 90726, 90728, and 90731), or were provided by Nestlé, Lausanne, Switzerland (ST11 [24]). L. lactis strains NZ9800 (8) and 302 (19) and Escherichia coli JM101 (New England Biolabs) were also used. Bacterial plasmids used in this study are listed in Table 1. S. thermophilus strains were grown in Elliker medium (Difco) supplemented with 10 g of beef extract and 9.5 g of ß-glycerophosphate per liter at 37°C. L. lactis strains were grown in M17 broth (Oxoid) supplemented with 5 g of glucose per liter (GM17) at 30°C. E. coli strains were grown in Luria-Bertani broth as described earlier (33) at 37°C. Erythromycin and chloramphenicol were used at 100 and 10 µg/ml, respectively, for E. coli or at 5 µg/ml for each of these antibiotics in S. thermophilus and L. lactis. If simultaneous selection for two antibiotics was desired, the final concentration of each of the antibiotics was 2.5 µg/ml. Nisin was used at a final concentration of 2 nM.

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TABLE 1. Plasmids used in this study

Plasmid DNA isolation, transformation, and cloning strategies.
Plasmid DNA was isolated from E. coli and S. thermophilus using the Wizard Plus Miniprep Kit (Promega) following the manufacturer's instructions, except that, for isolation of plasmid DNA from S. thermophilus, an incubation step at 37°C for 30 min in the presence of 30 mg of lysozyme/ml in the "cell resuspension solution" was applied prior to the standard isolation procedure. Plasmid DNA was isolated from L. lactis using a previously described method (29). Electrotransformation of E. coli (33), S. thermophilus (31), or L. lactis (41) was performed as previously described, except that for the latter GM17 broth supplemented with 2% (wt/vol) glycine was used as growth medium. Restriction enzymes and T4 DNA ligase were purchased from Roche Molecular Biochemicals and were used according to the manufacturer's instructions. PCR products were amplified using either the Expand Long Template PCR System (Roche Molecular Biochemicals) or Taq PCR Mastermix (QIAgen) with a Gene Amp PCR System 2400 Thermal Cycler (Perkin-Elmer Cetus, Norwalk, Conn.). Primers used in this study are listed in Table 2. The cadC_St (subscript St represents S. thermophilus) and/or cadA_St gene was amplified by PCR from the genome of S. thermophilus strain 4134 (Table 2). Appropriate restriction enzyme sites were incorporated in the PCR primers to facilitate cloning of the cadA_St gene on its own or the cadCA_St genes, including the assumed promoter region, downstream of the nisin-inducible promoter P_nisA of pNZ8048 (8) to generate pNZcadA or pNZcadCA, respectively. The cadC_St gene was cloned downstream of the constitutive promoter P₄₄ of pNZ44 (22) to generate p44cadC. To measure in vivo expression of cadC_St and cadA_St, two different translational fusions to the E. coli lacZ gene on plasmid pIR12 (25) were constructed. To generate a cadC_St-lacZ fusion, the assumed cadC_St promoter region and the first nine codons of the cadC_St gene were amplified by PCR (Table 2) and were introduced into pIR12 to produce plasmid pIRcadC. For the construction of a cadA_St-lacZ fusion plasmid, the cadC_St promoter region, the complete cadC_St gene, and the first nine codons of the cadA_St gene were amplified by PCR (Table 2) and were introduced into pIR12 to produce pIRcadCA. All plasmid constructions involving PCR were verified by DNA sequencing of relevant regions.

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TABLE 2. Primers used in this study

ß-Galactosidase assays.
Cells were grown in GM17 containing the appropriate antibiotics at 30°C to an optical density at 600 nm of 0.2 to 0.3. The culture was halved, and to one half of it CdCl₂ was added to a final concentration of 5 µM. Cells were grown for an additional 4 h, after which 4 ml was collected by centrifugation and resuspended in 1 ml of cold Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, and 1 mM MgSO₄, pH 7 [23]). Cells were then disrupted using a bead beater in the presence of 200 µl of glass beads (0.1-mm diameter). Glass beads and cell debris were removed by centrifugation at 14,000 rpm for 10 min in a tabletop centrifuge. ß-Galactosidase activity was measured at 23°C in Z buffer containing 0.8 mg of o-nitrophenyl-ß-D-galactopyranoside/ml. The reaction was stopped by the addition of an equal volume of 0.5 M Na₂CO₃. Specific ß-galactosidase activity was calculated as described previously (23).

Electrophoretic mobility shift assays (EMSA).
For the preparation of cell extracts, an overnight L. lactis cell culture (20 ml) was collected by centrifugation and resuspended in 1 ml of buffer containing 20 mM Tris, pH 8.0, and 1 mM dithiothreitol and was ruptured in a bead beater in the presence of 200 µl of glass beads (0.1-mm diameter). Samples were centrifuged at 14,000 rpm for 5 min in a tabletop centrifuge to remove cell debris. Protein concentrations of the supernatants were determined using the Bio-Rad protein assay kit (Bio-Rad). Gel retardation assays were performed as outlined below. PCR fragments were labeled with polynucleotide kinase using [-³²P]ATP (Amersham). DNA binding assays were carried out in 40-µl reaction volumes containing 50 mM Tris, pH 8.0, 10% (vol/vol) glycerol, 1 mM EDTA, 5 mM MgCl₂, 500 mM KCl, 2 mM dithiothreitol, 50 µg of bovine serum albumin/ml, 50 µg of calf thymus DNA/ml, 1 µl of labeled probe, and 10 µg of total protein of a particular cell lysate. Samples were incubated for 10 min at room temperature and, following the addition of 10 µl of 50% glycerol, were loaded on a 4% polyacrylamide gel containing 2.5% glycerol. Gels were run in Tris-acetate-EDTA buffer (0.04 M Tris-acetate, pH 7.5, and 2 mM EDTA) at 120 V for 3 h, dried, and exposed overnight at -70°C to X-Omat film (Kodak).

Sequence analysis.
DNA sequence determination was executed by MWG-Biotech (Ebersberg, Germany). Sequence assembly and alignments were performed using the DNASTAR 1996 release software package. Analysis of protein sequences for coiled-coil motifs was done using the Network Protein Sequence program @nalysis, found at the website http://npsa-pbil.ibcp.fr. Database searches were performed using the BLASTN or BLASTP program (2). Transcriptional terminator sequences were identified with the program mfold, version 3.1 (21, 43). Sequence data for S. thermophilus strain LMG18311, presented in Fig. 1A, were obtained from the Université catholique de Louvain Life Sciences Institute website at http://www.biol.ucl.ac.be/gene/genome/. (Sequencing of the S. thermophilus strain LMG18311 genome was supported by Walloon Region [BIOVAL grant no. 9813866].)

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FIG. 1. Genomic context of the cadmium resistance cassette of S. thermophilus 4134. (A) Schematic representation of the gene organization of the complete sequenced 15,874-bp fragment from the genome of S. thermophilus 4134 (St4134 [GenBank accession no. AJ315964]) in comparison to the relevant plasmid sequences of pAH82 (AF243383) and pNZ4000 (NC_002137) from L. lactis, pLI100 (NC_003383) from L. innocua, pER35 (NC_000937) from S. thermophilus, and the relevant genome region of S. thermophilus LMG18311 (LMG18311 [http://www.biol.ucl.ac.be/gene/genome/], Genemark prediction of ORFs of contig C112). ORFs are denoted by arrows, while insertion sequence elements are denoted by boxes. DNA sequence similarity to St4134 is indicated by color shading of ORFs, with the exception of LMG18311, where only protein sequences could be compared. Homologous regions are connected by a gray background. The hsdR ORF of St4134 contains an in-frame stop codon (denoted by a black bar within the ORF) and may not be expressed (Schirawski et al., unpublished). The figure is to scale. (B) Detail of the DNA sequence upstream of cadCSt comprising nucleotides -85 to +10 (nucleotides 12140 to 12045 of sequence represented by GenBank accession no. AJ315964). The -10 and -35 promoter regions are underlined, the ribosomal binding site (rbs) is in italics, and the inverted repeats (IR1 and IR2) are boldfaced and denoted with an arrow above the sequence. The start codon of cadCSt is in boldface and is indicated by a bent arrow.

Nucleotide sequence accession number.
The sequence determined from the S. thermophilus strain 4134 genome containing the cadC_St and cadA_St genes (15,874 bp) was deposited in GenBank (accession number AJ315964).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of a cadmium resistance cassette on the chromosome of S. thermophilus strain 4134.
In search of a type I restriction/modification system, a 15,874-bp fragment from the chromosome of S. thermophilus strain 4134 was completely sequenced (Fig. 1A). It contained three open reading frames (ORFs) that putatively code for the three subunits of a type I restriction/modification system (hsdR, hsdM, and hsdS) (J. Schirawski, G. F. Fitzgerald, and D. van Sinderen, unpublished data), a complete copy of the insertion element IS1191 (13), a partial copy each of the insertion elements ICESt1 (6) and IS1295L (GenBank accession no. AJ278471), two partial ORFs, and two ORFs with high sequence similarity to proteins of unknown function derived from lactococcal plasmids (30, 40). Furthermore, an ORF was identified (Fig. 1A) whose putative amino acid sequence of 705 amino acids is identical in sequence to the plasmid-encoded CadA proteins of L. lactis and L. innocua and showed significant similarity to CadA of S. aureus (64% amino acid identity), as well as to those of other bacteria (Table 3). Immediately upstream of this ORF, designated here cadA_St, a second ORF was located on the chromosome of S. thermophilus strain 4134, whose putative amino acid sequence of 129 amino acids is identical in sequence to the AsrR-type regulatory protein CadC from L. lactis and L. innocua and is highly similar to CadC from L. monocytogenes (52% amino acid identity) as well as to those from other bacteria (Table 3). The identified S. thermophilus homologue of CadC was therefore designated CadC_St. Although the cadmium resistance cassette described here has been identified earlier on plasmids of L. lactis and L. innocua, gene regulation or the mechanism of action of the cassette has never been studied.

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TABLE 3. Comparison of the putative CadASt and CadCSt protein sequences of S. thermophilus strain 4134 to the respective CadA and CadC protein sequences from other bacteria

The high sequence homology of the two ORFs cadC_St and cadA_St to their respective ORFs from plasmids identified in L. lactis and L. innocua (Table 3) induced us to investigate whether the sequence similarity would extend beyond the coding regions of these two ORFs. We found that the sequence similarity to the lactococcal plasmid pAH82 (30) extended over a stretch of about 10 kb of the sequenced genomic DNA from S. thermophilus. This homologous DNA region encompassed the three ORFs of the type I restriction/modification system, the cadCA_St genes, and two more ORFs (orf3 and orf4) (Fig. 1A). With the exception of the variable sequences of the hsdS gene (Schirawski et al., unpublished), the 10-kb stretch from S. thermophilus showed >95% DNA sequence homology to the corresponding stretch of the lactococcal plasmid pAH82. Downstream of the cadA gene from pLI100 (12) of L. innocua, an ORF was located that showed 87% DNA sequence homology to orf4, which is located upstream of cadC_St on the S. thermophilus 4134 genome (Fig. 1A). The two ORFs of unknown function (orf3 and orf4) and about 700 bp of upstream sequence also showed high sequence similarity to the lactococcal plasmid pNZ4000 (40) (Fig. 1A), while the three ORFs encoding the type I restriction/modification system showed (with the exception of the variable regions of the hsdS gene) high sequence similarity to the streptococcal plasmid pER35 (35) (Fig. 1A). These data suggest that the cadCA_St genes and surrounding DNA were introduced into the S. thermophilus genome by a lateral gene transfer event.

The potentially plasmid-derived sequences discovered on the genome of S. thermophilus 4134 are flanked by insertion elements previously identified for this organism (6, 13) (GenBank accession no. AJ278471) (Fig. 1A). These elements, in turn, are flanked by two only partially sequenced ORFs that have also been identified during the sequencing project of S. thermophilus strain LMG18311 (http://www.biol.ucl.ac.be/gene/genome/) (Fig. 1A). However, on the LMG18311 genome, these genes are situated within 2 kb of each other, while on the genome of strain 4134 they are separated by about 15 kb of sequence. This suggests that, in the genome of S. thermophilus strain 4134, which has been used in industrial fermentations, a sequence of about 2 kb has been deleted and replaced by insertion elements from S. thermophilus and by sequences derived from one or several plasmids of lactococcal, streptococcal, or listerial origin.

Analysis of the DNA sequence upstream of cadC_St revealed the presence of a typical promoter sequence found in LAB (9, 16), i.e., a -35 (TTGAAT) and an extended -10 region (TGATACAAT) (Fig. 1B). The stop codon of cadC_St partially overlaps the start codon of cadA_St, indicating that the two genes are translationally coupled. Downstream of cadA_St a putative rho-independent transcriptional terminator was identified (not shown). These findings suggest that a cadmium resistance cassette (cadCA_St), consisting of the cadC_St and cadA_St genes, is present on the chromosome of S. thermophilus strain 4134 and that the two genes appear to be organized in an operon.

S. thermophilus strains containing the cadCA_St genes are cadmium resistant.
A number of S. thermophilus strains were tested for their ability to grow in the presence of cadmium. Of 12 different S. thermophilus strains tested, only two (strains 4134 and 4116) were able to grow in the presence of 1 mM CdCl₂, while 10 strains (strains 4035, 4038, 4039, 4044, 4141, 90461, 90726, 90728, 90731, and ST11) proved to be cadmium sensitive (not shown). The cadmium-sensitive S. thermophilus strain 4035 was unable to grow in the presence of 0.25 mM CdCl₂, whereas strain 4134 grew well in the presence of up to 1.6 mM CdCl₂ (not shown). The strains were tested for presence of the cadCA_St cassette by PCR, using cadC_St- and cadA_St-specific primer combinations. PCR products of the expected sizes were obtained for strains 4134 and 4116 but not for any of the cadmium-sensitive strains (not shown), indicating that only strains containing the cadCA_St genes are able to grow in the presence of elevated levels of CdCl₂.

The cadCA_St cassette from S. thermophilus confers cadmium resistance to both L. lactis and S. thermophilus.
The putative cadmium resistance cassette from the chromosome of S. thermophilus 4134, including 271 bp of upstream sequence, was cloned into pNZ8048. The resulting plasmid, pNZcadCA, was introduced into the cadmium-sensitive strains L. lactis NZ9800 and S. thermophilus 90728. While the control strains containing pNZ8048 were unable to grow in the presence of 0.5 mM CdCl₂, the strain L. lactis NZ9800 containing pNZcadCA was cadmium resistant and was capable of growth in the presence of as much as 1.5 mM CdCl₂, while S. thermophilus 90728 containing pNZcadCA grew well in the presence of up to 1 mM CdCl₂ in the growth medium (not shown). This confirms that the cadCA cassette from S. thermophilus strain 4134 confers resistance against CdCl₂ to both S. thermophilus and L. lactis. Cadmium-dependent expression of the cad-lacZ fusions confirmed the apparent presence of all regulatory elements on the cloned fragment (see below).

The cadCA_St cassette also confers resistance to zinc.
Growth of the cadmium-resistant S. thermophilus strain 4134 was compared to growth of the cadmium-sensitive S. thermophilus strain 4035 in the presence of various concentrations of different metal ions. No difference in the growth capacities of the two strains was observed in the presence of various concentrations of the metal ions Cu²⁺, Ni²⁺, Co²⁺, Ag⁺, and Hg²⁺. However, strain 4134 was resistant to higher concentrations of Zn²⁺ (2 mM) than was strain 4035 (0.5 mM [data not shown]). Growth of the two L. lactis NZ9800 strains, containing pNZ8048 or containing pNZcadCA, was compared. The pNZcadCA-containing strain was able to grow in the presence of 5 mM Zn²⁺, whereas the control strain could not grow above Zn²⁺ concentrations of 1 mM (not shown). This demonstrates that the presence of the cadCA_St cassette not only confers resistance against cadmium but is also responsible for increased zinc resistance.

CadA_St protein is sufficient for cadmium resistance in L. lactis.
The cadA_St gene was cloned under the control of the nisin-inducible promoter of pNZ8048 to generate pNZcadA. pNZcadA was introduced into L. lactis strain NZ9800. In the absence of nisin, L. lactis NZ9800 containing pNZcadA was not able to grow in the presence of cadmium (0.5 mM), whereas the addition of nisin to the medium allowed growth of the strain in the presence of up to 1 mM Cd²⁺ (not shown). This is in line with the prediction that the CadA_St protein functions as a cadmium efflux transporter and shows that the CadC_St protein is not necessary for cadmium resistance when CadA_St is expressed.

CadC_St regulates CadA_St expression in response to cadmium in vivo.
A cadA_St-lacZ fusion was constructed by replacing the P_R promoter derived from bacteriophage r1t of pIR12 (25) by the putative cadCA_St promoter, the cadC_St gene, and the 5' end of the cadA_St gene, generating pIRcadCA and thus placing the expression of the cadA_St-lacZ fusion under the control of the cadCA_St promoter. pIRcadCA was introduced into L. lactis 302, and ß-galactosidase activity during growth of the strain was monitored in the absence or presence of 10 µM CdCl₂ (Fig. 2). ß-Galactosidase activity is at a basal, low level in the absence of cadmium but increases within 2 h after the addition of cadmium chloride, indicating that CadA expression in vivo is induced by the presence of cadmium.

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FIG. 2. ß-Galactosidase activity of L. lactis 302 containing pIRcadCA in the absence (open circles) or presence (solid squares) of 10 µM CdCl2.

When ß-galactosidase activity levels of L. lactis 302 containing pIRcadCA and of L. lactis 302 containing pIR12 were compared during growth of the strains in the absence of cadmium, a higher ß-galactosidase activity was measured for the strain containing pIRcadCA (Table 4). The P_R promoter present on pIR12 is derived from bacteriophage r1t and constitutes a very stringently regulated early lytic promoter, which is very tightly repressed by the r1t repressor, also encoded on pIR12 (25). In contrast, the promoter of the cadCA_St genes apparently allows a low-level basal transcription of the cadCA_St genes.

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TABLE 4. ß-Galactosidasc activity (Miller units) of L. lactis 302 containing different plasmidsa

A cadC_St-lacZ fusion was constructed in pIR12, generating pIRcadC. In contrast to strain L. lactis 302 containing pIRcadCA, which showed high ß-galactosidase activity only in the presence of cadmium, the strain containing pIRcadC, which lacks a functional CadC_St protein, showed high ß-galactosidase activity regardless of the presence or the absence of cadmium (Table 4), suggesting that CadC_St is a negative regulator of cadCA_St expression. In line with this interpretation is the finding that, in strain L. lactis 302 containing both pIRcadC and p44cadC (the latter plasmid expresses CadC constitutively), cadmium-dependent expression of cadC_St-lacZ is restored (Table 4). The activity of ß-galactosidase of L. lactis 302 containing both pIRcadC and p44cadC is lower than that of L. lactis 302 containing pIRcadCA, both in the presence and in the absence of cadmium. This probably reflects high expression of CadC_St from the constitutive promoter in p44cadC. CadC_St in high concentrations may bind more efficiently to the cadCA_St promoter and thus repress cadCA_St transcription more effectively, so that the negative regulatory effect cannot be completely alleviated by the presence of 5 µM cadmium.

CadC_St is a sequence-specific DNA binding protein.
The CadC_St protein was expressed in L. lactis NZ9800 under the control of the constitutive P₄₄ promoter in p44cadC. A cell extract from this strain served as a source of CadC_St protein for use in an EMSA. An EMSA was performed with a 358-bp fragment (fragment 1) containing 279 bp of sequence upstream of cadC_St (Fig. 3A). The mobility of this DNA fragment was retarded by the CadC_St-containing cell extract (Fig. 3A, lane 2) and was not retarded by a cell extract lacking CadC_St prepared from an overnight culture of L. lactis NZ9800 pNZ44 (Fig. 3A, lane 11), showing that CadC_St is a DNA binding protein. An unrelated control DNA fragment of 400 bp was not retained by the CadC_St-containing cell extract (Fig. 3A, lanes 12 and 13), confirming that CadC_St-directed DNA binding is sequence specific. Furthermore, retardation of the labeled promoter fragment by the CadC_St protein could be effectively reversed by competitive binding using increasing amounts of unlabeled DNA fragment 1 (Fig. 3A, lanes 2 to 8).

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FIG. 3. EMSA of promoter DNA fragment 1 (A) or fragment 3 (B) by a CadCSt-containing protein extract prepared from an overnight culture of L. lactis NZ9800 containing p44cadC. (A) Promoter fragment 1 (40 ng) was mixed with no protein extract (lane 1), CadCSt-containing protein extract (lane 2), or CadCSt-containing protein extract in the presence of 0.04, 0.2, 0.4, 1.0, 2.0, or 3.5 µg, respectively, of unlabeled promoter DNA (lanes 3 to 8). Lanes 9 and 10, same as lanes 1 and 2, respectively. Lane 11, labeled promoter fragment 1 in the presence of cell extract prepared from an overnight culture of L. lactis NZ9800 containing pNZ44. Lanes 12 and 13, 40 ng of a labeled 400-bp DNA fragment derived from an unrelated plasmid in the absence (lane 12) or presence (lane 13) of the CadCSt-containing protein extract. (B) Promoter fragment 3 (40 ng, lane 1) with the CadCSt-containing cell extract in the absence (lane 2) or presence (lanes 3 to 5) of 10, 100, or 1,000 µM CdCl2, respectively, in the binding reaction.

CadC_St binds its own promoter region at a repeated inverted repeat.
In search for a potential binding site of the CadC_St protein to its own promoter region, the sequence upstream of cadC_St was examined. The analysis revealed the presence of a 7-bp inverted perfect repeat (IR1) (ATTCAAAcaaacaTTTGAAT) spaced by 6 nt (lowercase letters). A second copy of this inverted repeat (which was imperfect at 1 nucleotide) was detected 45 bp upstream of the first (IR2) (ATTCAAAcattcaCTTGAAT) (Fig. 1B). An analysis of the complete 15,874-bp sequenced fragment from the S. thermophilus 4134 chromosome did not reveal any other copy of this inverted repeat. To ascertain whether the inverted repeats could serve as potential binding sites for the CadC_St protein, six fragments (fragments 2 to 7 [Table 2]) were generated by PCR, which represented 5'- and/or 3'-truncated versions of the 358-bp promoter fragment 1 and contained both (fragments 1, 2, 3, and 7), one (fragments 4 and 5), or no (fragment 6) copy of the inverted repeats (Fig. 4A). All seven fragments were tested in an EMSA by using the CadC_St-containing cell extract of L. lactis NZ9800 p44cadC (Fig. 4B). The mobility of fragments 1, 2, 3, and 7 was retarded by the presence of the cell extract, and fragment 4 exhibited only a partial mobility shift, while the presence of the CadC_St-containing cell extract did not influence the migration behavior of fragments 5 and 6 (Fig. 4B). The 5' end of the CadC_St binding site could be located to position -111 upstream of cadC_St, since the mobility of fragments 1, 2 and 3 but not of fragment 4 was efficiently retarded by CadC_St-containing cell extract, while the 3' end was mapped to position -15, since fragment 7, but not fragment 6, exhibited mobility shifts in the presence of CadC_St. Thus, the CadC_St binding region was localized to the promoter region of cadC_St (positions 12166 to 12070 of the sequence represented by GenBank accession no. AJ315964) and contains both inverted repeats.

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FIG. 4. EMSA of truncated promoter DNA fragments with a CadCSt-containing cell extract. (A) Representation of promoter fragments 1 to 7 used for CadCSt-DNA binding studies. Fragment names and their sizes are indicated on the left. On the right, the results of the EMSA (B) are stated. Gray rectangle, cadCSt ORF; black triangles, repeats; IR1, inverted repeat 1; IR2, inverted repeat 2; and -35 and -10, -35 and -10 promoter sequences. (B) EMSA of the labeled promoter DNA fragments 1 to 7 (A) as specified below the lanes. -, absence; +, presence of a CadCSt-containing cell extract. Lanes 1 to 12 were run for 3 h; lanes 13 and 14 were run for 1.5 h.

The CadC_St-DNA interaction is lost in the presence of cadmium.
An EMSA was performed using promoter fragment 3 and the CadC_St-containing cell extract (Fig. 3B). The fragment was retarded by the presence of the cell extract in the absence of cadmium. However, the presence of 1 mM CdCl₂ in the binding assay completely abolished the capacity of the cell extract to retard the fragment (Fig. 3B). Thus, the CadC_St protein seems to lose its DNA affinity in the presence of cadmium.

Top Abstract Introduction Materials and Methods Results Discussion References

 
This work is the first description of a cadmium resistance cassette from the genome of S. thermophilus. This cadmium resistance cassette, which consists of the two adjacent genes cadC_St and cadA_St, was identified on a 15,874-bp fragment from the genome of S. thermophilus strain 4134. A stretch of about 10 kb of this sequence, which includes the cadCA_St genes, shows very high DNA sequence homology (>90%) to plasmid pAH82 from L. lactis. The G+C content of the cassette is lower (34.5%) than the average G+C content of S. thermophilus (37.2 to 40.3%) (36) or that of L. monocytogenes and L. innocua (39 and 37%, respectively) (12) but is in line with the G+C content of L. lactis (35.4%) (4). Although the cadmium resistance cassette is widespread among different (mainly gram-positive) bacteria, it does not seem to be common in S. thermophilus. Out of 12 strains tested, only two appeared to contain the cassette. It is thus possible that the cadmium resistance cassette has originated from L. lactis and has entered the genome of S. thermophilus 4134 through a plasmid that was exchanged during industrial coculture. Intergeneric transfer of DNA from L. lactis to S. thermophilus during coculture in the manufacture of cheese was suggested to explain the presence of the insertion element iso-IS981 on the genome of S. thermophilus (13). Possessing the cadmium resistance cassette may have such a selective advantage for the bacteria (17) that some species acquire it through intergeneric transfer from only distantly related organisms, as has been proposed for the gram-negative bacterium S. maltophilia, which contains the cadmium resistance cassette usually found in gram-positive bacteria (1). In the case of S. thermophilus strain 4134, the selective advantage may even have been larger, because the acquired DNA also contains a type I restriction/modification system that is thought to act as a phage resistance cassette. Sequences adjacent to the 10-kb stretch showing homology to lactococcal plasmids contained the complete or partial S. thermophilus insertion sequences IS1191 (13), ICESt1 (6), and IS1195L (GenBank accession no. AJ278471). Sequence analysis of the regions next to the indicated IS elements identified potential ORFs with counterparts in the genome sequence of S. thermophilus LMG18311. This may indicate that the genomic region sequenced may be a result of multiple DNA integration events. Whether integration of the insertion sequences occurred independently or at the same time or even allowed insertion of lactococcal plasmid sequences is a matter of speculation.

The two genes of the cassette are putatively organized as a transcriptional unit in S. thermophilus, with a promoter sequence upstream of the cadC_St gene and a transcriptional terminator downstream of the cadA_St gene. Thus, gene organization seems similar to that found in other bacteria, with the exception of L. monocytogenes, where the gene order is reversed (18) and where the two chromosomally located genes are not adjacent (12).

The cadCA_St cassette including 279 bp of upstream sequence was shown to confer cadmium and zinc resistance to both S. thermophilus and L. lactis. The system is thus similar to the one identified on S. aureus plasmid pI258, which confers cadmium and zinc resistance (42), but is different from that of L. monocytogenes, which does not confer zinc resistance (18).

Expression of CadA_St was shown to be sufficient for cadmium resistance, confirming that CadA_St functions as the cadmium resistance protein. CadC_St was shown to be a trans-acting, sequence-specific, DNA binding and cadmium-dependent regulatory protein of cadCA_St expression. The specific CadC_St-DNA interaction is completely alleviated in the presence of cadmium in vitro. Thus, cadmium seems to have a more stringent role in preventing or reversing the CadC_St-DNA interaction than in affecting the interaction of CadC from S. aureus with its promoter DNA, which is only partly diminished in the presence of cadmium (10). It seems possible that CadC_St can either bind DNA or cadmium and can function thus as a cadmium sensor protein. The amino acid sequence contains three motifs with similarity to domains involved in binding to metal ions (26). Two of the metal binding motifs overlap the proposed helix-turn-helix motif involved in DNA binding (18). It has been suggested that one of the functions of the CadC protein is to deliver the metal ion to the CadA protein for export (42) or to function as a structural component for the cadmium efflux complex (42). Since we have shown that CadA_St is sufficient for cadmium resistance, we propose that CadC_St functions as a sensor of cadmium concentration. Metal binding to CadC_St may influence its capacity to act as a negative regulator through DNA-protein interactions. It may therefore be that the interaction of CadC_St with cadmium or with DNA is mutually exclusive.

CadC_St binds specifically to the cadCA_St promoter region at positions -111 to -15 upstream of cadC_St (positions 12166 to 12070 of the complete sequenced fragment [GenBank accession no. AJ315964]). The CadC_St DNA binding region includes the presence of two inverted repeats. Only fragments containing both inverted repeats were efficiently retarded in an EMSA (Fig. 4). It is likely that these inverted repeats constitute the core recognition sequence that is specifically recognized by the CadC_St protein. CadC_St might bind these elements independently or cooperatively and/or in a multimeric form. An analysis of the protein sequences of CadC of S. thermophilus and S. aureus indicated the presence of putative coiled-coil motifs (amino acids 10 to 25 of CadC_St and 12 to 27 of S. aureus CadC). These sequences may therefore represent a part of the CadC protein required for protein-protein interaction (5), potentially between CadC molecules. Since CadC_St does not bind efficiently to fragments that contained only IR1, it is tempting to speculate that CadC_St binds to both IR1 and IR2 as a dimer of dimers. The EMSA did not show the formation of two or more bound forms of the promoter fragments, as reported by Endo and Silver (10) for the interaction of CadC from S. aureus plasmid pI258 with the cadC promoter. It is possible that the difference in retardation behavior of the two systems is a result of the different purification states of the respective proteins. Additional bound forms of promoter DNA may be observed when purified CadC_St protein is used for the EMSA. Alternatively, CadC_St might bind as a multimer formed in solution, and multimer formation might be stabilized by other proteins present in the crude cell extracts.

Endo and Silver (10) reported for the CadC protein of pI258 of S. aureus sequence-specific binding to an inverted repeat (TCAAATAaaTATTTGA). We analyzed their sequence and found a second copy of the inverted repeat 45 bp upstream of the first (TCAAATAttTGCTTGC), which, as is the case for S. thermophilus, is an imperfect repeat. The CadC protein of S. aureus might only be able to bind to the imperfect repeat when a CadC dimer is already bound to the perfect inverted repeat. Alternatively, CadC binding affinity to the imperfect repeat might be lower.

We propose the following model for the regulation of expression of cadmium resistance in S. thermophilus (Fig. 5): at low cadmium concentrations, a low basal level of transcription allows the production of CadC_St protein, which binds (potentially as a dimer of dimers) to the inverted repeats IR1 and IR2 of the cadCA_St promoter region. This binding prevents the efficient establishment of a functional RNA polymerase-cadCA_St promoter DNA interaction and thus inhibits further transcription. At high cadmium concentrations, the binding of cadmium to the CadC_St protein leads to a conformational change, which results in the inability of CadC_St to bind to DNA. The CadC_St-promoter DNA complex is thus destroyed, making room for binding of RNA polymerase. This results in the production of cadCA_St transcript and the expression of CadA_St, which is responsible for the establishment of cadmium resistance.

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FIG. 5. Working model for the regulation of the expression of cadmium resistance in S. thermophilus. At low cadmium concentrations in the growth medium, most of the CadCSt binding sites (black triangles) will be occupied by CadCSt protein (dark gray circles), which possibly binds as a dimer of dimers and inhibits most of the cadCASt transcription. At high cadmium concentrations, binding of cadmium to the CadCSt protein is proposed to cause a conformational change in the CadCSt protein (gray triangles), which interferes with its DNA binding properties. As a consequence, CadCSt will release the promoter DNA and make room for binding of the RNA polymerase complex. Thus, transcription of the cadCASt cassette is activated, which leads to the production of CadASt protein and the establishment of cadmium resistance.

The most widely accepted approach for precise genetic improvement of starter cultures destined for consumer products is the conjugal transfer of naturally occurring plasmids responsible for key industrial traits, like lactose catabolism, citrate transport, proteinase production, polysaccharide production, bacteriocin production and immunity, and bacteriophage resistance. Bacteriophage attack of starter cultures is still one of the most eminent problems in the dairy industry and leads to significant economic losses worldwide. Hence, the transfer of plasmids conferring bacteriophage resistance into industrially relevant strains is of key interest (37). A difficulty with this approach is the paucity of easily selectable markers for phage resistance plasmids. Bacteriocin immunity has been used successfully for the transfer of phage resistance plasmids (7, 32) but resulting transconjugants produced bacteriocin, which could be undesirable in some industrial situations, particularly where a combination of starter strains is used. Recently, it was shown that the cadmium resistance cassette identified on the L. lactis plasmid pNP-40 could be used as a selectable marker for the introduction of phage resistance plasmids into industrial starter strains (37). It should be possible to use the cadmium resistance cassette identified here as a selectable marker to also improve thermophilic starter strains like S. thermophilus.

 
We thank S. Leach, University College Cork, for excellent technical advice and B. Mollet, Nestlé, for providing strain ST11. Sequence data for S. thermophilus strain LMG18311, presented in Fig. 1A, were obtained from the Université catholique de Louvain Life Sciences Institute website at http://www.biol.ucl.ac.be/gene/genome/. Sequencing of the S. thermophilus strain LMG18311 genome was supported by the Walloon Region (BIOVAL grant no. 9813866).

This work was supported by a European Commission biotechnology grant to J.S. (BIO-CT98-5097).

 
* Corresponding author. Present address: Max-Planck-Institute for Terrestrial Microbiology, Department of Organismic Interactions, Karl-von-Frisch-Str., 35043 Marburg, Germany. Phone: 49-6421-178510. Fax: 49-6421-178509. E-mail: schiraws{at}mailer.uni-marburg.de.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, November 2002, p. 5508-5516, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5508-5516.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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