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Applied and Environmental Microbiology, March 2008, p. 1642-1645, Vol. 74, No. 5
0099-2240/08/$08.00+0 doi:10.1128/AEM.01878-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
cadDX Operon of Streptococcus salivarius 57.I
Yi-Ywan M. Chen,1*
C. W. Feng,1
C. F. Chiu,1 and
Robert A. Burne2
Department of Microbiology and Immunology, Chang Gung University, Tao-Yuan, Taiwan, Republic of China,1
Department of Oral Biology, University of Florida, Gainesville, Florida2
Received 15 August 2007/
Accepted 19 December 2007
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ABSTRACT
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A CadDX system that confers resistance to Cd2+ and Zn2+ was identified in Streptococcus salivarius 57.I. Unlike with other CadDX systems, the expression of the cad promoter was negatively regulated by CadX, and the repression was inducible by Cd2+ and Zn2+, similar to what was found for CadCA systems. The lower G+C content of the S. salivarius cadDX genes suggests acquisition by horizontal gene transfer.
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INTRODUCTION
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The two best-known cadmium resistance systems of gram-positive bacteria are CadCA and CadBX (CadDX). CadCA, encoded by Staphylococcus aureus plasmid pI258, is composed of a repressor, CadC, and a P-type metal efflux ATPase, CadA (13). The resistance phenotype of CadCA is inducible by Cd2+, Pb2+, Bi3+, Zn2+, and Co2+ at different levels (17, 24, 25). The direct binding of these metal ions to dimeric CadC decreases the affinity of CadC to the operator and subsequently derepresses the expression (2, 3, 22, 23).
CadBX and the closely related CadDX are also plasmid-borne systems (5) where CadB is a membrane protein and CadX is a regulatory protein (16, 21). CadB and CadA differ in both sequence and function, but CadX and CadC share significant homology. However, CadX is essential for CadB-mediated resistance in Staphylococcus lugdunensis (5). When a related cadDX* (* represents a truncation in cadX) is complemented in trans with an intact cadX gene, cadmium resistance levels increase significantly, confirming the positive effect of CadX (7). Although additional cadDX operons with intact cadX have been described, functional analysis is limited to S. aureus carrying pUB101 (14).
A cadDX homolog was identified in the Streptococcus salivarius genome recently. In contrast to the CadX described previously (5, 7), S. salivarius CadX represses the cadDX expression. The possible regulatory mechanism is discussed.
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Characterization of the cad operon.
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A DNA fragment containing one partial open reading frame (ORF) and two complete ORFs was isolated from an S. salivarius subgenomic library (Fig. 1). The deduced amino acid sequence of the partial ORF was 87% identical to the carboxyl terminus of Enterococcus faecium TcrB (9), and the two complete ORFs are almost identical to the CadDX identified from the genomes of Streptococcus pyogenes MGAS strains (1). Additionally, ORF1 shared significant homology to S. aureus pBORa53 CadD (55% identity) (12) and pLUG10 CadB (56% identity). ORF2 shares 42% and 33% identity with pLUG10 CadX and pI258 CadC, respectively. Both the conserved cys-7 and ELC58VC60D metal binding motifs of CadC were found in the corresponding locations within ORF2 (22, 23). Thus, ORFs 1 and 2 were designated cadD and cadX, respectively.
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FIG. 1. Schematic diagram of the cad operon and the flanking region of S. salivarius 57.I. The truncated tcrB gene is indicated by a dashed-line arrow. The putative rho-independent terminators of the cad operon are indicated. The locations of  kan and erm in CWF5 and CWF10 are indicated by inverted triangles. The limits of the DNA sequence under EU021082 are indicated by vertical arrows. The size of each ORF in nucleotides (nt), the predicated molecular mass in kDa, and the pI of each gene product are shown. The distance between tcrB and cadD is shown.
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Similar to that of cadCA of Streptococcus thermophilus (19), the G+C content of this cadDX operon is approximately 34%, which is lower than that of S. salivarius 57.I based on its known genes (39 to 42%), suggesting that cadDX was introduced from other sources. However, examination of the flanking regions of the chromosomal cad loci that are most homologous to S. salivarius cadDX did not reveal any common region or insertion sequences. We also noticed that Neisseria meningitidis MC58 cadD (NC_003112.2), which is 95% identical to S. salivarius CadD at the amino acid level, is an orphan gene. Thus, these cadDX operons are likely from a common origin, and multiple integration, rearrangement, or deletion events occurred during evolution.
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cadDX confers cadmium and zinc resistance.
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The MICs of all metals tested in wild-type 57.I were 12 µM for Cd2+, 800 µM for Zn2+, 500 µM for Co2+, 1 mM for Cu2+, and 5 mM for Ni2+. Inactivation of cadDX by insertion of a polar mutation (kan) (15) significantly reduced the resistance to Cd2+ (2 µM) and Zn2+ (500 µM) but did not affect the resistance for Co2+, Cu2+, or Ni2+ (Fig. 2, CWF5).
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FIG. 2. Cadmium and zinc resistance in wild-type 57.I, a CadDX-deficient strain (CWF5), a CadX-deficient strain (CWF10), a CadD complementation strain (CWF18), and a CadX complementation strain (CWF16). Overnight cultures of each strain were diluted at 1:100 in fresh brain heart infusion containing various concentrations of CdCl2 (A) or ZnCl2 (B). After incubation for 16 h, the levels of growth were evaluated as final optical densities at 600 nm (O.D.600). Each data point shows the mean and standard deviation for three independent experiments.
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An intact cadD gene with its original promoter (pcad) was cloned onto the Escherichia coli-Streptococcus shuttle vector pDL278 (11) and transferred into CWF5 to investigate the function of CadD. The trans-complementation of cadD restored resistance to both metals (Fig. 2, CFW18), and the levels of resistance to Cd2+ and Zn2+ in this strain were more than nine- and sixfold higher, respectively, than those in the wild-type strain, presumably because of the increased copy numbers, confirming that CadD is responsible for the resistance.
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Localization of pcad.
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The transcription initiation site of the cadDX operon was determined by primer extension analysis with primers CadAS1290-IRD (5'-AGTAAGTCTACGGCTGTCC) and CadAS1330-IRD (5'-TTTCTGCTTTTTCTTTTGGCA), which are located 40 and 80 bases 3' of the cadD ATG codon, respectively. The signal observed with RNA isolated from CWF18 showed higher intensity than wild-type 57.I (data not shown), which is in agreement with the resistance phenotype. This signal (Fig. 3), 27 bases 5' of the cadD ATG codon, was consistently observed with both primers. A 
70-like promoter sequence (TTGACA-N17-TAGAAT) was mapped at an appropriate distance.
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FIG. 3. Primer extension analysis of pcad and the nucleotide sequences of the 5' flanking region of cadDX operon. Total cellular RNA (100 µg) isolated from CWF18 was incubated with IRD-800-labeled primer CadAS1290, and the cDNA was synthesized. The extended products were analyzed on a 12% gel alongside a DNA-sequencing reaction by using the same primer, and signals were detected on a LI-COR DNA sequencer (model 4000L). In the sequence below, the transcriptional start site of cadDX is indicated by a vertical arrow (+1) and the corresponding –10 and –35 regions are overlined. The sequences of the IRs are indicated by stars.
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CadX negatively regulated cadDX expression.
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The resistance phenotype in CWF18 indicates that CadD alone is sufficient for the resistance, which is opposite to what was found for the CadBX system (5). The function of CadX was further investigated by constructing a pcad-cat fusion strain in the wild type (CWF9) and a CadX-deficient strain (CWF10). Briefly, pcad was amplified from S. salivarius by PCR using primers CadS810SacI (5'-CCTCCATgagctcTTGATTA) and CadAS1250BamHI (5'-TTGAATCATggatccCCTCATTCAAATAT). Restriction sites (in lowercase) were incorporated for cloning. The pcad region was subsequently fused to the 5' end of a promoterless cat gene from S. aureus (10). The pcad-cat fusion was integrated into the lacZ locus via the integration vector pMC195 (6) to generate CWF9. The cadX locus in CWF9 was further insertionally inactivated by an erm marker (18) to generate CWF10. The levels of resistance to Cd2+ and Zn2+ were up-regulated in CWF10 compared to those in the wild-type strain, suggesting that CadX is a repressor of the operon (Fig. 2). Two cadX complementation strains, where cadX was driven by either pcad or a highly expressed S. salivarius promoter, pureI
21(6), were constructed. Both pcad-cadX and pureI21-cadX were transferred into CWF10 on pDL278 to generate CWF12 and CWF16, respectively. In CWF12, the resistance levels were similar to that observed in the parental strain, CWF10 (data not shown). It was predicted that CadX negatively regulated pcad expression, and only a very small amount of CadX was produced, although cadX was present in multiple copies. This also suggested that CadX represses pcad on the plasmid more effectively than the chromosomal pcad promoter. This problem was circumvented when cadX was driven by pureI21. As expected, resistance in CWF16 was lower than that in CWF10, confirming the negative effect of CadX (Fig. 2). We also noticed that the Cd2+ resistance level in CWF16 was lower than that in the wild-type strain, 57.I (8 µM versus 12 µM), presumably due to the multiple copies of CadX present in the system. CWF16 exhibited a similar pattern of resistance to Zn2+.
Consistent with the metal resistance pattern, pcad activity in CFW10 was more than 60-fold higher than that in CWF9, indicating that the regulation occurs mainly at the transcriptional level via CadX (Fig. 4). When CWF10 was complemented by pcad-cadX (CWF12), the chloramphenicol acetyltransferase (CAT) activity was 50-fold higher than that in CWF9, although a reduction in CAT activity compared to that in CWF10 was observed. The discrepancy was completely abolished in CWF16. No significant difference in CAT activity was observed between CWF9 and CWF16, confirming the negative effect of CadX.
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FIG. 4. CAT-specific activities in recombinant S. salivarius strains. The pcad-cat gene fusion was integrated into the lacZ locus in wild-type 57.I (CWF9) in a CadX-deficient strain (CWF10) and in CadX complementation strains (CWF12 and CWF16). The cadX gene in strain CWF12 was driven by its own promoter, pcad, whereas in CWF16, pcad was driven by the constitutively expressed pureI21 promoter (6). Total protein lysates used in assays were obtained from cultures grown to an optical density at 600 nm of 0.65. CAT activities were determined according to the methods of Shaw (20), and the specific activities (U) were calculated as nmole chloramphenicol acetylated min–1 mg total protein–1. Values shown are means and standard deviations of three independent experiments. The negative controls were reactions carried out in the absence of chloramphenicol.
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Based on the homology between CadC and CadX, we predicted that the negative nature of CadX in S. salivarius might be linked to conserved motifs found in the CadCA system. When the upstream regions of the known cadCA operons were compared with that of S. salivarius cadDX, a 7-bp inverted repeat (IR) separated by 6 bp (ATTCAA-N6-TTTGAAT), presumably the target site of CadC (19), was found in all cases. In both pI258 and S. thermophilus cadCA (19), two copies of this IR, separated by 25 bp, were observed, although a 1-base mismatch is present in both repeats in pI258. Only one perfect IR, 10 bp 5' of the cadD ATG codon, was found in S. salivarius. The +1 regions of the cad operons in both pI258 and S. salivarius are embedded within the IR (24); thus, binding of CadX may preclude the transcription of the operon. Alternatively, S. salivarius CadX may bind to sequences other than the IR and regulate the expression via a different mechanism.
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pcad is inducible by Cd2+ and Zn2+.
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A significant increase in CAT activity was observed in CWF9 when culture medium was supplemented with 2 µM CdCl2 (Fig. 5). Further increases were achieved with higher concentrations of CdCl2, and the maximum level was observed with 12 µM CdCl2, at which concentration slight inhibitions in growth were observed. The induction by ZnCl2 was less significant. The maximal induction was observed with 1 mM ZnCl2, but the CAT activity was less than 50% of that induced by CdCl2 (1.02 versus 2.68 U). Thus, the repression by CadX can be alleviated by the presence of Cd2+ and to a lesser degree by Zn2+. The lower degree of induction by Zn2+ may be due to the higher affinity of the metallated protein for pcad, as noted for CadC (4).
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FIG. 5. Induction of pcad by CdCl2 and ZnCl2. Overnight cultures of CWF9 in brain heart infusion containing different concentrations of CdCl2 or ZnCl2 were diluted at 1:100 in fresh brain heart infusion containing the same amount of metal ions and incubated to an optical density at 600 nm of 0.65. Expression of pcad under each condition was evaluated by measuring CAT specific activity. The specific activities were calculated as described in the legend for Fig. 4. Values shown are means and standard deviations of three independent experiments.
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In conclusion, the cadDX system confers resistance to cadmium and zinc in S. salivarius 57.I. This system is negatively regulated by CadX, which resembles CadC of the cadCA system (8).
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Nucleotide sequence accession number.
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The complete sequence of the cad operon has been deposited in GenBank under accession number EU021082.
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ACKNOWLEDGMENTS
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We thank J. A. Lemos and P. Fives-Taylor for critical review of the manuscript.
This work was supported by the National Science Council of Taiwan, grant NSC-942311-B182-007, and the Chang Gung Memorial Hospital of Taiwan, grant CMRPD34001.
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FOOTNOTES
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan. Phone: 886-3-2118800, ext. 3352. Fax: 886-3-2118700. E-mail: mchen{at}mail.cgu.edu.tw 
Published ahead of print on 28 December 2007.
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Applied and Environmental Microbiology, March 2008, p. 1642-1645, Vol. 74, No. 5
0099-2240/08/$08.00+0 doi:10.1128/AEM.01878-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
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CadX negatively regulated cadDX...
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