cadDX Operon of Streptococcus salivarius 57.I

A CadDX system that confers resistance to Cd²⁺ and Zn²⁺ wasidentified in Streptococcus salivarius 57.I. Unlike with otherCadDX systems, the expression of the cad promoter was negativelyregulated by CadX, and the repression was inducible by Cd²⁺and Zn²⁺, similar to what was found for CadCA systems. The lowerG+C content of the S. salivarius cadDX genes suggests acquisitionby horizontal gene transfer.

INTRODUCTION

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INTRODUCTION

The two best-known cadmium resistance systems of gram-positivebacteria are CadCA and CadBX (CadDX). CadCA, encoded by Staphylococcusaureus plasmid pI258, is composed of a repressor, CadC, anda P-type metal efflux ATPase, CadA (13). The resistance phenotypeof CadCA is inducible by Cd²⁺, Pb²⁺, Bi³⁺, Zn²⁺, and Co²⁺ atdifferent levels (17, 24, 25). The direct binding of these metalions to dimeric CadC decreases the affinity of CadC to the operatorand 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 regulatoryprotein (16, 21). CadB and CadA differ in both sequence andfunction, but CadX and CadC share significant homology. However,CadX is essential for CadB-mediated resistance in Staphylococcuslugdunensis (5). When a related cadDX* (* represents a truncationin cadX) is complemented in trans with an intact cadX gene,cadmium resistance levels increase significantly, confirmingthe positive effect of CadX (7). Although additional cadDX operonswith intact cadX have been described, functional analysis islimited to S. aureus carrying pUB101 (14).

A cadDX homolog was identified in the Streptococcus salivariusgenome recently. In contrast to the CadX described previously(5, 7), S. salivarius CadX represses the cadDX expression. Thepossible regulatory mechanism is discussed.

Characterization of the cad operon.

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Characterization of the cad...

A DNA fragment containing one partial open reading frame (ORF)and two complete ORFs was isolated from an S. salivarius subgenomiclibrary (Fig. 1). The deduced amino acid sequence of the partialORF was 87% identical to the carboxyl terminus of Enterococcusfaecium TcrB (9), and the two complete ORFs are almost identicalto the CadDX identified from the genomes of Streptococcus pyogenesMGAS strains (1). Additionally, ORF1 shared significant homologyto S. aureus pBORa53 CadD (55% identity) (12) and pLUG10 CadB(56% identity). ORF2 shares 42% and 33% identity with pLUG10CadX and pI258 CadC, respectively. Both the conserved cys-7and ELC⁵⁸VC⁶⁰D metal binding motifs of CadC were found in thecorresponding locations within ORF2 (22, 23). Thus, ORFs 1 and2 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 ${Omega}$ 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.

Similar to that of cadCA of Streptococcus thermophilus (19),the G+C content of this cadDX operon is approximately 34%, whichis lower than that of S. salivarius 57.I based on its knowngenes (39 to 42%), suggesting that cadDX was introduced fromother sources. However, examination of the flanking regionsof the chromosomal cad loci that are most homologous to S. salivariuscadDX 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 acidlevel, is an orphan gene. Thus, these cadDX operons are likelyfrom a common origin, and multiple integration, rearrangement,or deletion events occurred during evolution.

cadDX confers cadmium and zinc resistance.

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cadDX confers cadmium and...

The MICs of all metals tested in wild-type 57.I were 12 µMfor Cd²⁺, 800 µM for Zn²⁺, 500 µM for Co²⁺, 1 mMfor Cu²⁺, and 5 mM for Ni²⁺. Inactivation of cadDX by insertionof a polar mutation ( ${Omega}$ kan) (15) significantly reduced the resistanceto Cd²⁺ (2 µM) and Zn²⁺ (500 µM) but did not affectthe 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.

An intact cadD gene with its original promoter (pcad) was clonedonto the Escherichia coli-Streptococcus shuttle vector pDL278(11) and transferred into CWF5 to investigate the function ofCadD. The trans-complementation of cadD restored resistanceto both metals (Fig. 2, CFW18), and the levels of resistanceto Cd²⁺ and Zn²⁺ in this strain were more than nine- and sixfoldhigher, respectively, than those in the wild-type strain, presumablybecause of the increased copy numbers, confirming that CadDis responsible for the resistance.

Localization of pcad.

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Localization of pcad.

The transcription initiation site of the cadDX operon was determinedby primer extension analysis with primers CadAS1290-IRD (5'-AGTAAGTCTACGGCTGTCC)and CadAS1330-IRD (5'-TTTCTGCTTTTTCTTTTGGCA), which are located40 and 80 bases 3' of the cadD ATG codon, respectively. Thesignal observed with RNA isolated from CWF18 showed higher intensitythan wild-type 57.I (data not shown), which is in agreementwith the resistance phenotype. This signal (Fig. 3), 27 bases5' of the cadD ATG codon, was consistently observed with bothprimers. A ${sigma}$ ⁷⁰-like promoter sequence (TTGACA-N₁₇-TAGAAT) wasmapped 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.

CadX negatively regulated cadDX expression.

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CadX negatively regulated cadDX...

The resistance phenotype in CWF18 indicates that CadD aloneis sufficient for the resistance, which is opposite to whatwas found for the CadBX system (5). The function of CadX wasfurther investigated by constructing a pcad-cat fusion strainin the wild type (CWF9) and a CadX-deficient strain (CWF10).Briefly, pcad was amplified from S. salivarius by PCR usingprimers CadS810SacI (5'-CCTCCATgagctcTTGATTA) and CadAS1250BamHI(5'-TTGAATCATggatccCCTCATTCAAATAT). Restriction sites (in lowercase)were incorporated for cloning. The pcad region was subsequentlyfused to the 5' end of a promoterless cat gene from S. aureus(10). The pcad-cat fusion was integrated into the lacZ locusvia the integration vector pMC195 (6) to generate CWF9. ThecadX locus in CWF9 was further insertionally inactivated byan erm marker (18) to generate CWF10. The levels of resistanceto Cd²⁺ and Zn²⁺ were up-regulated in CWF10 compared to thosein the wild-type strain, suggesting that CadX is a repressorof the operon (Fig. 2). Two cadX complementation strains, wherecadX was driven by either pcad or a highly expressed S. salivariuspromoter, pureI ${Delta}$ 21(6), were constructed. Both pcad-cadX and pureI ${Delta}$ 21-cadXwere transferred into CWF10 on pDL278 to generate CWF12 andCWF16, respectively. In CWF12, the resistance levels were similarto 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, althoughcadX was present in multiple copies. This also suggested thatCadX represses pcad on the plasmid more effectively than thechromosomal pcad promoter. This problem was circumvented whencadX was driven by pureI ${Delta}$ 21. As expected, resistance in CWF16was lower than that in CWF10, confirming the negative effectof CadX (Fig. 2). We also noticed that the Cd²⁺ resistance levelin CWF16 was lower than that in the wild-type strain, 57.I (8µM versus 12 µM), presumably due to the multiplecopies of CadX present in the system. CWF16 exhibited a similarpattern of resistance to Zn²⁺.

Consistent with the metal resistance pattern, pcad activityin CFW10 was more than 60-fold higher than that in CWF9, indicatingthat the regulation occurs mainly at the transcriptional levelvia CadX (Fig. 4). When CWF10 was complemented by pcad-cadX(CWF12), the chloramphenicol acetyltransferase (CAT) activitywas 50-fold higher than that in CWF9, although a reduction inCAT activity compared to that in CWF10 was observed. The discrepancywas completely abolished in CWF16. No significant differencein CAT activity was observed between CWF9 and CWF16, confirmingthe 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 pureI ${Delta}$ 21 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.

Based on the homology between CadC and CadX, we predicted thatthe negative nature of CadX in S. salivarius might be linkedto conserved motifs found in the CadCA system. When the upstreamregions of the known cadCA operons were compared with that ofS. salivarius cadDX, a 7-bp inverted repeat (IR) separated by6 bp (ATTCAA-N₆-TTTGAAT), presumably the target site of CadC(19), was found in all cases. In both pI258 and S. thermophiluscadCA (19), two copies of this IR, separated by 25 bp, wereobserved, although a 1-base mismatch is present in both repeatsin pI258. Only one perfect IR, 10 bp 5' of the cadD ATG codon,was found in S. salivarius. The +1 regions of the cad operonsin both pI258 and S. salivarius are embedded within the IR (24);thus, binding of CadX may preclude the transcription of theoperon. Alternatively, S. salivarius CadX may bind to sequencesother than the IR and regulate the expression via a differentmechanism.

pcad is inducible by Cd²⁺ and Zn²⁺.

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pcad is inducible by...

A significant increase in CAT activity was observed in CWF9when culture medium was supplemented with 2 µM CdCl₂ (Fig.5). Further increases were achieved with higher concentrationsof CdCl₂, and the maximum level was observed with 12 µMCdCl₂, at which concentration slight inhibitions in growth wereobserved. The induction by ZnCl₂ was less significant. The maximalinduction was observed with 1 mM ZnCl₂, but the CAT activitywas less than 50% of that induced by CdCl₂ (1.02 versus 2.68U). Thus, the repression by CadX can be alleviated by the presenceof Cd²⁺ and to a lesser degree by Zn²⁺. The lower degree ofinduction by Zn²⁺ may be due to the higher affinity of the metallatedprotein 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.

In conclusion, the cadDX system confers resistance to cadmiumand zinc in S. salivarius 57.I. This system is negatively regulatedby CadX, which resembles CadC of the cadCA system (8).

Nucleotide sequence accession number.

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Nucleotide sequence accession...

The complete sequence of the cad operon has been deposited inGenBank under accession number EU021082.

ACKNOWLEDGMENTS

We thank J. A. Lemos and P. Fives-Taylor for critical reviewof the manuscript.

This work was supported by the National Science Council of Taiwan,grant NSC-942311-B182-007, and the Chang Gung Memorial Hospitalof Taiwan, grant CMRPD34001.

FOOTNOTES

* 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.

REFERENCES

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