ZuxAB gene fusions with the arsenic and cadmium resistance operons of Staphylococcus aureus

pC101, a novel shuttle vector between Escherichia coli and Staphylococcus aureus carrying the lux genes encoding luciferase from vibrio harueyi, selectable ampicillin and chloramphenicol markers and origins of replication for Gram-negative and Gram-positive bacteria has been constructed. The inducibility of the arsenic and cadmium operon from S. aureus plasmid ~I258 to different ions has been tested in E. coli and in S. aureus with two fusions in pC101: an arsB-1uxAB and a cadA-1uxAB transcriptional gene fusion. Patterns of induction are influenced by the host strain and are slightly different from previous reports using the b1a.Z gene as reporter gene.


Introduction
Plasmid ~1258 from Staphylococcus aureus contains the 3.5-kb cad operon conferring resistance to cadmium and zinc [ll and the 2.7-kb ars operon conferring resistance to arsenate, arsenite and antimony [2]. The arsenic efflux resistance operon consists of three genes in the following order: arsR, a regulatory gene; arsB, whose gene product is a membrane protein; and arsC, encoding an arsenate reductase converting intracellular arsenate to arsenite [2,3]. The cadA resistance operon contains two genes, the first cadC gene encodes a soluble protein and the second cadA gene encodes a membrane ATPase protein. CadA protein is a member of a protein family known as P-type ATPases 11,451. Such a protein is not encountered in cadmium resistance observed in Gram-negative bacteria although resistance proceeds also through cation efflux [6]. CadA is not sufficient to confer full resistance to cadmium and zinc; cadC must also be present [7]. The function of CadC in Cd2+ efflux is not clear and the regulator of the cadA operon has not been identified [8,9]. Gene expression studies in S. aweuS or Bacillus subtilis have been commonly realized by gene fusion with the S. aureus blaZ 232 [2,7,10] or cat genes [11] encoding fl-lactamase and chloramphenicol acetyltransferase, respectively, lux genes encoding luciferase used as reporter genes have been shown to be extremely valuable in determining gene expression [12] and provide a very sensitive, non-destructive alternative assay already successfully applied in B. subtilis [13,14].
In this work, we describe the construction of a novel shuttle vector between Escherichia coli and S. aureus containing the luxAB genes from Vibrio harveyi as reporter genes. An arsB-luxAB and a cadA-luxAB transcriptional fusion were constructed and used to study the regulation of the ars and cad operons.

Bacterial strains
The bacterial strains and plasmids used in this study are listed in Table 1. Cells were grown in Luria broth [15] with ampicillin (50/zg ml-1) or chloramphenicol (5/~g ml-1), when required. Cell growth was monitored by measuring the culture turbidity in a OD photometer.

Chemicals
Sodium arsenate, sodium arsenite and antimony potassium tartrate were used as oxyanions (from Sigma Chemical Co, St. Louis, MO). Bismuth sodium tartrate was from RSA Corp. (Ardsley, NY). Cadmium, cobalt, mercury and lead chloride were from Aldrich Chemical Co. (Bornem, Belgium).

Plasmid DNA preparations
Mini-preparation of plasmid DNA from E. coli was done by alkaline lysis method [15] and rapid isolation of plasmid DNA was done by the boiling method [16]. Mini-preparation and rapid isolation of plasmid DNA from S. aureus were described before [7].

DNA manipulations
Restriction endonuclease digestion of DNA was carried out following the manufacturer's instructions. Conversion of 5' or 3' protruding termini to blunt end was carried out with either DNA polymerase I (Klenow fragment) or T4 DNA polymerase respectively. Calf intestinal alkaline phosphatase was used to remove 5'-phosphate of the digested vector DNA to minimize self ligation  Luciferase activity assay Overnight cultures of cells containing the arsB-lur, AB or the cadA-luxAB fusion plasmids were washed twice with LB broth, suspended in LB and grown at 37°C to mid-log phase. Cells were induced by addition of increasing amounts of oxyanions or other cations for 60 to 120 min at 37°C. Uninduced cells were grown for the same period of time in the absence of heavy metal ions. Cells were diluted if necessary with ice-cold LB and 25 /zM n-decyl aldehyde (n-decanal, Sigma) was added to the sample. After vortexing for 3 s, the samples were immediately counted using the 3H channel in a Packard Tri-Carb liquid scintillation spectrometer (Packard Instrument Co., Downers Grove, IL) for 1 rain with a detection region selected between 0 and 2000 keV. The luciferase specific activity was defined as counts per min (cpm) per OD66 o unit.

Construction of a shuttle vector between E. coli and S. aureus containing the lux.4B reporter genes
This vector was constructed by cloning the replication origin of staphylococcal vector pSK265 into the Gram-negative promoter expression vector pQF70 [18] containing the luxA and luxB genes of Vibrio harveyi, lacking a transcriptional promoter, but each witff its own ribosomal binding site for protein synthesis initiation and lacking the genes luxCDE for reduction of fatty acids into aldehyde and therefore dependent upon exogenously added aldehyde. Plasmid pQF70 was cut with PvuII, dephosphorylated and ligated with pSK265 cut with HindIII (to remove its multiple cloning site) and blunt-ended. The ligation product was transformed into competent E. coli HB101 cells. Transformants were selected on ampicillin containing LB plates followed by checking the plasmid size on a 0.8% agarose electrophoresis gel. The orientation of the 2.7-kb pSK265 insert was checked by double digestion with PvulI and HindlII. Plasmids containing inserts in both orientations were obtained and termed pC100 and pC101, respectively (Fig. 1A). Both plasmids were electroporated into S. aureus strain RN4220. The stability of pC101 in E. coli and S. aureus was determined by growing the cells non-selectively for ten generations. 99% of E. coli and 100% of S. aureus cells retained the plasmid.

Construction of a transcription arsB-luxAB fusion using pClO1
The intact S. aureus 2.7-kb SalI ars determinant previously cloned into pUC19 forming pGJ103 [2] was digested with SalI and HindIII to generate two fragments of 1.262 kb and 1.438 kb. The smaller fragment containing the promoter, the intact arsR and a partial deletion of arsB (first 676 nt corresponding to 225 amino acids) was isolated by electro-elution from a 0.8% agarose gel and ligated with pC101, cut with SalI and HindIII and dephosphorylated. The ligation product was transformed in competent E. coli HB101 and ampicillin-resistant clones were selected. The cloned fragment was checked by size on a 0.8% agarose gel. Three clones were obtained all containing the same arsB-luxAB fusion. This construct was named pC200 (Fig. 1B). Plasmid pC200 was electroporated into S. aureus RN4220 and chloramphenicol-resistant clones containing the pC200 were selected.

Induction of the arsB-luxAB fusion in E. coli and S. aureus
E. coli HB101 (pC200) and S. aureus RN4220 (pC200) were induced as described in Materials and Methods in the presence of increasing concentrations of arsenite, arsenate, antimonite, manganese and bismuth. The patterns of induction ( Fig. 2A) in E. coli HB101 were similar to those observed by Ji and Silver [2] using a similar construction pGJ501, a 774-nt DNA fragment containing the intact arsR and the first 188 nt of arsB cloned into pQF70. Arsenite was the stronger inducer followed by arsenate and bismuth. The maximum light emission was obtained at 10 /xM arsenite. Arsenite, arsenate, bismuth and antimonite salts were all inducers of the S. aureus pi258 ars operon as shown with an arsB-lacZ fusion in S. aureus [2] and inducers of the E. coli R773 ars operon as shown by Wu and Rosen [19] with an arsD-bla gene fusion in E. coli HBI01. However, we currently do not know why antimonite did not function as inducer in either arsB-lux construction on pGJ501 and pC200 plasmid.
When the pC200 plasmid was placed into S. aureus RN4220 (Fig. 2B), the system was induced only by arsenite. This arsenite-specific response is different from pattern of induction for reduced arsenic uptake by the intact plasmid [20] or using a different 'reporter' gene with arsB-blaZ gene fusions in E. coli HB101 [2]. The arsenite-specific bioluminescent response may be related to the 70 x lower response of the ars-lux fusion in S. aureus. A lower level of bioluminescence in Gram-positive genera compared to Gram-negative genera was already reported before and explained as a consequence of an inadequate capacity to produce/generate reduced flavin mononucleotide in Gram-positive bacteria [13]. It is interesting to note that the S. xylus pSX267 ars operon, highly homologous to the pi258 ars operon [21] is also induced by arsenite as shown with an arsB-lip gene fusion in S. carnosus [22].
The vector plasmid pC101 without insert did not produce detectable light regardless of addition of heavy metals (data not shown). No measurable light could either be observed with plasmid pC200 when n-decyl aldehyde was not added to the assay mixture.

Construction of a transcription cadA-l~ fusion using pClO1
The S. aureus cadA 3.5-kb fragment previously cloned into plasmid pSK265 forming pGN114 [1] was digested with Sau96I to generate a 1085-bp fragment. This fragment containing the promoter, the intact cadC and the first 51 nt of cadA corresponding to 17 amino acids was isolated from a 1% agarose gel, blunt-ended and ligated with vector pC101 cut with HindIII, blunt-ended and dephosphorylated. Competent E. coli DH10B were transformed with the ligation product and ampicillin-resistant clones were screened by hy-bridization with the 21-nt oligonucleotide radioactive probe (data not shown). The cloned fragment was also checked by size on a 0.8% agarose gel. Three clones were obtained containing the desired cadA-lux.4B fusion. Clone 3 was further analyzed and the construct was termed pC300 (Fig. 1C). pC300 was electroporated in S. aureus RN4220 cells and chloramphenicol-resistant clones all contained pC300.

Induction of the cadA-lurAB fusion in E. coli and S. aureus
The induction of the cadA-luxAB fusion by heavy metals was first tested in E. coli DH10B as described in Materials and Methods. The system was only lightly induced by cadmium, bismuth and lead (Fig. 3A). Other E. coli strains (HB101, C600, S17/1)were electroporated with the pC300 and the inducibility of the cadA-luxAB fusion tested with cadmium as inducer (data not shown). Different Cd2+-induced levels of bioluminescence were observed depending on the E. coli host strain: e.g. in E. coli HB101 ceils the bioluminescence levels were about 66 x higher than with DH10B cells, but the Cd2+-induced/non-induced bioluminescence ratio remained poor (data not shown). In S. aureus RN4220 (pC300), the cadA-luxAB fusion showed a low background in the presence of any of the inducers (Fig. 3B). Cd 2+ was found to be the most efficient inducer, although higher levels of Cd 2+ were inhibitory. (Note that the strain RN4220(pC300) is cadmium-sensitive, since it lacks most of the cad.4 operon.) At high Bi 3+ and Pb 2÷ concentrations cadA-luxAB fusion was also induced, but at a lower level. This has to be related to earlier reports on the genetics of plasmid pi258 indicating a gene for marginal resistance to bismuth and lead salts mapping between bla and cadA [23]. Co 2+, Zn 2+, and Mn 2+ did not induce the cadA-luxAB fusion significantly even when the cells were exposed to high concentrations of Co 2+, Zn 2+, and Mn 2÷ in the experiments. Yoon et al. [8] constructed a translational cadA-blaZ fusion with the same first 51 nt from 5' end of cadA and also report a /3-1actamase activity when Cd 2÷, Bi 3÷ and Pb 2÷ were used as inducers. However, some differences between the aureus. E. coli HB101 and S. aureus RN4220 cells containing plasmid pC300 were grown and induced by the addition of indicated amounts by the addition of indicated levels of ions at 37°C for 60 rain and 120 min, respectively. The specific luciferase activity was measured as described in Materials and Methods. <3, cadmium; A, bismuth; zx, lead; D, zinc; I1, manganese; e, mercury. pKPY100 and the pC300 induction patterns were observed. The Cd 2+ concentration giving the maximum /3-1actamase activity was 10 x lower than the Cd 2+ concentration giving the maximum light emission. The Cd2+-induced light emission was much higher than the value obtained for Bi 3+ and Pb 2+, whereas the maximum fl-lactamase activity levels were about the same for Cd 2÷, Bi 3+ and Pb 2÷. Both differences can be explained if the maximal /3-1actamase activity is somehow limited in the/3-1actamase assay so that /3-1actamase activities became underestimated. The maximum light emission was observed at 20 /xM Cd 2÷, and to obtain a signal/noise ratio of 2 (as chosen limit of detection) 0.5 /xM Cd 2÷ (56 ppb) is required. Exposure of RN4220(pC300) cells with Cd 2÷, Bi 3+, or Pb 2÷ during 120 min instead of 60 min approximately doubled the maximum light emission (data not shown) but a 180-min Cd 2+ exposure did not further increase the light emission (data not shown). No measurable light could either be observed with pC300 if no n-decanal was added to the luciferase assay mixture. The shuttle vector pC101 containing the luxAB as reporter genes is a useful tool to detect promoter activities in a wide range of Gram-negative and Gram-positive bacteria, pC101 contains indeed origins of replication allowing self replication in Escherichia coli (pMB1) staphylococci (repC) and Pseudomonas (pRO1600). The different inducibility patterns of pC200 and pC300 in E. coli and S. aureus are not completely understood and are probably linked to the different cellular background. The cadA-luxAB fusion can be further used to test the regulation of the cadA operon. The specificity and sensitivity properties of the cadA-luxAB and the arsB-luxAB fusions can be further exploited to construct bacterial biosensors for analytical or environmental purposes.