Journal of Biotechnology, 19 (1991) 0 1991 Elsevier Science Publishers ADONIS 016816569100111H

BIOTEC

287-300 B.V. 0168-1656/91/$03.50

287

00628

Cloning and expression in Escherichia coli of mercuric ion resistance coding genes from Zymomonas mobilis T. Karunalcaran Department

of Microbiology Madurai (Received

20 May

and P. Gunasekaran

and Microbial Technology, School of Biological Kamaraj University, Madurai, India 1990;

revision

accepted

12 January

Sciences,

1991)

From a genomic library of Zymomonas mobilis prepared in Escherichia coli, two clones (carrying pZH4 and pZH5) resistant to the mercuric ion were isolated. On partial restriction analysis these two clones appeared to have the same 2.9 kb insert. Mercuric reductase activity was assayed from the Escherichia coli clone carrying pZH5 and it was Hg2+ -inducible, NADH dependent and also required 2mercaptoethanol for its activity. The plasmid pZH5 encoded three polypeptides, mercuric reductase (merA; 65 kDa), a transport protein (merT 18-17 kDa) and me& (15 kDa) as analysed by SDS-PAGE. Southern blot analysis showed the positive signal for the total DNA prepared from Hg’ Z. mobilis but not with the HgS strain which was cured for a plasmid (30 kb). These results were also confirmed by isolating this plasmid from Hg’ Z. mobilis and transforming into E. coli. Moreover the plasmid pZH5 also hybridized with the mer probes derived from Tn21. Zymomonas mobilis; Mercuric ion resistance; Cloning; Expression

Correspondence to: P. Gunasekaran, Dept. of Microbiology Biological Sciences, Madurai Kamaraj University, Madurai-625 Nomenclarure: Hgr/HgS. Mercury ion resistance/sensitive; lactoside; X-gal, 5-bromo-4-chloro-3-indolyl-8-D-palactoside.

and Microbial 021, India. Ap, Ampicillii;

Technology, IPTG,

Isopropyl

School thioga-

of

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Introduction

Bacteria have evolved a variety of means of resistance to different forms of mercury. Available data indicate that plasmid-encoded resistance to mercury is as common as antibiotic resistance (e.g. plasmids RlOO; P1258) (Summers, 1986; Silver and Misra, 1988). Plasmid-encoded resistance to mercuric ions is widespread in both Gram-positive and Gram-negative bacteria (Schottel et al., 1974; Clark et al., 1977; Weiss et al., 1977; Summers and Silver, 1978). Two general classes of plasmid-encoded mercury resistance have been defined (Schottel et al., 1974). Narrow-spectrum resistance involving the reduction of Hg2+ to metallic Hg” have been shown to provide resistance to several organomercurials including merbromin and fluorescein mercuric acetate. A broad-spectrum resistance to organomercurials such as merthiolate, phenylmercuric borate, methyhnercuric and ethyhnercuric salts has also been characterized. In a few cases resistance to mercuric ion toxicity has been demonstrated to be encoded by tranposable elements (Stanisich et al., 1977; Summers, 1986; Radford et al., 1981). It is known that Hg*+ is transported into the cytoplasm through a specific transport protein (merT product) where it is reduced to elemental Hg” by an intracellular mercuric reductase (merA product). Mercuric reductase is found to be a flavine adenine dinucleotide-containing a multimeric protein that uses NADPH as a cofactor (Schottel, 1978). Expression of the reductase system requires the induction by sub-inhibitory concentrations of Hg*+ (Summers and Silver, 1972; Foster et al., 1979). Zymomonas mobilis harbours naturally occurring plasmids (Tonomura et al., 1982; Stokes et al., 1983; Skotnicki et al., 1984) which cannot be eliminated completely from the cell. Probably these plasmids harbour some of the housekeeping genes such as resistance to various drugs and antibiotics (Skotnicki et al., 1983). Resistance to gentamycin, kanamycin and streptomycin has been localized on plasmid pRUT41 of Z. mobilis CP4 (Walia et al., 1983). However, there has been no consistency in the observations reported so far regarding the characterization of plasmids of Z. mobilis ATCC 10988 (Tonomura et al., 1982; Stokes et al., 1983; Skotnicki et al., 1984). This paper deals with the cloning and expression in E. coli of the genes from Z. mobilis which code for mercuric-ion resistance.

Materials and Methods Bacterial strains and plasmids Z. mobilis NRRL B-806 (Hg’ or Hg”) was used to prepare total DNA. E. coli strains DHSol, HBlOl and TGll were used as recipients. Vector pUC19 (Pharmacia) was used for the construction of the genomic library. Plasmid pDU1003 was a gift from T.J. Foster, UK and pPG102 was obtained from A.O. Summers, U.S.A.

289

Growth conditions 2. mobilis strains were grown in RM medium (glucose, 2.0%; yeast extract, 1.0%;

KH,PO,,

0.2%; pH 6.0) (Skotnicki et al., 1984) at 30°C without shaking. Hg’ 2. lo* cells on RM agar plates containing mercuric acetate (50 pg ml-‘) and incubating for 72 h. E. coli strains were grown aerobically in LB medium (tryptone, 1.0%; yeast extract, 0.5%; and NaCl, 1.0%; pH 7.4) with or without antibiotic Ap (50 pg ml-‘) or mercury salt (50 pg ml-‘) at 37°C. mobilis strains were isolated by plating

Genomic library of Z. mobilis in E. coli

Total DNA was isolated from Z. mobilis (Hg’) as described by Byun et al. (1986) and was partially digested with Sau3A, ethanol precipitated, suspended in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) buffer and layered onto a 5 to 40% (w/v) sucrose gradient. The gradient was centrifuged in a Beckman SW41 rotor at 26,000 rpm for 18 h at 5°C and then fractionated. The fractions were analysed for DNA content and size by electrophoresis on a 0.7% agarose gel. Fractions containing DNA fragments were pooled, ethanol precipitated and resuspended in TE buffer. The fragments were ligated into the dephosphorylated BamHI site of pUC19 with T4 DNA ligase (Boehringer Marmheim, F.R.G.) at 15°C for 18 h and used to transform E. coli DHSa. The transformants were selected on LB agar containing Ap (50 mg l-l), IPTG (40 mg 1-i) and X-Gal (20 mg 1-i). The white colonies were selected as recombinant clones. Screening for the recombinant clones

The E. coli recombinant clones expressing mercury resistance were selected on LB agar plates containing Ap (50 pg ml-‘) and mercuric acetate (50 pg ml-‘). The plates were incubated for 72 h. They were checked further for their resistance to mercuric chloride (50 pg ml-‘). The clones growing on these plates were selected as Hg’ clones. DNA manipulations

Rapid preparation of plasmid DNA from E. coli strains was carried out using the modified alkaline lysis method of Bimboim and Doly (1979). Plasmid DNA for cloning experiments was further purified by CsCl/EtBr gradient centrifugation. The DNA fragments from agarose gels were obtained by electroelution and further purification was achieved by passing them through an Elutip-d column (Schleicher & Schuell, Dassel, RFA). All cloning experiments were carried out according to the standard methods described by Maniatis et al. (1982). Southern hybridizations

The total DNA of Z. mobilis was digested with EcoRl endonuclease as specified by the suppliers and the fragments were seperated on 0.7% agarose gels. Southern

290

transfer to nitrocellulose (Hybond-Amersham) was performed according to the standard procedures (Maniatis et al., 1982). The DNA probe (pZH5) was labelled by nick translation using cu’2P-dCTP (specific activity 3000 Ci mMole-‘) by using a nick translation kit (Amersham) with specific activity of approx. lo8 cpm pg-‘. After prehybridization, the labelled probe was allowed to hybridize to Southern blots for 18 h at 55°C according to the standard procedure (Amersham). The blots were then washed, three times (for 5 min each) in 2 x SSPE and 0.5% SDS at 37”C, twice (for 10 min each) in 1 X SSPE at 50°C and finally rinsed in 1 X SSPE at room temperature. Autoradiographic analysis was used to determine the hybridization pattern. Assay of Hg-dependent NADPH

oxidation

Crude extracts of E. coli carrying the recombinant plasmid pZH5 were prepared at 4°C as follows. Cells from 150 ml culture grown in the presence of 5 pg of HgCl, per ml were harvested by centrifugation at 8000 rpm for 20 mm. The cell pellets were washed and resuspended in 1 ml of 50 mM phosphate buffer (pH 7.4) and disrupted with a Branson sonifier on ice. Debris was removed by centrifugation at 8000 r-pm for 15 min. Supematants were used as the crude extracts (Blaghen et al., 1983; Shiratori et al., 1989). Routine enzyme assays were carried out at 37’C in 50 mM Tris-HCl (pH 7.4) buffer supplemented with 100 PM NADPH, 10 mM 2-mercaptoethanol, 15 pg HgCl, with or without 10 ~1 crude enzyme extract. The oxidation was followed spectrophotometrically at 340 nm. Protein concentration was determind by the method of Lowry et al. (1951) with bovine serum albumin as a standard. Analysis of plasmid pZH.5 encoded proteins by SDS-PAGE E. coli (pZH5) was grown in LB medium with Ap (50 pg ml-‘) and HgCl, (5 pg ml-‘). Cells were collected by centrifugation, suspended in lysis buffer and broken by shaking with glass beads (Maynard and Kuramitsu, 1979). After centrifugation, the supematant was suspended in gel loading buffer and heated at 80 o C for 3 min. Samples were loaded onto an SDS-polyacrylamide gel (12% w/v) (Laernmli, 1970). Coomassie blue stained gels were analysed by LKB Ultroscan XL. Curing of plasmid in Z. mobilis

Mercury resistant Z. mobilis ATCC 10988 cells were grown in RM overnight with ethidium bromide (75 pg ml-‘), washed and resuspended in RM medium. After 20 h of growth, cells were plated on RM agar medium and individual colonies were scored on RM agar plates. After growing for 48 h, colonies were replicated on RM agar. Colonies that did not grow on HgCl, containing plates but grew on RM agar plates were selected as Hg” colonies (Miller, 1972).

291

Results

Cloning of Z. mobilis mercwy resistance genes in E. coli A genomic library of the total DNA of Z. mobilis was constructed by cloning Sau3A digested and size-fractionated fragments into the BamHI site of the plasmid pUC19. The recombinant clones (1100 clones) of E. coli DHSa were screened on the Ap, Hg-Acetate LB agar plates. Two clones displayed resistance to mercury. E. coli DHSa carrying pUC19 did not grow on the plates containing mercuric acetate (MIC 4 pg ml-‘). Both recombinant clones exhibited resistance to mercuric acetate up to 100 /.lg ml-‘, three times higher than the level of resistance exhibited by Z. mobilis. These clones were also resistant to mercuric chloride (60 ,clg ml-‘) on plates. Both the clones contained the same size fragment (2.9 kb) and showed a similar type of restriction pattern. Plasmid pZH5 was selected for further analysis. Its ability to confer mercuric resistance was confirmed by retransforming E. coli strains HBlOl, DHSa and TGll with pZH5. All transformants were found to inherit both the Ap and mercury resistance phenotype. This result along with that of NADPH oxidation presented in the subsequent section suggested that the mercury resistance gene of Z. mobilis was expressed in E. coli. Restriction mapping of the plasmid pZH5 Both the recombinant plasmids pZH4 and pZH5 exhibited the presence of a 2.9 kb insert fragment. Analysis of the results obtained from the single and double digestion of the plasmid pZH5 with restriction enzymes lead to the construction of the physical map of the fragment which is shown in Fig. 1. The plasmid pZH5

I

I

IO.51 10.6

2

I

1.1 1

I

I.6

I

I.3

I.9

I.3 I

3

I

I 1

23 2.2

I in

I

Hpa

IO.4 1

BgII

1.1

kb

I

Hind

III

I

SphI

IO.6

1

Sac I

IO.7

1

Ava II

I.6



Ikb



Fig. 1. Restriction mapping of pZH5. The thin line indicates the DNA insert which originated from plasmid of Z. mobilis; shadowed bar indicates the vector pUC19. HI- HpaI, BI-BglI, EI- EcoRI, SacI, KI- KpnI, Sp- SphI, HIII- Hi&III, A-AuaII, PI- &I.

the SI-

292

carried two sites for each of the enzymes Bg0 and &a1 in the insert. Hi&III, SphI, Sac1 and AuaII shared a single site each in the insert fragment. Mercuric reductase is coded by the plosmid pZHS

Crude enzyme extract was prepared from E. coli (pZH5) grown for 16 h in LB medium containing HgCl, (5 pg ml-‘) as described in Materials and Methods. When HgCl, (15 pg ml-‘) was added to an assay mixture with the cell extracts (7 m3 of ProtW, A,,, (A,, of reduced NADP) was decreased significantly. Half of the reaction mixture in which HgCl, had been omitted was removed, mixed with 15 pg of HgCl, per ml and incubated at 37’C. The remaining half of the reaction mixture was allowed to continue as before. NADPH oxidation was observed only in the half of the reaction mixture into which HgCl, had been added. In the absence of mercuric chloride, A,, did not change even after 75 min of incubation. Mercuric reductase activity was optimal with an NADPH concentration near 60 PM which also required the presence of 2-mercaptoethanol. This indicated that the Hg-dependent NADPH oxidation by the mercuric reductase (Fig. 2) was encoded by the plasmid pZH5. Study of the thermostability of the enzyme revealed that 60% of the activity remained after treatment at 80°C for 15 min, whereas the enzyme was completely inactivated after incubating at 100°C for 10 min. Incubation of the reaction mixture with the cell extract and HgCl, at 42°C has shown near maximal activity of the mercuric reductase. This enzymatic activity did not differ from that of other similar enzymes in Pseudomonas aeruginosu (Summers and Sugarman, 1974; Schottel,

HgC12

(mM1

Fig. 2. Effect of mercuric chloride on oxidation of NADPH by the crude extract of E. coli carrying l : 10 mM 2-mercaptoethanol; x X: 5 mM 2-mercaptoethanol; o0: 2 pZH5. l A), 5 mM (HmM 2-mercaptoethanol; E. coii carrying pUC19 with 2 mM (An ) and 10 mM 0) of 2-mercaptoethanol. to-

293

1978), Thiobacillus ferroxidans (Blaghen et al., 1983).

Hg’ +-inducible

(Olson et al., 1982) and Yersinia enterocolitica

polypeptides coded by the plasmid pZH5

The plasmid pZH5 was tested for the ability to direct the synthesis of Hg*+-inducible polypeptides in E. coli. The profile of mer polypeptides encoded by this plasmid had the molecular masses: 65 kDa, 18-17 kDa and 15 kDa on SDS-PAGE (Fig. 3). In addition, two more polypeptides of 44 and 42 kDa were also observed. The 65 kDa polypeptide was believed to be the mercuric reductase (merA) which has been reported previously for other mer operons (Fox and Walsh, 1982; Jackson and Summers, 1982a,b; Ni’Bhrian and Foster, 1986). Hg*+-inducible proteins of approx. 17 and 18 kDa were considered to be the product of merT, which could be resolved but frequently migrated as a doublet or as a single band (Ni’Bhrian and Foster, 1986). Yet another new protein migrated around 15 kDa and this could be the product of mer C. An intense band was also seen below the merC product and this might be analogous to either the product of merD or merP. In addition, the proteins of molecular mass 22 and 20 kDa were also detected. The polypeptides 44 and 42 kDa might be the dimer forms of the proteins 22 and 20 kDa and these proteins appeared not to have any role in regulation of the mer operon and indeed were reported previously for other mer systems (Barrineau and Summers, 1983; Ni’Bhrian et al., 1983; Ni’Bhrian and Foster, 1986).

-

MER A

- f3. LACTP rMASE

LMER -MER -MER

T C D or P (?I

Fig. 3. Hg*+-inducible polypeptides of the plasmid pZH5. Lane A: mobility of standard weight markers. Lane B: protein pattern of E. co/i carrying pUC19. Lane C: protein pattern carrying pZH5; the positions of merA, merT and me& are marked.

molecular of E. coli

294

Mercury resistance is coded by a megaplasmid of Z. mobilis Southern transfer and DNA hybridization were performed to confirm the origin of the 2.9 kb DNA insert in the plasmid pZH5. The total DNA of both Hg’ and Hg” strains of Z. mobilis was digested with EcoRI. The restricted fragments were separated on an agarose gel (0.7%), transferred to a nitrocellulose filter and hybridized to nick-translated pZH5 using (032P)-dCTP. Two positive signals were observed with the EcoRI restricted total DNA of Hg’ Z. mobilis. However, no such

E

F

G

D kb

- 23.1 - 9.4 - 6.5

-

2.3

Fig. 4. Southern blotting and DNA hybridization of pZH5. (a) Lane A: lambda Hind111 restricted fragments; Lane B: EcoRl digested total DNA of Z. mobilis (Hg’); Lane C: EcoRI digested total DNA of Z. mobilis (Hg’); Lane D: electroeluted 30.0 kb plasmid of Z. mobilis (Hg’); Lanes E, F, G: autoradiogram of the lanes B, C and D. (b) Lane A: pDU1003 (contains merRTPCAD of Tn21); Lane B: pPGlO2 (contains merC N-terminal part of merA of Tn21); Lane C: lambda Hi&III restricted fragments; Lanes D, E: autoradiogram of the lanes A, B.

295

ABCDEFG kl

Fig. 5. Plasmid profile of Z. mobih (Hg’ and HgS) strains. Lane A: fragments; Lane B: native plasmid profile of Z. mobilis (Hg’) ATCC 10988; of Z. mobilis (HgS); Lanes E, F, G: E. coli Hg’ transformants carrying

lambda Hind111 restricted Lanes C, D: plasmid profile the 30.0 kb plasmid.

hybridization signals were detected with the EcoRI digest of HgS Z. mobilis DNA (Fig. 4). These results suggested the absence of an homologous region in HgS Z. mobilis. This tempted us to look for the plasmid profile of the Hg’ and Hg” strains of Z. mobilis. A report (Ogale and Deobagkar, 1988) also suggested the presence of Hg’ in one of the plasmids. Three of 470 colonies, after curing with ethidium bromide (75 pg ml-‘), were found to be HgS. These colonies were grown individually to late-exponential phase in RM medium and the native plasmid profile was analysed in comparison with Hg’ Z. mobilis. From Fig. 5 it was clear that these mercury-sensitive colonies were cured for a megaplasmid of size 30 kb. In order to confirm further the origin of the insert fragment in pZH5, the total plasmids of Z. mobilis were analysed, which exhibited the presence of five plasmids 1.7, 2.9, 4.0, 30.0 and 69.0 kb (Fig. 5). Individual plasmids were isolated and analysed. A positive hybridization signal was obtained for the electroeluted 30 kb plasmid of Hg’ Z. mobilis (Fig. 4). These results indicated that the insert present in pZH5 had originated from this plasmid. Another line of evidence was obtained by electroeluting this 30 kb plasmid from Hg’ Z. mobilis and transforming it into E. coli DHScu. Transformants were analysed for the plasmid profile. On agarose gel electrophoresis, they were found to contain the plasmid corresponding to the plasmid of 30 kb size (Fig. 5). These transformants were resistant to HgCl, (60 pg ml-‘), the level that was twice that of the Hg’ Z. mobilis strain. However, these two, E. coli Hg’ transformant and the original clone carrying pZH5, were found to grow after a definite lag in LB medium containing 40 pg ml-’ HgCl,. Hence in the experiments, sub-inhibitory concentration (5 pg ml-‘) of HgCl, was used just to induce the plasmid coded Hg’ genes. During non-selective

296

conditions, only 19% of the population retained the Hg’ after 25 generations. This could be due to the instability of the origin of the replication of the Zymomonus plasmid (30 kb) in E. coli. The plasmid pZH5 also hybridized with pDU1003 (carrying the mer operon of Tn21) and pPG102 (carrying the merC and part of the N-terminus portion of merA of Tn21) (Fig. 4b). This could be possible only when pZHS shared DNA homology with these probes. The major difference between mer of Tn501 and mer of Tn21 is that the former lacks the mer C. The presence of the hybridization signal for merC and merA suggested that the 2.9 kb insert present in the pZH5 closely resembled the mer operon of the Tn21 transposon.

Discussion Two E. coli clones expressing mercuric ion resistance were isolated from a genomic library of Z. mobilis. Both the recombinant plasmids were found to exhibit a resistance level that was three times higher than that of the Hg’ Z. mobilis. These two clones contained the same size (2.9 kb) insert fragment and consequently one of the clones carrying pZH5 was used for further studies. To confirm recombinant plasmid pZH5 coded mercury resistance in E. coli the plasmid was isolated and transformed into E. cofi strains HBlOl, TGll and DHSa. All the transformants inherited resistance to both Ap and Hg. Mercuric reductase activity was assayed from the E. coli clone carrying pZH5 and it was the Hg2+ -inducible and NADPH-dependent which also required the thiol compound, 2-mercaptoethanol for its activity. No reductase activity could be detected in uninduced samples. These results along with those of the above, indicated that the expression of the gene coding formed resistance from its own promoter rather than the promoters in the vector. Moreover, this activity did not seem to differ from that of similar enzymes reported earlier in other bacterial species (Summers and Sugarman, 1974; Schottel, 1978; Olson et al., 1982; Blaghen et al., 1983). The plasmid pZH5 encoded three identified polypeptides (65 kDa, 17-18 kDa and 15 kDa) anologous to the products observed with other mer operons and two other unidentified polypeptides (44 and 42 kDa) in E. coli. Hg’+-inducible polypeptides 65 kDa were the mercuric reductase (merA) subunit and its clipped form, which had been reported previously in other mer systems (Fox and Walsh, 1982; Jackson and Summers, 1982a,b; Ni’Bhrian and Foster, 1986) and 17 and 18 kDa were the product of merT, the hydrophobic membrane protein likely to be responsible for uptake of Hg2+ ions (Ni’Bhrian and Foster, 1986; Silver and Misra, 1988). Yet another protein migrated around 15 kDa. This protein could not be the product of merR (Barrineau et al., 1984) as, being a regulatory protein like merR, was unlikely to be present in large amounts and the product of merR could not be detected even in a minicell system without amplification (Ni’Bhrian et al, 1983; Ni’Bhrian and Foster, 1986). Hence this polypeptide might be the Hg2+-inducible product of merC. A broad and intense band migrated below this merC product

297

which might be the product of either merD or merP. The unidentified polypeptides, 44 and 42 kDa appeared to be a dimer form of the proteins 22 and 20 kDa and they may not have any role in regulation of the mer operon. These products were indeed reported in other mer systems even in merA+, merA::TnS and merD::Tn5 backgrounds and they were suggested to be the artifacts produced by the use of multicopy mer plasmids. However, they were not seen in samples derived from low-copy-number mer plasmids (Ni’Bhrian et al., 1983; Ni’Bhrian and Foster, 1986). Such artifacts had also been described with X mer transducing phages in E. coli maxicells (Dempsey and McIntire, 1979). Two sources of evidence suggested the possibility of the Hg’ coded by 30 kb native plasmid of Z. mobilis: (a) by curing one of the native plasmids that lead to loss of Hg’ in Z. mobilis, (b) electroeluting the 30 kb plasmid and transforming it into E. coli DH5a made the E. coli transformant resistant to Hg2+. These results suggested that the origin of the Hg’ lies on the megaplasmid (30 kb) of Z. mobilis. Southern hybridization data suggested that a positive signal for hybridization could be obtained in the EcoRI digested total DNA of Z. mobifis resistant to mercury but not in the strain sensitive to mercury. Moreover pZH5 strongly exhibited hybridization with the electroeluted 30 kb plasmid. The plasmids of 30.0 and 69.0 kb were found to be related on restriction analysis and designated as el and e2 bands of pZM06 (Scordaki and Drainas, 1987). Restriction analysis and hybridization of pZM06 showed that this plasmid was not related to the other native plasmids of Z. mobilis ATCC 10988 (Scordaki and Drainas, 1987). However, inconsistent reports were available regarding the native plasmids of Z. mobilis. Recently Ogale and Deobagkar (1988) reported that HgCl, resistance in Z. mobilis was encoded by a high molecular weight plasmid but they did not specify the plasmid responsible for this. However, here two lines of evidence are presented to demonstrate the plasmid responsible for Hg’. The results presented suggested the presence of part of an operon containing merT, A, C, P or D genes in Z. mobilis. The mer operon of Tn501 is comprised of mer RTPAD genes while Tn21 of the plasmid RlOO has mer RTPCAD genes (Silver and Misra, 1988). Recently, Gilbert and Summers (1988) reported that merc-positive class of the mer locus was almost exclusively found on the plasmid of E. coli and related to facultative anaerobic bacteria that had a limited host-range. Hybridization of mer probes pDU1003 and pPG102 with pZH5 presented a positive signal and especially the hybridization with the merC and part of N-terminal portion of merA of the latter probe indicated that the merC-like gene might be present in the 2.9 kb DNA insert of pZH5. These results suggest that the Z. mobilis mer system resembled the mer of Tn21. However, so far no report is available to confirm the presence of a transposon in Z. mobilis. The Hg2+ resistance system is found mainly on plasmids and transposons in bacterial species (Summers and Silver, 1978; Foster, 1983; Robinson and Tuovinen, 1984; Silver and Misra, 1988) with a few exceptions (Witte et al., 1986; Wang et al., 1987). In Z. mobilis most of the plasmids are cryptic and no markers have been ascribed for these plasmids except the plasmid pRUT41 which was found to carry gentamycin, kanamycin and streptomycin markers (Walia et al., 1983). Recently

298

Ogale and Deobagkar (1988) reported the presence of mercuric resistance in a high molecular weight plasmid of Z. mobilis. In a different approach, mercuric ion resistance coding genes from Z. mobilis were cloned in E. coli; its homology with transposon Tn21, NADPH dependent mercuric reductase activity of the cloned Hg’ genes in E. coli and the Hg2+ -inducible polypeptides encoded by the recombinant plasmid pZH5 are presented here for the first time.

The authors thank Dr. C.P. Kurtzrnan, NRRL, Peoria, U.S.A. for the Z. mobilis strain, Dr. J. Baratti, CNRS, Marseille, France for facilities, Dr. T.J. Foster, University of Dublin, Dublin, Ireland for providing the plasmid pDU1003, Dr. A.O. Summers, University of Georgia, Athens, U.S.A. for providing the plasmid pPG102 and Dr. S. Silver, University of Chicago College of Medicine, Chicago, U.S.A. for helpful advice and CSIR-India for the financial support.

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Cloning and expression in Escherichia coli of mercuric ion resistance coding genes from Zymomonas mobilis.

From a genomic library of Zymomonas mobilis prepared in Escherichia coli, two clones (carrying pZH4 and pZH5) resistant to the mercuric ion were isola...
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