Cloning and comparison of mercury- and organomercurial-resistance determinants from a Pseudomonas stutzeri plasmid

Gene, 166 (1995) 77 82 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

77

GENE 09260

Cloning and comparison of mercury- and organomercurial-resistance determinants from a Pseudomonas stutzeri plasmid (Broad spectrum; transposon; mer; organomercurial lyase; inverted repeat)

Daniela Reniero, Enrica Galli and Paola Barbieri Dipartimento di Genetica e di Biologia dei Microrganismi, Milano, Italy

Received by A.M. Chakrabarty: 16 March 1995; Revised/Accepted: 26 June/5 July 1995; Received at publishers: 8 August 1995

SUMMARY

Plasmid pPB confers broad-spectrum mercury resistance (Hg R) to a Pseudomonas stutzeri strain. Two pPB regions, separated by 25-30 kb and sharing homology with Tn501 mer (Hg detoxification) genes, were cloned separately and each was shown to carry a cluster of functional and independently regulated mer genes. One of the two gene clusters conferred resistance only to inorganic mercury, and had a structure identical to the classical model of narrow-spectrum mer operons. In the other cluster a novel rnerB gene, not homologous to the other known merB, but with the same function, was mapped upstream from merA, interposed between an organomercurial-responsive regulatory element and transport genes. Evidence suggests that merB and the other structural mer genes might be transcribed from two distinct promoters. The presence of two inverted repeat-like elements, identical to those of Tn5053, upstream from merR suggests that the pPB broad-spectrum-gene cluster could be part of a transposon-like element.

INTRODUCTION

Mercury resistance (HgR), a feature of several bacteria isolated from different environments, has been widely studied at both the biochemical and genetic level (Misra, 1992; Silver and Walderhaug, 1992; Summers, 1992). A narrow spectrum operon confers resistance to inorganic Hg and typically contains merR, which is transcribed divergently from the other mer genes and encodes a represCorrespondence to: Dr. P. Barbieri, Dipartimento di Genetica e di Biologia dei Microrganismi, Universit/~ degli Studi di Milano, Via Celoria 26, 20133 Milano, Italia. Tel. (39-2) 266-05227; Fax (39-2) 266-4551; e-mail: [email protected]

Abbreviations: aa, amino acid(s); bp, base pair(s); A, deletion; HgR, Hg 2+ reductase; IPTG, isopropyl-13-D-thiogalactopyranoside; IR, inverted repeat(s);kb, kilobase(s) or 1000bp; LB, Luria-Bertani (medium); met, genes for mercury detoxification; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; PMA, phenylmercuric acetate; R, resistance/ resistant; RBS, ribosome-binding site(s); SDS, sodium dodecyl sulfate; Tn, transposon. SSD1 0378-1119(95)00546-3

sor/activator protein, and structural genes, which encode a transport system (merTP and, in some cases, an additional merC gene), mercuric reductase (merA), and a MerD protein, involved in down-regulation of the operon (Mukhopadhyay et al., 1991). Broad-spectrum resistance, i.e., resistance to organomercurials such as phenylmercuric acetate (PMA), methylmercuric chloride and thimerosal, requires the presence of an additional gene, merB, that codes for organomercurial lyase which hydrolizes the C-Hg bond before Hg 2+ reduction. In most cases merB is linked to the other mer genes and maps immediately downstream from merA (Griffin et al., 1987; Laddaga et al., 1987; Sedlmeir and Altenbuchner, 1992). pPB is an 80-kb indigenous plasmid which confers broad-spectrum Hg R to a Pseudomonas stutzeri strain originally isolated for its ability to degrade o-xylene (Baggi et al., 1987; Barbieri et al., 1989). In this work we report that pPB carries two functional mer operons, one conferring narrow-spectrum, the other broad-spectrum resistance; the structure and a part of nt sequence of the latter is compared with known met operons.

78 followed by a merA gene (Fig. 1). Cells carrying pPB105 (HindlII fragment from pPBI0, coordinates 1.5-4.3 in Fig. 1), grown in the presence of I P T G , were hypersensitive to HgC12 and P M A only when the HindlII fragment was cloned in one of the two possible orientations, suggesting that the pPB105 insert lacks a sequence involved in transcription of transport genes.

EXPERIMENTAL AND DISCUSSION

(a) Cloning and mapping of m e t genes Two non-adjacent pPB fragments, which were shown to share homology with the Tn501 merTPAD genes (Fig. 1), were separately cloned in pUC19 (Vieira and Messing, 1982) and transformed into E. coli JM109 (Yanisch-Perron et al., 1985). The host cells levels of resistance were then evaluated by the disk method in LB medium with 30 ~tg of HgC12 or PMA. Both pPB10 (PstI D fragment of pPB, coordinates 0.8-8.3 in pPB117 map) and pPB20 (KpnI C) conferred resistance to HgC12 but not to PMA. Subcloning from pPB10 and pPB20 led to the mapping, in both clusters, of functional transport genes

(b) Location of organomercurial resistance determinant P M A resistance was achieved by cloning a SalI-PstI fragment (pPB117, Fig. 1) that overlapped the pPB10 insert. Although pPB 117 showed no hybridization signals with the merB gene of pDU1358 (data not shown), it was found that its presence conferred resistance not only to HgC12 and PMA, but also to methylmercuric chloride

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Fig. 1. Restriction maps of plasmid pPB and its cloned fragments. The letters inside the physical circular map of pPB represent the restriction fragments obtained after digestion with PstI, KpnI and NotI. The dashed lines outside the pPB circular map represent the two regions homologous to the Tn501 merTPADgenes. The dotted line represents the region homologous to the Tn501 mpR,A genes. OP1, operon 1 (broad spectrum); OP2, operon 2 (narrow spectrum). The maps of pPB117 and of pPB20 are, respectively,clockwise and counterclockwisewith respect to the circular map of pPB. Orientation of the two gene clusters was revealed by subcloning and Southern hybridizations. The gene arrangement and the putative promoters are shown below the maps. With the exception of sequenced genes, gene size has been assigned in analogy to Tn21 or TnS01 genes. The presence of genes indicated in parentheses can be hypothesized but has not been investigated.

79 and thimerosal, and promoted Hg volatilization in all these compounds (data not shown). Thus, the genetic product of the organomercurial resistance determinant of pPB seems to be functionally analogous to that of the merB of already known systems. In pPB117 merB was mapped by deletion analysis and by complementation. A 0.9-kb HindIII fragment (coordinates 0.6-1.5) (pPB106) was found to be the minimum fragment able to complement the PMA sensitivity of the pPB10 insert. Complementation occurred only when the pPB106 insert was cloned with the lac promoter at the left end of the fragment and the host cells were induced with IPTG, suggesting that merB is transcribed from a promoter different from that of the transport genes but in the same direction. The direction of transcription of merB was confirmed by SDS-PAGE (data not shown). pPB106 was not homologous with other pPB regions (data not shown) so it can be supposed that only a single copy of this gene is present on pPB. Thus, pPB resembles the pDU1358 of Serratia mareescens (Griffin et al., 1987), from which two mer operons, one broad and one narrowspectrum resistance, have been cloned. Several broadspectrum resistance operons have been studied and merB has always mapped downstream from merA, in most cases contiguous (Griffin et al., 1987; Laddaga et al., 1987; Sedlmeier and Altenbuchner, 1992), or a few kb downstream (Wang et al., 1989). In our case merB mapped upstream from the transport genes, a gene order that is unreported, to the best of our knowledge, for any other broad-spectrum Hg R operon.

(c) Inducibility of Hg 2+ reductase (HgR) Inducibility of HgR was assayed in E. col±cells carrying different recombinant plasmids (Table I). In cells carrying p P B l l 7 HgR activity was inducible by both HgClz and PMA. The insert of pPB10 produced comparable levels of HgR activity regardless of its orientation. The second mer gene cluster was found to be independently regulated. In this case HgR was inducible by HgC12 while PMA seemed to be a less efficient inducer. In both gene clusters the deletion of merR resulted in high levels of HgR activity, suggesting that the product of the two merR genes can act as a repressor, although we cannot exclude an activating role. (d) The nt sequence of the beginning of the broadspectrum operon The nt sequence of both strands of the left end of the p P B l l 7 insert (broad-spectrum resistance) (Fig. 2) was determined directly from pPB117 and its deleted derivatives, using commercial kits with either the M13/pUC universal primer or custom designed oligos.

TABLE I Induction of HgR activity (nmol rain per mg-~ protein) in E. col± JM109 a Plasmid b

pPBll7 pPBll7A03 pPB10 pPB20 a pPB209 pPB206

Inducerc None

HgCI 2

PMA

18.0±13.49 132.3±9.84 207.3±77.63 16.8±21.18 14.6±11.46 182.3 ± 15.10

225.6± 18.78 149.6_+13.42 212.0±43.15 120.6± 16.00 165.6±37.39 268.6± 11.61

237.3 ±31.64 153.3+6.01 248.0+37.06 47.7±69.60 N.D. 271.3 ± 12.39

a Exponentially growing cells (A60on m approx. 0.6) induced with a sublethal concentration of HgCl 2 (5 ~g/ml) or PMA (3 p.g/ml) were harvested, washed, resuspended in a suitable amount of lysis buffer (20 mM Tris-HCl pH 8/150 mM KCI/5 mM MgC12/0.01% Triton X-100/1 mM DTT) and disrupted by vortexing with l/3vol, of glass beads (150-212 ram) (Sigma); cell debris and glass beads were removed by

centrifugationin a microfugefor 10 min at 4°C. The supernatants were kept on ice and used on the day of preparation. Total protein content was estimated by the BCA assay (Sigma) at 37°C, using bovine serum albumine as standard. HgR activitywas assayedas previouslydescribed (Barbieri et al., 1989). b pPBll7A03; 300-bp deletion at the Sail end of the pPBll7 insert, HgRPMAR. pPB209, carries the HindlII-NotI fragment (coordinates 1.5-4.8 in Fig. 1) subcloned from pPB20, HgRPMAs. pPB206, carries the AvaI fragment (coordinates 2.1 4.6 in Fig. 1) subcloned from pPB20, HgRPMAs. c Data are means±SD of three independent experiments; N.D., not determined. d Data are means± SD of five independent experiments.

The sequence, nt 40-600, shows 94% identity with that of Tn5053 (Kholodii et al., 1993). As in Tn5053, 25 nt, which strongly resemble the IR of the In2 integron (Brown et al., 1986), and 38 nt, almost identical to the IR of Tn21 (Misra et al., 1984), were found at the beginning of the operon. The merR gene is highly homologous to already known merR genes and the predicted 144-aa polypeptide showed 88, 90.3 and 94.4% identity with TnS01, pDU1358 and Tn5053 MerR, respectively. Eight of the ten C-terminal aa which make the pDU 1358 broadspectrum MerR interactable with aromatic organomercurials (Yu et al., 1994), are conserved in pPB broad-spectrum MerR. Downstream from merR there is a sequence corresponding to the typical PmerT, which, in this case, might function as a merB promoter. The alignment with known mer operons proceeds with a putative PmerR and breaks down at nt 656. Within the HindIII fragment, which complements the PMA sensitivity of pPB10, a sequence showing no homology with any known merB gene was found. The analysis revealed the presence of an ORF, with its putative RBS, starting at nt 685 and ending at nt 1344. The predicted 219-aa polypep-

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Fig. 2. Nucleotide sequence of 1740 bp of the pPB plasmid broad-spectrum mer gene cluster, including the merB region, from plasmid pPBll7. Sequencing terminated in the inerT gene. RBS and predicted aa sequences are shown. Asterisks (*) represent stop codons. The IR-like elements are marked above the sequence. The putative promoters of the structural genes, showing the typical 19-bp spacing and the dyad sequence (bold line above), and the putative PmerRare underlined. The element underlined with the dashed line is thought to be unfunctional (see sections d and e). The nt sequence has been deposited in the GenBank data library under accession No. U21000. c); on the contrary, the element identical to PmerR,which in this context seems to be incongruous, might represent the remains of a recombination event (see section e) and is probably unfuctional. Thus, in accordance with the data reported above, m e r B and the transport genes of the pPB broad-spectrum operon could be transcribed from two distinct promoters. In this case, m e r B could also be regulated independently of the other m e r genes, although this seems unlikely, since organomercurial lyase activity was found in HgC12induced E. coli cells carrying the whole operon (data not shown).

tide was aligned with the four available MerB aa sequences and a significant degree of similarity was found within the central 100 aa (Fig. 3). Three conserved Cys residues (C wS, C 129, C~71), thought to be important in the reaction mechanism (Misra, 1992), were found. The degree of similarity drastically decreases at the N- and C-terminal regions. H o m o l o g y with known m e r operons resumes beyond nt 1423, showing two elements identical to pDU1358 PmerTand PmerRfollowed by a m e r T gene highly homologous to that of both pDU1358 and T n 5 0 1 . This putative PmerTactually seems to be functional (see sections a and

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174 167 162 165 167

Fig. 3. Central part (aa 80-174) of the deduced aa sequence of the pPB MerB compared with those of Bacillus sp. (Ba, Wang et al., 1989), pDU1358 (pDU, Griffin et al., 1987), pi258 (pI, Laddaga et al., 1987) and S. lividans (St, Sedlmeier and Altenbuchner, 1992). Asterisks (*) indicate residues identical in all the sequences; (#) those conserved in two of the five sequences and present in pPB MerB; (V) conserved Cys residues.

81

(e) Conclusions

ACKNOWLEDGEMENTS

(1) Plasmid pPB carries two regions, separated by 25-30 kb, which harbour functional genes coding for Hgr~; only one of these two gene clusters confers broadspectrum resistance. In both regions mer genes spanned approx. 5 kb and were found to be in opposite orientations on the physical map of pPB. (2) The structure of the narrow-spectrum resistance gene cluster strongly resembles the one of the classical model of narrow-spectrum mer operons. On the contrary, the gene arrangement in the broad-spectrum resistance operon does not correspond to any other so far reported, the organomercurial resistance determinant having been mapped between the regulatory gene and the transport genes. (3) The pPB organomercurial resistance determinant is not homologous to any other known merB gene, however it codes for an organomercurial lyase activity and the predicted polypeptide has, at least in its central part, a significant degree of similarity with other MerB proteins. Thus, it can be considered a novel merB gene. The position, and the presence itself, of this peculiar version of merB flanked by sequences which otherwise are highly homologous to those of already known mer operons, strongly suggest that this determinant has been secondarily acquired, by an insertional event, from an unknown source. This hypothesis might also explain the presence of promoters both upstream and downstream from merB; in particular the element identical to PmerR found downstream from merB may represent the remains of the original operon after the insertion of merB itself. (4) Gene transposition and/or duplication are not uncommon in Pseudomonas and related bacteria and, together with recombination, are considered important mechanisms for the evolution of microorganisms. Such mechanisms can also account for the presence of two mer operons on pPB, such operons not conferring specific selective advantage to the cells but a hypothetical, and not demonstrated, more efficient detoxification of Hg 2+ . Recombination events are also suggested by the presence of some conserved restriction sites in the terminal part of the two operons. (5) The presence on pPB of IR-like elements, and of sequences sharing homology with the Tn501 tnpR and tnpA genes (Fig. 2), together with the observation of rather frequent rearrangements in the pPB map (not shown in the present work) and the high frequency of formation of pPB::RP4 hybrids when RP4 was used to mobilize pPB (Barbieri et al., 1989), suggest that the pPB broad-spectrum mer operon could be, or have been, part of a transposon-like structure, considering that mer genes are often part of transposons.

This work was supported by the Ministero dell'Universitfi e della Ricerca Scientifica e Tecnologica (40%) and by the Consiglio Nazionale delle Ricerche (CNR), Rome, grant No. 9201197 PF70 of the P.F. Biotecnologie e Biostrumentazione. Part of the experiments were carried out in the Department of Microbiology and Immunology, University of Illinois (Chicago, IL, USA), in the laboratory of Prof. S. Silver, whom we thank for his useful suggestions. We thank F. Baldi (Univeritfi di Siena) for his assistance in volatilization assays. P. Veronesi collaborated in the experimental work and U. Pasotti in computerized analyses.

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Yanisch-Perron, C., Vieira, J. and Messing, J.: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33 (1985) 103-119. Yu, H., Mukhopadhyay, D. and Misra, T.K.: Purification and characterization of a novel organometallic receptor protein regulating the expression of the broad spectrum mercury-resistant operon of plasmid pDU1358. J. Biol. Chem. 269 (1994) 15697-15702.