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Cloning o f meta l - res is tance conferr ing genes f rom an Acidocel la strain

S. Ghosh, N. R. Mahapatra and P. C. Banerjee*

Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta-700032, India E-mail : iichbio@giascl01.vsnl.net.in

Metal resistance is regarded as the most suitable phenotypic trait for selection of genetically engineered bioleaching bacteria. Since the expression of metal resistance conferring genes is very limited in heterologous systems, search for these genes from homologous and related biomining bacteria is envisaged to be rewarding for genetic manipulation of this group of microorganisms. Acidocella strain GS 19h, a bacterium having high resistance to cadmium and zinc, was chosen for cloning of metal resistance genes from its plasmids. Purified plasmid preparation from this bacterium was partially digested with Sau3AI, ligated at the BamHI site of pBluescriptlI KS+/-, and transferred into E. coli DH5c~ strain by transformation. The E. coli derivatives containing cloned segments were tested for metal resistance and a few cadmium resistant colonies were found to contain a recombinant plasmid having a 0.8 kb insert DNA. This DNA fragment was sequenced and analyzed for common sequences in other sources from gene data banks. Although partial homologies with a part of various transposons, merR genes and others were detected, it appears that the cloned gene is a novel one with no apparent similarity with the existing cadmium, copper, nickel, or zinc resistance-conferring genes.

1. INTRODUCTION

In the process of developing improved strains for leaching of ores employing acidophilic bacteria such as Thiobacillus ferrooxidans, construction of plasmid vectors is essential [ 1-5]. In the case of acidophiles, metal resistance is considered to be the most suitable phenotypic trait of such vectors [4-6]. But due to very low to non-existent levels of expression of this phenotypic characteristic in heterologous systems [7, 8], it is important to identify and isolate

* Corresponding author. This work was partly supported by the Department of Science and Technology, Govt. of India. Authors are grateful to Mr. Suvobrata Nandi and Mr. Chirajyoti Deb for their assistance in the study. S. G. and N. R. M. are thankful to Labonya Prova Bose Trust, Calcutta and Council of Scientific and Industrial Research, New Delhi for providing them with fellowships.

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metal resistance encoding genes from T. ferrooxidans and other acidophiles of the same habitat, and preferably from their plasmids. It is presumed that such genes might be useful for genetic engineering of leaching microbes for the development of this biotechnology [6, 9, 10]. In the case of T. ferrooxidans, the evidence for plasmid-mediated metal resistance was only circumstantial. For example, among the T. ferrooxidans isolates four strains containing a 20 kb plasmid were highly resistant to UO2 + whereas in one strain absence of the plasmid coincided with a reduction in uranium resistance [11 ]. This well-studied leaching bacterium has so far been reported to contain its zinc, arsenic [12], mercury [13] and copper [14] - resistance determinants only in the chromosome. On the other hand, the resistance determinants for arsenic and mercury in an acidophilic heterotroph of acidic mine environment Acidiphilium multivorum AIU301 have been found to be encoded in its 56 kb plasmid [15]. The resistances to Cd 2+, Cu 2+ and Zn 2+ of another acidophilic heterotrophic bacterium Acidocella sp. strain GS 19h have also been observed to be plasmid-mediated [ 16]. Thus, unlike T. ferrooxidans the acidophilic heterotrophs may harbour metal-resistance genes on their plasmids, which may be developed for construction of plasmid vectors for acidophiles. The present piece of work was undertaken to clone the metal resistant genes present in the plasmids of Acidocella strain GS 19h via construction of a miniplasmid library.

2. MATERIALS AND METHODS

2.1. Bacterial strains, plasmids, and culture conditions Acidocella species strain GS 19h [ 17], E. coli DH5~ [ 18] and pBluescript II KS+/- [ 18] were

used in this study. The Acidocella strain was grown at 30~ with shaking in MGY medium [g 1-1 composition: (NH4)2SO4, 2.0; K2HPO4, 0.25; MgSO4.7H20,0.25; KC1, 0.1; glucose, 1.0; and yeast extract, 0.1] of pH 3. The E. coli cells were grown in LB broth [19]. Selection of transformants of E. coli DH5~ was made on LB-agar containing ampicillin (100~tg ml 1) or CdSO4 (8 mM and 10 mM) or ZnSO4 (12 and 16 raM) or CuSO4 (12 and 16 mM).

2.2. Isolation, purification and electrophoresis of plasmid DNA Plasmid DNAs were isolated, in general, by the alkaline lysis method as described

previously [ 16, 20], and purified by CsCl-ethidium bromide gradient centrifugation [ 19] or by polyethylene glycol treatment [21 ] whenever required. For nucleotide sequencing, isolation of plasmid DNA was carried out by strictly following the Perkin Elmer protocol [21 ] excepting that E. coli cells were grown in LB broth instead of 'terrific broth'. Plasmid DNAs were electrophoresed in 0.5-1.0% (w/v) agarose gels and detected aider ethidium bromide (0.5 ~tg ml "1) staining as usual [19].

2.3. Construction of miniplasmid library About 1 pg of total plasmid DNA from Acidocella strain GS19h was digested with Sau3AI

in order to get the most DNA fragments of size ranging between 0.5-6.0 kb. After dephosphorylation by calf intestinal phosphatase DNA fragments were ligated with BamHI digested pBluescript II KS +/- by T4 DNA ligase as described previously [19]. The ligated DNA samples were used to transform E. coli DH5a cells made competent by CaCI2 treatment

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[ 19] and the transformants containing recombinant DNA molecules were selected on LB-agar with ampilicillin and requisite amounts of X-gal (5-bromo-4-chloro-3-indolyl-D-galacto- pyranoside) and IPTG (isopropyl 13-D-thiogalactopyranoside). The transformed colonies were then checked for their metal resistance characteristics.

2.4. DNA sequencing Nucleotide sequence of the DNA fragment inserted at the BamHI site of the vector plasmid

pBluescript II KS +/- was determined by ABI Prism Model 377 DNA sequencer (Perkin Elmer). Universal M13 primer was used for sequencing reaction.

3. RESULTS

3.1. Metal resistance characteristics of the transformants The transformants were observed to be sensitive to 12 mM CuSO4. But 13% of the total

white colonies were resistant to CdSO4 and about 5% of the same to ZnSO4 - the MIC values being 10 mM and 16 mM respectively while those for E. coli DH5a (pBluescript II KS+/-) were 2 mM and 10 mM respectively.

3.2. Plasmid profile of the clones Metal resistant E. coli transformants were of different types based on the sizes of the

recombinant DNAs. From some Cd2+-resistant transformants a recombinant plasmid designated as pSGX was isolated, and subjected to further studies. The size of pSGX was determined to be 3.7 kb as it gives 3.7 kb, 2.9 kb and 0.8 kb DNA bands on partial digestion with PstI and NotI (Figure 1, lane 6).

3.3. Analysis of the nucleotide sequence of the cloned DNA fragment The recombinant plasmid pSGX contains 814 bp DNA insert. The nucleotide sequence is

shown in Figure 2. The forward sequence contains 149 A, 162 T, 242 G and 261 C bases; the overall (G+C) mol% being 61.9 which is almost the same as that of the chromosomal DNA [ 17]. About 80 restriction enzymes including AluI, AvaI, DpnI, DraI, EcoRI, EcoRII, HaelI, HaelII, Sau3AI, SmaI and TaqI have cutting site(s) in the cloned region. On the other hand, about 150 enzymes do not have any restriction site in this piece of DNA. These include some enzymes commonly used in cloning experiments such as BamHI, BgllI, BgllII, ClaI, Dram, EcoRV, HaeI, HpaI, KpnI, NdeI, NotI, PstI, PvuI, PvulI, SaclI, SalI, SfiI, SpeI, SwaI, XbaI and XhoI.

3.4. Sequence similarities of the cloned DNA with other genes DNA sequence similarities between the cloned DNA sequence in pSGX and other sequences in the EMBL database were examined. It was found that the cloned plasmid region has insignificant sequence homology with other gene/DNA sequences in comparison with their large sizes vis-a-vis the small fraction of nucleotides with which the cloned DNA shows similarity. The maximum number of 347 bases (from nucleotide 464-814) showed 61.4 % homology with a T. ferrooxidans plasmid of 19.8 kb size. Majority of the other genes and DNA sequences (viz Pseudomonus aeruginosa multiresistance 13-1actamase transposon

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Tn1412, phage P1 darA operon, plasmid pMER05, mercury resistance transposons Tn5053 and Tn552, Rhodospirillum rubrum plasmid pKY1, glucosidase gene of Salmonella typhimurium, Enterobacter aerogens R plasmid etc.) also showed 57-63 % similarity with the cloned DNA within the base 470 and 814. P. fluorescens merR gene, A. faecalis merR gene, Enterobactor aerogens mer gene for regulation, P. testosteroni merR gene, and Rhizobium sp. pNG have 55-58 % similarity with the first portion (from base 20-325) of the cloned DNA. The middle 135 bp portion (from base 315-450) of the cloned DNA segment did not show similarity with any of the reported genes to a significant extent. Thus, the cloned DNA although showing some homologies with a variety of different genes including some mercury resistance transposons and plasmids, it may be regarded as a novel one expressing Cd 2+ resistance in E. coli DH5a.

k b I ...... 2 ....... ~ .... 4 ........ 5 .......... 6 ....... 7 ......... k b

2.3 2.0

3.5

2.0 1.9 1.5 1.3

0.9 0.8

Figure 1. Agarose (1%, w/v) gel electrophoretogram showing generation of a ca. 0.8 kb insert DNA from recombinant clone pSGX through double digestion with restriction enzymes. Lane 1, X HindIII digest; lane 2, chromosomal DNA of E. coli DH5a; lane 3, pSGX DNA; lane 4, PstI digested pSGX (< indicates open circular form); lane 5, NotI digested pSGX; lane 6, PstI and NotI digested pSGX(~ indicates position of the cloned DNA frgament); lane 7, ~. HindIII plus EcoRI digest. Numbers on both sides indicate molecular sizes of linear DNA markers.

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1 G A T C T T A G T C G T C T T G T T T T T C G C G T T A C G C G A C T G G C G G 40 SD

41 ATTTCGCTGATCTATGGCGCCGCCGAAATCGCGCGCCTCA 80 ORFI-~ M

81 CCTTCATCTGCCGTGCCCGGGAACTCGGCTTCTCTCTCGA 120

121 CGAGGTACGCGGCCTTCTCAGCCTGGCCGAAAGAGATGAA 160

161 CGCCACTGTGAGGACGTGAAACAAGCTGCTATCCGCCATC 200

201 G T C A G G A C G T G C G C C G C A A G A T C G C C G A C C T G C G G G C G G T 240

241 CGAGGTCACTCTGGGAACCCTCATTCGGCAATGCGAAGCA 280 SD ORF2--) M

281 CGCGGGCCGGCGGAATGCCCCTTGATCGAAGCGCTATCTC 320

321 AACCGAAAGCGGCAGCGCCCGCGCCTTGAAGCGTCGCCGT 360

361 TTAAAACACCACCGGATGATCCGTCCATAATCTCAAACTA 400

401 GCCAGATCAGAGCGATGCTGAGCAACGATGCGCGACACCG 440 SD ORF3--) M

441 TCGGTGCATTGATATTGTAAAGCCGAGCATTTCGGCCCCG 480

481 G A C T T G C G G C C A G T G A T G A C G C T C T C G G C G A T C T C G C G G C 520

521 GTTTGGCGGCATCGAGTTCTTGCGACGCCCACCGATGCGA 560

561 C T T T C G G C G C G G G C G G C T G C A A G G C C G G C G G A G G T C C G T T 600 SD

601 CCCGGATCATGGCACGCTCGAATTCGGCGAAGCTGCCGAC 640 ORF--)4 M

641 CATCTGCATCATCATTCGGCCAGCCGGCGTCGTGGTGTCG 680

681 A T G T T C T C G G T T A G C G A C C G G A A G C C T G C C C C G G C T T C C G 720

721 CGATGCGCTCCATGATGTGCAGCACGTCCTTCAGTGAGCG 760

761 TGACAGCCGGTCGAGCTTCCAGACAACGACGGTATCCCCC 800

801 TCCCGCAGATGATC 814 4r

Figure 2. Nucleotide sequence of the 814 bp cloned DNA fragment from Acidocella strain GS19h

plasmid. 'M' and '*' represent the underlined start and stop codons respectively; 'SD' represents the

putative ribosome binding site; 'CI indicates direction of transcription.

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3.5. Analysis of the deduced amino acid sequences Deduced amino acid sequences (3 each from the forward and reverse directions of the

nucleotide sequence) revealed that there are four complete open reading frames (ORFs) which are indicated in Figure 2, and are presented in Table 1. The putative proteins encoded by open reading frames (ORFs) were searched for similarity to other proteins from the EMBL database with program FASTA.

Table 1. Deduced amino acid sequences of the predicted ORFs

ORF Nucleotides

from to Amino acid sequence

1 54

2 271

3 415

4 609

176 MAPPKSRASPSSAVPGNSASLSTRYAAFSAWPKEMNATVRT

387 MRSTRAGGMPLDRSAISTESGSARALKRRRLKHHRMIRP

732 MLSNDARHRRCIDIVKPSISAPDLRPVMTLSAISRRLAASSSCD

AHRCDFRRGRLQGRRRSVPGSWHARIRRSCRPSASSFGQPAS

WCRCSRLATGSLPRLPRCAP

809 MARSNSAKLPTICIIIRPAGVVVSMFSVSDRKPAPASAMRSMM

CSTSFSERDSRSSFQTTTVSPSRR

ORFI (Nucleotide sequence : 54-176) The ORF1 codes for a small peptide of 41 amino acids. This ORF has a Shine-Dalgamo (SD)- like sequence (GGA) 12 base upstream of the ATG start codon. The putative polypeptide contains 8 serine(19.5%), 8 alanine (19.5%), 5 proline (12.2%), 3 arginine (7.3%). Apart from the N-terminal, it contains only another methionine residue.

ORF2 (Nucleotide sequence : 271- 387) This ORF encodes a even smaller peptide with 39 amino acid residues, and is oriented in the same direction as ORF1. It has a SD like region (GGGAA) 13 base upstream of the start codon. This polypeptide also contains 9 arginine (23.1%), 5 serine (12.8%), 4 alanine (10.3%), 2 methionine (excepting N terminal), and 2 consecutive histidine residues.

ORF3 (Nucleotide sequence : 415-732) This ORF encodes 106 amino acid residues,has a SD like sequence (AGAG), 2 bases upstream of the start codon and it is oriented in the same direction as ORF1. Notably, there are 21 arginine (19.8%), 17 serine (16%), 11 alanine (10.4%), 9 proline (8.5%), 7 cysteine (6.6%), and 3 histidine residues.

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ORF4 (Nucleotide sequence : 609-809) This ORF encodes only 67 amino acid residues and is oriented in the same direction as ORF1. It has a SD like sequence (GGAGG), 14 base upstream of the start codon. The putative polypeptide contains 15 serine (22.4%), 8 arginine (11.9%), 6 alanine (8.9%), 5 threonine (7.4%), and 2 cysteine residues.

4. DISCUSSION

The nucleotide sequence analyses of the 814 bp cloned DNA fragment from a plasmid of Acidocella strain GS19h show that the sequence does not have any homology with the conventional Cd 2+, Zn 2+ or Cu 2+ resistance genes. The deduced amino acid sequences also do not show any such similarity. It is however of interest to note that all the ORFs, especially ORFs 2 and 3 contain substantial amounts of the basic amino acid arginine. Additionally ORF3 contains 7 cysteine residues, the amino acid responsible for metal binding capacity of metallothioneins. As mentioned earlier, the nucleotide sequence corresponding to the ORF3 has homology with various genes including regulatory protein of mercury resistance operon. Therefore, it may be suggested that (i) in this acidophilic bacterium, bound arginine along with cysteine may play the same role as free histidine and metallothioneins do in other systems for relieving the cells from the heavy metal toxicity [22-25], and (ii) the resistance in this acidophilic heterotroph may arise via small proteins which have regulatory roles in gene expression.

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