Mercuric reductase structural genes from plasmid R100 and transposon Tn501: functional domains of the enzyme

The nucleotide sequence for the 2240 bp of plasmid R100 following the merC gene of the mercuric resistance operon has been determined and compared with the homologous sequence of transposon Tn501. The sequences following merC and preceding the next structural gene merA are unrelated between R100 and Tn501 and differ in length, with 72 bp in Tn501 and 509 bp in R100. The R100 sequence has a potential open reading frame (ORF) for a 140 amino acid polypeptide with a reasonable translational start signal preceding it. The merA genes contain 1686 (Tn501) and 1695 (R100) bp respectively. When optimally aligned, the merA sequences differ in 18% of their positions. These differences were clustered in specific regions. In addition, there was one nucleotide triplet in the Tn501 sequence which has no counterpart in the R100 sequence and one dodecyl-nucleotide sequence in the R100 sequence without counterpart in Tn501. Thus the predicted merA polypeptide of Tn501 contains 561 amino acids and the R100 counterpart contains 564 amino acids. Comparison of the R100 mercuric reductase sequences with that for human glutathione reductase [Krauth-Siegel et al.: Eur. J. Biochem. 121 (1982) 259-267], for which there is a 2 A resolution electron density map [Thieme et al.: J. Mol. Biol. 152 (1981) 763-782] shows a strong homology, with 26% identical amino acids and many conservative substitutions. This homology allows the conclusion that the active site of these enzymes and the contact positions for flavin adenine dinucleotide (FAD) and NADPH are highly conserved, while the amino- and carboxyl-terminal sequences differ.     

Restriction pattern and polypeptide homology among plasmid-borne mercury resistance determinants

The structural and functional properties of mercury resistance determinants cloned from a series of independently isolated conjugative plasmids were compared with those of the prototype HgR determinants from Tn501 and plasmid R100 (containing Tn21). Restriction endonuclease mapping classified the HgR determinants into at least three different but related structural groups which are distantly related to those from Tn501 and R100. These relationships were confirmed by the functional analysis of sub-clones and gamma delta insertion mutations and from the polypeptides specified by the cloned HgR determinants. Each mercury resistance clone synthesized polypeptides equivalent in size to the merA, merT, and merP gene products. However, those for merA and merT showed considerable size variation. No polypeptide equivalent to merD or merC of R100 was detected.     

Tn5037-a Tn21-like mercury transposon, detected in Thiobacillus ferrooxidans

The 6645-bp mercury resistance transposon of the chemolithotrophic bacterium Thiobacillus ferrooxidans was cloned and sequenced. This transposon, named Tn5037, belongs to the Tn21 branch of the Tn21 subgroup, many members of which have been isolated from clinical sources. Having the minimum set of the genes (merRTPA), the mercury resistance operon of Tn5037 is organized similarly to most of the Gram-negative bacteria mer operons and is closest to that of Thiobacillus 3.2. The operator-promoter region of the mer operon of Tn5037 also has the common (Tn21/Tn501-like) structure. However, its inverted, presumably MerR protein binding repeats in the operator/promoter element are two base pairs shorter than in Tn21/Tn501. In the merA region, this transposon shares 77.4, 79.1, 83.2 and 87.8% identical bases with Tn21, Tn501, T. ferrooxidance E-15, and Thiobacillus 3.2, respectively. No inducibility of the Tn5037 mer operon was detected in the in vivo experiments. The transposition system (terminal repeats plus gene tnpA) of Tn5037 was inactive in Escherichia coli K12, in contrast to its resolution system (res site plus gene tnpR). However, transposition of Tn5037 in this host was provided by the tnpA gene of Tn5036, a member of the Tn21 subgroup. Sequence analysis of the Tn5037 res site suggested its recombinant nature.     

Nucleotide sequence of a gene from the Pseudomonas transposon Tn501 encoding mercuric reductase

We have determined the nucleotide sequence of the merA gene from the mercury-resistance transposon Tn501 and have predicted the structure of the gene product, mercuric reductase. The DNA sequence predicts a polypeptide of Mr 58 660, the primary structure of which shows strong homologies to glutathione reductase and lipoamide dehydrogenase, but mercuric reductase contains as additional N-terminal region that may form a separate domain. The implications of these comparisons for the tertiary structure and mechanism of mercuric reductase are discussed. The DNA sequence presented here has an overall G+C content of 65.1 mol%, typical of the bulk DNA of Pseudomonas aeruginosa from which Tn501 was originally isolated. Analysis of the codon usage in the merA gene shows that codons with C or G at the third position are preferentially utilized.     

Mercuric reductase enzyme from a mercury-volatilizing strain of Thiobacillus ferrooxidans.

Cell-free mercury volatilization activity (mercuric reductase) was obtained from a mercury-volatilizing Thiobacillus ferrooxidans strain, and the properties of intact-cell and cell-free activities were compared with those determined by plasmid R100 in Escherichia coli. Intact cells of T. ferrooxidans volatilized mercury at pH 2.5, whereas cells of E. coli did not. Cell-free enzyme preparations from both bacteria functioned best at or above neutral pH and not at all at pH 2.5. The T. ferrooxidans mercuric reductase was a soluble enzyme that was dependent upon added NAD(P)H. The enzyme activity was stable at 80 degrees C, required an added thiol compound, and was stimulated by EDTA. Antisera against purified mercuric reductases from transposon Tn501 and plasmid R831 (which inactivated mercuric reductases from a wide range of enteric and pseudomonad strains) did not inactivate the enzyme from T. ferrooxidans.     

The merR regulatory gene in Thiobacillus ferrooxidans is spaced apart from the mer structural genes

Two distinct merR genes, which regulate expression of the mercuric ion resistance gene (mer), of Thiobacillus ferrooxidans strain E-15 have been cloned, sequenced and termed merR1 and merR2. As a result of gene walking around two merR genes, it was found that these two genes were quite close in distance. The nucleotide sequence of the region (5,001 base pairs; PstI-EcoRI fragment) containing the merR genes was determined. Between the two merR genes, there were five potential open reading frames (ORFs). Two of these were identified as merC genes, and the other three as ORFs 1 to 3. ORFs 1 to 3 show significant homology to merA, tnsA from transposon Tn7, and merA, respectively. Both merR genes consist of a 408 bp ORF coding for 135 amino acids. Their gene products, MerR1 and MerR2, differed at three amino acid positions, and shared 56-57% and 32-38% identity with the MerRs from other Gram-negative and Gram-positive bacteria, respectively. Competitive primer extension analysis revealed that both regulatory genes were expressed in the host cells. These merR genes were located more than 6 kb from either end of the mer structural genes (merC-merA). This is the first example of merR being separated from the mer structural genes. The two merC genes, each of which coded for a 140-amino-acid protein, appeared to be functionally active because Escherichia coli cells carrying these merC genes on plasmid vectors showed hypersensitivity to HgCl2. However, ORFs 1 and 3, which were homologous to merA, seemed to be inactive both structurally and enzymatically. The gene arrangement in this region took on a mirror image, with the truncated tnsA as the symmetrical centre. It is suggested that the Tn7-like factor may have participated in gene duplication events of the mer region, and in its chromosomal integration.     

Plasmid and transposon transfer to Thiobacillus ferrooxidans.

The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and pUB307 were transferred to acidophilic, obligately chemolithotrophic Thiobacillus ferrooxidans from Escherichia coli by conjugation. A genetic marker of kanamycin resistance was expressed in T. ferrooxidans. Plasmid RP4 was transferred back to E. coli from T. ferrooxidans. The broad-host-range IncQ vector pJRD215 was mobilized to T. ferrooxidans with the aid of plasmid RP4 integrated in the chromosome of E. coli SM10. pJRD215 was stable, and all genetic markers (kanamycin/neomycin and streptomycin resistance) were expressed in T. ferrooxidans. By the use of suicide vector pSUP1011, transposon Tn5 was introduced into T. ferrooxidans. The influence of some factors on plasmid transfer from E. coli to T. ferrooxidans was investigated. Results showed that the physiological state of donor cells might be important to the mobilization of plasmids. The transfer of plasmids from E. coli to T. ferrooxidans occurred in the absence of energy sources for both donor and recipient.     

Nucleotide sequence of the gene encoding the nitrogenase iron protein of Thiobacillus ferrooxidans.

The DNA sequence was determined for the cloned Thiobacillus ferrooxidans nifH and part of the nifD genes. A putative T. ferrooxidans nifH promoter was identified whose sequences showed perfect consensus with those of the Klebsiella pneumoniae nif promoter. Two putative consensus upstream activator sequences were also identified. The amino acid sequence was deduced from the DNA sequence. In a comparison of nifH DNA sequences from T. ferrooxidans and eight other nitrogen-fixing microbes, a Rhizobium sp. isolated from Parasponia andersonii showed the greatest homology (74%) and Clostridium pasteurianum (nifH 1) showed the least homology (54%). In a comparison of the amino acid sequences of the Fe proteins, the Rhizobium sp. and Rhizobium japonicum showed the greatest homology (both 86%) and C. pasteurianum (nifH 1 gene product) demonstrated the least homology (56%) to the T. ferrooxidans Fe protein.     

The region of the IncN plasmid R46 coding for resistance to beta-lactam antibiotics, streptomycin/spectinomycin and sulphonamides is closely related to antibiotic resistance segments found in IncW plasmids and in Tn21-like transposons.

The nucleotide sequence of a 2.5 kb segment of the pKM101 (R46) genome has been determined. The 1.3 kb from a BamHI site at 153 to base 1440 differs by only 2 bases from a part of the published sequence of the aadB (gentamicin resistance) gene region including the coding region for the N-terminal 70 amino acids of the predicted aadB product. The same sequence has been found 5'-to the dhfrII gene of R388 and to the aadA gene of Tn21 (R538-1). Three open reading frames are located in this region, two on the same strand as the resistance genes and one on the complementary strand. The latter predicts a polypeptide of 337 amino acids, whose N-terminal segment is 40% homologous to the predicted product of an open reading frame of 179 amino acids located next to the dhfrI gene of Tn7. The oxa2 (oxacillin resistance) gene predicts a long polypeptide commencing with (the N-terminal) 70 amino acids of the aadB product. A similar arrangement is found in the aadA gene of R538-1. The N-terminal segment of an aadA gene is located 3'- to oxa2, separated by 36 bases. Sequences surrounding the BamHI site are identical to sequences 5'- to the tnpM gene of Tn21 and homology ceases where homology between Tn21 and Tn501 commences. The possibility that this antibiotic resistance segment is a discrete mobile DNA element is discussed.     

DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721

DNA sequences that encode the tnpR genes and internal resolution (res) sites of transposons Tn21 and Tn501, and the res site and the start of the tnpR gene of Tn1721 have been determined. There is considerable homology between all three sequences. The homology between Tn21 and Tn501 extends further than that between Tn1721 and Tn501 (or Tn21), but in the homologous regions, Tn1721 is 93% homologous with Tn501, while Tn21 is only 72-73% homologous. The tnpR genes of Tn21 and Tn501 encode proteins of 186 amino acids which show homology with the tnpR gene product of Tn3 and with other enzymes that carry out site-specific recombination. However, in all three transposons, and in contrast to Tn3, the tnpR gene is transcribed towards tnpA gene, and the res site is upstream of both. The res site of Tn3 shows no obvious homology with the res regions of these three transposons. Just upstream of the tnpR gene and within the region that displays common homology between the three elements, there is a 50 bp deletion in Tn21, compared to the other two elements. A TnpR- derivative of Tn21 was complemented by Tn21, Tn501 and Tn1721, but not by Tn3.     

The nucleotide sequence of the tnpA gene of Tn21.

The nucleotide sequence of the tnpA gene of Tn21 is presented. The transposase encoded by this gene is exactly the same length (988 amino acids) as the Tn501 transposase (4), and shows 72% homology overall with this protein, with greater homology towards the C-terminus. The sequence of the transposase is discussed in the context of the evolution of Class II transposable elements and of the characteristics of the enzyme's action.     

The nucleotide sequence of the tnpA gene completes the sequence of the Pseudomonas transposon Tn501.

The nucleotide sequence of the gene (tnpA) which codes for the transposase of transposon Tn501 has been determined. It contains an open reading frame for a polypeptide of Mr = 111,500, which terminates within the inverted repeat sequence of the transposon. The reading frame would be transcribed in the same direction as the mercury-resistance genes and the tnpR gene. The amino acid sequence predicted from this reading frame shows 32% identity with that of the transposase of the related transposon Tn3. The C-terminal regions of these two polypeptides show slightly greater homology than the N-terminal regions when conservative amino acid substitutions are considered. With this sequence determination, the nucleotide sequence of Tn501 is fully defined. The main features of the sequence are briefly presented.     

The distribution and divergence of DNA sequences related to the Tn21 and Tn501 mer operons

The mercury resistance (mer) operons of the Gram-negative bacterial transposons, Tn21 and Tn501, are phenotypically indistinguishable and have extensive DNA identity. However, Tn21 mer has an additional coding region (merC) in the middle of the operon which is lacking in Tn501 and there is also a discrete region of the mercuric ion reductase gene (merA) which differs markedly between the two operons. DNA fragment probes were used to determine the distribution of specific mer coding regions in two distinct collections of mercury-resistant (Hgr) Gram-negative bacteria. Colony blot hybridization analysis showed that merC-positive operons occur almost exclusively in Escherichia, although merC-negative operons can also be found in this genus. The merC-negative operons were found in Citrobacter, Klebsiella, and Enterobacter and in some Pseudomonas. Most of the Pseudomonas did not hybridize detectably with either of the two operons studied, indicating that they harbor an unrelated or more distantly related class of mercury resistance locus. Southern hybridization patterns demonstrated that the merC-positive mer operon is well conserved at the DNA level, whereas the merC-negative operons are much less conserved. The presence of merC also correlated with conservation of a specific variant region of the merA gene and with an antibiotic resistance pattern similar to that of Tn21. Tn501 appears to be an atypical example of the merC-negative subgroup of Hgr loci.     

Phylogeny of mercury resistance (mer) operons of gram-negative bacteria isolated from the fecal flora of primates.

Nine polymorphic mer loci carried by 185 gram-negative fecal bacterial strains from humans and nonhuman primates are described. The loci were characterized with specific intragenic and intergenic PCR primers to amplify distinct regions covering approximately 80% of the typical gram-negative mer locus. These loci were grouped phylogenetically with respect to each other and with respect to seven previously sequenced mer operons from gram-negative bacteria (the latter designated loci 1, 2, 3, 6, 7, 8, and delta 8 by us here for the purpose of this analysis). Six of the mer loci recovered from primates are similar either to these previously sequenced mer loci or to another locus recently observed in environmental isolates (locus 4), and three are novel (loci 5, 9, and 10). We have observed merC, or a merC-like gene, or merF on the 5' side of merA in all of the loci except that of Tn501 (here designated mer locus 6). The merB gene was observed occasionally, always on the 3' side of merA. Unlike the initial example of a merB-containing mer locus carried by plasmid pDU1358 (locus 8), all the natural primate loci carrying merB also had large deletions of the central region of the operon (and were therefore designated locus delta 8). Four of the loci we describe (loci 2, 5, 9, and 10) have no region of homology to merB from pDU1358 and yet strains carrying them were phenylmercury resistant. Two of these loci (loci 5 and 10) also lacked merD, the putative secondary regulator of operon expression. Phylogenetic comparison of character states derived from PCR product data grouped those loci which have merC into one clade; these are locus 1 (including Tn21), locus 3, and locus 4. The mer loci which lack merC grouped into a second clade: locus 6 (including Tn501) and locus 2. Outlying groups lacked merD or possessed merB. While these mer operons are characterized by considerable polymorphism, our ability to discern coherent clades suggests that recombination is not entirely random and indeed may be focused on the immediate 5' and 3' proximal regions of merA. Our observations confirm and extend the idea that the mer operon is a genetic mosaic and has a predominance of insertions and/or deletions of functional genes immediately before and after the merA gene. chi sites are found in several of the sequenced operons and may be involved in the abundant reassortments we observe for mer genes.     

Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia coli

The nucleotide sequence of the recA gene of Thiobacillus ferrooxidans has been determined. No SOS box characteristic of LexA-regulated promoters could be identified in the 196-bp region upstream from the coding region. The cloned T. ferrooxidans recA gene was expressed in Escherichia coli from both the lambda pR and lac promoters. It was not expressed from the 2.2-kb of T. ferrooxidans DNA preceding the gene. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and Pseudomonas aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with proteolytic activity have been substituted, the cloned protein has retained protease activity towards the lambda and E. coli LexA repressors.     

Insertion sites and the terminal nucleotide sequences of the Tn4 transposon.

The nucleotide sequences at the ends of the Tn4 transposon (mercury spectinomycin and sulfonamide resistance) have been determined. They are inverted repeated sequences of 38 nucleotides with three mismatched base pairs. These sequences are strongly homologous with the terminal sequences of Tn501 (mercury resistance) but less so with those of Tn3 (ampicillin resistance). The Tn4 transposon generates pentanucleotide members (Tn3, Tn1000, Tn501, Tn551, IS2) with the exception of Tn1721 and bacteriophage Mu. Among the three Tn4 insertion sites examined here, two of them occurred near a nonanucleotide sequence in perfect homology with part of the terminal inverted-repeat sequence of Tn4 and the third insertion occurred near a sequence of partial homology to one end of Tn4. All three insertions were in the same orientation such that IRb is proximal to its homologous sequence on the recipient DNA.