Overexpression of MerT, the mercuric ion transport protein of transposon Tn501, and genetic selection of mercury hypersensitivity mutations

The small (116 amino acids) inner membrane protein MerT encoded by the transposon Tn501 has been overexpressed under the control of the bacteriophage T7 expression system. Random mutants of MerT were made and screened for loss of mercuric ion hypersensitivity. Several mutantmerT genes were selected and sequenced: Cys24Arg and Cys25Tyr mutations abolish mercury resistance, as do charge-substitution mutations in the first predicted transmembrane helix (Glyl4Arg, Glyl5Arg, Gly27Arg, Ala18Asp), and the termination mutations Trp66Ter and Cys82Ter.     

Characterisation of Tn501, a transposon determining resistance to mercuric ions

Summary The transposon encoding resistance to mercuric ions, Tn501, is 5.2 (±0.1)×10⁶ daltons and is bounded by small inverted repeats. The restriction sites for the restriction endonucleases EcoRI, HindIII and SalGI have been mapped on the element.     

The role of cysteine residues in the transport of mercuric ions by the Tn501 MerT and MerP mercury-resistance proteins

Each cysteine residue in the MerT and MerP polypeptides of bacterial transposon Tn 501 was replaced by serine, and the mercury-resistance phenotypes of the mutants were determined in Escherichia coli . Cys−24 and Cys−25 in the first transmembrane region of MerT were essential for transport of mercuric ions through the cytoplasmic membrane, and mutations Cys−76-Ser, Cys−82-Ser or Gly−38-Asp in MerT or Cys−36-Ser in MerP all reduced transport and resistance. Deletion of the merP gene slightly reduced mercuric ion resistance and transport, whereas a Cys−33-Ser mutation in MerP appears to block transport of mercuric ions by MerT. The effects of deleting merP on mutations in merT were tested. The 116-amino-acid MerT protein is sufficient for mercuric ion transport across the cytoplasmic membrane.     

C-terminal cysteines of Tn501 mercuric ion reductase.

Mercuric ion reductase (MerA) catalyzes the reduction of Hg(II) to Hg(0) as the last step in the bacterial mercury detoxification pathway. A member of the flavin disulfide oxidoreductase family, MerA contains an FAD prosthetic group and redox-active disulfide in its active site. However, the presence of these two moieties is not sufficient for catalytic Hg(II) reduction, as other enzyme family members are potently inhibited by mercurials. We have previously identified a second pair of active site cysteines (Cys558 Cys559 in the Tn501 enzyme) unique to MerA, that are essential for high levels of mercuric ion reductase activity [Moore, M. J., & Walsh, C. T. (1989) Biochemistry 28, 1183; Miller, S. M., et al. (1989) Biochemistry 28, 1194]. In this paper, we have examined the individual roles of Cys558 and Cys559 by site-directed mutagenesis of each to alanine. Phenotypic analysis indicates that both merA mutations result in a total disruption of the Hg(II) detoxification pathway in vivo, while characterization of the purified mutant enzymes in vitro shows each to have differential effects on catalytic function. Compared to wild-type enzyme, the C558A mutant shows a 20-fold reduction in kcat and a 10-fold increase in Km, for an overall decrease in catalytic efficiency of 200-fold in kcat/Km. In contrast, mutation of Cys559 to alanine results in less than a 2-fold reduction in kcat and an increase in Km of only 4-5 fold for an overall decrease in catalytic efficiency of only ca. 10-fold in vitro. From these results, it appears that Cys558 plays a more important role in forming the reducible complex with Hg(II), while both Cys558 and Cys559 seem to be involved in efficient scavenging (i.e., tight binding) of Hg(II).     

Nucleotide sequences at the ends of the mercury resistance transposon, Tn501.

The nucleotide sequences at the ends of the mercury-resistance transposon, Tn501, have been determined. The terminal sequences are inverted repeated sequences 38 nucleotide pairs in length, which differ in 3 nucleotide pairs. The transposon is flanked by directly repeated sequences of 5 nucleotide pairs, originating from a single pentanucleotide sequence in the recipient replicon. There is no obvious homology between recipient replicons at the site of insertion of the transposon. The structures of the ends of Tn501 are compared with those of other transposons and insertion sequences. The use of Tn501 to locate an EcoRI site within a genetically defined sequence of interest is discussed.     

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.     

Introduction of the mercury transposon Tn501 into Rhizobium japonicum strains 31 and 110

Abstract Transposon Tn 501 , which encodes resistance to mercuric ions, was introduced into Rhizobium japonicum 110 and 31 by conjugal transfer. The transposon donor plasmid (pMD100) was able to mobilize into R. japonicum , but could not be maintained. Hg²⁺-resistant colonies were recovered at a frequency of 1.9 × 10⁻⁸/recipient for strain 110, and 1.7 × 10⁻⁷/recipient for strain 31. Presence of Tn 501 in Hg-resistant isolates was verified by Southern analysis and demonstrating transposition of Hg resistance. Transposon mutagenesis has been used to generate auxotrophic mutations at low frequency.     

Tn5563, a transposon encoding putative mercuric ion transport proteins located on plasmid pRA2 of Pseudomonas alcaligenes

Sequence analysis of pRA2, an endogenous 33-kb plasmid from Pseudomonas alcaligenes NCIB 9867 (strain P25X), revealed the presence of a 6256-bp transposon of the Tn3 family, designated Tn5563. Tn5563, which is flanked by two 39-bp inverted repeats, encodes a transposase, a resolvase, and two open reading frames which share amino acid sequence similarities with the mercuric ion transport proteins MerT and MerP encoded by several mer operons. However, no other mer operon genes were found on Tn5563. Sequencing of a RP4::Xln hybrid plasmid indicates possible interactions between pRA2 and the P25X chromosome mediated by Tn5563.     

Ordering of the flagellar genes in Pseudomonas aeruginosa by insertions of mercury transposon Tn501.

The flagellar genes of Pseudomonas aeruginosa PAO cluster on the chromosome at two distinct regions, region I and region II. The order of the flagellar cistrons in this organism was established by using transducing phage G101 and plasmids FP5 and R68.45. A method to insert transposon Tn501 near the fla genes was devised. We obtained two strains in which Tn501 was inserted at sites close to the flagellar cistrons in region II. We isolated Fla mutants in which the chromosomal segment between the two Tn501 insertion sites was deleted. Using Tn501-encoded mercury resistance as an outside marker, we determined the order of 9 of the 11 flagellar cistrons in region II as follows: puuF-region I-flaG-flaC-flaI-flaH-flaD-flaB-flaA-flaF-flaE-pur-67. By using phage G101-mediated transduction, the mutation converting monoflagellated bacteria into the multiflagellated (mfl) form was closely linked to the five fla cistrons in region I. Using mfl as an outside marker, we determined the order of the five cistrons as follows: puuF-flaV-flaZ-flaW-flaX-flaY-region II. The mfl mutation was shown to be either located within the flaV cistron or linked very closely to this cistron. No linkage was observed in transductions between any of the fla cistrons in region I and any of the fla cistrons in region II.     

The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system

Summary The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes.     

Genes encoding mercuric reductases from selected gram-negative aquatic bacteria have a low degree of homology with merA of transposon Tn501

An investigation of the Hg2+ resistance mechanism of four freshwater and four coastal marine bacteria that did not hybridize with a mer operonic probe was conducted (T. Barkay, C. Liebert, and M. Gillman, Appl. Environ. Microbiol. 55:1196-1202, 1989). Hybridization with a merA probe, the gene encoding the mercuric reductase polypeptide, at a stringency of hybridization permitting hybrid formation between evolutionarily distant merA genes (as exists between gram-positive and -negative bacteria), detected merA sequences in the genomes of all tested strains. Inducible Hg2+ volatilization was demonstrated for all eight organisms, and NADPH-dependent mercuric reductase activities were detected in crude cell extracts of six of the strains. Because these strains represented random selections of bacteria from three aquatic environments, it is concluded that merA encodes a common molecular mechanism for Hg2+ resistance and volatilization in aerobic heterotrophic aquatic communities.     

Genes encoding mercuric reductases from selected gram-negative aquatic bacteria have a low degree of homology with merA of transposon Tn501.

An investigation of the Hg2+ resistance mechanism of four freshwater and four coastal marine bacteria that did not hybridize with a mer operonic probe was conducted (T. Barkay, C. Liebert, and M. Gillman, Appl. Environ. Microbiol. 55:1196-1202, 1989). Hybridization with a merA probe, the gene encoding the mercuric reductase polypeptide, at a stringency of hybridization permitting hybrid formation between evolutionarily distant merA genes (as exists between gram-positive and -negative bacteria), detected merA sequences in the genomes of all tested strains. Inducible Hg2+ volatilization was demonstrated for all eight organisms, and NADPH-dependent mercuric reductase activities were detected in crude cell extracts of six of the strains. Because these strains represented random selections of bacteria from three aquatic environments, it is concluded that merA encodes a common molecular mechanism for Hg2+ resistance and volatilization in aerobic heterotrophic aquatic communities.     

Mutagenesis of the N- and C-terminal cysteine pairs of Tn501 mercuric ion reductase: consequences for bacterial detoxification of mercurials

Mercuric ion reductase (the merA gene product) is a unique member of the class of FAD and redox-active disulfide-containing oxidoreductases by virtue of its ability to reduce Hg(II) to Hg(0) as the last step in bacterial detoxification of mercurials. In addition to the active site redox-active disulfide, formed between Cys135 and Cys140 in Tn501 MerA, the protein products of the three merA gene sequences published to date have two additional conserved pairs of cysteines, one near the N-terminus (Cys10Cys13 in Tn501 MerA) and another near the C-terminus (Cys558Cys559 in Tn501 MerA). Neither of these pairs is found in other members of this enzyme family. To assess the possible roles of these peripheral cysteines in the Hg(II) detoxification pathway, we have constructed and characterized one single mutant, Cys10Ala13, and two double mutants, Ala10Ala13 and Ala558Ala559. The N-terminal mutants are fully functional in vivo as determined by HgCl2 resistance studies, showing the N-terminal cysteine pair to be dispensable. In contrast, the Ala558Ala559 mutant is defective for HgCl2 resistance in vivo and Hg(SR)2 reduction in vitro, thereby implicating Cys558 and/or Cys559 in Hg(II) reduction by the wild-type enzyme. Other activities, such as NADPH/thio-NADP+ transhydrogenation, NADPH oxidation, and DTNB reduction, are unimpaired in this mutant.     

A Tn21 terminal sequence within Tn501: complementation of tnpA gene function and transposon evolution

Summary The prokaryotic mercury-resistance transposon Tn501 contains a sequence, 80 nucleotides from one end, which is identical with an inverted terminal repeat (IR) of Tn21. This Tn21 IR sequence is used when Tn21 complements a TnpA^- derivative of Tn501, but not when Tn501 is used for the complementation. Complementation by Tn1721 shows a preference for the normal Tn501 IRs. The element (Tn820) transposed when Tn21 is used to complement a Hg^- TnpR^- TnpA^- Res^- deletion mutant of Tn501 contains the Tn21 IR sequence at one terminus and a Tn501 IR at the other. Transposition of Tn820 can be complemented by Tn501 and Tn1721, but at a much lower frequency than transposition of the parental element (Tn819) which has two Tn501 IRs. The relationship between the transposition functions of Tn501, Tn21 and Tn1721, and available nucleotide sequence data suggest that Tn501 evolved by the transposition of a Tn21-like element into another transposable element (similar to that found within Tn1721) followed by deletion of the Tn21-like transposition functions.Abbreviations used (IR) Inverted repeat - (Cb) carbenicillin - (Cm) chloramphenicol - (Sm) streptomycin - (Su) sulphonamide - (Tc) tetracycline - (Tp) trimethoprim     

Detection and characterization of Tn2501, a transposon included within the lactose transposon Tn951

The DNA sequence spanning coordinates 9.9 to 16.4 kilobases of the lactose transposon Tn951 ( Cornelis et al., Mol. Gen. Genet. 160:215-224, 1978) constitutes a transposable element by itself. Unlike Tn951 ( Cornelis et al., Mol. Gen. Genet. 184:241-248, 1981), this element, called Tn2501 , transposes in the absence of any other transposon. Transposition of Tn2501 proceeds through transient cointegration and duplicates 5 base pairs of host DNA. Tn2501 is flanked by nearly perfect inverted repeats (44 of 48), related to the inverted repeats of Tn21 ( Zheng et al., Nucleic Acids Res. 9:6265-6278, 1982). Unlike Tn21 , Tn2501 does not confer mercury resistance.