Tn5037, a Tn21-like Mercury Resistance transposon from Thiobacillus ferrooxidans

The 6645-bp mercury resistance transposon of the chemolithotrophic bacterium Thiobacillus ferrooxidanswas cloned and sequenced. This transposon, named Tn5037, belongs to the Tn21branch of the Tn21subgroup, many members of which have been isolated from clinical sources. Having the minimum set of the genes (merRTPA), the mercury resistance operon of Tn5037is organized similarly to most of the Gram-negative bacteria meroperons and is closest to that of ThiobacillusT3.2. The operator-promoter region of the meroperon of Tn5037also 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. ferrooxidansE-15, and ThiobacillusT3.2, respectively. No inducibility of the Tn5037 meroperon was detected in the in vivo experiments. The transposition system (terminal repeats plus gene tnpA) of Tn5037was inactive in Escherichia coliK12, in contrast to its resolution system (ressite plus gene tnpR). However, transposition of Tn5037in this host was provided by the tnpAgene of Tn5036, a member of the Tn21subgroup. Sequence analysis of the Tn5037 ressite suggested its recombinant nature.     

Structure and function of Tn5467, a Tn21-like transposon located on the Thiobacillus ferrooxidans broad-host-range plasmid pTF-FC2.

A 3.5-kb region of plasmid pTF-FC2, which contains a transposon-like element designated Tn5467, has been sequenced, and its biological activity has been investigated. The transposon is bordered by two 38-bp inverted repeat sequences which have sequence identity in 37 of 38 and in 38 of 39 bp to the tnpA distal and tnpA proximal inverted repeats of Tn21, respectively. Within these borders, open reading frames with amino acid similarity to a glutaredoxin-like protein, a MerR regulatory protein, and a multidrug-resistant-membrane transport-like protein were found. The gene for the glutaredoxin-like protein was expressed in Escherichia coli and enabled growth of a glutathione-requiring E. coli trxA gshA mutant on minimal medium and the reduction of methionine sulfoxide to methionine. In addition, there were two regions which, when translated, had homology to 85% of the N-terminal region of the Tn21 resolvase (tnpR) and to 15% of the C terminus of the Tn21 transposase (tnpA). A region containing res-like sites was located immediately upstream of the partial tnpR gene. Neither the partial transposase nor the resolvase genes of Tn5467 were biologically active, but Tn5467 was transposed and resolved when the Tn21 transposase and resolvase were provided in trans. Tn5467 appears to be a defective transposon which belongs to the Tn21 subgroup of the Tn3 family.     

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.     

The resolvase protein from the transposon Tn21

Summary The tac promoter was inserted into Tn21 upstream of the tnpR gene and the resultant plasmid was used to generate substantial amounts of resolvase. This protein was purified to homogeneity. The protein was characterized by amino acid sequence studies (which showed that an open-reading frame previously identified by DNA sequencing had been correctly assigned to the tnpR gene) and by molecular weight measurements (which demonstrated that the only active for of the protein in solution was dimeric). Pure Tn21 resolvase catalysed site-specific recombinations between directly repeated res sites from Tn21 or Tn1721 but not from Tn3 nor on inverted res sites from Tn21.     

Oxidation of arsenopyrite by Thiobacillus ferrooxidans detected by a mineral electrode

Summary A new electrochemical study of arsenopyrite biooxidation was based on process detection by arsenopyrite electrode. The rate of reaction was evaluated as the exchange current density calculated from polarization curves. Obtained data were used for determination of released electrons from mineral and for evaluation of reaction mechanism of its oxidation.     

Ferrous iron-dependent volatilization of mercury by the plasma membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe(2+) medium (pH 2.5) supplemented with 6 microM Hg(2+). In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 microM Hg(2+). When incubated for 3 h in a salt solution (pH 2.5) with 0.7 microM Hg(2+), resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe(2+) was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30 degrees C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe(2+)-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 microM Hg(2+) and 1 mM Fe(2+), plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe(2+)-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe(2+)-dependent mercury volatilization activity of the plasma membrane.     

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.     

Thiobacillus ferrooxidans, a facultative hydrogen oxidizer

The type strain (ATCC 23270) and two other strains of Thiobacillus ferrooxidans were able to grow by hydrogen oxidation, a feature not recognized before. When cultivated on H2, a hydrogenase was induced and the strains were less extremely acidophilic than during growth on sulfidic ores. Cells of T. ferrooxidans grown on H2 and on ferrous iron showed 100% DNA homology. Hydrogen oxidation was not observed in eight other species of the genus Thiobacillus and in Leptospirillum ferrooxidans.     

Cloning and expression of Thiobacillus ferrooxidans mercury ion resistance genes in Escherichia coli.

A search of various domestic isolates of Thiobacillus ferrooxidans revealed that some were fairly resistant to mercury ion. A proportion of mercury-resistant clones were able to volatilize mercury, and their corresponding gene was localized not in the plasmid DNA but in chromosomal DNA. This mercury ion resistance gene was cloned in Escherichia coli. E. coli carrying the recombinant plasmid was able to grow in the presence of more than 40 micrograms of HgCl2 per ml. Deletion analysis of the recombinant plasmid showed that the entire coding sequence of the mercury ion resistance gene was located within a 2.3-kilobase fragment of the chromosomal DNA from strain E-15. At least two polypeptides (molecular mass, 56 and 16 kDa, respectively) were coded by this fragment.     

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.     

Thiobacillus ferrooxidans, a facultative hydrogen oxidizer.

The type strain (ATCC 23270) and two other strains of Thiobacillus ferrooxidans were able to grow by hydrogen oxidation, a feature not recognized before. When cultivated on H2, a hydrogenase was induced and the strains were less extremely acidophilic than during growth on sulfidic ores. Cells of T. ferrooxidans grown on H2 and on ferrous iron showed 100% DNA homology. Hydrogen oxidation was not observed in eight other species of the genus Thiobacillus and in Leptospirillum ferrooxidans.     

Sensitivity of Iron-Oxidizing Bacteria, Thiobacillus ferrooxidans and Leptospirillum ferrooxidans, to Bisulfite Ion

When grown on iron-salt medium supplemented with the bisulfite ion, Leptospirillum ferrooxidans was much more sensitive to the ion than was Thiobacillus ferrooxidans. The causes of the sensitivity of L. ferrooxidans to the bisulfite ion were studied. The bisulfite ion completely inhibited the iron-oxidizing activities of L. ferrooxidans and T. ferrooxidans at 0.02 and 0.2 mM, respectively. A trapping reagent for the bisulfite ion, formaldehyde, completely reversed the inhibition. The treatment of intact cells with 1.0 mM bisulfite ion for 1 h and washing the bisulfite ion from the cells had no harmful effects on the iron-oxidizing activity of T. ferrooxidans. However, the treatment of L. ferrooxidans with 0.1 mM bisulfite ion for 1 h completely destroyed the iron-oxidizing activity. T. ferrooxidans had sulfite:ferric ion oxidoreductase activity. In contrast, a quite low level of sulfite:ferric ion oxidoreductase activity was found in L. ferrooxidans, suggesting that it is much more difficult for L. ferrooxidans to oxidize the bisulfite ion to the less harmful sulfate than it is for T. ferrooxidans. These results suggest that the sensitivity of L. ferrooxidans to the bisulfite ion is due to a lack of an active sulfite:ferric ion oxidoreductase and the sensitivity of its iron oxidase to bisulfite ion.     

A Tn7-Like Transposon Is Present in the glmUS Region of the Obligately Chemoautolithotrophic Bacterium Thiobacillus ferrooxidans

The region downstream of the Thiobacillus ferrooxidans ATCC 33020 atp operon was examined, and the genes encoding N-acetylglucosamine-1-uridyltransferase (glmU) and glucosamine synthetase (glmS) were found. This atpEFHAGDC-glmUS gene order is identical to that of Escherichia coli. The T. ferrooxidans glmS gene was shown to complement E. coli glmS mutants for growth on minimal medium lacking glucosamine. A Tn7-like transposon, Tn5468, was found inserted into the region immediately downstream of the glmS gene in a manner similar to the site-specific insertion of transposon Tn7 within the termination region of the E. coli glmS gene. Tn5468 was sequenced, and Tn7-like terminal repeat sequences as well as several open reading frames which are related to the Tn7 transposition genes tnsA, tnsB, tnsC, and tnsD were found. Tn5468 is the closest relative of Tn7 to have been characterized to date. Southern blot hybridization indicated that a similar or identical transposon was present in three T. ferrooxidans strains isolated from different parts of the world but not in two Thiobacillus thiooxidans strains or a Leptospirillum ferrooxidans strain. Since T. ferrooxidans is an obligately acidophilic autotroph and E. coli is a heterotroph, ancestors of the Tn7-like transposons must have been active in a variety of physiologically different bacteria so that their descendants are now found in bacteria that occupy very different ecological niches.     

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     

Transposition of IST2 in Thiobacillus ferrooxidans

The genome of Thiobacillus ferrooxidans contains at least two different repetitive DNA elements. One of these elements, termed IS T2 has been sequenced and shown to exhibit the characteristics of a typical prokaryotic insertion sequence. Furthermore, preliminary evidence has implicated IS T2 in genomic rearrangements, although the mechanism of rearrangement, whether by transposition or recombination, has not been established. In this report we provide evidence from detailed restriction enzyme analyses and DNA sequencing data that support a model of transposition, consistent with the notion that IS T2 is a mobile insertion sequence.     

Conduction of pEC22, a plasmid coding for MR.EcoT22I, mediated by a resident Tn3-like transposon, Tn5396.

pEC22 is a small plasmid that encodes the restriction-modification system MR.EcoT22I. Restriction and functional analysis of the plasmid identified the positions of genes encoding that system. The plasmid is able to be conducted by conjugal plasmids, a process mediated by a transposon contained within pEC22. This cryptic transposon, called Tn5396, was isolated from pEC22 and partially sequenced. The sequence of Tn5396 is for the most part typical of transposons of the Tn3 family and is most similar to that of Tn1000. The transposon differs from closely related transposons in that it lacks well-conserved sequences in the inverted-repeat region and has an unusually long terminal inverted repeat. Consideration of regions of internal sequence similarity in this and other transposons in the Tn3 family supports a theory of the mechanism by which the ends of Tn3-like transposons may maintain substantial identity between their inverted repeats over the course of evolutionary time.     

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.     

Tn3702, a conjugative transposon in Enterococcus faecalis

Enterococcus faecalis strain D434 was found to carry on its chromosome a determinant encoding tetracycline-minocycline resistance (Tcr-Mnr) and to harbor both an R plasmid and a cryptic conjugative plasmid, pIP1141. The determinant coding for Tcr-Mnr was located on a conjugative transposon, designated Tn3702. The transposition of Tn3702 on to both pIP1141 and the hemolysin plasmid pIP964 yielded different derivatives each of which contained an 18.5-kilobase insert. The structure of Tn3702 is similar to that of the conjugative transposon Tn916.