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 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.     

Evolution of complex resistance transposons from an ancestral mercury transposon.

The molecular interrelationship of a transposon family which confers multiple antibiotic resistance and is assumed to have been generated from an ancestral mercury transposon was analyzed. Initially, the transposons Tn2613 (7.2 kilobases), encoding mercury resistance, and Tn2608 (13.5 kilobases), encoding mercury, streptomycin, and sulfonamide resistances, were isolated and their structures were analyzed. Next, the following transposons were compared with respect to their genetic and physical maps: Tn2613 and Tn501, encoding mercury resistance; Tn2608 and Tn21, encoding mercury, streptomycin, and sulfonamide resistance; Tn2607 and Tn4, encoding streptomycin, sulfonamide, and ampicillin resistance; and Tn2603, encoding mercury, streptomycin, sulfonamide, and ampicillin resistance. The results suggest that the transposons encoding multiple resistance were evolved from an ancestral mercury transposon.     

Association of the strA-strB genes with plasmids and transposons in the present-day bacteria and in bacterial strains from permafrost

Transposons closely related to the streptomycin resistance transposon of modem bacteria, Tn5393, were detected in the bacterial isolates from permafrost resistant to streptomycin. Many transposons studied were located on the medium-size plasmids with a narrow host range. None of the streptomycin-resistant strains isolated from permafrost contained small plasmids carrying the strA-strB genes and related to the broad host range plasmid RSF1010.     

Characterization of Tn3926, a new mercury-resistance transposon from Yersinia enterocolitica

A new transposon coding for mercury resistance (HgR), Tn3926, has been found in a strain of Yersinia enterocolitica, YE138A14. The element has a size of 7.8 kb and transposes to conjugative plasmids belonging to different incompatibility groups. A restriction map has been established. DNA-DNA hybridization indicates that Tn3926 displays homology with both Tn501 and Tn21; the greatest homology is shown with the regions of these transposons that encode HgR. Weaker homology is observed between Tn3926 sequences and those regions of Tn501 and Tn21 that encode transposition functions. Complementation experiments indicate that the Tn3926 transposase mediates transposition of Tn21, albeit somewhat inefficiently, but not of Tn501, while the resolvase mediates resolution of transposition cointegrates formed via Tn21, Tn501, or Tn1721.     

Hybridization of DNA probes with whole-community genome for detection of genes that encode microbial responses to pollutants: mer genes and Hg2+ resistance

Nucleic acids extracted from microbial biomass without prior culturing were hybridized with probes representing four mer operons to detect genes encoding adaptation to Hg2+ in whole-community genomes. A 29-fold enrichment in sequences similar to the mer genes of transposon Tn501 occurred during adaptation in a freshwater community. In an estuarine community, all four mer genes were only slightly enriched (by three- to fivefold), suggesting that additional, yet uncharacterized, mer genes encoded adaptation to Hg2+.     

Hybridization of DNA probes with whole-community genome for detection of genes that encode microbial responses to pollutants: mer genes and Hg2+ resistance.

Nucleic acids extracted from microbial biomass without prior culturing were hybridized with probes representing four mer operons to detect genes encoding adaptation to Hg2+ in whole-community genomes. A 29-fold enrichment in sequences similar to the mer genes of transposon Tn501 occurred during adaptation in a freshwater community. In an estuarine community, all four mer genes were only slightly enriched (by three- to fivefold), suggesting that additional, yet uncharacterized, mer genes encoded adaptation to Hg2+.     

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.     

Four genes, two ends, and a res region are involved in transposition of Tn5053: a paradigm for a novel family of transposons carrying either a mer operon or an integron

The complete nucleotide sequence of an 8447 bp-long mercury-resistance transposon (Tn 5053 ) has been determined. Tn 5053 is composed of two modules: (i) the mercury-resistance module and (ii) the transposition module. The mercury-resistance module carries a mer operon, merRTPFAD , and appears to be a single-ended relic of a transposon closely related to the classical mercury-resistance transposons Tn 21 and Tn 501 . The transposition module of Tn 5053 is bounded by 25 bp terminal inverted repeats and contains four genes involved in transposition, i.e. tniA, tniB, tniQ , and tniR . Transposition of Tn 5053 occurs via cointegrate formation mediated by the products of the tniABQ genes, followed by site-specific cointegrate resolution. This is catalysed by the product of the tniR gene at the res region, which is located upstream of tniR . The same pathway of transposition is used by Tn 402 (Tn 5090 ) which carries the integron of R751. Transposition genes of Tn 5053 and Tn 402 are interchangeable. Sequence analysis suggests that Tn 5053 and Tn 402 are representatives of a new family of transposable elements, which fall into a recently recognized superfamily of transposons including retroviruses, insertion sequences of the IS 3 family, and transposons Tn 552 and Tn 7 . We suggest that the tni genes were involved in the dissemination of integrons.     

Structure of haloacetate-catabolic IncP-1beta plasmid pUO1 and genetic mobility of its residing haloacetate-catabolic transposon

The self-transmissible plasmid pUO1 from Delftia acidovorans strain B carries two haloacetate-catabolic transposons, TnHad1 and TnHad2, and the mer genes for resistance to mercury. The complete 67,066-bp sequence of pUO1 revealed that the mer genes were also carried by two Tn402/Tn5053-like transposons, Tn4671 and Tn4672, and that the pUO1 backbone regions shared 99% identity to those of the archetype IncP-1beta plasmid R751. Comparison of pUO1 with three other IncP-1beta plasmids illustrated the importance of transposon insertion in the diversity and evolution of this group of plasmids. Mutational analysis of the four outermost residues in the inverted repeats (IRs) of TnHad2, a Tn21-related transposon, revealed a crucial role of the second residue of its IRs in transposition.     

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.     

Distribution of transposons Tn5044 and Tn5070 with unusual mer operons in environmental bacterial populations

The distribution of unusual mercury resistance transposons, Tn5044 and Tn5070, was examined. A characteristic feature of Tn5044 is temperature sensitivity of its mercury operon and the presence in the mer operon of the gene homologous to RNA polymerase a subunit. Structural organization of mercury operon Tn5070, containing minimum gene set (merRTPA), differs from mer operons of both Gram-negative and Gram-positive bacteria. None of more than two thousand environmental bacterial strains displaying mercury resistance and isolated from the samples selected from different geographical regions hybridized to Tn5040- and Tn5070-specific probes. A concept on the existence of cosmopolite, endemic, and rare transposons in environmental bacterial populations was formulated.     

Structure analysis of a class II transposon encoding the mercury resistance of the Gram-positive Bacterium bacillus megaterium MB1, a strain isolated from minamata bay, Japan.

A unique transposon was found in the chromosome of Bacillus megaterium MB1, a Gram-positive bacterium isolated from mercury-polluted sediments of Minamata Bay, Japan. The transposon region of a 14.5kb DNA fragment was amplified by PCR using a single PCR primer designed from the nucleotide sequence of an inverted repeat of class II transposons. The molecular analysis revealed that the PCR-amplified DNA fragment encodes a transposition module similar to that of Tn21. The transposon also encodes a broad-spectrum mercury resistance region having a restriction endonuclease map identical to that of Bacillus cereus RC607, a strain isolated from Boston Harbor, USA. The result of a phylogenetic analysis of the amino acid sequence of putative resolvase of the transposon showed that the transposon is phylogenetically closer to the transposons of Gram-positive bacteria than those of Gram-negative bacteria. Besides the transposition module and mer operon, the transposon encodes a mobile genetic element of bacterial group II introns between the resolvase gene and mer operon. The intron, however, does not intervene in any exon gene. The discovery of this newly found combination of the complex mobile elements may offer a clue to understanding the horizontal dissemination of broad-spectrum mercury resistance among microbes.     

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.     

Transposon Tn21, Flagship of the Floating Genome

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.     

Localization of camphor degradative plasmids on the chromosome of Pseudomonas putida strains PaW]

Camphor degradative plasmids (CAM, pRK1) are preferentially situated on chromosomes of Pseudomonas putida strains PaW. After having been transferred into Cam+ strains, the TOL plasmid pWWO dissociates into the cryptic plasmid pWWO-8 and chromosome-borne transposon Tn4651. The opposite situation, i.e. reconstruction of the TOL plasmid pWWO from the cryptic plasmid pWWO-8 and chromosome-borne catabolic operons of the pWWO plasmid has been described. Cam- derivatives of the CAM plasmid were obtained in vivo which contain the TOL plasmid transposons Tn4651 or Tn4652 as obligatory structural elements. These plasmids as well as pWWO-8 determine conjugational mobilization of chromosome-located cam operons followed by their integration into the chromosome of recipient.     

Transposon Tn21, flagship of the floating genome.

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.