Gennady Kholodii

Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases.

DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase‐like proteins. Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044. Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans. To our knowledge, this is the first case when a site‐specific recombination system outside a transposon has been shown to be involved in transposition.

Mercury resistance transposons of gram-negative environmental bacteria and their classification.

A total of 29 mercury resistance transposons were isolated from mercury-resistant environmental strains of proteobacteria collected in different parts of Eurasia and the USA and tested for hybridization with probes specific for transposase genes of known mercury resistance transposons. 9 were related to Tn21 in this test, 12 were related to Tn5053, 4 to Tn5041 and 1 to Tn5044; three transposons were negative in this test. Restriction mapping and DNA sequencing revealed that 12 transposons were identical or nearly identical to their corresponding relatives while the rest showed varying divergence from their closest relatives. Most of these previously unknown transposons apparently arose as a result of homologous or site-specific recombination. One of these, Tn5046, was completely sequenced, and shown to be a chimera with the mer operon and the transposition module derived from the transposons related to Tn5041 and to Tn5044, respectively. Transposon Tn5070, showing no hybridization with the specific probes used in this study, was also completely sequenced. The transposition module of Tn5070 was most closely related to that of Tn3 while the mer operon was most closely related to that of plasmid pMERPH. The merR of Tn5070 is transcribed in the same direction as the mer structural genes, which is typical for mer operons of gram-positive bacteria. Our data suggest that environmental bacteria may harbor many not yet recognized mercury resistance transposons and warrant their further inventory.

Intercontinental spread of promiscuous mercury- resistance transposons in environmental bacteria

We demonstrate that horizontal spread of mer operons similar to worldwide spread of antibiotic‐resistance genes in medically important bacteria occurred in bacteria found in ores, soils and waters. The spread was mediated by different transposons and plasmids. Some of the spreading transposons were damaged in different ways but this did not prevent their further spread. Certain transposons are mosaics composed of segments belonging to distinct sequence types. These mosaics arose as a result of homologous and site‐specific recombination. Our data suggest that the mercury‐resistance operons of Gram‐negative environmental bacteria can be considered as a worldwide population composed of a relatively small number of distinct recombining clones shared, at least partially, by environmental and clinical bacteria.

Tn5044, a novel Tn3 family transposon coding for temperature-sensitive mercury resistance

We report the discovery and characterization of the mercury resistance transposon, Tn5044, from a Xanthomonas strain from the Kamchatka peninsula. In addition to the standard set of merRTPCAD genes, the mer operon of Tn5044 contains a gene named sigY that encodes the RNA polymerase sigma factor-like protein. Mercury resistance determined by Tn5044 is expressed at low (30 degrees C) but not at elevated temperatures (37 degrees C). None of the mer operon genes downstream of merA is responsible for the temperature-sensitive mercury resistance. The transposition module of Tn5044 is closely related to those of Tn1412 isolated from medical sources and to Tn5563 and ISXc5 from environmental sources. However, Tn5044 differs from these transposons in that it has unusually long terminal inverted repeats. Sequence analysis of the transposase (tnpA) genes places Tn5044 and its close relatives into the Tn3 subgroup of the Tn3 family. However, the orientation of their resolvase and transposase genes is unusual for the Tn3 family: tnpR is proximal to the end of the transposon, while divergently transcribed tnpA is oriented inwardly. The region between tnpA and tnpR genes is unusually large and contains two short conserved open reading frames. In addition to the complete set of sequence motifs common to true resolvases, the resolvase of Tn5044 and its close relatives possesses a C-terminal extension showing no homology to known proteins. Despite this peculiarity, Tn5044 resolvase can resolve cointegrates formed during Tn5044 transposition controlled by tnpA. Genetic data suggest that the extension is essential for TnpR functioning.

Tn5041-like transposons: molecular diversity, evolutionary relationships and distribution of distinct variants in environmental bacteria.

A detailed study on the geographic distribution, molecular diversity and evolutionary relationships of 24 closely related variants of the Tn5041 transposon found among 182 mercury resistant environmental Gram-negative strains from the IMG-Hg Reference Collection is reported here. RFLP analysis, followed by the determination of partial DNA sequences, identified 14 distinct types of these transposons, which differed from each other by 1-7 single-event DNA polymorphisms. No polymorphisms were detected at the right arm of the transposons except an insertion of a new mobile DNA element carrying a mer operon (named the mer2 cassette) within the Tn5041 mer operon. According to the model presented here, the insertion occurred via homologous recombination with a circular form of the mer2 cassette. A total of 8 point mutations, 1 internal deletion, 2 end-involving deletions, 3 mosaic regions and 2 insertions were detected at the left arm of the transposons. The insertions were a transposon closely related to Tn21 but lacking the integron and a new group II intron (named INT5041C). Inspection of the geographic distribution of the Tn5041 variants suggested that at least three long-distance waves of dissemination of these variants had occurred, accompanied by homologous recombination between different Tn5041 lineages. Movements of circular DNAs by homologous recombination as a source of mosaic genes and new mer genes, and formation of unusual mosaics ending or beginning at the Tn5041 att site are discussed.

Tn5060 from the Siberian permafrost is most closely related to the ancestor of Tn21 prior to integron acquisition

A Tn21-related mercury resistance transposon, Tn5060, has been isolated from Pseudomonas sp. strain A19-1 from a 8,000-10,000-year-old Siberian permafrost sample, and sequenced. Like Tn21, the element transposes to different plasmids at a frequency of 10(-2)-10(-3) per target plasmid transfer. Comparison of the complete Tn5060 DNA sequence (8,667 bp) with that of Tn21 (19,672 bp) shows that Tn5060 does not contain integron In2 and deviates from Tn21 in four nucleotide positions. These and other comparative data demonstrate that Tn5060 is the most closely related of the characterized mercury resistances to the as yet hypothetical immediate ancestor of Tn21, TnX.

Tn5044-conferred mercury resistance depends on temperature: the complexity of the character of thermosensitivity.

Escherichia coli K12 containing the transposon Tn5044 mer operon (merR, T, P, C, and A genes) is resistant to mercuric chloride at 30°C but sensitive to this compound at 37–41.5°C. We have studied the mechanism underlying the temperature-sensitive nature of this mercury resistance phenotype, and found that the expression of the Tn5044 merA gene coding for mercuric reductase (MerA) is severely inhibited at non-permissive temperatures. Additionally, MerA showed a considerably reduced functional activity in vivo at non-permissive temperatures. However, the temperature-sensitive character of the functioning of this enzyme in cell extracts, where it interacted with one of the low-molecular weight SH compounds rather than with the transport protein MerT (as is the case in vivo), was not apparent. These data suggest that the temperature-sensitive mercury resistance phenotype should stay under control at two stages: when the merA gene is expressed and when its product interacts with MerT to accept the mercuric ion.

A novel gene, ardD, determines antirestriction activity of the non-conjugative transposon Tn5053 and is located antisense within the tniA gene

Abstract The mercury-resistance transposon Tn5053 inhibits restriction activity of the type I restriction-modification endonuclease EcoKI in Escherichia coli K12 cells. This is the first report of antirestriction activity of a non-conjugative transposon. The gene (ardD) coding for the antirestriction protein has been cloned. The ardD gene is located within the tniA gene, coding for transposase, on the complementary strand. The direction of transcription is opposite to transcription of the tniA gene.