Mercury Resistance Transposon Research Articles

Identification of A Novel Arsenic Resistance Transposon Nested in A Mercury Resistance Transposon of Bacillus sp. MB24.

A novel TnMERI1-like transposon designated as TnMARS1 was identified from mercury resistant Bacilli isolated from Minamata Bay sediment. Two adjacent ars operon-like gene clusters, ars1 and ars2, flanked by a pair of 78-bp inverted repeat sequences, which resulted in a 13.8-kbp transposon-like fragment, were found to be sandwiched between two transposable genes of the TnMERI1-like transposon of a mercury resistant bacterium, Bacillus sp. MB24. The presence of a single transcription start site in each cluster determined by 5′-RACE suggested that both are operons. Quantitative real time RT-PCR showed that the transcription of the arsR genes contained in each operon was induced by arsenite, while arsR2 responded to arsenite more sensitively and strikingly than arsR1 did. Further, arsenic resistance complementary experiments showed that the ars2 operon conferred arsenate and arsenite resistance to an arsB-knocked out Bacillus host, while the ars1 operon only raised arsenite resistance slightly. This transposon nested in TnMARS1 was designated as TnARS1. Multi-gene cluster blast against bacteria and Bacilli whole genome sequence databases suggested that TnMARS1 is the first case of a TnMERI1-like transposon combined with an arsenic resistance transposon. The findings of this study suggested that TnMERI1-like transposons could recruit other mobile elements into its genetic structure, and subsequently cause horizontal dissemination of both mercury and arsenic resistances among Bacilli in Minamata Bay.

Z/I1 Hybrid Virulence Plasmids Carrying Antimicrobial Resistance genes in S. Typhimurium from Australian Food Animal Production.

Knowledge of mobile genetic elements that capture and disseminate antimicrobial resistance genes between diverse environments, particularly across human–animal boundaries, is key to understanding the role anthropogenic activities have in the evolution of antimicrobial resistance. Plasmids that circulate within the Enterobacteriaceae and the Proteobacteria more broadly are well placed to acquire resistance genes sourced from separate niche environments and provide a platform for smaller mobile elements such as IS26 to assemble these genes into large, complex genomic structures. Here, we characterised two atypical Z/I1 hybrid plasmids, pSTM32-108 and pSTM37-118, hosting antimicrobial resistance and virulence associated genes within endemic pathogen Salmonella enterica serovar Typhimurium 1,4,[5],12:i:-, sourced from Australian swine production facilities during 2013. We showed that the plasmids found in S. Typhimurium 1,4,[5],12:i:- are close relatives of two plasmids identified from Escherichia coli of human and bovine origin in Australia circa 1998. The older plasmids, pO26-CRL125 and pO111-CRL115, encoded a putative serine protease autotransporter and were host to a complex resistance region composed of a hybrid Tn21-Tn1721 mercury resistance transposon and composite IS26 transposon Tn6026. This gave a broad antimicrobial resistance profile keyed towards first generation antimicrobials used in Australian agriculture but also included a class 1 integron hosting the trimethoprim resistance gene dfrA5. Genes encoding resistance to ampicillin, trimethoprim, sulphonamides, streptomycin, aminoglycosides, tetracyclines and mercury were a feature of these plasmids. Phylogenetic analyses showed very little genetic drift in the sequences of these plasmids over the past 15 years; however, some alterations within the complex resistance regions present on each plasmid have led to the loss of various resistance genes, presumably as a result of the activity of IS26. These alterations may reflect the specific selective pressures placed on the host strains over time. Our studies suggest that these plasmids and variants of them are endemic in Australian food production systems.

Variable plasmid fitness effects and mobile genetic element dynamics across Pseudomonas species.

Mobile genetic elements (MGE) such as plasmids and transposons mobilise genes within and between species, playing a crucial role in bacterial evolution via horizontal gene transfer (HGT). Currently, we lack data on variation in MGE dynamics across bacterial host species. We tracked the dynamics of a large conjugative plasmid, pQBR103, and its Tn5042 mercury resistance transposon, in five diverse Pseudomonas species in environments with and without mercury selection. Plasmid fitness effects and stability varied extensively between host species and environments, as did the propensity for chromosomal capture of the Tn5042 mercury resistance transposon associated with loss of the plasmid. Whereas Pseudomonas fluorescens and Pseudomonas savastanoi stably maintained the plasmid in both environments, the plasmid was highly unstable in Pseudomonas aeruginosa and Pseudomonas putida, where plasmid-free genotypes with Tn5042 captured to the chromosome invaded to higher frequency under mercury selection. These data confirm that plasmid stability is dependent upon the specific genetic interaction of the plasmid and host chromosome rather than being a property of plasmids alone, and moreover imply that MGE dynamics in diverse natural communities are likely to be complex and driven by a subset of species capable of stably maintaining plasmids that would then act as hubs of HGT.

PIP40a, a type 1 IncC plasmid from 1969 carries the integrative element GIsul2 and a novel class II mercury resistance transposon

The 167.5kb sequence of the conjugative IncC plasmid pIP40a, isolated from a Pseudomonas aeruginosa in 1969, was analysed. pIP40a confers resistance to kanamycin, neomycin, ampicillin, sulphonamides and mercuric ions, and several insertions in a type 1 IncC backbone were found, including copies of IS3, Tn1000 and a novel mercury resistance transposon, Tn6182. The antibiotic resistance genes were in two locations. Tn6023, containing the aphA1 kanamycin and neomycin resistance gene, is in a partial copy of Tn1/Tn2/Tn3 (blaTEM, ampicillin resistance) in the kfrA gene, and the sul2 sulphonamide resistance gene is in the integrative element GIsul2 in the position of ARI-B islands. The 11.5kb class II transposon Tn6182 is only distantly related to other class II transposons, with at most 33% identity between the TnpA of Tn6182 and TnpA of other group members. In addition, the inverted repeats are 37bp rather than 38bp, and the likely resolution enzyme is a tyrosine recombinase (TnpI). Re-annotation of GIsul2 revealed genes predicted to confer resistance to arsenate and arsenite, but resistance was not detected. The location of GIsul2 confirms it as the progenitor of the ARI-B configurations seen in many IncC plasmids isolated more recently. However, GIsul2 has integrated at the same site in type 1 and type 2 IncC plasmids, indicating that it targets this site. Analysis of the distribution of GIsul2 revealed that it in addition to its chromosomal integration site at the 3′-end of the guaA gene, it has also integrated into other plasmids, increasing its mobility.

IncA/C Conjugative Plasmids Mobilize a New Family of Multidrug Resistance Islands in Clinical Vibrio cholerae Non-O1/Non-O139 Isolates from Haiti

ABSTRACTMobile genetic elements play a pivotal role in the adaptation of bacterial populations, allowing them to rapidly cope with hostile conditions, including the presence of antimicrobial compounds. IncA/C conjugative plasmids (ACPs) are efficient vehicles for dissemination of multidrug resistance genes in a broad range of pathogenic species of Enterobacteriaceae. ACPs have sporadically been reported in Vibrio cholerae, the infectious agent of the diarrheal disease cholera. The regulatory network that controls ACP mobility ultimately depends on the transcriptional activation of multiple ACP-borne operons by the master activator AcaCD. Beyond ACP conjugation, AcaCD has also recently been shown to activate the expression of genes located in the Salmonella genomic island 1 (SGI1). Here, we describe MGIVchHai6, a novel and unrelated mobilizable genomic island (MGI) integrated into the 3′ end of trmE in chromosome I of V. cholerae HC-36A1, a non-O1/non-O139 multidrug-resistant clinical isolate recovered from Haiti in 2010. MGIVchHai6 contains a mercury resistance transposon and an integron In104-like multidrug resistance element similar to the one of SGI1. We show that MGIVchHai6 excises from the chromosome in an AcaCD-dependent manner and is mobilized by ACPs. Acquisition of MGIVchHai6 confers resistance to β-lactams, sulfamethoxazole, tetracycline, chloramphenicol, trimethoprim, and streptomycin/spectinomycin. In silico analyses revealed that MGIVchHai6-like elements are carried by several environmental and clinical V. cholerae strains recovered from the Indian subcontinent, as well as from North and South America, including all non-O1/non-O139 clinical isolates from Haiti.

Mercury resistance transposons in Bacilli strains from different geographical regions.

A total of 65 spore-forming mercury-resistant bacteria were isolated from natural environments worldwide in order to understand the acquisition of additional genes by and dissemination of mercury resistance transposons across related Bacilli genera by horizontal gene movement. PCR amplification using a single primer complementary to the inverted repeat sequence of TnMERI1-like transposons showed that 12 of 65 isolates had a transposon-like structure. There were four types of amplified fragments: Tn5084, Tn5085, Tn(d)MER3 (a newly identified deleted transposon-like fragment) and Tn6294 (a newly identified transposon). Tn(d)MER3 is a 3.5-kb sequence that carries a merRETPA operon with no merB or transposase genes. It is related to the mer operon of Bacillus licheniformis strain FA6-12 from Russia. DNA homology analysis shows that Tn6294 is an 8.5-kb sequence that is possibly derived from Tn(d)MER3 by integration of a TnMERI1-type transposase and resolvase genes and in addition the merR2 and merB1 genes. Bacteria harboring Tn6294 exhibited broad-spectrum mercury resistance to organomercurial compounds, although Tn6294 had only merB1 and did not have the merB2 and merB3 sequences for organomercurial lyases found in Tn5084 of B. cereus strain RC607. Strains with Tn6294 encode mercuric reductase (MerA) of less than 600 amino acids in length with a single N-terminal mercury-binding domain, whereas MerA encoded by strains MB1 and RC607 has two tandem domains. Thus, Tn(d)MER3 and Tn6294 are shorter prototypes for TnMERI1-like transposons. Identification of Tn6294 in Bacillus sp. from Taiwan and in Paenibacillus sp. from Antarctica indicates the wide horizontal dissemination of TnMERI1-like transposons across bacterial species and geographical barriers.

Tn6249, a new Tn6162 transposon derivative carrying a double-integron platform and involved with acquisition of the blaVIM-1 metallo-β-lactamase gene in Pseudomonas aeruginosa.

The In70.2 integron platform appears to be a conserved structure involved in the dissemination of the blaVIM-1 metallo-β-lactamase gene in Pseudomonas aeruginosa. The genetic context of the In70.2 integron platform from P. aeruginosa VR-143/97, the VIM-1-producing index strain isolated in Italy in 1997, was fully characterized by a next-generation sequencing approach refined by conventional sequencing. The In70.2 integron platform from VR-143/97 was found to be associated with a defective Tn402-like transposon inserted into the urf2 gene of a Tn3 family transposon of an original structure, named Tn6249, which also carried a partially deleted mer operon and an In90 integron platform in a tail-to-tail orientation. Tn6249 was inserted into a PACS171b-like genomic island, which was in turn inserted into the endA gene of the Pseudomonas chromosomal backbone. Tn6249 showed a similar structure and a conserved location with respect to that of Tn6060, a Tn3 family transposon associated with In70.2 and carrying a double-integron platform, which was detected in a VIM-1-producing P. aeruginosa strain isolated in Australia in 2008. Both Tn6249 and Tn6060 are apparently derived from Tn6162, a mercury resistance transposon carrying an integron platform, which was found in P. aeruginosa isolates from different geographic locations. The conservation of the genetic context of Tn6249 and Tn6060 suggests an in situ evolution of these elements after the insertion of a Tn6162-like ancestor into the PACS171b-like genomic island (GI) present in the genome of a successful widespread P. aeruginosa clonal lineage.

Antirestriction activity of the mercury resistance nonconjugative transposon Tn5053 is controlled by the protease ClpXP

When transformed into Escherichia coli K12 strains, the mercury resistance transposon Tn5053@ exhibits high antirestriction activity against the EcoKI type I restriction and modification system. The products of the genes merR and ardD contribute to the antirestriction activity of Tn5053. The merR gene encodes the MerR protein, the transcription regulator of the mer operon genes. The ardD gene is responsible for ArdD protein synthesis and is located within the tni operon. In the following study, it was demonstrated that the antirestriction activity of the transposon Tn5053 is absent in E. coli K12 strains with the mutant genes clpX, clpP, and recA. The antirestriction effect of Tn5053 is not enhanced by 2-aminopurine. The Tn5053 antirestriction activity is not altered in E. coli K12 with the mutant dam gene; however, it is decreased in the E. coli K12 mutD. It is assumed that the activities of the MerR and ArdD proteins lead to the formation of a significant amount of unmodified DNA in the bacterial cell, causing the SOS-dependent reduction of the EcoKI (R2M2S) enzyme activity associated with ClpXP-induced proteolysis of the R-subuinit.

Complete Nucleotide Sequence and Analysis of Two Conjugative Broad Host Range Plasmids from a Marine Microbial Biofilm

The complete nucleotide sequence of plasmids pMCBF1 and pMCBF6 was determined and analyzed. pMCBF1 and pMCBF6 form a novel clade within the IncP-1 plasmid family designated IncP-1 ς. The plasmids were exogenously isolated earlier from a marine biofilm. pMCBF1 (62 689 base pairs; bp) and pMCBF6 (66 729 bp) have identical backbones, but differ in their mercury resistance transposons. pMCBF1 carries Tn5053 and pMCBF6 carries Tn5058. Both are flanked by 5 bp direct repeats, typical of replicative transposition. Both insertions are in the vicinity of a resolvase gene in the backbone, supporting the idea that both transposons are “res-site hunters” that preferably insert close to and use external resolvase functions. The similarity of the backbones indicates recent insertion of the two transposons and the ongoing dynamics of plasmid evolution in marine biofilms. Both plasmids also carry the insertion sequence ISPst1, albeit without flanking repeats. ISPs1is located in an unusual site within the control region of the plasmid. In contrast to most known IncP-1 plasmids the pMCBF1/pMCBF6 backbone has no insert between the replication initiation gene (trfA) and the vegetative replication origin (oriV). One pMCBF1/pMCBF6 block of about 2.5 kilo bases (kb) has no similarity with known sequences in the databases. Furthermore, insertion of three genes with similarity to the multidrug efflux pump operon mexEF and a gene from the NodT family of the tripartite multi-drug resistance-nodulation-division (RND) system in Pseudomonas aeruginosa was found. They do not seem to confer antibiotic resistance to the hosts of pMCBF1/pMCBF6, but the presence of RND on promiscuous plasmids may have serious implications for the spread of antibiotic multi-resistance.

A novel gene,ardD, determines antirestriction activity of the non-conjugative transposon Tn5053and is located antisense within thetniAgene

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.

Evolution of IncHI2 plasmids via acquisition of transposons carrying antibiotic resistance determinants

To investigate the relationships between IncHI2 plasmids conferring resistance to antibiotics isolated in Australia and those from other countries. PCR, restriction digestion, cloning and DNA sequencing were used to characterize transposons and determine their location in IncHI2 plasmids recovered from Salmonella enterica isolates from Australian animals. Tn10, carrying the tet(B) tetracycline resistance determinant, was found in IncHI2 plasmids pSRC26 and pSRC125 recovered from S. enterica serovar Typhimurium from Australian cattle and in IncHI2 plasmids from serovar Infantis isolates from chickens. Its location was the same as seen in the IncHI2 reference plasmid R478. The location of Tn1696-related mercury and multiple antibiotic resistance transposons was also the same in all of the Australian plasmids, and the mer end was in the same position as the mer module in R478. However, R478 has lost the tnp end (including most of the integron) and some adjacent sequence. The sequence adjacent to the tnp end of the Tn1696-related transposons in the Australian plasmids is in the bla(CMY-8)-carrying plasmid pK29, but only 22 bp from the transposon inverted repeat remains. These plasmids all belong to the same evolutionary lineage. Neither transposon was found in TP116, which represents a separate lineage. Transposon locations are useful markers for lineages of closely related plasmids. The configuration surrounding the Tn1696-like transposons in the Australian IncHI2 plasmids is ancestral to those found in R478 and pK29, each of which has part of the transposon and adjacent sequence replaced by other resistance genes.

Sequencing and comparative analysis of IncP-1α antibiotic resistance plasmids reveal a highly conserved backbone and differences within accessory regions

Although IncP-1 plasmids are important for horizontal gene transfer among bacteria, in particular antibiotic resistance spread, so far only three plasmids from the subgroup IncP-1α have been completely sequenced. In this study we doubled this number. The three IncP-1α plasmids pB5, pB11 and pSP21 were isolated from bacteria of two different sewage treatment plants and sequenced by a combination of next-generation and capillary sequencing technologies. A comparative analysis including the previously analysed IncP-1α plasmids RK2, pTB11 and pBS228 revealed a highly conserved plasmid backbone (at least 99.9% DNA sequence identity) comprising 54 core genes. The accessory elements of the plasmid pB5 constitute a class 1 integron interrupting the parC gene and an IS6100 copy inserted into the integron. In addition, the tetracycline resistance genes tetAR and the ISTB11-like element are located between the klc operon and the trfA-ssb operon. Plasmid pB11 is loaded with a Tn5053-like mercury resistance transposon between the parCBA and parDE operons and contains tetAR that are identical to those identified in plasmid pB5 and the insertion sequence ISSP21. Plasmid pSP21 harbours an ISPa7 element in a Tn402 transposon including a class 1 integron between the partitioning genes parCBA and parDE. The IS-element ISSP21 (99.89% DNA sequence identity to ISSP21 from pB11), inserted downstream of the tetR gene and a copy of ISTB11 (identical to ISTB11 on pTB11) inserted between the genes pncA and pinR. On all three plasmids the accessory genes are almost always located between the backbone modules confirming the importance of the backbone functions for plasmid maintenance. The striking backbone conservation among the six completely sequenced IncP-1α plasmids is in contrast to the much higher diversity within the IncP-1β subgroup.

Purification and function analysis of a broad-spectrum organomercurial lyase (MerB3) from mercury resistance transposon, TnMERI1

Purification and function analysis of a broad-spectrum organomercurial lyase (MerB3) from mercury resistance transposon, TnMERI1

Transposons Related to Tn1696in IncHI2 Plasmids in Multiply Antibiotic ResistantSalmonella entericaSerovar Typhimurium from Australian Animals

Conjugative IncHI2 plasmids carrying tetracycline, trimethoprim, and sulphonamide resistance genes were recovered from two multiply antibiotic resistant Salmonella enterica serovar Typhimurium isolates from Australian food-producing animals. Transposons related to the mercury resistance transposon Tn1696 were identified in both IncHI2 plasmids. These transposons contained an In4-type class 1 integron that carried a dfrA5 trimethoprim resistance gene cassette and the sul1 sulfonamide resistance gene. These integrons were located in the same position as In4 in Tn1696. The integron from one isolate includes a large transposon-like structure containing four IS26 and the strAB, sul2, bla(TEM), and aphA1 genes conferring resistance to streptomycin, sulphonamides, ampicillin, kanamycin, and neomycin, respectively. This structure is flanked by an 8-bp duplication, but it includes both the aphA1-containing transposon Tn4352 and a transposon, Tn6029, carrying genes derived from RSF1010 and from Tn2. However, Tn4352 and Tn6029 overlap, sharing one IS26 copy. This suggests that they do not move by a standard transpositional mechanism. A circular intermediate, carrying only the region containing the resistance gene(s) and one of the IS26 bounding it, is proposed as an intermediate.

Tn502 and Tn512 are res site hunters that provide evidence of resolvase-independent transposition to random sites.

In this study, we report on the transposition behavior of the mercury(II) resistance transposons Tn502 and Tn512, which are members of the Tn5053 family. These transposons exhibit targeted and oriented insertion in the par region of plasmid RP1, since par-encoded components, namely, the ParA resolvase and its cognate res region, are essential for such transposition. Tn502 and, under some circumstances, Tn512 can transpose when par is absent, providing evidence for an alternative, par-independent pathway of transposition. We show that the alternative pathway proceeds by a two-step replicative process involving random target selection and orientation of insertion, leading to the formation of cointegrates as the predominant product of the first stage of transposition. Cointegrates remain unresolved because the transposon-encoded (TniR) recombination system is relatively inefficient, as is the host-encoded (RecA) system. In the presence of the res-ParA recombination system, TniR-mediated (and RecA-mediated) cointegrate resolution is highly efficient, enabling resolution both of cointegrates involving functional transposons (Tn502 and Tn512) and of defective elements (In0 and In2). These findings implicate the target-encoded accessory functions in the second stage of transposition as well as in the first. We also show that the par-independent pathway enables the formation of deletions in the target molecule.

Expanded-Spectrum β-Lactamase PER-1 in an Environmental Aeromonas media Isolate from Switzerland

Aeromonas spp. are environmental reservoirs of resistance determinants to different classes of antibiotic molecules (1, 3, 7). Intrinsic resistance to β-lactams among this genus may arise from the expression of chromosomal β-lactamases and/or efflux pumps (5). Acquired β-lactamases conferring resistance to narrow-spectrum β-lactams, such as OXA-type enzymes and the penicillinases TEM-1 and SHV-1, have been identified in that genus as well as in a single report of the extended-spectrum β-lactamase (ESBL) TEM-24 (2, 4). The aim of this study was to evaluate the prevalence of ESBLs among Aeromonas sp. strains recovered from several rivers, lakes, and activated sludges from the southern part of the Swiss Alps. Water samples were collected between 2002 and 2005 from the rivers Ticino and Vedeggio, from the lakes Cadagno and Lugano, and from activated sludges, all located in the Tessin county of Switzerland. The water samples were plated and growing bacteria identified by conventional biochemical methods (API-NE system; bioMerieux, Marcy-l'Etoile, France). All of the 50 Aeromonas sp. strains recovered were tested for ESBL detection by a synergy test using disks of ticarcillin-clavulanic acid, ceftazidime, cefotaxime, and aztreonam. The susceptibility testing of isolates was performed using Etest strips (AB Biodisk, Solna, Sweden). A single isolate, A72, recovered from an activated sludge located in Bioggio, displayed an ESBL phenotype. This isolate was resistant to most β-lactams, except cephamycins and carbapenems (Table ​(Table1).1). PCR amplifications performed as described previously (9), using primers specific for ESBL-encoding genes, followed by sequencing identified the blaPER-1 gene. Genotyping of isolate A72, according to the results of gyrB sequencing (10), identified Aeromonas media, a waterborne species known to cause diarrhea in humans (6). TABLE 1. MICs of antimicrobial agents for Aeromonas media A72, E. coli J53 harboring natural plasmid p72, and E. coli reference strain J53 A 70-kb plasmid (pAM) harboring the blaPER-1 gene was transferred by conjugation into the Escherichia coli J53 recipient strain, using a selection made of ceftazidime (10 μg/ml) and sodium azide (100 μg/ml). Transconjugant E. coli J53(pAM) showed an antimicrobial susceptibility pattern mirroring that of A. media A72 (Table ​(Table11). PCR mapping performed as described previously (9) identified the blaPER-1 gene as part of a Tn1213 composite transposon, as previously found (9). In addition, this Tn1213 structure was bracketed by the aphA6 and strA genes (encoding resistance to aminoglycosides) of transposon Tn5393d, as already observed in an Alcaligenes faecalis clinical isolate producing PER-1 recovered from Italy. In that latter isolate, the Tn5393d transposon was inserted into a Tn501 backbone carrying a mercury resistance transposon (8) that was not identified in A. media A72 by PCR mapping. Our study constitutes the first identification of an ESBL-producing environmental Aeromonas sp. strain. The occurrence of blaPER-1 on a conjugative and broad-host-range plasmid, inside a genetic context similar to that described for an A. faecalis clinical isolate recovered in a neighboring country, may indicate that Aeromonas spp. may act as vehicles for spreading such an antibiotic resistance determinant. These findings also strengthen the role of Aeromonas spp. as reservoirs or vectors of antimicrobial resistance determinants of clinical relevance, as recently exemplified by the identification of the plasmid-mediated quinolone resistance qnrS2 gene in Aeromonas punctata and A. media from the Seine River, Paris, France (1).

Facilities for transcription and mobilization of an exon-less bacterial group II intron nested in transposon Tn MERI1

Transcription and mobilization facilities of a group II intron, B.me.I1, which was classified within a group IIB subclass and nested in a broad-spectrum mercury resistance transposon Tn MERI1 found from the chromosome of Bacillus megaterium strain MB1 were investigated. Though B.me.I1 does not intervene in any recognizable exon gene, the splicing ability of B.me.I1 in Escherichia coli was confirmed by applying RT-PCR with RNA template transcribed by using a T7 RNA polymerase-promoter expression system. The homing activity of B.me.I1 was confirmed by using an intron-less allele fragment as a specific integration site which was cloned from Tn 5084, a Tn MERI1-family mercury resistance transposon. The transcription and splicing of B.me.I1 in the original host, the strain MB1, were also observed as its natural property. Primer-walking and 5′ RACE analyses showed that the transcription start site and the putative promoter region of B.me.I1 were located on the antisense strand of tnpR gene of Tn MERI1. Therefore, it is cleared that Tn MERI1 provides a specific integration site and facilities for transcription of B.me.I1. From these results, it is considered that these genetic facilities given by the transposon Tn MERI1 confer splicing and homing capability on B.me.I1, even though the group II intron is not associated with any exon gene.

Increased abundance of IncP-1beta plasmids and mercury resistance genes in mercury-polluted river sediments: first discovery of IncP-1beta plasmids with a complex mer transposon as the sole accessory element.

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1beta replicon-specific sequences detected in total community DNA. The isolation of IncP-1beta plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1beta plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1beta plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTDeltaP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1beta plasmids R751 and pB8. However, this is the first example of an IncP-1beta plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.

Dissemination and Persistence of bla CTX-M-9 Are Linked to Class 1 Integrons Containing CR1 Associated with Defective Transposon Derivatives from Tn 402 Located in Early Antibiotic Resistance Plasmids of IncHI2, IncP1-α, and IncFI Groups

This study analyzes the diversity of In60, a class 1 integron bearing CR1 and containing bla(CTX-M-9), and its association with Tn402, Tn21, and classical conjugative plasmids among 45 CTX-M-9-producing clinical strains (41 Escherichia coli strains, 2 Klebsiella pneumoniae strains, 1 Salmonella enterica strain, and 1 Enterobacter cloacae strain). Forty-five patients in a Spanish tertiary care hospital were studied (1996 to 2003). The diversity of In60 and association of In60 with Tn402 or mercury resistance transposons were investigated by overlapping PCR assays and/or hybridization. Plasmid characterization included comparison of restriction fragment length polymorphism patterns and determination of incompatibility group by PCR-based replicon typing, sequencing, and hybridization. CTX-M-9 plasmids belonged to IncHI2 (n = 26), IncP-1alpha (n = 10), IncFI (n = 4), and IncI (n = 1) groups. Genetic platforms containing bla(CTX-M-9) were classified in six types in relation to the In60 backbone and in eight subtypes in relation to Tn402 derivatives. They were associated with Tn21 sequences when located in IncP-1alpha or IncHI2 plasmids. Our study identified bla(CTX-M-9) in a high diversity of CR1-bearing class 1 integrons linked to different Tn402 derivatives, often to Tn21, highlighting the role of recombination events in the evolution of antibiotic resistance plasmids. The presence of bla(CTX-M-9) on broad-host-range IncP-1alpha plasmids might contribute to its dissemination to hosts that were not members of the family Enterobacteriaceae.

Participation of the recA determinant in the transposition of class II transposon mini-Tn MERI1

As an initial step to understand the mobile nature of class II mercury resistance transposon Tn MERI1, the effect of the recA gene on translocation of mini-Tn MERI1 was evaluated. A higher transposition frequency in the LE392 strain (2.4 ± 1.2 × 10 −5) than in the recA-deficient DH1 strain (1.2 ± 0.8 × 10 −6) indicated participation of the recA gene in mini-Tn MERI1 transposition. Introduction of the recA gene into the DH1 strain complemented the transposition frequency at the same level as in LE392 and confirmed participation of the recA gene in transposition. However, treatment of cells by stress agents, including irradiation of up to 3000 J m −2 UV doses, did not alter the transposition frequency and suggested independence of RecA from the SOS stress response. Further analysis of transconjugants indicated participation of RecA in the resolution of the cointegrate structure of the transposon. These results suggested that RecA is a constitutive cellular factor that increases translocation of mini-Tn MERI1 and may participate in dissemination of Tn MERI1-like transposons.