Two aberrant mercury resistance transposons in the Pseudomonas stutzeri plasmid pPB

The two mer operons of the Pseudomonas stutzeri OX plasmid pPB and their flanking regions have been sequenced and found to be part of two aberrant transposons. The narrow spectrum mer operon is almost identical to that of Tn501, but is associated with the remnants of Tn5053 tni genes rather than the Tn501 transposition module. The broad spectrum mer operon shows an overall homology with that of Tn5053, but differs from it in the presence of a merB gene, absent in Tn5053, and a merC gene instead of a merF. The pPB broad spectrum mer operon is associated with an incomplete Tn5053-like transposition module and with the Tn501 tnp genes, which are proximal, respectively, to the end and to the beginning of the mer operon. A hypothesis about pPB evolution is presented.     

Mobility and Generation of Mosaic Non-Autonomous Transposons by Tn3-Derived Inverted-Repeat Miniature Elements (TIMEs)

Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons – Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.     

Distribution and Replication of the Pathogenicity Plasmid pPATH in Diverse Populations of the Gall-Forming Bacterium Pantoea agglomerans

Pantoea agglomerans has been transformed from a commensal bacterium into two related gall-forming pathovars by acquisition of pPATH plasmids containing a pathogenicity island (PAI). This PAI harbors an hrp/hrc gene cluster, type III effectors, and phytohormone biosynthetic genes. DNA typing by pulsed-field gel electrophoresis revealed two major groups of P. agglomerans pv. gypsophilae and one group of P. agglomerans pv. betae. The pPATH plasmids of the different groups had nearly identical replicons (98% identity), and the RepA protein showed the highest level of similarity with IncN plasmid proteins. A series of plasmids, designated pRAs, in which the whole replicon region (2,170 bp) or deleted derivatives of it were ligated with nptI were generated for replicon analysis. A basic 929-bp replicon (pRA6) was sufficient for replication in Escherichia coli and in nonpathogenic P. agglomerans. However, the whole replicon region (pRA1) was necessary for expulsion of the pPATH plasmid, which resulted in the loss of pathogenicity. The presence of direct repeats in the replicon region suggests that the pPATH plasmid is an iteron plasmid and that the repeats may regulate its replication. The pPATH plasmids are nonconjugative but exhibit a broad host range, as shown by replication of pRA1 in Erwinia, Pseudomonas, and Xanthomonas. Restriction fragment length polymorphism analyses indicated that the PAIs in the two groups of P. agglomerans pv. gypsophilae are similar but different from those in P. agglomerans pv. betae. The results could indicate that the pPATH plasmids evolved from a common ancestral mobilizable plasmid that was transferred into different strains of P. agglomerans.     

Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer

The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the OmegaStr(r)/Spc(r) gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 x 10(-4) per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.     

Molecular Characterization of an Aberrant Mercury Resistance Transposable Element from an Environmental Acinetobacter Strain

We present the complete nucleotide sequence of a mer operon located on a 60-kb conjugative plasmid pKLH2 from an environmental bacterium, Acinetobacter calcoaceticus , isolated from a mercury mine. The pKLH2 mer operon has essentially the same gene organization as that of Tn21 and Tn501 from clinical bacteria. The pKLH2 mer operon nucleotide sequence shows 85.5% identity with the Tn501 and 80.9% identity with the Tn21 sequences. Vestigial sequences have been found at the ends of the pKLH2 mer operon, indicating that the pKLH2 mer operon was once a part of a Tn21-like transposon, which had committed suicide by an aberrant resolution event.     

Mobilization of vancomycin resistance by transposon-mediated fusion of a VanA plasmid with an Enterococcus faecium sex pheromone-response plasmid

A striking feature of recent outbreaks of vancomycin-resistant (Vm^R) enterococci is the apparent horizontal dissemination of resistance determinants. The plasmids pHKK702 and pHKK703 from Enterococcus faecium clinical isolate R7 have been implicated in the conjugal transfer of Vm^R. pHKK702 is a 41-kb plasmid that contains an element indistinguishable from the glycopeptide-resistance transposon Tn1546. pHKK703 is an approx. 55-kb putative sex pheromone-response plasmid that is required for conjugative mobilization of pHKK702. During experiments in which strain R7 was used as a donor, a highly conjugative Vm^R transconjugant was isolated that formed constitutive cellular aggregates. Restriction analyses and DNA hybridizations revealed that the transconjugant harbored a single plasmid of approx. 92 kb and this plasmid (pHKK701) was composed of DNA from both pHKK702 and pHKK703. Results from DNA sequence analyses showed that a 39-kb composite transposon (Tn5506) from pHKK702 had inserted into pHKK703. The left end of Tn5506 contained a single insertion sequence (IS) element, IS1216V2, whereas the right end was composed of a tandem IS structure consisting of the novel 1065-bp IS1252 nested within an IS1216V1 element. Transposition of Tn5506 from pHKK702 to pHKK703 created an 8-bp target sequence duplication at the site of insertion and interrupted an ORF (ORFX) that was 91% identical to that of prgX, a gene proposed to negatively regulate sex pheromone response of the E. faecalis plasmid, pCF10. We propose that the interruption of ORFX by Tn5506 led to the constitutive cellular aggregation phenotype and thereby enhanced the efficiency with which Vm^R was transferred. Similar IS1216V-mediated transposition events may contribute to the horizontal spread of glycopeptide resistance among enterococci in nature.     

Novel mercury resistance determinants carried by IncJ plasmids pMERPH and R391

Summary HgCl₂ resistance (Hg^r) in a strain of Pseudomonas putrefaciens isolated from the River Mersey was identified as plasmid-borne by its transfer to Escherichia coli in conjugative matings. This plasmid, pMERPH, could not be isolated and was incompatible with the chromosomally integrated IncJ Hg^r plasmid R391. pMERPH and R391 both express inducible, narrow-spectrum mercury resistance and detoxify HgCl₂ by volatilization. The cloned mer determinants from pMERPH (pSP100) and R391 (pSP200) have very similar restriction maps and express identical polypeptide products. However, these features show distinct differences from those of the Tn501 family of mer determinants. pSP100 and pSP200 failed to hybridize at moderate stringency to merRTPA and merC probes from Tn501 and Tn21, respectively. We conclude that the IncJ mer determinants are only distantly related to that from Tn501 and its closely homologous relatives and that it identifies a novel sequence which is relatively rare in bacteria isolated from natural environments.     

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.     

Heat Curing of a Sym Plasmid in a Fast-Growing Rhizobium sp. That Is Able to Nodulate Legumes and the Nonlegume Parasponia sp

Genes involved in nodulation of both legumes and the nonlegume Parasponia sp., as well as nitrogenase genes, reside on a large plasmid in a fast-growing Rhizobium sp. from Lablab purpureus. This plasmid can be cured by incubation at elevated temperatures and can be mobilized by the P1 group plasmid RP1::Tn501.     

Comparative Genomics of Community-Acquired ST59 Methicillin-Resistant Staphylococcus aureus in Taiwan: Novel Mobile Resistance Structures with IS1216V

Methicillin-resistant Staphylococcus aureus (MRSA) with ST59/SCCmecV and Panton-Valentine leukocidin gene is a major community-acquired MRSA (CA-MRSA) lineage in Taiwan and has been multidrug-resistant since its initial isolation. In this study, we studied the acquisition mechanism of multidrug resistance in an ST59 CA-MRSA strain (PM1) by comparative genomics. PM1’s non-β-lactam resistance was encoded by two unique genetic traits. One was a 21,832-bp composite mobile element structure (MES_PM1), which was flanked by direct repeats of enterococcal IS1216V and was inserted into the chromosomal sasK gene; the target sequence (att) was 8 bp long and was duplicated at both ends of MES_PM1. MES_PM1 consisted of two regions: the 5′-end side 12.4-kb region carrying Tn551 (with ermB) and Tn5405-like (with aph[3′]-IIIa and aadE), similar to an Enterococcus faecalis plasmid, and the 3′-end side 6,587-bp region (MES_cat) that carries cat and is flanked by inverted repeats of IS1216V. MES_cat possessed att duplication at both ends and additional two copies of IS1216V inside. MES_PM1 represents the first enterococcal IS1216V-mediated composite transposon emerged in MRSA. IS1216V-mediated deletion likely occurred in IS1216V-rich MES_PM1, resulting in distinct resistance patterns in PM1-derivative strains. Another structure was a 6,025-bp tet-carrying element (MES_tet) on a 25,961-bp novel mosaic penicillinase plasmid (pPM1); MES_tet was flanked by direct repeats of IS431, but with no target sequence repeats. Moreover, the PM1 genome was deficient in a copy of the restriction and modification genes (hsdM and hsdS), which might have contributed to the acquisition of enterococcal multidrug resistance.     

Transposition of a DNA fragment flanked by two inverted Tn1 sequences

The 32 Md fragment (derived from plasmid RP4::Tn1) carrying the Km^r gene and flanked by two inverted Tn1 elements is capable of recA-independent translocation to other plasmids. We designated this new transposon Tn1755. In various crosses, frequencies of Tn1755 transposition to plasmids Co1B-R3, R15 and F′ColVBtrp varied from 2.5 to 90% of the frequencies of Tn1 transposition. Tn1755 can integrate into various sites of the recipient plasmids. We failed to observe transposition of another RP4::Tn1 fragment flanked by two opposingly oriented Tn1 transposons and harboring the Tc^r gene. Presumably, to form a new transposable structure, other features must also be of importance.     

Construction of a Slime Negative Transposon Mutant in Staphylococcus epidermidis Using the Enterococcus faecalis Transposon Tn917

The slime-producing Staphylococcus epidermidis strain sensu strictu CNS23 was transformed by protoplast transformation with the plasmid pTV1 which carries transposon Tn917. Using this transposon mutagenesis system we obtained the Tn917-inserted mutant CT512, which has lost the ability to produce slime. A single insertion of the trasposon Tn917 into the chromosome of CT512 could be detected by Southern hybridization. This mutant showed a significantly higher stability concerning its slime-negative phenotype compared with spontaneous slime-negative mutants of S. epidermidis strain CNS23. In slime-ELISA no slime-associated antigen could be detected in extracts of the transposon mutant. Compared to slime-positive S. epidermidis strains, CT512 lacked in accumulative growth in microtiter tube test.     

Comparative Genomics of the IncA/C Multidrug Resistance Plasmid Family

Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.Antimicrobial compounds have been used extensively in agriculture since the 1960s not only to treat and prevent disease in plants, fruits, vegetables, and animals but also to promote growth in fish, poultry, and other livestock (42). The risk of transferring antimicrobial drug resistance to nonresistant bacteria and the propagation of multidrug-resistant (MDR) bacteria from agricultural to clinical and/or community-associated settings are being debated by research, regulatory, and health authorities (27, 28). In this context, the recent discovery of a group of self-transferable IncA/C antimicrobial resistance plasmids, which are widely distributed among agricultural nontyphoidal Salmonella enterica isolates from the United States (24, 45) has caused considerable concern in the public health community. Similar IncA/C plasmids were identified in an MDR isolate from Madagascar of Yersinia pestis, the causative agent of the plague (16), and MDR strains of Vibrio cholerae O139 from China (34), as well as in MDR isolates of the fish pathogen Photobacterium damselae subsp. piscicida from the United States and Japan (21). While the IncA/C group of MDR plasmids seems to be efficient in collecting antimicrobial resistance traits and mobilizing them across geographical and taxonomical borders, little is known about the evolutionary origin of these plasmids or the genetic basis for their spread.The IncA/C reference plasmid, pRA1, was isolated in 1971 from the fish pathogen Aeromonas liquefaciens, later renamed Aeromonas hydrophila, as a transferable antimicrobial resistance plasmid conferring resistance to sulfonamides and tetracyclines (2). The repA gene of pRA1, located at the origin of replication and responsible for encoding the replication initiation protein A, has been sequenced (25) and is used for PCR-based replicon typing of IncA/C plasmids (7). repA genes from all sequenced IncA/C plasmids to date share at least 98% nucleotide sequence identity.To better understand the evolutionary origin of IncA/C plasmids, pRA1 was isolated, sequenced, and compared to all IncA/C plasmid sequences currently available. In addition to pRA1, a pRA1-derived cryptic IncA/C plasmid, designated pRAx, was also sequenced and included in the analysis. pRAx was isolated from Escherichia coli D7-3, a strain that was obtained through the conjugative transfer of pRA1 from A. hydrophila in 1980 (30). While the laboratory history of the pRAx-carrying strain E. coli D7-3 since the conjugative plasmid acquisition is unknown, pRAx was included in this study as it tested positive for the repA reference gene from pRA1 (100% nucleotide sequence identity) but negative for 11 out of 12 additional IncA/C marker genes that were shown to be part of a conserved plasmid backbone shared by recently isolated IncA/C plasmids (45).     

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.It is widely recognized that mobile genetic elements contribute to genome plasticity and have been a driving force in the emergence and spread of resistance determinants within and between bacterial species; their impact is ongoing (10, 51). Significant among these elements are various classes of plasmids, transposons, and integrons which may lack resistance determinants or carry one or multiple determinants. Resistance determinants that have become globally dispersed in environmental and clinically significant bacteria include mercury(II) resistance (2, 17), evident even in ancient bacteria (27), and antibiotic resistance, which has increased in dominance since the advent of the antibiotic era (23, 40).This paper concerns the mercury resistance (mer) transposons Tn502 and Tn512, whose sequence organization and transpositional behavior show that they are new members of a family of elements exemplified by the mer transposon Tn5053 (22). These elements are closely related to those in the Tn402 family, which contain an integron (intI) recombination system (14, 36). Members of the two families differ in the positions of the mer or intI determinants (modules) near one end of the transposition (tni) module. The latter module contains four genes (tniABQR), and the entire transposon is bounded by 25-bp inverted-repeat termini (IRi and IRt). TniA, TniB, and TniQ are required to form the transpositional cointegrate, which is then resolved by the action of TniR (a serine resolvase) on a resolution (res) sequence located between tniR and tniQ (22). The transposon in its new location is flanked by 5-bp direct repeats (DRs) (20, 22). TniA, which contains a D,D(35)E transposase catalytic motif, is thought to function cooperatively with TniB, a putative nucleotide-binding protein, as the active TniAB transposase (21, 36). Studies of TniA conducted in vitro show binding to the IRs and to additional 19-bp repeat sequences that make up the complex termini of the transposon (21). The precise role of TniQ is unknown.An unexpected and unique feature of Tn5053 and Tn402 is that they depend on externally coded accessory functions for efficient transposition, namely, a res site served by a cognate resolvase (25). As a consequence, these transposons exhibit a strong transpositional bias for some target res sites (20, 25, 26) and have aptly been described as “res site hunters” (25). One such efficient interaction involves the res-ParA multimer resolution system of plasmid RP1 (IncPα); other plasmid- or transposon-encoded systems are less efficient or are refractory. Although the role of the external resolvase remains obscure, its capacity to bind to its cognate res is an essential requirement whereas its catalytic activity is not (20). For each interaction system, the target sites typically cluster in a single part of res but not necessarily within the same subregion and, on occasion, can lie in the vicinity of res. Typically, the transposon is in a single orientation with IRi closest to the resolvase gene. In one study, Tn402 clustered at two target sites, one within res and one nearby, and the orientations were different at the two sites (20).The experimentally observed target preference described above also occurs in natural associations of Tn5053/Tn402-like elements and became evident on sequencing class 1 integrons, which were often found positioned close to different res-resolvase gene regions (6, 20, 25). Most Tn402 family elements are comprised of an intI module that is flanked on the left by IRi and on the right by a 3′ conserved sequence (3′-CS) (13). In others, a remnant tni gene cluster may be present instead of the 3′-CS, and IRt occurs at the right flank. The structure of the latter category of integrons strongly indicated that they are defective transposons that were presumably capable of relocation provided that tni functions were supplied in trans (6, 32). The movement of In33 (Tn2521) from a chromosomal to a plasmid location appears to have been such an in trans event (30, 42), and others involving In0 and In2 are demonstrated in this study. In contrast, the integrons that lack the IRt end appear to be nonmobile remnants of Tn402-like transposons; they belong to several lineages, including those in which the incurred deletions are attributable to acquired insertion sequences (6). More recently, intact Tn5053/Tn402-like transposons and class 1 integrons have increasingly been detected in the res-parA region of IncP plasmids (39), which are arguably the most promiscuous of known plasmids (50). These various experimental and natural interactions provide insight into the dispersal pathways possible for Tn5053/Tn402-like elements.The res-hunting attribute is a striking feature that is experimentally supported by studies of four family members (namely, Tn5053 [22, 25], Tn402 [20, 26], and in this study, Tn502 [48] and Tn512). Another facet of the transposition of Tn502 is explored here. It concerns the observation that loss of the preferred par target region in RP1 does not abolish transposition of Tn502 (48), contrary to the finding with Tn5053 (25, 26) and, in this study, Tn512. The continued, low-frequency transposition of Tn502 involved at least three dispersed locations (48); however, nothing is known about the nature of these sites or about the features and requirements of the transposition process. Here we address these issues and uncover the existence of an alternative, par-independent pathway that is employed by Tn502 and is available to Tn512 under some circumstances. The study also provides information on the roles of the TniR and host (RecA) recombination systems in the resolution of transpositional cointegrates and on the ability of the par-independent transposition pathway to generate plasmid deletions.     

Characterization of thePac25I Restriction-Modification Genes Isolated from the Endogenous pRA2 Plasmid ofPseudomonas alcaligenesNCIB 9867

Genes for the class IIPseudomonas alcaligenesNCIB 9867 restriction-modification (R-M) system,Pac25I, have been cloned from its 33-kb endogenous plasmid, pRA2. ThePac25I endonuclease and methylase genes were found to be aligned in a head-to-tail orientation with the methylase gene preceding and overlapping the endonuclease gene by 1 bp. The deduced amino acid sequence of thePac25I methylase revealed significant similarity with theXcyI,XmaI,Cfr9I, andSmaI methylases. High sequence similarity was displayed between thePac25I endonuclease and theXcyI,XmaI, andCfr9I endonucleases which cleave between the external cytosines of the recognition sequence (i.e., 5′-C↓CCGGG-3′) and are thus perfect isoschizomers. However, no sequence similarity was detected between thePac25I endonuclease and theSmaI endonuclease which cleaves between the internal CpG of the recognition sequence (i.e., 5′-CCC↓GGG-3′). Both thePac25I methylase and endonuclease were expressed inEscherichia coli.An open reading frame encoding a protein which shows significant similarity to invertases and resolvases was located immediately upstream of thePac25I R-M operon. In addition, a transposon designated Tn5563was located 1531 bp downstream of the R-M genes. The location on a self-transmissible plasmid as well as the close association with genes involved in DNA mobility suggests horizontal transfer as a possible mode of distribution of this family of R-M genes in various bacteria.     

Internal size variations in Tn1546-like elements due to the presence of IS1216V

In this study, internal size variations in the VanA gene cluster Tn1546, encoding resistance to glycopeptides, is described. Studies of previously uncharacterized size variations of an internal region, encoding the vanX and vanY genes of Tn1546, revealed that these variations were due to the presence of the IS sequence, IS1216V. This IS sequence has previously been found integrated in Tn1546. Integration of the IS1216V element created both deletions and a duplication in a non-essential region of Tn1546. In several isolates, the entire vanY gene was deleted, proving that this gene is non-essential for vancomycin resistance.     

Nucleotide sequence and expression of the organomercurial-resistance determinants from a Pseudomonas K-62 plasmid pMR26

pMRA17 cloned from Pseudomonas K-62 plasmid pMR26 specified the resistance to both organic and inorganic mercurials. DNA sequence of this broad-spectrum resistant mer operon was determined. The 5504-bp sequence includes six open reading frames (ORFs), five of which were identified as merR, merT, merP, merA and merB in order by analysis of deletion mutants and by comparison with the DNA and amino acid (aa) sequences of previously sequenced mer operons. The merB encoding organomercurial lyase showed a less identity than the other mer genes with those from other broad-spectrum resistance operons. The remaining ORF named merE, located between merA and merB, had no significant homology with the published mer genes and seemed to be a new gene which may involve in phenylmercury resistance. Induction experiments and maxicell analyses of the mer-polypeptides revealed that pMRA17 mer operon expressed mercurial-inducible phenotype and the merB and merE as well as the merA were under the control of MerR which could activate not only by mercuric ion but also by organomercurials.© 1997 Elsevier Science B.V. All rights reserved.     

A Multicentre Hospital Outbreak in Sweden Caused by Introduction of a vanB2 Transposon into a Stably Maintained pRUM-Plasmid in an Enterococcus faecium ST192 Clone

The clonal dissemination of VanB-type vancomycin-resistant Enterococcus faecium (VREfm) strains in three Swedish hospitals between 2007 and 2011 prompted further analysis to reveal the possible origin and molecular characteristics of the outbreak strain. A representative subset of VREfm isolates (n = 18) and vancomycin-susceptible E. faecium (VSEfm, n = 2) reflecting the spread in time and location was approached by an array of methods including: selective whole genome sequencing (WGS; n = 3), multi locus sequence typing (MLST), antimicrobial susceptibility testing, virulence gene profiling, identification of mobile genetic elements conferring glycopeptide resistance and their ability to support glycopeptide resistance transfer. In addition, a single VREfm strain with an unrelated PFGE pattern collected prior to the outbreak was examined by WGS. MLST revealed a predominance of ST192, belonging to a hospital adapted high-risk lineage harbouring several known virulence determinants (n≥10). The VREfm outbreak strain was resistant to ampicillin, gentamicin, ciprofloxacin and vancomycin, and susceptible to teicoplanin. Consistently, a vanB2-subtype as part of Tn1549/Tn5382 with a unique genetic signature was identified in the VREfm outbreak strains. Moreover, Southern blot hybridisation analyses of PFGE separated S1 nuclease-restricted total DNAs and filter mating experiments showed that vanB2-Tn1549/Tn5382 was located in a 70-kb sized rep _17/pRUM plasmid readily transferable between E. faecium. This plasmid contained an axe-txe toxin-antitoxin module associated with stable maintenance. The two clonally related VSEfm harboured a 40 kb rep _17/pRUM plasmid absent of the 30 kb vanB2-Tn1549/Tn5382 gene complex. Otherwise, these two isolates were similar to the VREfm outbreak strain in virulence- and resistance profile. In conclusion, our observations support that the origin of the multicentre outbreak was caused by an introduction of vanB2-Tn1549/Tn5382 into a rep _17/pRUM plasmid harboured in a pre-existing high-risk E. faecium ST192 clone. The subsequent dissemination of VREfm to other centres was primarily caused by clonal spread rather than plasmid transfer to pre-existing high-risk clones.     

Integration of a transposable DNA sequence which mediates ampicillin resistance into Clo DF13 plasmid DNA: determination of the site and orientation of TnA insertions.

The isolation and characterization of Clo DF13 plasmids containing a transposable DNA sequence (TnA) that specifies for ampicillin resistance is described. The particular transposon is derived from the R plasmid pRI30, and is designated Tn901. In order to determine the site and orientation of Tn901 insertions into the Clo DF13 genome, we made use of restriction endonucleases and heteroduplex mapping. For this purpose, Clo DF13 plasmid DNA and DNA of Clo DF13::Tn901 plasmids were digested with endonucleases HincII, PstI, BamH-I, SalI, and HpaI or with a combination of two of these enzymes. By analysis of the resulting fragmentation patterns, the physical maps of Clo DF13 DNA and Tn901 DNA could be derived. Furthermore, the site and orientation of Tn901 insertions into the Clo DF13 genome could be determined by this approach. The data obtained were verified by heteroduplex mapping. Analysis of 33 independently isolated Clo DF13 recombinant plasmids showed that insertion of Tn901 had occurred at 31 different sites. No preference with respect to the orientation of Tn901 was observed. Insertion of Tn901 into a segment of about 20% of the Clo DF13 genome resulted in the loss of cloacin production, indicating that the structural gene coding for cloacin is located in this area. The sites of Tn901 insertions within Clo DF13 were more or less scattered; however, no Tn901 insertion sites were found in two distinct areas comprising 11 and 17%, respectively, of the Clo DF13 genome. Transposition of Tn901 DNA to the copy mutant Clo DF13-rep3 showed that the β-lactamase activity and the minimal inhibitory concentration of ampicillin were correlated to the number of plasmid copies per cell.     

Nested Deletions from a Fixed Site as an Aid to Nucleotide Sequencing: an in vitro System Using Tn3 Transposase

We have previously constructed a cloning/sequencing vector,with an in vivo system capable of creating nested deletionsfrom the end of transposon Tn3, which is useful for sequencinglarge DNAs. Here we report an in vitro system which uses anammonium sulfate fraction of extract from E. coli cells harboringa Tn3 transposase overproducer plasmid to generate nested deletions.A key feature of the procedure is exhaustive digestion of thereaction products with a restriction enzyme that cleaves onlybetween the Tn3 "right" terminus and the cloned fragment. Thisstep reduces the noise level due to mechanisms other than deletionsfrom the Tn3 terminus, and facilitates detection and isolationof the desired deletion products. This system enables us tosave at least 2 days' time when obtaining the necessary deletionscompared with the in vivo system.