Tn3-like Transposon Research Articles

Recruitment of CRISPR-Cas systems by Tn7-like transposons

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.

Evolution of class 1 integrons: Mobilization and dispersal via food-borne bacteria.

Class 1 integrons have played a major role in the global dissemination of antibiotic resistance. Reconstructing the history of class 1 integrons might help us control further spread of antibiotic resistance by understanding how human activities influence microbial evolution. Here we describe a class 1 integron that represents an intermediate stage in the evolutionary history of clinical integrons. It was embedded in a series of nested transposons, carried on an IncP plasmid resident in Enterobacter, isolated from the surface of baby spinach leaves. Based on the structure of this integron, we present a modified hypothesis for integron assembly, where the ancestral clinical class 1 integron was captured from a betaproteobacterial chromosome to form a Tn402-like transposon. This transposon then inserted into a plasmid-borne Tn21-like ancestor while in an environmental setting, possibly a bacterium resident in the phyllosphere. We suggest that the qacE gene cassette, conferring resistance to biocides, together with the mercury resistance operon carried by Tn21, provided a selective advantage when this bacterium made its way into the human commensal flora via food. The integron characterized here was located in Tn6007, which along with Tn6008, forms part of the larger Tn6006 transposon, itself inserted into another transposable element to form the Tn21-like transposon, Tn6005. This element has previously been described from the human microbiota, but with a promoter mutation that upregulates integron cassette expression. This element we describe here is from an environmental bacterium, and supports the hypothesis that the ancestral class 1 integron migrated into anthropogenic settings via foodstuffs. Selection pressures brought about by early antimicrobial agents, including mercury, arsenic and disinfectants, promoted its initial fixation, the acquisition of promoter mutations, and subsequent dissemination into various species and pathogens.

Draft Genome Sequences of Two Highly Erythromycin-Resistant Streptococcus gallolyticus subsp. gallolyticus Isolates Containing a Novel Tn916-Like Element, Tn6331.

ABSTRACTRecently, we reported the draft genome sequence of Streptococcus gallolyticus NTS31106099. It was found to contain a previously unknown putative Tn916-like conjugative transposon, Tn6263. Here, we report the draft genome sequences of two other clinical isolates, NTS31301958 and NTS31307655. Both of them contain another novel element, Tn6331, which is highly similar to Tn6263.

Sequence of the R1 plasmid and comparison to F and R100

The R1 antibiotic resistance plasmid, originally discovered in a clinical Salmonella isolate in London, 1963, has served for decades as a key model for understanding conjugative plasmids. Despite its scientific importance, a complete sequence of this plasmid has never been reported. We present the complete genome sequence of R1 along with a brief review of the current knowledge concerning its various genetic systems and a comparison to the F and R100 plasmids. R1 is 97,566 nucleotides long and contains 120 genes. The plasmid consists of a backbone largely similar to that of F and R100, a Tn21-like transposon that is nearly identical to that of R100, and a unique 9-kb sequence that bears some resemblance to sequences found in certain Klebsiella oxytoca strains. These three regions of R1 are separated by copies of the insertion sequence IS1. Overall, the structure of R1 and comparison to F and R100 suggest a fairly stable shared conjugative plasmid backbone into which a variety of mobile elements have inserted to form an “accessory” genome, containing multiple antibiotic resistance genes, transposons, remnants of phage genes, and genes whose functions remain unknown.

Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements

Copper-based antimicrobial compounds are widely used to control plant bacterial pathogens. Pathogens have adapted in response to this selective pressure. Xanthomonas citri pv. citri, a major citrus pathogen causing Asiatic citrus canker, was first reported to carry plasmid-encoded copper resistance in Argentina. This phenotype was conferred by the copLAB gene system. The emergence of resistant strains has since been reported in Réunion and Martinique. Using microsatellite-based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copper-resistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. In order to investigate this pattern more closely, we sequenced six copper-resistant X.citri pv.citri strains from Argentina, Martinique and Réunion, together with reference copper-resistant Xanthomonas and Stenotrophomonas strains using long-read sequencing technology. Genes involved in copper resistance were found to be strain dependent with the novel identification in X.citri pv.citri of copABCD and a cus heavy metal efflux resistance-nodulation-division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3-like transposons and included in a conjugative plasmid. This indicates the system's great versatility. The mining of all available bacterial genomes suggested that, within the bacterial community, the spread of copper resistance associated with mobile elements and their plasmid environments was primarily restricted to the Xanthomonadaceae family.

Co-spread of metal and antibiotic resistance within ST3-IncHI2 plasmids from E. coli isolates of food-producing animals.

Concerns have been raised in recent years regarding co-selection for antibiotic resistance among bacteria exposed to heavy metals, particularly copper and zinc, used as growth promoters for some livestock species. In this study, 25 IncHI2 plasmids harboring oqxAB (20/25)/blaCTX-M (18/25) were found with sizes ranging from ∼260 to ∼350 kb and 22 belonged to the ST3-IncHI2 group. In addition to blaCTX-M and oqxAB, pcoA-E (5/25) and silE-P (5/25), as well as aac(6′)-Ib-cr (18/25), floR (16/25), rmtB (6/25), qnrS1(3/25) and fosA3 (2/25), were also identified on these IncHI2 plasmids. The plasmids carried pco and sil contributed to increasing in the MICs of CuSO4 and AgNO3. The genetic context surrounding the two operons was well conserved except some variations within the pco operon. The ~32 kb region containing the two operons identified in the IncHI2 plasmids was also found in chromosomes of different Enterobacteriaceae species. Further, phylogenetic analysis of this structure showed that Tn7-like transposon might play an important role in cross-genus transfer of the sil and pco operons among Enterobacteriaceae. In conclusion, co-existence of the pco and sil operons, and oqxAB/blaCTX-M as well as other antibiotic resistance genes on IncHI2 plasmids may promote the development of multidrug-resistant bacteria.

Diversification of broad host range plasmids correlates with the presence of antibiotic resistance genes.

The IncP-1ε subgroup is a recently identified phylogenetic clade within IncP-1 plasmids, which plays an important role in the spread of antibiotic resistance and degradation of xenobiotic pollutants. Here, four IncP-1ε plasmids were exogenously captured from a petroleum-contaminated habitat in China and compared phylogenetically and genomically with previously reported IncP-1ε and other IncP-1 plasmids. The IncP-1ε plasmids can be clearly subdivided into two subclades, designated as ε-I and ε-II, based on phylogenetic analysis of backbone proteins TraI and TrfA. This was further supported by comparison of concatenated backbone genes. Moreover, the two subclades differed in the transposon types, phenotypes and insertion locations of the accessory elements. The accessory genes on ε-I plasmids were inserted between parA and traC, and harbored ISPa17 and Tn402-like transposon modules, typically carrying antibiotic resistance genes. In contrast, the accessory elements on ε-II plasmids were typically located between trfA and oriV, and contained IS1071, which was commonly inserted within the Tn501-like transposon, typically harboring a cluster of genes encoding mercury resistance and/or catabolic pathways. Our study is one of the first to compare IncP-1 plasmid genomes from China, expands the available collection of IncP-1ε plasmids and enhances our understanding of their diversity, biogeography and evolutionary history.

Complete sequence and detailed analysis of the first indigenous plasmid from Xanthomonas oryzae pv. oryzicola.

BackgroundBacterial plasmids have a major impact on metabolic function and adaptation of their hosts. An indigenous plasmid was identified in a Chinese isolate (GX01) of the invasive phytopathogen Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of rice bacterial leaf streak (BLS). To elucidate the biological functions of the plasmid, we have sequenced and comprehensively annotated the plasmid.MethodsThe plasmid DNA was extracted from Xoc strain GX01 by alkaline lysis and digested with restriction enzymes. The cloned and subcloned DNA fragments in pUC19 were sequenced by Sanger sequencing. Sequences were assembled by using Sequencher software. Gaps were closed by primer walking and sequencing, and multi-PCRs were conducted through the whole plasmid sequence for verification. BLAST, phylogenetic analysis and dinucleotide calculation were performed for gene annotation and DNA structure analysis. Transformation, transconjugation and stress tolerance tests were carried out for plasmid function assays.ResultsThe indigenous plasmid from Xoc strain GX01, designated pXOCgx01, is 53,206-bp long and has been annotated to possess 64 open reading frames (ORFs), including genes encoding type IV secretion system, heavy metal exporter, plasmid stability factors, and DNA mobile factors, i.e., the Tn3-like transposon. Bioinformatics analysis showed that pXOCgx01 has a mosaic structure containing different genome contexts with distinct genomic heterogeneities. Phylogenetic analysis indicated that the closest relative of pXOCgx01 is pXAC64 from Xanthomonas axonopodis pv. citri str. 306. It was estimated that there are four copies of pXOCgx01 per cell of Xoc GX01 by PCR assay and the calculation of whole genome shotgun sequencing data. We demonstrate that pXOCgx01 is a self-transmissible plasmid and can replicate in some Xanthomonas spp. strains, but not in Escherichia coli DH5α. It could significantly enhance the tolerance of Xanthomonas oryzae pv. oryzae PXO99A to the stresses of heavy metal ions. The plasmid survey indicated that nine out of 257 Xoc Chinese isolates contain plasmids.ConclusionspXOCgx01 is the first report of indigenous plasmid from Xanthomonas oryzae pv. oryzicola, and the first completely sequenced plasmid from Xanthomonas oryzae species. It is a self-transmissible plasmid and has a mosaic structure, containing genes for macromolecule secretion, heavy metal exportation, and DNA mobile factors, especially the Tn3-like transposon which may provide transposition function for mobile insertion cassette and play a major role in the spread of pathogenicity determinants. The results will be helpful to elucidate the biological significance of this cryptic plasmid and the adaptive evolution of Xoc.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0562-x) contains supplementary material, which is available to authorized users.

Genotyping of virulence plasmid from Vibrio parahaemolyticus isolates causing acute hepatopancreatic necrosis disease in shrimp.

Acute hepatopancreatic necrosis disease (AHPND) has caused severe mortalities in farmed penaeid shrimp throughout SE Asia and Mexico. The causative agent of AHPND is the marine bacterium Vibrio parahaemolyticus, which secretes PirA- and PirB-like binary toxin that caused deterioration in the hepatopancreas of infected shrimp. The genes responsible for the production of this toxin are located in a large plasmid residing within the bacterial cells. We analyzed the plasmid sequence from the whole genome sequences of AHPND-V. parahaemolyticus isolates and identified 2 regions that exhibit a clear geographical variation: a 4243-bp Tn3-like transposon and a 9-bp small sequence repeat (SSR). The Tn3-like transposon was only found in the isolates from Mexico and 2 unspecified Central American countries, but not in SE Asian isolates from China, Vietnam, and Thailand. We developed PCR methods to characterize AHPND-V. parahaemolyticus isolates as either Mexican-type or SE Asian-type based on the presence of the Tn3-like transposon. The SSR is found within the coding region of a hypothetical protein and has either 4, 5, or 6 repeat units. SSRs with 4 repeat units were found in isolates from Vietnam, China, and Thailand. SSRs with 5 repeat units were found in some Vietnamese isolates, and SSRs with 6 repeat units were only found in the Mexican isolates.

A TALE of transposition: Tn3-like transposons play a major role in the spread of pathogenicity determinants of Xanthomonas citri and other xanthomonads.

ABSTRACTMembers of the genus Xanthomonas are among the most important phytopathogens. A key feature of Xanthomonas pathogenesis is the translocation of type III secretion system (T3SS) effector proteins (T3SEs) into the plant target cells via a T3SS. Several T3SEs and a murein lytic transglycosylase gene (mlt, required for citrus canker symptoms) are found associated with three transposition-related genes in Xanthomonas citri plasmid pXAC64. These are flanked by short inverted repeats (IRs). The region was identified as a transposon, TnXax1, with typical Tn3 family features, including a transposase and two recombination genes. Two 14-bp palindromic sequences within a 193-bp potential resolution site occur between the recombination genes. Additional derivatives carrying different T3SEs and other passenger genes occur in different Xanthomonas species. The T3SEs include transcription activator-like effectors (TALEs). Certain TALEs are flanked by the same IRs as found in TnXax1 to form mobile insertion cassettes (MICs), suggesting that they may be transmitted horizontally. A significant number of MICs carrying other passenger genes (including a number of TALE genes) were also identified, flanked by the same TnXax1 IRs and delimited by 5-bp target site duplications. We conclude that a large fraction of T3SEs, including individual TALEs and potential pathogenicity determinants, have spread by transposition and that TnXax1, which exhibits all of the essential characteristics of a functional transposon, may be involved in driving MIC transposition. We also propose that TALE genes may diversify by fork slippage during the replicative Tn3 family transposition. These mechanisms may play a crucial role in the emergence of Xanthomonas pathogenicity.

Full-Length Genome Sequence of Type M/emm83 Group A Streptococcus pyogenes Strain STAB1101, Isolated from Clustered Cases in Brittany.

Here, we announce the complete annotated genome sequence of a Streptococcus pyogenes M/emm83 strain, STAB1101, isolated from clustered cases in homeless persons in Brittany (France). The genome is composed of 1,709,790 bp, with a G+C content of 38.4% and 1,550 identified coding sequences (CDS), and it harbors a Tn916-like transposon.

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.

Complete Genome Sequence of Streptococcus pyogenes M/emm44 Strain STAB901, Isolated in a Clonal Outbreak in French Brittany.

We report the complete genome sequence of an invasive isolate of Streptococcus pyogenes M/emm44, belonging to a clonal outbreak that occurred in French Brittany. The genome is composed of 1,795,608 bp, with a GC content of 38.5%, has 1,358 identified coding sequences (CDSs), and harbors a novel Tn916-like transposon (Tn6253).

Heteromeric transposase elements: generators of genomic islands across diverse bacteria.

Horizontally acquired genetic information in bacterial chromosomes accumulates in blocks termed genomic islands. Tn7-like transposons form genomic islands at a programmed insertion site in bacterial chromosomes, attTn7. Transposition involves five transposon-encoded genes (tnsABCDE) including an atypical heteromeric transposase. One transposase subunit, TnsB, is from the large family of bacterial transposases, the second, TnsA, is related to endonucleases. A regulator protein, TnsC, functions with different target site selecting proteins to recognize different targets. TnsD directs transposition into attTn7, while TnsE encourages horizontal transmission by targeting mobile plasmids. Recent work suggests that distantly related elements with heteromeric transposases exist with alternate targeting pathways that also facilitate the formation of genomic islands. Tn6230 and related elements can be found at a single position in a gene of unknown function (yhiN) in various bacteria as well as in mobile plasmids. Another group we term Tn6022-like elements form pathogenicity islands in the Acinetobacter baumannii comM gene. We find that Tn6022-like elements also appear to have an uncharacterized mechanism for provoking internal transposition and deletion events that serve as a conduit for evolving new elements. As a group, heteromeric transposase elements utilize diverse target site selection mechanisms adapted to the spread and rearrangement of genomic islands.

Characterization of pFOX-7a, a conjugative IncL/M plasmid encoding the FOX-7 AmpC-type β-lactamase, involved in a large outbreak in a neonatal intensive care unit.

FOX-type enzymes are a lineage of AmpC-type β-lactamases from Aeromonas spp. whose genes have been mobilized to plasmids spreading among Enterobacteriaceae, where they can be responsible for resistance to extended-spectrum cephalosporins and β-lactamase inhibitor combinations. Little is known about the genetic context and plasmid vehicles of bla(FOX) determinants. Here, we have characterized a plasmid encoding the FOX-7 β-lactamase, which was involved in a large outbreak caused by two Klebsiella pneumoniae clones in a neonatal intensive care unit. Plasmid transferability was tested in conjugation experiments using Escherichia coli recipients. Plasmids from different strains were compared by restriction profiling and PCR mapping. The complete sequence of pFOX-7a plasmid was determined by a next-generation sequencing approach followed by gap filling using PCR and sequencing. An apparently identical conjugative plasmid encoding FOX-7 was detected in representatives of the K. pneumoniae clones that caused the outbreak and in sporadic FOX-7-producing strains of other species from the same ward. The plasmid, named pFOX-7a, has an IncL/M-type backbone and two separate resistance modules including a Tn3-like transposon and a novel Tn1696 derivative, named Tn6234, which carries an integron platform, a hybrid (but still functional) mercury resistance module and a novel putative transposon of original structure, named Tn6240, associated with the bla(FOX-7) gene. pFOX-7a is the first completely characterized plasmid encoding a FOX-type β-lactamase. The bla(FOX-7) gene was associated with a putative transposable element of original structure, which was likely involved in its mobilization from the Aeromonas metagenome.

Evolution and genetic diversity of the Spain23F-ST81 clone causing adult invasive pneumococcal disease in Barcelona (1990–2012)

ObjectivesWe aimed to analyse the clinical epidemiology and genetic diversity of invasive pneumococcal disease (IPD) episodes attributed to the Spain23F-ST81 (PMEN1) clone.MethodsFifty-eight (2.7%) of 2117 invasive pneumococci isolated from adult patients during the 1990–2012 period shared a PFGE pattern related to the PMEN1 clone. The genotype was confirmed by multilocus sequence typing. The pbp2x, pbp1a, pbp2b and pspA genes were PCR-amplified and sequenced. Polymorphisms in the pspC gene were identified by PCR restriction fragment length polymorphism. The presence of transposons with erythromycin and tetracycline resistance determinants was detected by PCR.ResultsThe prevalence of the PMEN1 clone increased from 0.8% in 1991 to 6.2% in 2001, and decreased to 0% in 2010–12, concomitant with the introduction of the seven-valent pneumococcal conjugate vaccine for children. A total of 93.1% of patients had pneumonia, meningitis or peritonitis; 87.9% of patients had associated underlying diseases, mainly cancer, chronic obstructive pulmonary disease and diabetes. Two closely related sequence types (STs) (ST81, n = 52; ST85, n = 6) were detected, with different serotypes: 23F (n = 42), 19A (n = 9) and 19F (n = 6). All the isolates were resistant to penicillin, co-trimoxazole and chloramphenicol. All the isolates also shared the same pbp1a allele, whereas multiple alleles of pbp2b, pbp2x, pspA and pspC were detected. Of the isolates, 89.7% were tetracycline resistant and 60.3% (n = 35) were macrolide resistant, and resistance was associated with different Tn916-like transposons.ConclusionsAdult IPD caused by this clone was mainly detected in patients with underlying conditions, and genetic variability was observed among PMEN1 isolates collected in our area over the past 20 years.

Structures of a Bifunctional Cell Wall Hydrolase CwlT Containing a Novel Bacterial Lysozyme and an NlpC/P60 dl-Endopeptidase

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).

Characterization of plasmid pAX22, encoding VIM-1 metallo-β-lactamase, reveals a new putative mechanism of In70 integron mobilization

VIM-type enzymes are among the most widespread acquired metallo-β-lactamases among Gram-negative pathogens. Integron In70 is a class 1 integron that has emerged as a successful genetic support for blaVIM-1 (one of the most prevalent blaVIM allelic variants) in Gram-negative non-fermenters, and is usually chromosome borne. The objective of this study was to characterize plasmid pAX22 from Achromobacter xylosoxidans AX22, which represents the only In70-harbouring plasmid known so far, to gather insights into the mechanisms of evolution and dissemination of In70-like elements. The complete sequence of pAX22 was obtained by pyrosequencing and assembled with Roche Newbler software. The draft sequence, completed using a PCR-based strategy, was annotated via the BASys tool and compared with known sequences using BLAST algorithms. The backbone of pAX22 showed significant similarity with that of pNOR-2000, a blaVIM-2-harbouring plasmid from Pseudomonas aeruginosa, and with the TnCP23 transposon. The three elements differed from each other mainly by the class 1 integron cassette arrays and by some integron-associated structures. In pAX22, In70 was associated with a novel putative transposon, Tn7017, composed of a defective Tn402-like transposon carrying In70 and the ISPa17 insertion sequence. Plasmid pAX22 belongs to a lineage of plasmids circulating among Gram-negative non-fermenters. In70 was probably acquired by pAX22 by transposition of Tn7017, revealing a novel putative mechanism of In70 mobilization. Our results highlight the potential role that ISPa17 could have in mobilizing defective Tn402-like transposons carrying class 1 integrons.

Comparative genomics of IncP-1ε plasmids from water environments reveals diverse and unique accessory genetic elements

The goal of this study was to determine and compare the complete genome sequences of three new broad-host-range conjugative plasmids. Plasmids pMLUA1, pMLUA3 and pMLUA4 were previously recovered from estuarine water by exogenous plasmid isolation and ranged in size from ∼55 to 59kb. Comparative genomics showed that their backbone region was identical to the prototype pKJK5 and other IncP1-ε plasmids captured from soils. The accessory region was inserted between the tra region and parA, and presented the typical IncP-1ε ISPa17 and Tn402-like transposon modules. Nevertheless, new class 1 integrons were identified (In794, carrying aadA5 and In795, carrying qacF5-aadA5), as well as a composite transposon IS26-msr(E)-mph(E)-IS26 carrying genes that confer resistance to macrolides. A new insertion sequence, termed ISUnCu17, was also identified on pMLUA3. The architecture of the accessory regions implies the occurrence of multiple insertions and deletions. These data support the notion that IncP-1 plasmids from the ε subgroup are proficient in the capture of diverse genetic elements, including antibiotic resistance genes, and thus may contribute to the co-selection of several resistance determinants. This study constitutes the first report of completely sequenced IncP-1ε plasmids from water environments, and enhances our understanding of the geographic distribution and genetic diversity of these replicons.

IncP-1ε Plasmids are Important Vectors of Antibiotic Resistance Genes in Agricultural Systems: Diversification Driven by Class 1 Integron Gene Cassettes

The role of broad-host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5′-nuclease assay for real-time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridization. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.