Putative Composite Transposon Research Articles

Highly conserved composite transposon harbouring aerobactin iuc3 in Klebsiella pneumoniae from pigs.

Klebsiella pneumoniae is an important opportunistic pathogen associated with severe invasive disease in humans. Hypervirulent K. pneumoniae, which are K. pneumoniae with several acquired virulence determinants such as the siderophore aerobactin and others, are more prominent in countries in South and South-East Asia compared to European countries. This Klebsiella pathotype is capable of causing liver abscesses in immunocompetent persons in the community. K. pneumoniae has not been extensively studied in non-human niches. In the present study, K. pneumoniae isolated from caecal samples (n=299) from healthy fattening pigs in Norway were characterized with regard to population structure and virulence determinants. These data were compared to data from a previous study on K. pneumoniae from healthy pigs in Thailand. Lastly, an in-depth plasmid study on K. pneumoniae with aerobactin was performed. Culturing and whole-genome sequencing was applied to detect, confirm and characterize K. pneumoniae isolates. Phylogenetic analysis described the evolutionary relationship and diversity of the isolates, while virulence determinants and sequence types were detected with Kleborate. Long-read sequencing was applied to obtain the complete sequence of virulence plasmids harbouring aerobactin. A total of 48.8 % of the investigated Norwegian pig caecal samples (n=299) were positive for K. pneumoniae. Acquired virulence determinants were detected in 72.6 % of the isolates, the most prominent being aerobactin (69.2 %), all of which were iuc3. In contrast, only 4.6 % of the isolates from Thailand harboured aerobactin. The aerobactin operon was located on potentially conjugative IncFIBK/FIIK plasmids of varying sizes in isolates from both countries. A putative, highly conserved composite transposon with a mean length of 16.2 kb flanked by truncated IS3-family IS407-group insertion sequences was detected on these plasmids, harbouring the aerobactin operon as well as several genes that may confer increased fitness in mammalian hosts. This putative composite transposon was also detected in plasmids harboured by K. pneumoniae from several countries and sources, such as human clinical samples. The high occurrence of K. pneumoniae harbouring aerobactin in Norwegian pigs, taken together with international data, suggest that pigs are a reservoir for K. pneumoniae with iuc3. Truncation of the flanking ISKpn78-element suggest that the putative composite transposon has been permanently integrated into the plasmid, and that it is no longer mobilizable.

Insertion sequences and other mobile elements associated with antibiotic resistance genes in Enterococcus isolates from an inpatient with prolonged bacteraemia.

Insertion sequences (ISs) and other transposable elements are associated with the mobilization of antibiotic resistance determinants and the modulation of pathogenic characteristics. In this work, we aimed to investigate the association between ISs and antibiotic resistance genes, and their role in the dissemination and modification of the antibiotic-resistant phenotype. To that end, we leveraged fully resolved Enterococcus faecium and Enterococcus faecalis genomes of isolates collected over 5 days from an inpatient with prolonged bacteraemia. Isolates from both species harboured similar IS family content but showed significant species-dependent differences in copy number and arrangements of ISs throughout their replicons. Here, we describe two inter-specific IS-mediated recombination events and IS-mediated excision events in plasmids of E. faecium isolates. We also characterize a novel arrangement of the ISs in a Tn1546-like transposon in E. faecalis isolates likely implicated in a vancomycin genotype–phenotype discrepancy. Furthermore, an extended analysis revealed a novel association between daptomycin resistance mutations in liaSR genes and a putative composite transposon in E. faecium , offering a new paradigm for the study of daptomycin resistance and novel insights into its dissemination. In conclusion, our study highlights the role ISs and other transposable elements play in the rapid adaptation and response to clinically relevant stresses such as aggressive antibiotic treatment in enterococci.

A Novel Plasmid pALWVS1.4 from Acinetobacter lwoffii Strain VS15, Carrying the Chloramphenicol Resistance Gene

Sequencing and analysis of the genome of the chloramphenicol-resistant strain Acinetobacter lwoffii VS15, isolated from permafrost, revealed a circular plasmid 11 964 bp in length, designated pAL-WVS1.4. Apart from the genes supporting the plasmid maintenance and mobilization, pALWVS1.4 contains the cflA gene encoding the membrane protein of the MFS transporter family. Close homologues of cflA were found in bacteria of the genus Psychrobacter, but were absent in the Acinetobacter genomes. The cflA gene is flanked by two copies of IS elements of the IS4 family, indicating that it was acquired by A. lwoffii VS15 via horizontal transfer within this putative composite transposon. A recombinant Escherichia coli strain expressing CflA was obtained and it was shown that under conditions of induced expression, the strain was resistant to chloramphenicol.

A novel sulfonamide resistance mechanism by two-component flavin-dependent monooxygenase system in sulfonamide-degrading actinobacteria

Sulfonamide-degrading bacteria have been discovered in various environments, suggesting the presence of novel resistance mechanisms via drug inactivation. In this study, Microbacterium sp. CJ77 capable of utilizing various sulfonamides as a sole carbon source was isolated from a composting facility. Genome and proteome analyses revealed that a gene cluster containing a flavin-dependent monooxygenase and a flavin reductase was highly up-regulated in response to sulfonamides. Biochemical analysis showed that the two-component monooxygenase system was key enzymes for the initial cleavage of sulfonamides. Co-expression of the two-component system in Escherichia coli conferred decreased susceptibility to sulfamethoxazole, indicating that the genes encoding drug-inactivating enzymes are potential resistance determinants. Comparative genomic analysis revealed that the gene cluster containing sulfonamide monooxygenase (renamed as sulX) and flavin reductase (sulR) was highly conserved in a genomic island shared among sulfonamide-degrading actinobacteria, all of which also contained sul1-carrying class 1 integrons. These results suggest that the sulfonamide metabolism may have evolved in sulfonamide-resistant bacteria which had already acquired the class 1 integron under sulfonamide selection pressures. Furthermore, the presence of multiple insertion sequence elements and putative composite transposon structures containing the sulX gene cluster indicated potential mobilization. This is the first study to report that sulX responsible for both sulfonamide degradation and resistance is prevalent in sulfonamide-degrading actinobacteria and its genetic signatures indicate horizontal gene transfer of the novel resistance gene.

Transferable Plasmid-Borne mcr-1 in a Colistin-Resistant Shigella flexneri Isolate.

ABSTRACTSince the initial discovery of mcr-1 in an Escherichia coli isolate from China, the gene has also been detected in Klebsiella pneumoniae and Salmonella enterica but is rarely reported in other Enterobacteriaceae. Here, we report the isolation and identification of a Shigella flexneri strain harboring mcr-1 from stool samples in a pig farm in China from 2009. The MIC of colistin for the isolate is 4 μg/ml. Conjugation assays showed that the donor S. flexneri strain has functional and transferable colistin resistance. Sequencing revealed that mcr-1 was present on a putative composite transposon flanked by inverted repeats of ISApl1.IMPORTANCE There are four species of Shigella, and Shigella flexneri is the most frequently isolated species in low- and middle-income countries (LMICs). In this study, we report a functional, transferable, plasmid-mediated mcr-1 gene in S. flexneri. We have shown that mcr-1 is located on a novel composite transposon which is flanked by inverted repeats of ISApl1. The host strain is multidrug resistant, and this multidrug resistance is also transferable. The finding of a functional mcr-1 gene in S. flexneri, a human-associated Enterobacteriaceae family member, is a cause for concern as infections due to S. flexneri are the main Shigella infections in most low- and middle-income countries.

The genetic diversity of cereulide biosynthesis gene cluster indicates a composite transposon Tnces in emetic Bacillus weihenstephanensis.

BackgroundCereulide is a cyclic dodecadepsipeptide ionophore, produced via non-ribosomal peptide synthetases (NRPS), which in rare cases can lead to human death. Early studies had shown that emetic toxin formation belongs to a homogeneous group of Bacillus cereus sensu stricto and the genetic determinants of cereulide (a 24-kb gene cluster of cesHPTABCD) are located on a 270-kb plasmid related to the Bacillus anthracis virulence plasmid pXO1.ResultsThe whole genome sequences from seven emetic isolates, including two B. cereus sensu stricto and five Bacillus weihenstephanensis strains, were compared, and their inside and adjacent DNA sequences of the cereulide biosynthesis gene clusters were analyzed. The sequence diversity was observed, which classified the seven emetic isolates into three clades. Different genomic locations of the cereulide biosynthesis gene clusters, plasmid-borne and chromosome-borne, were also found. Potential mobile genetic elements (MGEs) were identified in the flanking sequences of the ces gene cluster in all three types. The most striking observation was the identification of a putative composite transposon, Tnces, consisting of two copies of ISces element (belonging to IS6 family) in opposite orientations flanking the ces gene cluster in emetic B. weihenstephanensis. The mobility of this element was tested by replacing the ces gene cluster by a KmR gene marker and performing mating-out transposition assays in Escherichia coli. The results showed that Tnces::km transposes efficiently (1.04 × 10-3 T/R) and produces 8-bp direct repeat (DR) at the insertion sites.ConclusionsCereulide biosynthesis gene clusters display sequence diversity, different genomic locations and association with MGEs, in which the transposition capacity of a resistant derivative of the composite transposon Tnces in E. coli was demonstrated. Further study is needed to look for appropriate genetic tools to analysis the transposition of Tnces in Bacillus spp. and the dynamics of other MGEs flanking the ces gene clusters.

Analysis of Novel Iron-Regulated, Surface-Anchored Hemin-Binding Proteins in Corynebacterium diphtheriae

Corynebacterium diphtheriae utilizes hemin and hemoglobin (Hb) as iron sources during growth in iron-depleted environments, and recent studies have shown that the surface-exposed HtaA protein binds both hemin and Hb and also contributes to the utilization of hemin iron. Conserved (CR) domains within HtaA and in the associated hemin-binding protein, HtaB, are required for the ability to bind hemin and Hb. In this study, we identified and characterized two novel genetic loci in C. diphtheriae that encode factors that bind hemin and Hb. Both genetic systems contain two-gene operons that are transcriptionally regulated by DtxR and iron. The gene products of these operons are ChtA-ChtB and ChtC-CirA (previously DIP0522-DIP0523). The chtA and chtB genes are carried on a putative composite transposon associated with C. diphtheriae isolates that dominated the diphtheria outbreak in the former Soviet Union in the 1990s. ChtA and ChtC each contain a single N-terminal CR domain and exhibit significant sequence similarity to each other but only limited similarity with HtaA. The chtB and htaB gene products exhibited a high level of sequence similarity throughout their sequences, and both proteins contain a single CR domain. Whole-cell binding studies as well as protease analysis indicated that all four of the proteins encoded by these two operons are surface exposed, which is consistent with the presence of a transmembrane segment in their C-terminal regions. ChtA, ChtB, and ChtC are able to bind hemin and Hb, with ChtA showing the highest affinity. Site-directed mutagenesis showed that specific tyrosine residues within the ChtA CR domain were critical for hemin and Hb binding. Hemin iron utilization assays using various C. diphtheriae mutants indicate that deletion of the chtA-chtB region and the chtC gene has no affect on the ability of C. diphtheriae to use hemin or Hb as iron sources; however, a chtB htaB double mutant exhibits a significant decrease in hemin iron use, indicating a role in hemin transport for HtaB and ChtB.

Tn 5393 d, a Complex Tn 5393 Derivative Carrying the PER-1 Extended-Spectrum β-Lactamase Gene and Other Resistance Determinants

In Alcaligenes faecalis FL-424/98, a clinical isolate that produces the PER-1 extended-spectrum beta-lactamase, the bla(PER-1) gene was found to be carried on a 44-kb nonconjugative plasmid, named pFL424, that was transferred to Escherichia coli by electroporation. Investigation of the genetic context of the bla(PER-1) gene in pFL424 by means of a combined cloning and PCR mapping approach revealed that the gene is associated with a transposonlike element of the Tn3 family. This 14-kb element is a Tn5393 derivative of original structure, named Tn5393d, which contains the transposition module and the strAB genes typical of other members of the Tn5393 lineage plus additional resistance determinants, including the bla(PER-1) gene and a new allelic variant of the aphA6 aminoglycoside phosphotransferase gene, named aphA6b, whose product is active against kanamycin, streptomycin, and amikacin. Tn5393d apparently originated from the consecutive insertion of two composite transposons into a Tn5393 backbone carrying the aphA6b and the bla(PER-1) genes, respectively. The putative composite transposon carrying bla(PER-1), named Tn4176, is made of two original and nonidentical insertion sequences of the IS4 family, named IS1387a and IS1387b, of which one is interrupted by the insertion of an original insertion sequence of the IS30 family, named IS1066. In pFL424, Tn5393d is inserted into a Tn501-like mercury resistance transposon. Transposition of Tn5393d or modules thereof containing the bla(PER-1) gene from pFL424 to small multicopy plasmids or to a bacterial artificial chromosome was not detected in an E. coli host harboring both replicons.

Bap-dependent biofilm formation by pathogenic species of Staphylococcus: evidence of horizontal gene transfer?

The biofilm-associated protein (Bap) is a surface protein implicated in biofilm formation by Staphylococcus aureus isolated from chronic mastitis infections. The bap gene is carried in a putative composite transposon inserted in SaPIbov2, a mobile staphylococcal pathogenicity island. In this study, bap orthologue genes from several staphylococcal species, including Staphylococcus epidermidis, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus simulans and Staphylococcus hyicus, were identified, cloned and sequenced. Sequence analysis comparison of the bap gene from these species revealed a very high sequence similarity, suggesting the horizontal gene transfer of SaPIbov2 amongst them. However, sequence analyses of the flanking region revealed that the bap gene of these species was not contained in the SaPIbov2 pathogenicity island. Although they did not contain the icaADBC operon, all the coagulase-negative staphylococcal isolates harbouring bap were strong biofilm producers. Disruption of the bap gene in S. epidermidis abolished its capacity to form a biofilm, whereas heterologous complementation of a biofilm-negative strain of S. aureus with the Bap protein from S. epidermidis bestowed the capacity to form a biofilm on a polystyrene surface. Altogether, these results demonstrate that Bap orthologues from coagulase-negative staphylococci induce an alternative mechanism of biofilm formation that is independent of the PIA/PNAG exopolysaccharide.

Horizontal gene transfer and host specificity of beta-haemolytic streptococci: the role of a putative composite transposon containing scpB and lmb.

Beta-haemolytic streptococci are important human and animal pathogens: their genetic traits that are associated with the ability to infect human hosts remain, however, unclear. The surface protein, Lmb, mediates the adherence of Streptococcus agalactiae to human laminin. For further analysis of the corresponding gene, the adjacent genomic regions were sequenced. Lmb is localized on a putative composite transposon of 16 kb and is flanked by two copies of a novel insertion sequence element (ISSag2). It harbours the genes scpB and lmb, which are 98% identical with the respective genes of Streptococcus pyogenes. Analysis of the distribution of these genes and ISSag2 among 131 streptococcal strains revealed that all of the human isolates, but only 20% (12 of 61) of the animal isolates, contained scpB and lmb or their homologues. To investigate if the putative transposon can be mobilized, an erythromycin resistance marker was incorporated into the lmb gene of S. agalactiae. Screening for mutant strains with a regained susceptibility for erythromycin identified strains with a deletion of scpB, lmb, and one copy of ISSag2. We hypothesize that a horizontal gene transfer caused the exchange of scpB and lmb and that the ability of S. pyogenes, S. agalactiae and group C and G streptococcal strains to colonize or infect human hosts is dependent on their presence.

Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10.

The nucleotide sequences of the 27,939-bp-long upstream and 9,448-bp-long downstream regions of the carAaAaBaBbCAc(ORF7)Ad genes of carbazole-degrading Pseudomonas sp. strain CA10 were determined. Thirty-two open reading frames (ORFs) were identified, and the car gene cluster was consequently revealed to consist of 10 genes (carAaAaBaBbCAcAdDFE) encoding the enzymes for the three-step conversion of carbazole to anthranilate and the degradation of 2-hydroxypenta-2,4-dienoate. The high identities (68 to 83%) with the enzymes involved in 3-(3-hydroxyphenyl)propionic acid degradation were observed only for CarFE. This observation, together with the fact that two ORFs are inserted between carD and carFE, makes it quite likely that the carFE genes were recruited from another locus. In the 21-kb region upstream from carAa, aromatic-ring-hydroxylating dioxygenase genes (ORF26, ORF27, and ORF28) were found. Inductive expression in carbazole-grown cells and the results of homology searching indicate that these genes encode the anthranilate 1,2-dioxygenase involved in carbazole degradation. Therefore, these ORFs were designated antABC. Four homologous insertion sequences, IS5car1 to IS5car4, were identified in the neighboring regions of car and ant genes. IS5car2 and IS5car3 constituted the putative composite transposon containing antABC. One-ended transposition of IS5car2 together with the 5' portion of antA into the region immediately upstream of carAa had resulted in the formation of IS5car1 and ORF9. In addition to the insertion sequence-dependent recombination, gene duplications and presumed gene fusion were observed. In conclusion, through the above gene rearrangement, the novel genetic structure of the car gene cluster has been constructed. In addition, it was also revealed that the car and ant gene clusters are located on the megaplasmid pCAR1.

Spontaneous tandem amplification and deletion of the shiga toxin operon in Shigella dysenteriae 1.

Only one species of Shigella, Shigella dysenteriae 1, has been demonstrated to produce Shiga toxin (Stx). Stx is closely related to the toxins produced by Shiga toxin-producing Escherichia coli (STEC). In STEC, these toxins are often encoded on lambdoid bacteriophages and are major virulence factors for these organisms. Although the bacteriophage-encoded stx genes of STEC are highly mobile, the stx genes in S. dysenteriae 1 have been believed to be chromosomally encoded and not transmissible. We have located the toxin genes of S. dysenteriae 1 to a region homologous to minute 30 of the E. coli chromosome, within a 22.4 kbp putative composite transposon bracketed by IS600 insertion sequences. This region is present in all the S. dysenteriae 1 strains examined. Tandem amplification occurs via the flanking insertion sequences, leading to increased toxin production. The global regulatory gene, fnr, is located within the stx region, allowing deletions of the toxin genes to be created by anaerobic growth on chlorate-containing medium. Deletions occur by recombination between the flanking IS600 elements. Lambdoid bacteriophage genes are found both upstream and within the region, and we demonstrate the lysogeny of Shigella species with STEC bacteriophages. These observations suggest that S. dysenteriae 1 originally carried a Stx-encoding lambdoid prophage, which became defective due to loss of bacteriophage sequences after IS element insertions and rearrangements. These insertion sequences have subsequently allowed the amplification and deletion of the stx region.