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PubMed2006

Combinatorial genetic evolution of multiresistance.

The explosion in genetic information, whilst extending our knowledge, might not necessary increase our conceptual understanding on the complexities of bacterial genetics, or why some antibiotic resistant genotypes such as blaCTX-M-15 and blaVIM-2 appear to dominate. However, the information we have thus far suggests that clinical isolates have 'hijacked' plasmids, primarily built of backbone-DNA originating from environmental bacteria. Additionally, the combinatorial presence of other elements such as transposons, integrons, insertion sequence (IS) elements and the 'new' ISCR (IS common region) elements have also contributed to the increase in antibiotic resistance - an antibiotic resistant cluster composing four or five genes has become commonplace. In some instances, the presence of antibiotics themselves, such as fluoroquinolones, can mediate a bacterial SOS cell response, subsequently amplifying and/or augmenting the transfer of large genetic entities therefore, potentially promoting long-term detrimental effects.

Anti-Bacterial Agents↗

PubMed2006

Incidence and behaviour of Tn916-like elements within tetracycline-resistant bacteria isolated from root canals.

INTRODUCTION: Tetracycline resistance is commonly found in endodontic bacteria. One of the most common tetracycline-resistance genes is tet(M), which is often encoded on the broad-host-range conjugative transposon Tn916. This study aimed to determine whether tet(M) was present in bacteria isolated from endodontic patients at the Eastman Dental Institute and whether this gene was carried on the transferable conjugative transposon Tn916. METHODS: The cultivable microflora isolated from 15 endodontic patients was screened for resistance to tetracycline. Polymerase chain reactions for tet(M) and for unique regions of Tn916 were carried out on the DNA of all tetracycline-resistant bacteria. Filter-mating experiments were used to see if transfer of any Tn916-like elements could occur. RESULTS: Eight out of 15 tetracycline-resistant bacteria isolated were shown to possess tet(M). Furthermore, four of these eight were shown to possess the Tn916-unique regions linked to the tet(M) gene. Transfer experiments demonstrated that a Neisseria sp. donor could transfer an extremely unstable Tn916-like element to Enterococcus faecalis. CONCLUSIONS: The tet(M) gene is present in the majority of tetracycline-resistant bacteria isolated in this study and the conjugative transposon Tn916 has been shown to be responsible for the support and transfer of this gene in some of the bacteria isolated.

Bacteria↗

PubMed1998

An origin of transfer (oriT) on the conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3.

Previous analysis of the Tra1 region of the conjugative element pRS01 from Lactococcus lactis subsp. lactis ML3 suggested that an origin of transfer (oriT) was present. Deletion derivatives of this cloned Tra1 region were assayed for mobilization in the presence of the wild-type pRS01 element in trans. The pRS01 oriT was localized to a 446-nucleotide segment in the intergenic region between open reading frames ltrD and ltrE. Sequence analysis of this region revealed a cluster of direct and inverted repeat structures characteristic of oriT regions associated with other conjugative systems.

Amino Acid Sequence↗

PubMed1986

Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri.

A plasmid of about 140 megadaltons has been associated with the invasiveness of Shigella flexneri. Upon subculturing in liquid media of fully virulent isolates of Shigella flexneri 2a YSH6000, which contains only a 230-kilobase-pair (kbp) plasmid in addition to 3.3- and 4.2-kbp cryptic plasmids characteristic to all S. flexneri strains, loss of invasiveness, loss of Congo red binding activity (Pcr), and complete loss of, or a deletion, or even a single-site IS insertion in the plasmid occurred simultaneously. This was ascribed to the fact that, once a noninvasive Pcr- cell has emerged, it overgrows the wild type as a consequence of its selective advantage in artificial media. A deletion map of the 230-kbp plasmid was made by analyzing SalI digests of 39 deletion derivatives plus 1 formed by insertion of an IS1-like element in independently isolated, noninvasive Pcr- mutants. Of 39 deletion derivatives, 16 belonged to a single type, and 6 belonged to another, suggesting deletion hot spots. The deletion map was confirmed and extended by analyzing 359 SalI-generated partial digests of the wild-type plasmid cloned into pBR322. Three copies of IS1-like elements were found on three different SalI fragments by Southern hybridization. Segments required for the Pcr+ phenotype seemed to occur at several different locations in the plasmid. Each of 28 representative Pcr- mutants were negative by the Sereny test. Hence, many, or possibly all, Pcr determinants were required for full virulence.

Chromosome Mapping↗

PubMed1994

A fourth secreted aspartyl proteinase gene (SAP4) and a CARE2 repetitive element are located upstream of the SAP1 gene in Candida albicans.

Candida albicans secreted aspartyl proteinases (Sap), products of the SAP genes, which are presumed to act as virulence factors. In the C. albicans strain WO-1, the ability to secrete Sap1 is regulated with switch phenotype, another putative virulence factor. KpnI restriction fragment length polymorphisms differentiate between several distinct SAP1 alleles in laboratory and clinical strains. Both SAP1 alleles from strain WO-1 along with their 5'- and 3'-flanking regions were cloned and sequenced, as were both alleles from another strain, SS. The 5'-flanking regions were remarkably similar in all four of the sequenced alleles over approximately 1,500 nucleotides. S1 analysis revealed that both alleles of WO-1 are transcribed. Characterization of the one allele from strain WO-1 identified a 284-nucleotide insertion flanked by 8-bp direct repeats that shows homology to the CARE2 repetitive element and that is not present in the other alleles. Characterization of the SAP1 alleles also identified a fourth SAP gene (SAP4) that includes an extended leader sequence. SAP4 is positioned upstream, in tandem to SAP1, in all strains tested and may encode another closely related secreted aspartyl proteinase.

Alleles↗

PubMed1996

Site-specific insertion of IS1301 and distribution in Neisseria meningitidis strains.

The insertion element IS1301 has been shown to mediate capsule phase variation in Neisseria meningitidis found in N. serogroup B by reversible insertional inactivation of the siaA gene. We have determined the target site specificity of this element by cloning and sequencing the insertion sites of 12 identical IS1301 copies found in N. meningitidis B1940. A target consensus core of 5'-AYTAG-3' was identified, with the central TA being duplicated following insertion. Additional features around the target sites, including extended palindromic symmetry, stem-loop formation, and the high incidence of AT tracts, indicate that other factors, such as DNA secondary structure, are involved in target recognition. The left inverted repeat of an IS1016-like element acts as a hot spot for insertion, with one insertion element combination located upstream of their gene. According to further sequence analysis, we were able to place IS1301 in the IS5 subgroup within the IS4 family of elements. A survey of 135 Neisseria strains indicated the presence of IS1301 in 27.9 to 33.3% of N. meningitides serogroup B, C, and W135 strains and in 86.7% of serogroup Y strains. IS1301 did not occur in serogroup A strains, in Neisseria gonorrhoeae, or in apathogenic Neisseria spp.

Amino Acid Sequence↗

PubMed1982

A cloned immunoglobulin cDNA fragment enhances transposition of IS elements into recombinant plasmids.

Evidence is presented indicating that a novel DNA sequence arrangement generated by in vitro recombination may elicit high frequency transpositions of IS elements. A 109 bp Bam HI fragment of the cDNA for the immunoglobulin kappa light chain from MOPC 321 myeloma was cloned into the Bam HI site of pBR313. The cloned fragment extends from the codon for Gly 57 to the V-J junction. Insertions of IS1 or IS5 were identified in 6 of 50 plasmid DNAs isolated from freshly transformed clones. Additional transposition events were detected after subculturing for several growth cycles. Three independent insertions of IS1 occurred in the promoter region of the TcR operon. All IS5 and the remaining IS1 insertions were located in the TcR region upstream to the cloned DNA sequence. Sequences homologous to the ends of IS1, or corresponding to the consensus sequence at the target site of IS5 are present near the estimated sites of insertion of IS1 or IS5 respectively. Bacteria harboring recombinant plasmids carrying the cloned DNA in either orientation grew at a reduced rate relative to cells harboring pBR313, suggesting that fused gene products made from the two types of plasmid were inhibitory to cell growth. IS insertions, which relieved this inhibitory effect and thereby provided a selective advantage, were found exclusively in plasmids carrying the cloned DNA in only one of the two orientations. The fact that IS elements were not observed in the other type of recombinant plasmid indicates that selective pressure alone is not sufficient to account for the frequent IS insertions observed and that sequences at a distance from the site of IS insertion may be critical in the regulation of transposition frequency.

Animals↗

PubMed1994

An unusual type of cointegrate formation between a Bacteroides plasmid and the excised circular form of an integrated element (NBU1).

Evidence for an unusual type of cointegrate formation was found as the result of analyzing three integration events that fused a mobilization-deficient Bacteroides plasmid (pEG920) with the excised circular form of a nonreplicating Bacteroides element (NBU1). NBU1 is capable of inserting itself into DNA segments, but the cointegrates were the result of invasion of NBU1 by pEG920, not vice versa. The same site on pEG920 was involved in all cases. Sequence analysis of the cointegrates suggested that the integration events may have been the result of a multistep process in which a conjugative transposon was involved.

Bacteroides↗

PubMed1981

Cointegrates between bacteriophage P1 DNA and plasmid pBR322 derivatives suggest molecular mechanisms for P1-mediated transduction of small plasmids.

We characterized cointegrates formed in an Escherichia coli rec+ strain between bacteriophage P1 genomes and small plasmids related to pBR322. The partners were, on the one hand, either phage P1 DNA, which carries one copy of IS1, or phage P1-15 DNA, a derivative which lacks the IS1, and, on the other hand, plasmids containing either a split IS1 or no. In the presence of IS1 sequences on both partners, cointegrates were usually formed by reciprocal recombination between SI1 sequences. Cointegrates between P1 and a plasmid carrying no IS1 sequence were formed by transpositional cointegration mediated by IS1 of P1. Cointegrates between P1-15 and small plasmid containing a split IS1 were formed by one of three ways: (a) acquisition of an IS1 by P1-15 followed by reciprocal recombination between IS1 sequences, (b) transpositional cointegration mediated by the split IS1 element, Tn2657, or (c) involvement of the invertible segment carried on P1-15 DNA. Most cointegrates segregated into the small plasmids and phage P1 derivatives. A comparison of the phenomenon studied and of their frequencies allowed us to conclude that cointegrate formation is a molecular mechanism involved in the transduction of plasmids smaller than those packageable into P1 virions, although it does not seem to be the only process used.

Bacteriophages↗

PubMed1997

Comparison of three restriction endonucleases in IS1245-based RFLP typing of Mycobacterium avium.

IS1245-based restriction fragment length polymorphism (RFLP) analysis has been proposed recently for molecular typing of Mycobacterium avium isolates. As there is no standardised method with respect to the optimal restriction enzyme, three restriction endonucleases were tested for analysis of 17 human isolates. The restriction endonucleases, selected on the basis of the physical maps of IS1245 and of the highly homologous IS1311, were BsaAI, that cleaves IS1245, PvuII, that cleaves IS1311, and NruI, that cleaves both IS1245 and IS1311. All the restriction endonucleases yielded polymorphic and complex RFLP patterns. However, BsaAI- and NruI-generated bands were more evenly distributed and easier to detect than PvuII-generated bands, most of which clustered in a narrow zone of the fingerprint. In some cases, DNA digestion with BsaAI or NruI yielded probe-specific restriction fragments of molecular size lower than expected. Moreover, digestion with NruI, which was expected to generate the highest numbers of bands in all the isolates, yielded fewer bands than were obtained with BsaAI or PvuII in 14 and 5 isolates, respectively. These findings might suggest the existence of unidentified IS1245-related insertion element(s) in M. avium isolates. Computer analysis of the IS1245-based RFLP patterns of M. avium isolates showed that the restriction endonucleases were capable, although with minor differences, of defining distinct banding patterns and clusters of identical or highly related isolates, thus confirming IS1245-based RFLP analysis as a useful technique for epidemiological studies.

DNA Transposable Elements↗

PubMed1995

Tn5384, a composite enterococcal mobile element conferring resistance to erythromycin and gentamicin whose ends are directly repeated copies of IS256.

We have identified a 26-kb mobile element from Enterococcus faecalis CH116, designated Tn5384, which confers resistance to erythromycin and to high levels of gentamicin. Tn5384 is a composite element containing three copies of insertion element IS256. Two of the IS256 copies flank the aac6'-aph2" bifunctional aminoglycoside-modifying-enzyme gene in the inverted orientation, forming a structure similar to staphylococcal gentamicin resistance transposon Tn4001. One of the IS256 elements involved in the Tn4001-like structure also forms the left end of Tn5384, the right end of which is a directly repeated insertion of IS256 approximately 23 kb downstream of the leftmost insertion. Insertions of Tn5384 into enterococcal plasmid pLRM1 have been found associated with 8- and 9-bp duplications of the target sequence.

Base Sequence↗

PubMed1990

Nucleotide-sequence of insertion element IS15 delta IV from plasmid pBP11.

The nucleotide sequence of an insertion element in R-factor R1767 derivative pBP11 was determined. It is almost overall identical with IS15 delta, IS26 and IS46. Like IS46 it flanks one end of the sul-bla determinant and is involved in amplification of the resistance cassette. The significance for this process of a palindrome comprising part of IS15 delta IV is discussed.

Base Sequence↗

PubMed1990

[Expression of homologous and heterologous retrotransposons in a model system of yeast cells].

Yeast cells Saccharomyces cerevisiae were transformed by the recombinant plasmids containing the Yeast retrotransposon Ty and the Drosophila mobile element gypsy under the control of a strong Yeast promoter. The exogenous Ty-element induces the complete cycle of Ty-retrotransposition including the TyRNA synthesis, formation of virus-like particles, synthesis of all reverse transcriptase intermediates in the virus-like particles with the subsequent circles formation and transposition. The Drosophila mobile element gypsy is capable of inducing the formation of the virus-like particles containing RNA, DNA and proteins of the Ty-retrotransposon only. The Ty-circles and induction of transposition were not observed. The obtained data demonstrates the existence of the multistep repression system for Ty-transposition cycle. The possibility and efficiency of using the model to study the mechanism for retrotransposon transposition is discussed.

DNA Transposable Elements↗

PubMed1993

Transposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants.

The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants. The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements. In the primary transformants Ac transposition probably occurred during plant regeneration or early development. Other R0 transformants segregated kanamycin-resistant plants at a low frequency (< or = 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from one to six and almost all transposition events were independent. Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number. The frequency of Ac transposition, from different loci, remained low (< or = 7%) from R0 to R3 generations when only one Ac copy was present.(ABSTRACT TRUNCATED AT 250 WORDS)

Agrobacterium tumefaciens↗

PubMed1989

Sequence and characteristics of IS900, an insertion element identified in a human Crohn's disease isolate of Mycobacterium paratuberculosis.

The complete sequence of an insertion element IS900 in Mycobacterium paratuberculosis is reported. This is the first characterised example of a mycobacterial insertion element. IS900 consists of 1451bp of which 66% is G + C. It lacks terminal inverted and direct repeats, characteristic of Escherichia coli insertion elements but shows a degree of target sequence specificity. A single open reading frame (ORF 1197) coding for 399 amino acids is predicted. This amino acid sequence, and to a lesser extent the nucleotide sequence, show significant homologies to IS110, an insertion element of Streptomyces coelicolor A3(2). It is proposed that IS900, IS110, and similar insertion elements recently identified in disease isolates of Mycobacterium avium are members of a phylogenetically related family. IS900 will provide highly specific markers for the precise identification of Mycobacterium paratuberculosis, useful in defining its relationship to animal and human diseases.

Amino Acid Sequence↗

PubMed1987

Stable integration and expression of a bacterial gene in the mosquito Anopheles gambiae.

Foreign DNA was successfully introduced into the germline of the African mosquito vector of malaria Anopheles gambiae. Stable integration of genes into the germlines of insects had been achieved previously only in Drosophila melanogaster and related species and required the use of the P element transposon. In these experiments with Anopheles gambiae, the plasmid pUChsneo was used, which contains the selectable marker neo gene flanked by P element inverted repeats. Mosquitoes injected with this plasmid were screened for resistance to the neomycin analog G-418. A single event of plasmid insertion was recovered. Integration appears to be stable and, thus far, resistance to G-418 has been expressed for eight generations. The transformation event appears to be independent of P.

Anopheles↗

PubMed1997

[A mobile genetic element on a Bordetella pertussis chromosome stimulates cointegrate formation in Escherichia coli strains].

This work continues a series of reports devoted to the study of a repeated sequence isolated from the Bordetella pertussis chromosome (RSBP--Repeated Sequence of Bordetella Pertussis). The RSBP was earlier demonstrated to have a structure similar to IS elements and exhibit some properties of mobile genetic elements. The results of this work demonstrate the ability of this novel genetic element to stimulate the formation of cointegrates between independent replicons. We have studied the structure of five cointegrates formed by integration of a resident RSBP-containing plasmid into three different sites of a recipient replicon. The studied cointegrates are capable of resolution with the release of a resident plasmid independent of the host cell RecA function.

Bordetella pertussis↗

PubMed1984

Circular copies of mobile dispersed genetic elements in cultured Drosophila melanogaster cells.

Extrachromosomal supercoiled circular copies of mobile dispersed genetic elements mdg1, mdg3 and copia have been isolated from 67J25D and Kc lines of cultured Drosophila melanogaster cells. With the exception of the copia element in the line Kc, all circular molecules contain only one long terminal repeat. Both whole-length circular copies and molecules with deletion from the internal segment of the copia element have been detected in the line 67J25D. Mdg3-specific circular molecules in the line Kc are represented by the deleted molecules only. Similar copies of mdg3 are also present in chromosomal DNA of flies and cultured cells. The analysis of the mdg3 transcription in both cell lines suggests that extrachromosomal molecules of mdg may be created by reverse transcription.

Animals↗