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PubMed2006

ISfinder: the reference centre for bacterial insertion sequences.

ISfinder (www-is.biotoul.fr) is a dedicated database for bacterial insertion sequences (ISs). It has superseded the Stanford reference center. One of its functions is to assign IS names and to provide a focal point for a coherent nomenclature. It is also the repository for ISs. Each new IS is indexed together with information such as its DNA sequence and open reading frames or potential coding sequences, the sequence of the ends of the element and target sites, its origin and distribution together with a bibliography where available. Another objective is to continuously monitor ISs to provide updated comprehensive groupings or families and to provide some insight into their phylogenies. The site also contains extensive background information on ISs and transposons in general. Online tools are gradually being added. At present an online Blast facility against the entire bank is available. But additional features will include alignment capability, PsiBLAST and HMM profiles. ISfinder also includes a section on bacterial genomes and is involved in annotating the IS content of these genomes. Finally, this database is currently recommended by several microbiology journals for registration of new IS elements before their publication.

DNA Transposable Elements↗
PubMed2000

Double-hairpin elements in the mitochondrial DNA of allomyces: evidence for mobility.

The mitochondrial DNA (mtDNA) of the chytridiomycete fungus Allomyces macrogynus contains 81 G+C-rich sequence elements that are 26-79 bases long and can be folded into a unique secondary structure consisting of two stem-loops. At the primary sequence level, the conservation of these double-hairpin elements (DHEs) is variable, ranging from marginal to complete identity. Forty of these DHEs are inserted in intergenic regions, 35 in introns, and 6 in variable regions of rRNA genes. Ten DHEs are inserted into other DHE elements (twins); two even form triplets. A comparison of DHE sequences shows that loop regions contain more sequence variation than helical regions and that the latter often contain compensatory base changes. This suggests a functional importance of the DHE secondary structure. We further identified nine DHEs in a 4-kb region of Allomyces arbusculus, a close relative of A. macrogynus. Eight of these DHEs are highly similar in sequence (90%-100%) to those in A. macrogynus, but only five are inserted at the same positions as in A. macrogynus. Interestingly, DHEs are also found in the mtDNAs of other chytridiomycetes, as well as certain zygomycete and ascomycete fungi. The overall distribution pattern of DHEs in fungal mtDNAs suggests that they are mobile elements.

Base Sequence↗
PubMed2000

Isolation and analysis of a circular form of the IncJ conjugative transposon-like elements, R391 and R997: implications for IncJ incompatibility.

The incompatibility between the chromosomally integrating, conjugative transposon-like, IncJ elements R997 (ampicillin resistant) and R391 (kanamycin resistant) was examined by constructing strains harbouring both elements. Unusually, recA(+) strains harbouring the resistance determinants of both elements could be isolated but all strains lacked detectable extrachromosomal DNA. The phenotypic characteristics and transfer patterns observed suggested the formation of recombinant hybrids rather than strains harbouring both elements independently. Formation of strains harbouring two IncJ elements in a recA background was thus examined and resulted in the visualisation of extrachromosomal DNA. When R391 was transferred to a recA strain containing integrated R997, both elements co-existed stably and resulted in the isolation of a plasmid of 93.9 kb. When R997 was transferred to a recA strain harbouring an integrated R391, a plasmid of 85 kb was isolated. Comparison of restriction patterns for both elements revealed many common and several distinct fragments indicating a close physical relationship. These data suggest that although IncJ elements normally integrate at a unique site in the Escherichia coli chromosome, they possess the ability for autonomous replication which becomes manifest in a recA background when this site is occupied. This observation has implications for the nature of the incompatibility associated with IncJ elements and also provides a reliable method for isolating IncJ elements for molecular characterisation.

Ampicillin Resistance↗
PubMed1992

Evidence of incorporation of the chromosomal beta-lactamase gene of Enterococcus faecalis CH19 into a transposon derived from staphylococci.

We recently reported the chromosomal location of the staphylococcal beta-lactamase gene in four strains of Enterococcus faecalis. Transfer of this gene from strain CH19 to an enterococcal recipient was accompanied by transfer of numerous other antimicrobial resistance determinants in the absence of detectable plasmid DNA. A restriction map developed by comparing digestions of the regions surrounding the beta-lactamase gene in donor and recipient chromosomes resembles published maps of previously described staphylococcal beta-lactamase transposons, particularly in the area of the structural gene and its downstream region. In addition, DNA sequence analysis of the region immediately downstream of the beta-lactamase gene from both CH19 and its transcipient, CX19, revealed the presence of a 121-bp inverted repeat region found in Tn552 and Tn4002, two previously described staphylococcal beta-lactamase transposons. These results suggest that the chromosomal beta-lactamase gene of E. faecalis CH19 is incorporated into a transposonlike element derived from staphylococci.

Base Sequence↗
PubMed1999

Comparative genomics and evolutionary dynamics of Saccharomyces cerevisiae Ty elements.

The availability of the complete genome sequence of Saccharomyces cerevisiae provides the unique opportunity to study an entire genomic complement of retrotransposons from an evolutionary perspective. There are five families of yeast retrotransposons, Ty1-Ty5. We have conducted a series of comparative sequence analyses within and among S. cerevisiae Ty families in an effort to document the evolutionary forces that have shaped element variation. Our results indicate that within families Ty elements vary little in terms of both size and sequence. Furthermore, intra-element 5'-3' long terminal repeat (LTR) sequence comparisons indicate that almost all Ty elements in the genome have recently transposed. For each family, solo LTR sequences generated by intra-element recombination far outnumber full length insertions. Taken together, these results suggest a rapid genomic turnover of S. cerevisiae Ty elements. The closely related Ty1 and Ty2 are the most numerous elements in the genome. Phylogenetic analysis of full length insertions reveals that reverse transcriptase mediated recombination between Ty1 and Ty2 elements has generated a number of hybrid Ty1/2 elements. These hybrid Ty1/2 elements have similar genomic structures with chimeric LTRs and chimeric TYB (pol) genes. Analysis of the levels of nonsynonymous (Ka) and synonymous (Ks) nucleotide variation indicates that Ty1 and Ty2 coding regions have been subject to strong negative (purifying) selection. Distribution of Ka and Ks on Ty1, Ty2 and Ty1/2 phylogenies reveals evidence of negative selection on both internal and external branches. This pattern of variation suggests that the majority of full length Ty1, Ty2 and Ty1/2 insertions represent active or recently active element lineages and is consistent with a high level of genomic turnover. The evolutionary dynamics of S. cerevisae Ty elements uncovered by our analyses are discussed with respect to selection among elements and the interaction between the elements and their host genome.

Amino Acid Sequence↗
PubMed1987

Distribution and abundance of insertion sequences among natural isolates of Escherichia coli.

A reference collection of 71 natural isolates of Escherichia coli (the ECOR collection) has been studied with respect to the distribution and abundance of transposable insertion sequences using DNA hybridization. The data include 1173 occurrences of six unrelated insertion sequences (IS1, IS2, IS3, IS4, IS5 and IS30). The number of insertion elements per strain, and the sizes of DNA restriction fragments containing them, is highly variable and can be used to discriminate even among closely related strains. The occurrence and abundance of pairs of unrelated insertion sequences are apparently statistically independent, but significant correlations result from stratifications in the reference collection. However, there is a highly significant positive association among the insertion sequences considered in the aggregate. Nine branching process models, which differ in assumptions regarding the regulation of transposition and the effect of copy number on fitness, have been evaluated with regard to their fit of the observed distributions. No single model fits all copy number distributions. The best models incorporate no regulation of transposition and a moderate to strong decrease in fitness with increasing copy number for IS1 and IS5, strong regulation of transposition and a negligible to weak decrease in fitness with increasing copy number for IS3, and less than strong regulation of transposition for IS2, IS4 and IS30.

Base Sequence↗
PubMed2001

Molecular analysis of the Doppia transposable element of maize.

Doppia (Dop) transposable elements were first identified from element termini found in the upstream portions of certain alleles of the pl1 and r1 loci of maize. At the r1 locus, these Dop end sequences are present in a region called sigma, which functions as the promoter for the S genes of the R-r haplotype, and which is required for efficient epigenetic modification of the S genes during paramutation. In order to better understand the significance of the Dop element sequences at R-r, and to investigate the Dop-encoded products that might regulate r1 genes in this haplotype, we have cloned a more complete Dop element, Dop4. The Dop4 element can encode two proteins that have strong sequence similarity to the TnpA and TnpD proteins of the well characterized maize transposable element En/Spm. The DOPA protein, which is similar to TnpA of En/Spm, is shown to bind to short, subterminal repeat motifs located in the Dop element ends. Like TnpA, DOPA promotes intermolecular associations between DNA molecules. In contrast to the activity of TnpA, which is a transcriptional repressor of En/Spm, DOPA activates expression of reporter genes driven by either the Dop promoter or sigma in transient expression assays.

Amino Acid Sequence↗
PubMed1989

Insertion of L1 elements into sites that can form non-B DNA. Interactions of non-B DNA-forming sequences.

Three rat L1 element integration (target) sites chosen at random can adopt non-B DNA structures in vitro at normal bacterial superhelical densities. These target sites contain, respectively, short, mixed (AT)n tracts that we show can form one or more cruciforms, short (GT)n tracts, or polypurine:polypyrimidine regions. These sites share no sequence homology, and a non-B DNA structure appears to be the only feature common to them all. When the right end of the L1Rn3 element which forms a complex series of non-B DNA structures including two triplexes, and its target site which undergoes cruciform extrusion, are present on the same supercoiled molecule, they compete for available supercoil energy. The amount of non-B DNA formed at each site varies with pH, the concentration of cations, and the size of the topological domain. The implication of our findings for recombination of L1 elements and for the effect of these elements on contiguous DNA sequences is discussed.

Acetaldehyde↗
PubMed2002

R391: a conjugative integrating mosaic comprised of phage, plasmid, and transposon elements.

The conjugative, chromosomally integrating element R391 is the archetype of the IncJ class of mobile genetic elements. Originally found in a South African Providencia rettgeri strain, R391 carries antibiotic and mercury resistance traits, as well as genes involved in mutagenic DNA repair. While initially described as a plasmid, R391 has subsequently been shown to be integrated into the bacterial chromosome, employing a phage-like integration mechanism closely related to that of the SXT element from Vibrio cholerae O139. Analysis of the complete 89-kb nucleotide sequence of R391 has revealed a mosaic structure consisting of elements originating in bacteriophages and plasmids and of transposable elements. A total of 96 open reading frames were identified; of these, 30 could not be assigned a function. Sequence similarity suggests a relationship of large sections of R391 to sequences from Salmonella, in particular those corresponding to the putative conjugative transfer proteins, which are related to the IncHI1 plasmid R27. A composite transposon carrying the kanamycin resistance gene and a novel insertion element were identified. Challenging the previous assumption that IncJ elements are plasmids, no plasmid replicon was identified on R391, suggesting that they cannot replicate autonomously.

Bacteriophages↗
PubMed1985

Structure of an amplifiable DNA sequence in Streptomyces lividans 66.

Spontaneous chloramphenicol-sensitive mutants of Streptomyces lividans 66 had previously been shown to be very unstable and to yield arginine auxotrophic mutants at a frequency of 25% of spores; the Arg- mutants had amplified a particular 5.7 kb DNA sequence to over one hundred tandem copies per genome. In this paper we report the cloning of the amplifiable region from amplified and wild-type strains. This showed that the amplifiable fragment is already present as a duplication in wild type cells. Hybridisation experiments also demonstrated that in the amplified strains there was a deletion of neighbouring DNA sequences to one side of the amplifiable element; sequences to the other side remain intact.

DNA Restriction Enzymes↗
PubMed2001

Molecular genetics of SaPI1--a mobile pathogenicity island in Staphylococcus aureus.

The Staphylococcus aureus gene for toxic shock toxin (tst) is carried by a 15 kb mobile pathogenicity island, SaPI1, that has an intimate relationship with temperate staphylococcal phage 80alpha. During phage growth, SaPI1 is excised from its unique chromosomal site, attC, replicates autonomously, interferes with phage growth, and is efficiently encapsidated into special small phage heads commensurate with its size. Upon transfer to a recipient organism, SaPI1 integrates at attC by means of a self-coded integrase. One or more phage functions are required for excision, autonomous replication and encapsidation of the element and, thus, the overall relationship between SaPI1 and 80alpha is similar to that between coliphages P4 and P2. Among other staphylococcal phages tested, only phi13 interacts with SaPI1, inducing excision but not replication or transfer of the element.

Attachment Sites, Microbiological↗
PubMed2003

Genetic variability of the frameshift region in IS911 transposable elements from Escherichia coli clinical isolates.

The IS911 bacterial transposable element has been analyzed for its mechanism of transposition and for the way it controls the expression of its genes by programmed -1 translational frameshifting. In the present study the prevalence of IS911 has been determined in the Enterobacteriaceae family and in other Gram-negative bacilli. Three variants, found in Escherichia coli clinical isolates and having mutations in the region implicated in frameshifting, were functionally characterized. All three were altered in their frameshifting and transposition abilities, suggesting that the frameshift region of IS911 may constitute a target for mutations reducing the transposition frequency of this mobile element in natural populations of E. coli.

DNA Transposable Elements↗
PubMedDate not supplied

Permanent fixation of a transposable element insert in the A2 gene of maize (Zea mays L.).

Transposable elements are considered to be responsible for creating genetic variation that contributes to evolutionary change. The pervasiveness of transposable elements in certain breeding lines of maize suggests that part of the observed genetic variation in those lines might be the result of transposition activity. Stable genetic variation often results from the allelic differences created by footprints generated in the host genes upon element excision; such variation can also result from insertions that are permanently fixed at a particular locus. The 1.3-kb element within the a2-m1 (class II state) allele of maize is one example of a stable insertion. Though the l element never excises from the A2 gene, it interacts with the TNPA (transposase A) product of the En/Spm element. In this study, we tested whether continued interaction of the l element with TNPA would lead to excision of l or other change of this allele. Our screening of 220,000 kernels did not yield any new states of a2-m1 in the presence of an active autonomous En/Spm element, indicating that this l element is highly stable and permanently fixed at this locus. The probable implications of l-element stability are discussed.

Base Sequence↗
PubMed2000

Genetic and transcriptional analysis of a regulatory region in streptococcal conjugative transposon Tn5252.

In an attempt to increase our understanding of the mechanisms of conjugal transposition among gram-positive bacteria, we analyzed the genetic and structural properties of a 1.2-kb DNA fragment at the left end of the streptococcal conjugative transposon Tn5252. The sequence data revealed four short open reading frames. Polypeptides likely to correspond to two of these genes were identified. Transcriptional start sites and the promoter sequences of three transfer-related genes in the left terminal region of the element were identified. The deduced amino acid sequence of one of these, ORF3, was found to be similar to that of several prokaryotic transcriptional regulator proteins. Insertion mutagenesis at this locus reduced the transfer of the element by three orders of magnitude. The presence of a multicopy plasmid carrying ORF3 in a donor cell carrying Tn5252 with a mutated copy of ORF3 or an unaltered element also reduced the transfer frequency of the element similarly. Gel mobility shift assays showed that the ORF3 protein was capable of binding to not only other discrete sites at the left end of the element but also its own promoter, suggesting autoregulation. These results indicate that the ORF3 protein is involved in the regulation of the conjugative transposition of the element.

Amino Acid Sequence↗
PubMed1990

Characterization of plasmids and plasmid-borne macrolide resistance from Lactobacillus sp. strain 100-33.

Lactobacillus sp. strain 100-33 is resistant to macrolides, lincosamides, and streptogramin B-type antibiotics (MLSR) and appears to contain several major and minor plasmids. One of these plasmids, pLAR33, is approximately 18 kbp in size. When cells of strain 100-33 were protoplasted and regenerated, an MLSS isolate was derived. The derivative, designated strain ES1, contained a unique plasmid complement in which it had apparently lost the major plasmids of the parental strain, including pLAR33, and retained only a minor plasmid seen in low concentrations in strain 100-33. The MLSR determinant was cloned from plasmid DNA of strain 100-33 on a 3-kbp EcoRV fragment into pBR322 and localized to pLAR33. The determinant expressed macrolide and lincosamide resistance in Escherichia coli HB101, was localized to approximately 1 kbp on the cloned sequence, and is apparently under the control of its own promoter. MLSR electroporants were derived from strain ES1 electroporated with plasmid DNA from strain 100-33; these MLSR isolates had acquired a plasmid complement similar to that of strain 100-33, including pLAR33. Endonuclease digestion and Southern analysis of plasmid DNA from both strains indicated that the major plasmids are multimeric and deleted forms of one archetypal extrachromosomal element.

Anti-Bacterial Agents↗
PubMed1994

Distribution and hybridization patterns of the insertion element IS900 in clinical isolates of Mycobacterium paratuberculosis.

Reference strains and 31 clinical isolates of M. paratuberculosis, mainly from goats, were analysed for restriction fragment length polymorphism (RFLP). Restriction digests of bacterial DNA were hybridized with a repetitive insertion sequence, IS900, to obtain banding patterns for comparison of strains. Twenty-five of the 31 field-strains hybridized with IS900, and five hybridization patterns were identified. It was not possible to identify specific patterns for goat strains of M. paratuberculosis. Four hybridization patterns were similar, whereas the fifth pattern of a sheep strain diverged considerably in position and number of bands. Six goat strains failed to hybridize with IS900, and the absence of IS900 was verified by the polymerase chain reaction and hybridization with an oligonucleotide probe. The six IS900-negative goat strains had diverging phenotypic properties, and the identification of these strains is discussed. The present study shows that M. paratuberculosis strains infecting goats are genetically similar to cattle strains and that IS900 is a specific genetic element for identification of M. paratuberculosis.

Animals↗