Mobile DNA Elements Research Articles

Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104

The presence and genetic content of integrons was investigated in eight Salmonella enterica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sul1 and qacEΔ1 genes characteristic of integrons. The first integron encoded the ant (3″)-Ia gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 β-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.

Construction and characterization of a Bacteroides thetaiotaomicron recA mutant: transfer of Bacteroides integrated conjugative elements is RecA independent.

We report the construction and analysis of a Bacteroides thetaiotaomicron recA disruption mutant and an investigation of whether RecA is required for excision and integration of Bacteroides mobile DNA elements. The recA mutant was deficient in homologous recombination and was more sensitive than the wild-type strain to DNA-damaging agents. The recA mutant was also more sensitive to oxygen than the wild type, indicating that repair of DNA contributes to the aerotolerance of B. thetaiotaomicron. Many Bacteroides clinical isolates carry self-transmissible chromosomal elements known as conjugative transposons. These conjugative transposons can also excise and mobilize in trans a family of unlinked integrated elements called nonreplicating Bacteroides units (NBUs). The results of a previous study had raised the possibility that RecA plays a role in excision of Bacteroides conjugative transposons, but this hypothesis could not be tested in Bacteroides spp. because no RecA-deficient Bacteroides strain was available. We report here that the excision and integration of the Bacteroides conjugative transposons, as well as NBU1 and Tn4351, were unaffected by the absence of RecA activity.

Escape From Evolutionary Stasis by Transposon-mediated Deleterious Mutations

Evolution within a rugged fitness landscape is limited by the tendency for organisms to become trapped on local optima resulting in evolutionary stasis. It is presently unclear how founder populations escape from an adaptive peak to found a new species. Insertion sequences, transposons and other mobile DNA elements are found in all species of eukaryotes, bacteria and archaebacteria, where they have been sought and are usually considered to be genomic parasites or selfish genes, However, many transposons and other mobile repetitive DNA are remarkably species or phyla-specific, indicating that infection with transposable elements coincides with speciation events and is involved in promoting evolutionary change. We propose here a model in which transposable elements are involved in speciation events by their ability to produce irreversible deleterious mutations that promote escape from evolutionary stasis. We have constructed a genetic algorithm designed to model both spontaneous and transposon-mediated mutations in populations of asexual digital organisms. We use this model to investigate the effect of transposon-mediated mutations on the rate of evolution of digital organisms as they compete for resources within an artificial adaptive landscape. In the absence of transposon mutations the seed organisms quickly evolve to occupy the nearest adaptive peak but thereafter evolutionary stasis ensues and adjacent empty peaks are left unoccupied. In the presence of transposon mutations, evolution is again dominated by stasis but is punctuated by bursts of rapid evolution in which consecutive unoccupied adaptive peaks are filled with organisms derived from single transposition events. Rapid evolutionary events leading to founding of new biological species, may be similarly initiated by irreversible deleterious mutations induced by transposition.

The fungal mitochondrial genome project: evolution of fungal mitochondrial genomes and their gene expression

The goal of the fungal mitochondrial genome project (FMGP) is to sequence complete mitochondrial genomes for a representative sample of the major fungal lineages; to analyze the genome structure, gene content, and conserved sequence elements of these sequences; and to study the evolution of gene expression in fungal mitochondria. By using our new sequence data for evolutionary studies, we were able to construct phylogenetic trees that provide further solid evidence that animals and fungi share a common ancestor to the exclusion of chlorophytes and protists. With a database comprising multiple mitochondrial gene sequences, the level of support for our mitochondrial phylogenies is unprecedented, in comparison to trees inferred with nuclear ribosomal RNA sequences. We also found several new molecular features in the mitochondrial genomes of lower fungi, including: (1) tRNA editing, which is the same type as that found in the mitochondria of the amoeboid protozoan Acanthamoeba castellanii; (2) two novel types of putative mobile DNA elements, one encoding a site-specific endonuclease that confers mobility on the element, and the other constituting a class of highly compact, structured elements; and (3) a large number of introns, which provide insights into intron origins and evolution. Here, we present an overview of these results, and discuss examples of the diversity of structures found in the fungal mitochondrial genome.

The Drosophila P-element KP repressor protein dimerizes and interacts with multiple sites on P-element DNA.

Drosophila P elements are mobile DNA elements that encode an 87-kDa transposase enzyme and transpositional repressor proteins. One of these repressor proteins is the 207-amino-acid KP protein which is encoded by a naturally occurring P element with an internal deletion. To study the molecular mechanisms by which KP represses transposition, the protein was expressed, purified, and characterized. We show that the KP protein binds to multiple sites on the ends of P-element DNA, unlike the full-length transposase protein. These sites include the high-affinity transposase binding site, an 11-bp transpositional enhancer, and, at the highest concentrations tested, the terminal 31-hp inverted repeats. The DNA binding domain was localized to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal binding motif. We also demonstrate that the KP repressor protein can dimerize and contains two protein-protein interaction regions and that this dimerization is essential for high-affinity DNA binding.

A widespread transposable element masks expression of a yeast copper transport gene.

The trace element copper (Cu) is essential for cell growth. In this report we describe the identification of a new component of the high-affinity Cu transport machinery in yeast, encoded by the CTR3 gene. Ctr3p is a small intracellular cysteine-rich integral membrane protein that restores high-affinity Cu uptake, Cu, Zn superoxide dismutase activity, ferrous iron transport, and respiratory proficiency to strains lacking the CTR1 (Cu transporter 1) gene. In most commonly used Saccharomyces cerevisiae laboratory strains, expression of CTR3 is abolished by a Ty2 transposon insertion that separates the CTR3 promoter from the transcriptional start sites by 6 kb. In strains that do not possess a Ty2 transposon at the CTR3 locus, expression of CTR3 is repressed by copper and activated by copper starvation. In such strains inactivation of both CTR1 and CTR3 is required to generate lethal copper-deficient phenotypes. Although Ctr1p and Ctr3p can function independently in copper transport, the expression of both proteins provides maximal copper uptake and growth rate under copper-limiting conditions. These results underscore the importance of mobile DNA elements in the alteration of gene function and phenotypic variation.

DNA transposition: Assembly of a jumping gene machine

Transposition of the mobile DNA element Mu is stringently controlled by the assembly of an elaborate ‘jumping gene machine’, which is inactive until all the pieces are in place.

Bacterial insertion sequences.

While DNA has a property of being fundamentally stable as invariable genetic information, studies on gene expression and gene organization have revealed that the genome is often subject to dynamic changes. Some of these changes are brought about by mobile genetic elements which have been found in prokaryotic and eukaryotic genomes so far studied. Insertion sequences (ISs) are bacterial mobile DNA elements which cause various kinds of genome rearrangements, such as deletions, inversions, duplications, and replicon fusions, by their ability to transpose. These were discovered during investigation of mutations that are highly polar in the galactose and lactose operons of Escherichia coli K−12 (Jordan etal. 1968; Malamy 1966, 1970; Shapiro 1969) and in the early genes of bacteriophage λ (Brachet et al. 1970). Many of these mutations were shown by electron microscope heteroduplex analysis to be insertions of distinct segments of DNA which are hence called insertion sequences (Fiandt et al. 1972; Hirsch et al. 1972; Malamy et al. 1972). It was later shown that the transcription of flanking genes can originate from promoters located within an IS or from hybrid promoters created by the insertion event or by the IS-mediated genome rearrangements. An important note here is that the finding of IS elements as mobile elements to new loci to turn genes either off or on would re-evaluate the controlling elements described in maize by McClintock (1956, 1965) (see Starlinger and Saedler 1976).

Identification of Sequences Which Regulate the Expression of Drosophila melanogaster Doc Elements

Long interspersed nuclear elements (LINEs) are mobile DNA elements which propagate by reverse transcription of RNA intermediates. LINEs lack long terminal repeats, and their expression is controlled by promoters located inside to the transcribed region of unit-length DNA copies. Doc elements constitute one of the seven families of LINEs found in Drosophila melanogaster. Plasmids in which the chloramphenicol acetyltransferase (CAT) gene is preceded by DNA segments from different Doc family members were used as templates for transient expression assays in Drosophila S2 cells. Transcription is initiated at the 5' end of Doc elements within hexamers fitting the consensus (C/G)AYTCG and is regulated by a DNA region which is located approximately 20 base pairs (bp) downstream from the RNA start site(s). The region includes a sequence (RGACGTGY motif, or DE2) which stimulates transcription in other Drosophila LINEs, and two adjacent elements, DE1 and DE3. Moving the downstream region either 4 bp away from, or 5 bp closer to the RNA start site region inhibited transcription. Sequences located approximately 200 bp downstream from the Doc 5' end repressed CAT expression in an orientation- and position-dependent manner. The inhibition reflects impaired translation of the CAT gene possibly consequent to the interaction of specific Doc RNA sequences with a cellular component.

Two copies of a DNA element, ?Wendy?, in the chloroplast chromosome of Chlamydomonas reinhardtii between rearranged gene clusters

We have characterized two copies of a 2.4 kb DNA element that we call 'Wendy', in the chloroplast chromosome of Chlamydomonas reinhardtii. The two copies of Wendy reside in different single-copy regions at opposite positions in the chloroplast genome. Like many mobile DNA elements, both copies of Wendy are bordered by inverted repeats and contain several additional degenerate copies of these repeat sequences in direct or inverted orientation. In addition, four basepairs are repeated in direct orientation. Two major open reading frames (ORFs) are predicted from the DNA sequence of Wendy I. These ORFs are co-transcribed from a promoter inside the element. The deduced amino acid sequence of the larger of these ORFs shares some weak similarities with sequence motifs of transposases and integrases of other mobile elements. Wendy II appears to be altered relative to Wendy I by point mutations and small deletions and insertions which destroy the ORFs. The leader sequence of the Wendy transcript is nearly identical with the leader sequence of the rbcL transcript of C. reinhardtii, but not of C. moewusii (where the complete Wendy was also undetectable). Furthermore, both copies of Wendy are bracketed by gene clusters that are separated in C. reinhardtii but are contiguous in C. moewusii where they exist in an inverted orientation compared with C. reinhardtii. Wendy was not found in any of the completely sequenced chloroplast genomes of rice, tobacco, pine, Euglena or Marchantia, nor in any other GenBank entry. Our results suggest that Wendy has invaded C. reinhardtii after divergence from other species. Subsequent Wendy-dependent illegitimate homologous or site-specific recombination events or both may have contributed to scrambling of the C. reinhardtii chloroplast genome relative to genomes of other species.

Botulinal neurotoxin C1 complex genes, clostridial neurotoxin homology and genetic transfer in Clostridium botulinum

The botulinal neurotoxins (BoNT) associate with non-toxic proteins (ANTP) by non-covalent bonds to form large complexes. In C. botulinum C, the BoNT C1 locus consists of six genes which are organized in three clusters. Cluster 1 encompasses the genes of BoNT C1 and ANTP/139 which could be involved in the resistance of the BoNT C1 to the acidic pH and protease degradation. The second cluster consists of three genes which encode hemagglutinin components. The last gene encodes a DNA binding protein (Orf22) which might regulate the BoNT C1 complex gene expression. BoNT and tetanus toxin (TeTx) display similar structure and mechanism of action at the molecular level. Their identity at the amino acid level range from 34 to 96.8%, indicating that the clostridial neurotoxin genes probably derive from a common ancestor. The fact that Clostridium other than C. botulinum such as C. butyricum and C. baratii can produce a BoNT suggests that the BoNT genes can be transferred between Clostridium strains. The toxigenic C. butyricum strains seem to derive from originally non-toxic strains by neurotoxin gene transfer from C. botulinum E, probably including a mobile DNA element. In C. botulinum C and D the gene encoding the exoenzyme C3 has been localized in a transposon-like element of 21.5 kbp. Transposons could be involved in BoNT gene transfer in C. botulinum.

An intragenic tandem duplication of genomic DNA is responsible for the f3N mutation of Drosophila melanogaster.

Among the numerous X chromosome-linked forked bristle (f) mutations described in Drosophila melanogaster, one designated f3N exhibits the unusual property of reverting spontaneously to wild type at an inordinate frequency, a frequency that can be increased with x-ray irradiation. In contrast to the f mutants described thus far, all of which are associated with the insertion of mobile DNA elements, f3N is associated with an intragenic duplication of 2.8 kb of genomic DNA that resolves to the normal sequence when reversions occur. Consideration is given to intrachromosomal recombination as the mechanism of reversion and truncation of the forked protein as a cause for the mutant phenotype.

Segmented structure of protein sequences and early evolution of genome by combinatorial fusion of DNA elements

A theory of an early stage of genome evolution by combinatorial fusion of circular DNA units is suggested, based on protein sequence "fossil" evidence. The evidence includes preference of protein sequence lengths for certain sizes--multiples of 123 aa for eukaryotes and multiples of 152 aa for prokaryotes. At the DNA level these sizes correspond to 350-450 base pairs--the known optimal range for DNA ring closure. The methionine residues repeatedly appear along the sequences with the same period of about 120 aa (in eukaryotes), presumably marking the sites of insertion of the early genes--rings of protein-coding DNA. No torsional constraint in this DNA results in very sharp estimate of the helical periodicity of the early DNA, indistinguishable from the experimental mean value for extant DNA. According to the combinatorial fusion theory, based on the above evidence, in the pregenomic, prerecombinational stage the genes and the noncoding sequences existed in form of autonomously replicating DNA rings of close to standard size, randomly segregating between dividing cells, like modern plasmids do. In the recombinational early genomic stage the rings started to fuse, forming larger DNA molecules consisting of several unit genes connected in various combinations and forming long protein-coding sequences (combinatorial fusion). This process, which involved, perhaps, noncoding sequences as well, eventually resulted in the formation of large genomes. The dispersed circular DNA--or, rather, evolutionarily advanced derivatives thereof--may still exist in the form of various mobile DNA elements.

The molecular analysis of brown eye color mutations isolated from geographically discrete populations of Drosophila melanogaster.

A large proportion of spontaneous mutations in Drosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessive brown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caught Drosophila melanogaster females, bw mutations were isolated from seven separate geographic sites distributed among Japan, California. Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in the bw gene. The remainder are inseparable from wild-type bw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneous bw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.

Plasmid evolution by acquisition of mobile gene cassettes: plasmid pIE723 contains the aadB gene cassette precisely inserted at a secondary site in the IncQ plasmid RSF1010

Gene cassettes are mobile DNA elements which contain a specific recombination site, a 59-base element, recognized by the site-specific recombination system of integrons. Gene cassettes are normally found inserted at a unique site in an integron, downstream of a promoter which directs transcription of the cassette-associated genes. However, insertion of a gene cassette into a secondary site in a plasmid which does not contain an integron is also formally possible. Sequence analysis of the aadB gene in pIE723, a plasmid closely related to the IncQ plasmid RSF1010, revealed the presence of the complete aadB cassette inserted at a secondary site downstream of a known RSF1010 promoter. The site of insertion of the aadB cassette in RSF1010 conformed to the consensus for secondary sites recognized by the integron integrase (Int), and it is likely that the cassette was inserted via a single Int-mediated recombination event between the 59-base element of a free, circular aadB cassette and a secondary site in RSF1010. The cassette-associated recombination site was inactivated by the insertion, and Int-mediated excision of the aadB cassette from this non-specific location was not detectable, indicating that the cassette is stably inserted. The movement of gene cassettes to secondary sites is likely to play an important role in the acquisition of new genes by bacterial and plasmid genomes.

Comparative evaluation of molecular typing of strains from a national epidemic due to Salmonella brandenburg by rRNA gene and IS200 probes and pulsed-field gel electrophoresis.

In Switzerland in 1992 there was a prolonged series of outbreaks of human salmonellosis caused by a previously rare serotype, Salmonella brandenburg. In order to examine the genotypic basis of the epidemic, molecular typing was applied to representative strains of this serovar isolated between 1983 and 1992. These included sporadic human isolates up to 1985, isolates from unrelated geographical areas, and Swiss isolates from humans, animals, and meat products isolated after 1991. Plasmid profiling was not found to be applicable to S. brandenburg, but chromosomal typing was accomplished by analyzing restriction fragment length polymorphisms with DNA probes for three marker loci; the 16S and 23S rRNA genes and sites of insertion of the mobile DNA element IS200. The macrorestriction profiles of the whole genome were examined by pulsed-field gel electrophoresis, which proved to be the most discriminatory of the typing methods. The study demonstrated the comparative value and complementary relationship between these typing methods for epidemiological purposes. All approaches concurred in identifying the 1992 isolates as a single genotypic clone, which was present in multiple (food) vehicles of infection. They were distinct from sporadic isolates of this serovar and from strains of S. brandenburg isolated in other countries.

IS1139 from Streptococcus salivarius: Identification and Characterization of an Insertion Sequence-like Element Related to Mobile DNA Elements from Gram-Negative Bacteria

An insertion sequence-like element, IS1139, was cloned and sequenced from Streptococcus salivarius ATCC 25975 chromosome. This insertion sequence-like element is 1168 bp long and is delimited by inverted repeats of 29 bp and by a duplicated sequence of 6 bp. This IS possesses an open reading frame that codes for a putative transposase of 339 amino acids which has, respectively, 94, 35, 33, and 30% amino-acid identity with the transposases of IS1161 from S. salivarius ATCC 25975, IS4351 from Bacteroides fragilis , IS30 from Escherichia coli, and IS1086 from Alcaligenes eutrophus. Sequence analysis revealed that these transposases may have evolved from a common ancestral gene. Southern hybridization of restriction endonuclease-digested genomic DNA from 21 strains of oral streptococci, using a probe specific to the transposase-encoding gene (tnpA), revealed that IS1139 is found in two strains of S. salivarius, ATCC 25975 and ATCC 13419, in eight and two copies, respectively.

Integrons: novel DNA elements which capture genes by site-specific recombination.

Integrons are unusual DNA elements which include a gene encoding a site-specific DNA recombinase, a DNA integrase, and an adjacent site at which a wide variety of antibiotic resistance and other genes are found as inserts. One or more genes can be found in the insert region, but each gene is part of an independent gene cassette. The inserted genes are expressed from a promoter in the conserved sequences located 5' to the genes, and integrons are thus natural expression vectors. A model for gene insertion in which circular gene cassettes are inserted individually via a single site-specific recombination event has been proposed and verified experimentally. The gene cassettes include a gene coding region and, at the 3' end of the gene an imperfect inverted repeat, a 59-base element. The 59-base elements are a diverse family of elements which function as sites recognized by the DNA integrase. Site-specific insertion of individual genes thus represents a further mechanism which contributes to the evolution of the genomes of Gram-negative bacteria and their plasmids and transposons. Members of the most studied class of integrons, which include the sulI gene in the conserved sequences, are believed to be mobile DNA elements on the basis that they are found in many independent locations, and a discrete boundary is found at the outer end of the 5'-conserved segment. However, the length of the 3'-conserved segment is variable in the integrons examined to date, and it is likely that this variability has arisen as the result of insertion and deletion events. Though the true extent of the 3'-conserved segment remains to be determined, it seems likely that these integrons are mobile DNA elements. The second known class of integrons comprises members of the Tn7 transposon family.

Chromosomal genotypes (evolutionary lines) of Salmonella berta

The copy number and location of the insertion sequence IS 200, a mobile DNA element, was established across a collection of Salmonella berta. All strains contained one common site, assumed to be present in the evolutionary ancestor of this serovar. With one exception, all strains, including recent outbreak isolates from the UK and sporadic isolates of world-wide distribution, were representatives of a single genotypic clone which carried three common IS 200 insertion sites. This clone has acquired diverse combinations of plasmids, reflecting its actual or recent distribution and host. A single isolate, belonging to a second, minor genotypic clone was characterised by two IS 200 insertion sites.

Chromosomal genotypes (evolutionary lines) ofSalmonella berta

The copy number and location of the insertion sequence IS200, a mobile DNA element, was established across a collection of Salmonella berta. All strains contained one common site, assumed to be present in the evolutionary ancestor of this serovar. With one exception, all strains, including recent outbreak isolates from the UK and sporadic isolates of world-wide distribution, were representatives of a single genotypic clone which carried three common IS200 insertion sites. This clone has acquired diverse combinations of plasmids, reflecting its actual or recent distribution and host. A single isolate, belonging to a second, minor genotypic clone was characterised by two IS200 insertion sites.