The IStron CdISt1 of Clostridium difficile: molecular symbiosis of a group I intron and an insertion element

The IStron CdISt1 was first discovered as an insertion into the tcdA gene of the clinical isolate C34. It combines structural and functional properties of a group I intron at its 5'-end with those of an insertion element at its 3'-end. Up to date four different types could be found, mainly differing in their IS-element portions. Contrasting classical group I introns, CdISt1 is always integrated in ORFs encoding bacterial protein. In case CdISt1 had only the IS-element function such insertion would inactivate the protein encoded by the host gene. It is only due to the self-splicing activity of the group I intron parts that CdISt1 integration does not abolish protein function. Both elements seem to exist in molecular symbiosis and CdISt1 could thus be a prototype of a novel class of genetic elements. Moreover, integration of the CdISt1 into the genome could be advantageous for the bacterium, a motor function for evolution of bacterial proteins is discussed. In clinical practice CdISt1 might well serve as a tool for epidemiological studies of C. difficile infections.     

Isolation and characterization of functional insertion sequences of rhizobia

Rhizobia are a group of bacteria that form nodules on the roots of legume host plants. The sequenced genomes of the rhizobia are characterized by the presence of many putative insertion sequences (IS) elements. However, it is unknown whether these IS elements are functional and it is therefore relevant to assess their transposition activity. In this work, several functional insertion sequences belonging to the IS1256, IS3, IS5, IS166, and IS21 families were captured from Rhizobium tropici, Rhizobium sp. NGR234 and Sinorhizobium meliloti, using pGBG1 as a trapping system. In silico analysis shows that homologs of rhizobia mobile elements are present in distantly related genomes, suggesting that Rhizobium IS elements are prone to genetic transfer.     

DO PHAGES EFFICIENTLY SHUTTLE TRANSPOSABLE ELEMENTS AMONG PROKARYOTES?

The distribution, dynamics, and evolution of insertion sequences (IS), the most frequent class of prokaryotic transposable elements, are conditioned by their ability to horizontally transfer between cells. IS horizontal transfer (HT) requires shuttling by other mobile genetic elements. It is widely assumed in the literature that these vectors are phages and plasmids. By examining the relative abundance of IS in 454 plasmid and 446 phage genomes, we found that IS are very frequent in plasmids but, surprisingly, very rare in phages. Our results indicate that IS rarity in phages reflects very strong and efficient postinsertional purifying selection, mainly caused by a higher density of deleterious insertion sites in phages compared to plasmids. As they do not tolerate IS insertions, we conclude that phages may be rather poor vectors of IS HT in prokaryotes, in sharp contrast with the conventional view.     

ISWpi1 from Wolbachia pipientis defines a novel group of insertion sequences within the IS5 family

Insertion sequences are transposable elements that can represent substantial proportions of prokaryotic genomes and play a substantial role in shaping host genome evolution. As such, evaluating and understanding insertion sequence diversity is an important task to fulfill, because it is expected to yield new insight into the evolution of bacterial transposable elements and contribute to improve genome annotations. Here, I characterized an insertion sequence, termed ISWpi1, for which the taxonomic distribution appears to be restricted to the obligate intracellular alpha-Proteobacterium Wolbachia pipientis. ISWpi1 exhibits approximately 46% identity at the amino acid level with members of the IS1031 group of insertion sequences from the IS5 family. However, the IS1031 group is characterized by a transposase gene encoded by a single open reading frame, whereas the ISWpi1 transposase gene consists of two overlapping open reading frames presumably translated as a single protein via programmed translational frameshifting. Such structure suggests that ISWpi1 may instead be related to the IS427 group of insertion sequences from the IS5 family. Altogether, these data indicate that ISWpi1 extends the known spectrum of diversity of the IS5 family, and I propose to define a novel group of insertion sequences within the IS5 family typified by ISWpi1. Probable transpositional activity, relevant insertion site preferences and taxonomic specificity make ISWpi1 a promising tool for experimentally manipulating W. pipientis bacteria, especially in light of the increasing interest in developing these bacteria as tools for controlling insect disease vectors and agricultural pests.     

Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli

We have studied the spatial distribution of IS1 elements in the genomes of natural isolates comprising the ECOR reference collection of Escherichia coli. We find evidence for nonrandomness at three levels. Many pairs of IS1 elements are in much closer proximity (< 10 kb) than can be accounted for by chance. IS1 elements in close proximity were identified by long-range PCR amplification of the genomic sequence between them. Each amplified region was sequenced and its map location determined by database screening of DNA hybridization. Among the ECOR strains with at least two IS1 elements, 54% had one or more pairs of elements separated by < 10 kb. We propose that this type of clustering is a result of "local hopping," in which we assume that a significant proportion of tranposition events leads to the insertion of a daughter IS element in the vicinity of the parental element. A second level of nonrandomness is found in strains with a modest number of IS1 elements that are mapped through the use of inverse PCR to amplify flanking genomic sequences: in these strains, the insertion sites tend to be clustered over a smaller region of chromosome than would be expected by chance. A third level of nonrandomness is observed in the composite distribution of IS elements across strains: among 20 mapped IS1 elements, none were found in the region of 48-77 minutes, a significant gap. One region of the E. coli chromosome, at 98 min, had a cluster of IS1 elements in seven ECOR strains of diverse phylogenetic origin. We deduce from sequence analysis that this pattern of distribution is a result of initial insertion in the most recent common ancestor of these strains and therefore not a hot spot of insertion. Analysis using long- range PCR with primers for IS2 and IS3 also yielded pairs of elements in close proximity, suggesting that these elements may also occasionally transpose by local hopping.      

Survey of group I and group II introns in 29 sequenced genomes of the Bacillus cereus group: insights into their spread and evolution

Group I and group II introns are different catalytic self-splicing and mobile RNA elements that contribute to genome dynamics. In this study, we have analyzed their distribution and evolution in 29 sequenced genomes from the Bacillus cereus group of bacteria. Introns were of different structural classes and evolutionary origins, and a large number of nearly identical elements are shared between multiple strains of different sources, suggesting recent lateral transfers and/or that introns are under a strong selection pressure. Altogether, 73 group I introns were identified, inserted in essential genes from the chromosome or newly described prophages, including the first elements found within phages in bacterial plasmids. Notably, bacteriophages are an important source for spreading group I introns between strains. Furthermore, 77 group II introns were found within a diverse set of chromosomal and plasmidic genes. Unusual findings include elements located within conserved DNA metabolism and repair genes and one intron inserted within a novel retroelement. Group II introns are mainly disseminated via plasmids and can subsequently invade the host genome, in particular by coupling mobility with host cell replication. This study reveals a very high diversity and variability of mobile introns in B. cereus group strains.     

Mitogenome rearrangement in the cold-water scleractinian coral Lophelia pertusa (Cnidaria, Anthozoa) involves a long-term evolving group I intron

Group I introns are genetic insertion elements that invade host genomes in a wide range of organisms. In metazoans, however, group I introns are extremely rare, so far only identified within mitogenomes of hexacorals and some sponges. We sequenced the complete mitogenome of the cold-water scleractinian coral Lophelia pertusa, the dominating deep sea reef-building coral species in the North Atlantic Ocean. The mitogenome (16,150 bp) has the same gene content but organized in a unique gene order compared to that of other known scleractinian corals. A complex group I intron (6460 bp) inserted in the ND5 gene (position 717) was found to host seven essential mitochondrial protein genes and one ribosomal RNA gene. Phylogenetic analysis supports a vertical inheritance pattern of the ND5-717 intron among hexacoral mitogenomes with no examples of intron loss. Structural assessments of the Lophelia intron revealed an unusual organization that lacks the universally conserved ωG at the 3′ end, as well as a highly compact RNA core structure with overlapping ribozyme and protein coding capacities. Based on phylogenetic and structural analyses we reconstructed the evolutionary history of ND5-717, from its ancestral protist origin, through intron loss in some early metazoan lineages, and into a compulsory feature with functional implications in hexacorals.     

Acquisition of insertion sequences and the GBSi1 intron by Streptococcus agalactiae isolates correlates with the evolution of the species

The prevalence of insertion sequences IS1548, IS861, IS1381, and ISSa4 and of the group II intron GBSi1 within Streptococcus agalactiae human isolates strongly correlates with the genetic lineages obtained by multilocus sequence typing. Our results yielded an evolutionary scheme for the acquisition of these genetic elements linked to the ecosystems from which the isolates were obtained.     

USING RDNA GENES TO UNDERSTAND THE ORIGIN AND EVOLUTION OF SPLICEOSOMAL AND GROUP I INTRONS

Ribosomal DNA genes in lichen algae and lichen fungi are astonishingly rich in spliceosomal and group I introns. We use phylogenetic, secondary structure, and biochemical analyses to understand the evolution of these introns. Despite the widespread distribution of spliceosomal introns in nuclear pre-mRNA genes, their general mechanism of origin remains an open question because few proven cases of recent and pervasive intron origin have been documented. The lichen introns are valuable in this respect because they are undoubtedly of a "recent" origin and limited to the Euascomycetes. Our analyses suggest that rDNA spliceosomal introns have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into r RNAs. We propose that the spliceosome itself (and not an external agent; e.g. transposable elements, group II introns) has given rise to the introns. The rDNA introns are found most often between the flanking sequence G (78%) - intron-G (72%), and their clustered positions on secondary structures suggest that particular r RNA regions are preferred sites (i.e., proto-splice sites) for insertion. Mapping of intron positions on the newly available tertiary structures show that they are found most often in exposed regions of the ribosomes. This again is consistent with an intron origin through reverse-splicing. Remarkably, the distribution and phylogenetic relationships of most group I introns in nuclear rDNA genes are also consistent with a reverse-splicing origin. These data underline the value of lichens as a model system for understanding intron origin and stress the importance of RNA-level processes in the spread of these sequences in nuclear coding regions.     

Transposition of a bacterial insertion sequence in chloroplasts

Bacterial transposable elements (IS elements, transposons) represent an important determinant of genome structure and dynamics, and are a major force driving genome evolution. Here, we have tested whether bacterial insertion sequences (IS elements) can transpose in a prokaryotic compartment of the plant cell, the plastid (chloroplast). Using plastid transformation, we have integrated different versions of the Escherichia coli IS element IS 150 into the plastid genome of tobacco ( Nicotiana tabacum ) plants. We show that IS 150 is faithfully mobilized inside the chloroplast, and that enormous quantities of transposition intermediates accumulate. As synthesis of the IS 150 transposase is dependent upon programmed ribosomal frame shifting, our data indicate that this process also occurs in chloroplasts. Interestingly, all insertion events detected affect a single site in the plastid genome, suggesting that the integration of IS 150 is highly sequence dependent. In contrast, the initiation of the transposition process was found to be independent of the sequence context. Finally, our data also demonstrate that plastids lack the capacity to repair double-strand breaks in their genomes by non-homologous end joining, a finding that has important implications for genome stability, and which may explain the peculiar immunity of the plastid to invading promiscuous DNA sequences of nuclear and mitochondrial origin.     

Dispersal and evolution of the Sinorhizobium meliloti group II RmInt1 intron in bacteria that interact with plants

Group II introns are both self-splicing RNAs and mobile retroelements found in bacterial and archaeal genomes and in organelles of eukaryotes. They are thought to be the ancestors of eukaryote spliceosomal introns and non-long terminal repeat retrotransposons. We show here that RmInt1, a bacterial group II intron first described in the nitrogen-fixing symbiont of alfalfa (Medicago sativa) Sinorhizobium meliloti, is also present in other Sinorhizobium and Rhizobium species. The intron-homing sites in these species are IS elements of the ISRm2011-2 group as in S. meliloti, but ectopic insertion is also observed. We present evidence that these related bacteria have acquired RmInt1 by vertical inheritance from a common ancestor and by independent horizontal transfer events. We also show that RmInt1 is mobile in related taxa of bacteria that interact with plants and tends to evolve toward an inactive form by fragmentation, with loss of the 3' terminus including the intron-encoded protein. Our results provide an overview of the evolution and dispersion of a bacterial group II intron.     

Clostridium difficile IStron CdISt1: discovery of a variant encoding two complete transposase-like proteins

Screening a Clostridium difficile strain collection for the chimeric element CdISt1, we identified two additional variants, designated CdISt1-0 and CdISt1-III. In in vitro assays, we could prove the self-splicing ribozyme activity of these variants. Structural comparison of all known CdISt1 variants led us to define four types of IStrons that we designated CdISt1-0 through CdISt1-III. Since CdISt1-0 encodes two complete transposase-like proteins (TlpA and TlpB), we suggest that it represents the original genetic element, hypothesized before to have originated by fusion of a group I intron and an insertion sequence element.     

Identification of new transposable genetic elements in Burkholderia pseudomallei using subtractive hybridisation

A subtraction library of Burkholderia pseudomallei was constructed by subtractive hybridisation of B. pseudomallei genomic DNA with Burkholderia thailandensis genomic DNA. Two clones were found to have significant sequence similarity to insertion sequences which have previously not been found in B. pseudomallei (designated ISA and ISB); and two clones showed sequence similarity to different regions of Burkholderia cepacia IS407 that has recently been detected in B. pseudomallei. The former, though possibly non-functional, represents new transposable genetic elements of B. pseudomallei. All three sequences were found to be present in multi-copy in the genomes of a number of B. pseudomallei strains and in B. thailandensis, which are the first transposable elements identified in this species.     

Genome-wide comparison of cyanobacterial transposable elements, potential genetic diversity indicators

Transposable elements are widely distributed in archaea, bacteria and eukarya domains. Considerable discrepancies of transposable elements in eukaryotes have been reported, however, the studies focusing on the diversity of transposable element systems in prokaryotes were scarce. Understanding the transposable element system in cyanobacteria by the genome-wide analysis will greatly improve the knowledge of cyanobacterial diversity. In this study, the transposable elements of seventeen cyanobacterial genomes were analyzed. The abundance of insertion sequence (IS) elements differs significantly among the cyanobacterial genomes examined. In particular, water bloom forming Microcystis aeruginosa NIES843 was shown to have the highest abundance of IS elements reaching 10.85% of the genome. IS family is a widely acceptable IS classification unit, and IS subfamily, based on probe sequences, was firstly proposed as the basic classification unit for IS element system, therefore both IS family and IS subfamily were suggested as the two hierarchical units for evaluating the IS element system diversity. In total, 1980 predicted IS elements, within 21 IS families and 132 subfamilies, were identified in the examined cyanobacterial genomes. Families IS4, IS5, IS630 and IS200-605 are widely distributed, and therefore supposed to be the ancestral IS families. Analysis on the intactness of IS elements showed that the percentage of the intact IS differs largely among these cyanobacterial strains. Higher percentage of the intact IS detected in the two hot spring cyanobacterial strains implied that the intactness of IS elements may be related to the genomic stabilization of cyanobacteria inhabiting in the extreme environments. The frequencies between IS elements and miniature inverted-repeat transposable elements (MITEs) were shown to have a linear positive correlation. The transposable element system in cyanobacterial genomes is of hypervariability. With characterization of easy definition and stability, IS subfamily is considered as a reliable lower classification unit in IS element system. The abundance of intact IS, the composition of IS families and subfamilies, the sequence diversity of IS element nucleotide and transposase amino acid are informative and suitable as the indicators for studies on cyanobacterial diversity. Practically, the transposable system may provide us a new perspective to realize the diversity and evolution of populations of water bloom forming cyanobacterial species.