Analysis of insertion sequences in thermophilic cyanobacteria: exploring the mechanisms of establishing, maintaining, and withstanding high insertion sequence abundance

Insertion sequences (ISs) are simple mobile genetic elements capable of relocating within a genome. Through this transposition activity, they are known to create mutations which are mostly deleterious to the cell, although occasionally they are beneficial. Two closely related isolates of thermophilic Synechococcus species from hot spring microbial mats are known to harbor a large number of diverse ISs. To explore the mechanism of IS acquisition within natural populations and survival in the face of high IS abundance, we examined IS content and location in natural populations of Synechococcus by comparing metagenomic data to the genomes of fully sequenced cultured isolates. The observed IS distribution in the metagenome was equivalent to the distribution in the isolates, indicating that the cultured isolates are appropriate models for the environmental population. High sequence conservation between IS families shared between the two isolates suggests that ISs are able to move between individuals within populations and between species via lateral gene transfer, consistent with models for IS family accumulation. Most IS families show evidence of recent activity, and interruption of critical genes in some individuals was observed, demonstrating that transposition is an ongoing mutational force in the populations.     

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.     

New insertion sequences of Sulfolobus: functional properties and implications for genome evolution in hyperthermophilic archaea

Analyses of complete genomes indicate that insertion sequences (ISs) are abundant and widespread in hyperthermophilic archaea, but few experimental studies have measured their activities in these hosts. As a way to investigate the impact of ISs on Sulfolobus genomes, we identified seven transpositionally active ISs in a widely distributed Sulfolobus species, and measured their functional properties. Six of the seven were found to be distinct from previously described ISs of Sulfolobus, and one of the six could not be assigned to any known IS family. A type II 'Miniature Inverted-repeat Transposable Element' (MITE) related to one of the ISs was also recovered. Rates of transposition of the different ISs into the pyrEF region of their host strains varied over a 250-fold range. The Sulfolobus ISs also differed with respect to target-site selectivity, although several shared an apparent preference for the pyrEF promoter region. Despite the number of distinct ISs assayed and their molecular diversity, only one demonstrated precise excision from the chromosomal target region. The fact that this IS is the only one lacking inverted repeats and target-site duplication suggests that the observed precise excision may be promoted by the IS itself. Sequence searches revealed previously unidentified partial copies of the newly identified ISs in the Sulfolobus tokodaii and Sulfolobus solfataricus genomes. The structures of these fragmentary copies suggest several distinct molecular mechanisms which, in the absence of precise excision, inactivate ISs and gradually eliminate the defective copies from Sulfolobus genomes.     

Causes of insertion sequences abundance in prokaryotic genomes

Insertion sequences (ISs) are the smallest and most frequent transposable elements in prokaryotes where they play an important evolutionary role by promoting gene inactivation and genome plasticity. Their genomic abundance varies by several orders of magnitude for reasons largely unknown and widely speculated. The current availability of hundreds of genomes renders testable many of these hypotheses, notably that IS abundance correlates positively with the frequency of horizontal gene transfer (HGT), genome size, pathogenicity, nonobligatory ecological associations, and human association. We thus reannotated ISs in 262 prokaryotic genomes and tested these hypotheses showing that when using appropriate controls, there is no empirical basis for IS family specificity, pathogenicity, or human association to influence IS abundance or density. HGT seems necessary for the presence of ISs, but cannot alone explain the absence of ISs in more than 20% of the organisms, some of which showing high rates of HGT. Gene transfer is also not a significant determinant of the abundance of IS elements in genomes, suggesting that IS abundance is controlled at the level of transposition and ensuing natural selection and not at the level of infection. Prokaryotes engaging in obligatory associations have fewer ISs when controlled for genome size, but this may be caused by some being sexually isolated. Surprisingly, genome size is the only significant predictor of IS numbers and density. Alone, it explains over 40% of the variance of IS abundance. Because we find that genome size and IS abundance correlate negatively with minimal doubling times, we conclude that selection for rapid replication cannot account for the few ISs found in small genomes. Instead, we show evidence that IS numbers are controlled by the frequency of highly deleterious insertion targets. Indeed, IS abundance increases quickly with genome size, which is the exact inverse trend found for the density of genes under strong selection such as essential genes. Hence, for ISs, the bigger the genome the better.     

Functional analysis of unique class II insertion sequence IS1071

Various xenobiotic-degrading genes on many catabolic plasmids are often flanked by two copies of an insertion sequence, IS1071. This 3.2-kb IS element has long (110-bp) terminal inverted repeats (IRs) and a transposase gene that are phylogenetically related to those of the class II transposons. However, the transposition mechanism of IS1071 has remained unclear. Our study revealed that IS1071 was only able to transpose at high frequencies in two environmental beta-proteobacterial strains, Comamonas testosteroni and Delftia acidovorans, and not in any of the bacteria examined which belong to the alpha- and gamma-proteobacteria. IS1071 was found to have the functional features of the class II transposons in that (i) the final product of the IS1071 transposition was a cointegrate of its donor and target DNA molecules connected by two directly repeated copies of IS1071, one at each junction; (ii) a 5-bp duplication of the target sequence was observed at the insertion site; and (iii) a tnpA mutation of IS1071 was efficiently complemented by supplying the wild-type tnpA gene in trans. Deletion analysis of the IS1071 IR sequences indicated that nearly the entire region of the IRs was required for its transposition, suggesting that the interaction between the transposase and IRs of IS1071 might be different from that of the other well-characterized class II transposons.     

Insertion sequences in prokaryotic genomes

Insertion sequences (ISs) are small DNA segments that are often capable of moving neighbouring genes. Over 1500 different ISs have been identified to date. They can have large and spectacular effects in shaping and reshuffling the bacterial genome. Recent studies have provided dramatic examples of such IS activity, including massive IS expansion during the emergence of some pathogenic bacterial species and the intimate involvement of ISs in assembling genes into complex plasmid structures. However, a global understanding of their impact on bacterial genomes requires detailed knowledge of their distribution across the eubacterial and archaeal kingdoms, understanding their partition between chromosomes and extra-chromosomal elements (e.g. plasmids and viruses) and the factors which influence this, and appreciation of the different transposition mechanisms in action, the target preferences and the host factors that influence transposition. In addition, defective (non- autonomous) elements, which can be complemented by related active elements in the same cell, are often overlooked in genome annotations but also contribute to the evolution of genome organisation.     

Insertion sequence diversity in archaea.

Insertion sequences (ISs) can constitute an important component of prokaryotic (bacterial and archaeal) genomes. Over 1,500 individual ISs are included at present in the ISfinder database (www-is.biotoul.fr), and these represent only a small portion of those in the available prokaryotic genome sequences and those that are being discovered in ongoing sequencing projects. In spite of this diversity, the transposition mechanisms of only a few of these ubiquitous mobile genetic elements are known, and these are all restricted to those present in bacteria. This review presents an overview of ISs within the archaeal kingdom. We first provide a general historical summary of the known properties and behaviors of archaeal ISs. We then consider how transposition might be regulated in some cases by small antisense RNAs and by termination codon readthrough. This is followed by an extensive analysis of the IS content in the sequenced archaeal genomes present in the public databases as of June 2006, which provides an overview of their distribution among the major archaeal classes and species. We show that the diversity of archaeal ISs is very great and comparable to that of bacteria. We compare archaeal ISs to known bacterial ISs and find that most are clearly members of families first described for bacteria. Several cases of lateral gene transfer between bacteria and archaea are clearly documented, notably for methanogenic archaea. However, several archaeal ISs do not have bacterial equivalents but can be grouped into Archaea-specific groups or families. In addition to ISs, we identify and list nonautonomous IS-derived elements, such as miniature inverted-repeat transposable elements. Finally, we present a possible scenario for the evolutionary history of ISs in the Archaea.     

Characterization of a class II defective transposon carrying two haloacetate dehalogenase genes from Delftia acidovorans plasmid pUO1

The two haloacetate dehalogenase genes, dehH1 and dehH2, on the 65-kb plasmid pUO1 from Delftia acidovorans strain B were found to be located on transposable elements. The dehH2 gene was carried on an 8.9-kb class I composite transposon (TnHad1) that was flanked by two directly repeated copies of IS1071, IS1071L and IS1071R. The dehH1 gene was also flanked by IS1071L and a truncated version of IS1071 (IS1071N). TnHad1, dehH1, and IS1071N were located on a 15.6-kb class II transposon (TnHad2) whose terminal inverted repeats and res site showed high homology with those of the Tn21-related transposons. TnHad2 was defective in transposition because of its lacking the transposase and resolvase genes. TnHad2 could transpose when the Tn21-encoded transposase and resolvase were supplied in trans. These results demonstrated that Tn Had2 is a defective Tn21-related transposon carrying another class I catabolic transposon.     

Local hopping mobile DNA implicated in pseudogene formation and reductive evolution in an obligate cyanobacteria-plant symbiosis

Background

Insertion sequences (ISs) are approximately 1 kbp long “jumping” genes found in prokaryotes. ISs encode the protein Transposase, which facilitates the excision and reinsertion of ISs in genomes, making these sequences a type of class I (“cut-and-paste”) Mobile Genetic Elements. ISs are proposed to be involved in the reductive evolution of symbiotic prokaryotes. Our previous sequencing of the genome of the cyanobacterium ‘Nostoc azollae’ 0708, living in a tight perpetual symbiotic association with a plant (the water fern Azolla), revealed the presence of an eroding genome, with a high number of insertion sequences (ISs) together with an unprecedented large proportion of pseudogenes. To investigate the role of ISs in the reductive evolution of ‘Nostoc azollae’ 0708, and potentially in the formation of pseudogenes, a bioinformatic investigation of the IS identities and positions in 47 cyanobacterial genomes was conducted. To widen the scope, the IS contents were analysed qualitatively and quantitatively in 20 other genomes representing both free-living and symbiotic bacteria.

Results

Insertion Sequences were not randomly distributed in the bacterial genomes and were found to transpose short distances from their original location (“local hopping”) and pseudogenes were enriched in the vicinity of IS elements. In general, symbiotic organisms showed higher densities of IS elements and pseudogenes than non-symbiotic bacteria. A total of 1108 distinct repeated sequences over 500 bp were identified in the 67 genomes investigated. In the genome of ‘Nostoc azollae’ 0708, IS elements were apparent at 970 locations (14.3%), with 428 being full-length. Morphologically complex cyanobacteria with large genomes showed higher frequencies of IS elements, irrespective of life style.

Conclusions

The apparent co-location of IS elements and pseudogenes found in prokaryotic genomes implies earlier IS transpositions into genes. As transpositions tend to be local rather than genome wide this likely explains the proximity between IS elements and pseudogenes. These findings suggest that ISs facilitate the reductive evolution in for instance in the symbiotic cyanobacterium ‘Nostoc azollae’ 0708 and in other obligate prokaryotic symbionts.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1386-7) contains supplementary material, which is available to authorized users.     

Neighboring plasmid sequences can affect Mini-Mu DNA transposition in the absence of expression of the bacteriophage Mu semi-essential early region

Bacteriophage Mu DNA, like other transposable elements, requires DNA sequences at both extremities to transpose. It has been previously demonstrated that the transposition activity of various transposons can be influenced by sequences outside their ends. We have found that alterations in the neighboring plasmid sequences near the right extremity of a Mini-Mu, inserted in the plasmid pSC101, can exert an influence on the efficiency of Mini-Mu DNA transposition when an induced helper Mu prophage contains a polar insertion in its semi-essential early region (SEER). The SEER of Mu is known to contain several genes that can affect DNA transposition, and our results suggest that some function(s), located in the SEER of Mu, may be required for optimizing transposition (and thus, replication) of Mu genomes from restrictive locations during the lytic cycle.     

A survey of bacterial insertion sequences using IScan

Bacterial insertion sequences (ISs) are the simplest kinds of bacterial mobile DNA. Evolutionary studies need consistent IS annotation across many different genomes. We have developed an open-source software package, IScan, to identify bacterial ISs and their sequence elements—inverted and target direct repeats—in multiple genomes using multiple flexible search parameters. We applied IScan to 438 completely sequenced bacterial genomes and 20 IS families. The resulting data show that ISs within a genome are extremely similar, with a mean synonymous divergence of K_s = 0.033. Our analysis substantially extends previously available information, and suggests that most ISs have entered bacterial genomes recently. By implication, their population persistence may depend on horizontal transfer. We also used IScan's ability to analyze the statistical significance of sequence similarity among many IS inverted repeats. Although the inverted repeats of insertion sequences are evolutionarily highly flexible parts of ISs, we show that this ability can be used to enrich a dataset for ISs that are likely to be functional. Applied to the thousands of genomes that will soon be available, IScan could be used for many purposes, such as mapping the evolutionary history and horizontal transfer patterns of different ISs.     

Stimulation of transposition of the Mycobacterium tuberculosis insertion sequence IS6110 by exposure to a microaerobic environment

The Mycobacterium tuberculosis-specific insertion sequence IS6110/986 has been widely used as a probe because of the multiple polymorphism observed among different strains. To investigate transposition of IS6110, a series of artificially constructed composite transposons containing IS6110 and a kanamycin resistance marker were constructed. The composite transposons were inserted into a conditionally replicating, thermosensitive, Escherichia coli-mycobacterial shuttle vector and introduced into M. smegmatis mc2155. Lawns of transformants were grown at the permissive temperature on kanamycin-supplemented agar and subsequently prevented from further growth by shifting to the non-permissive temperature. Under normal atmospheric conditions, kanamycin-resistant papillae appeared after only about 5-6 weeks of incubation. However, these events were not associated with transposon mobilization. In contrast, lawns that were exposed to a 48 h microaerobic shock generated kanamycin-resistant papillae after only 6-14 days. These events were generated by conservative transposition of the IS6110 composite transposon into the M. smegmatis chromosome, with loss of the shuttle vector. In common with other IS3 family elements, transposition of IS6110 is thought to be controlled by translational frameshifting. However, we were unable to detect any significant frameshifting within the putative frameshifting site of IS6110, and the level of frameshifting was not affected by microaerobic incubation. The finding that transposition of IS6110 is stimulated by incubation at reduced oxygen tensions may be relevant to transposition of IS6110 in M. tuberculosis harboured within TB lesions.     

植物CACTA转座子的研究进展

转座子是染色体上可移动的DNA序列,根据转座机制可将其分为：通过RNA中间体进行转座的逆转录座子（Retrotransposon）和通过DNA中间体进行转座的转座子（Transposon）。En／Spm家族转座子是后者中的一类,它的末端反向重复序列（Terminal inverted repeats,TIRs）具有保守的5个碱基CACTA,所以通常又称为CACTA转座子。除此之外,其靶位点一般为3bp的同向重复（Target site duplication,TSD）;亚末端区域分布着若干正向或反向的重复序列（Subterminal repeat,STR）。迄今为止,CACTA转座子仅发现于植物基因组。过去一直认为由于其相对保守的转座机制而拷贝较少,但最近研究发现,该因子多拷贝存在于某些禾本科植物基因组中。由于该家族在基因组中分布的广泛性,具有用作分子指纹的应用前景。本文就其结构、转座机制和应用前景等做一综述。     

Progress in Plant CACTA Elements

Transposable elements are DNA fragments that can insert new chromosomal locations. On the basis of the mechanism of transposition, transposable elements were divided into two classes. Class 1 elements were retroelements that used reverse transposase to transpose by an RNA intermediate. Class 2 elements or DNA transposons transposed directly from DNA to DNA. Of the Class 2 elements, CACTA superfamily, so far identified exclusively in plants and previously regarded as low-copy-transposon for the conserved mechanism of propagation, recently received considerable interest because of their increasing evidence reiterating their high copies in some plant genomes. This article aimed at outlining CACTA elements with regard to their structure, transposition, and utilization.     

Orientation of IS50 transposase gene and IS50 transposition.

Reversal of transposase gene orientation with respect to the nonidentical ends of IS50 strongly decreased IS50 transposition in both Dam- and Dam+ hosts. In either orientation, IS50 transposase expression was unaffected. These effects were independent of the surrounding DNA context. This shows that the efficiency of IS50 transposition is dependent on transposase gene orientation. The transposition frequencies of transposons utilizing inverted IS50 inside ends (IE), IE-IE transposons, were lower than either outside end (OE)-IE or OE-OE transposons.     

Transposases are responsible for the target specificity of IS1397 and ISKpn1 for two different types of palindromic units (PUs)

Insertion sequences (IS)1397 and ISKpn1, found in Escherichia coli and Klebsiella pneumoniae, respectively, are IS3 family members that insert specifically into short palindromic repeated sequences (palindromic units or PUs). In this paper, we first show that although PUs are naturally absent from extrachromosomal elements, both ISs are able to transpose from the chromosome or from a plasmid into PUs artificially introduced into target plasmids. We also show that ISKpn1 target specificity is restricted to K.pneumoniae Z¹ PU type, whereas IS1397 target specificity is less stringent since the IS targets the three E.coli Y, Z¹ and Z² PU types indifferently. Experiments of transposition of both ISs driven by both transposases demonstrate that the inverted repeats flanking the ISs are not responsible for this target specificity, which is entirely due to the transposase itself. Implications on ISs evolution are presented.     

Evolution of cooperation with shared costs and benefits

The quest to determine how cooperation evolves can be based on evolutionary game theory, in spite of the fact that evolutionarily stable strategies (ESS) for most non-zero-sum games are not cooperative. We analyse the evolution of cooperation for a family of evolutionary games involving shared costs and benefits with a continuum of strategies from non-cooperation to total cooperation. This cost-benefit game allows the cooperator to share in the benefit of a cooperative act, and the recipient to be burdened with a share of the cooperator's cost. The cost-benefit game encompasses the Prisoner's Dilemma, Snowdrift game and Partial Altruism. The models produce ESS solutions of total cooperation, partial cooperation, non-cooperation and coexistence between cooperation and non-cooperation. Cooperation emerges from an interplay between the nonlinearities in the cost and benefit functions. If benefits increase at a decelerating rate and costs increase at an accelerating rate with the degree of cooperation, then the ESS has an intermediate level of cooperation. The game also exhibits non-ESS points such as unstable minima, convergent-stable minima and unstable maxima. The emergence of cooperative behaviour in this game represents enlightened self-interest, whereas non-cooperative solutions illustrate the Tragedy of the Commons. Games having either a stable maximum or a stable minimum have the property that small changes in the incentive structure (model parameter values) or culture (starting frequencies of strategies) result in correspondingly small changes in the degree of cooperation. Conversely, with unstable maxima or unstable minima, small changes in the incentive structure or culture can result in a switch from non-cooperation to total cooperation (and vice versa). These solutions identify when human or animal societies have the potential for cooperation and whether cooperation is robust or fragile.     

Symbiotic DNA in eukaryotic genomes

The recent explosive growth of molecular genetic databases has yielded increasingly detailed insights into the evolutionary dynamics of eukaryotic genomes. DNA sequences with the self-encoded ability to transpose and replicate are unexpectedly abundant and widespread in eukaryotic genomes. They seem to be sexual parasites. By dispersing themselves among the chromosomes, they increase their transmission rates and can invade outcrossing populations despite reducing host fitness. Once established, molecular parasites may themselves be parasitized by other elements, and through selection for reduced virulence may become beneficial genes. Elements have been isolated at various stages in this progression, from transposons that regulate their own transposition rates, to fundamental components of eukaryotic cytology, such as telomeres.