Wide-Range Distribution of Insertion Sequences Identified in B. halodurans among Bacilli and a New Transposon Disseminated in Alkaliphilic and Thermophilic Bacilli

All of the insertion sequences (ISs) except for IS663 and agroup II intron identified in the alkaliphilic Bacillus haloduransC-125 genome were also detected in nine other strains of thesame species by PCR and Southern blot analysis. The transposaseof IS653 identified in the genomes of the 10 strains of B. haloduranswas found to have become the most diversified of all ISs identifiedin the genomes of 10 strains. A new IS element designated IS661belonging to the IS1380 family with inverted repeats (IRs) 17bp in length was present within IS658 identified in the genomeof B. halodurans A59. In addition, a new transposon designatedTn3271bh was identified within the IS642 element in the A59genome, which is similar to a transposon identified in thermophilicGeobacillus stearothermophilus T-6. The new transposon, Tn3271bh,generated an 8-bp duplication of the target site sequence andcarries a 21-bp IR. On the other hand, all kinds of ISs exceptfor IS643 and IS658 were distributed in the genome of obligatelyalkaliphilic Bacillus alcalophilus. Three ISs (IS652, IS653,and IS660) and a group II intron (Bh.Int) were widely dispersedin other Bacillus species without a correlation with the phylogeneticplacement based on 16S rDNA sequences.     

Identification and distribution of new insertion sequences in the genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831.

Six kinds of new insertion sequences (ISs), IS667 to IS672, a group II intron (Oi.Int), and an incomplete transposon (Tn852loi) were identified in the 3,630,528-bp genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831. Of 19 ISs identified in the HTE831 genome, 7 were truncated, indicating the occurrence of internal rearrangement of the genome. All ISs except IS669 generated a 4- to 8-bp duplication of the target site sequence, and these ISs carried 23- to 28-bp inverted repeats (IRs). Sequence analysis revealed that four ISs (IS669, IS670, IS671, and IS672) were newly identified as belonging to separate IS families (IS200/IS605, IS30, IS5, and IS3, respectively). IS667 and IS668 were also characterized as new members of the ISL3 family. Tn8521oi, which belongs to the Tn3 family as a new member, generated a 5-bp duplication of the target site sequence and carried complete 38-bp IRs. Of the eight protein-coding sequences (CDSs) identified in Tn8521oi, three CDSs (OB481, OB482, and OB483) formed a ger gene cluster, and two other paralogous gene clusters were found in the HTE831 genome. Most of the ISs and the group II intron widely distributed throughout the genome were inserted in noncoding regions, while two ISs (IS667-08 and IS668-02) and Oi.Int-04 were inserted in the coding regions.     

An insertion sequence-dependent plasmid rearrangement in Aeromonas salmonicida causes the loss of the type three secretion system

Aeromonas salmonicida, a bacterial fish pathogen, possesses a functional Type Three Secretion System (TTSS), which is essential for its virulence. The genes for this system are mainly located in a single region of the large pAsa5 plasmid. Bacteria lose the TTSS region from this plasmid through rearrangements when grown in stressful growth conditions. The A. salmonicida genome is rich in insertion sequences (ISs), which are mobile DNA elements that can cause DNA rearrangements in other bacterial species. pAsa5 possesses numerous ISs. Three IS11s from the IS256 family encircle the rearranged regions. To confirm that these IS11s are involved in pAsa5 rearrangements, 26 strains derived from strain A449 and two Canadian isolates (01-B526 and 01-B516) with a pAsa5 rearrangement were tested using a PCR approach to determine whether the rearrangements were the result of an IS11-dependent process. Nine out of the 26 strains had a positive PCR result, suggesting that the rearrangement in these strains were IS-dependent. The PCR analysis showed that all the rearrangements in the A449-derived strains were IS11-dependent process while the rearrangements in 01-B526 and 01-B516 could only be partially coupled to the action of IS11. Unidentified elements that affect IS-dependent rearrangements may be present in 01-B526 and 01-B516. Our results suggested that pAsa5 rearrangements involve IS11. This is the first study showing that ISs are involved in plasmid instability in A. salmonicida.     

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.     

IS<Emphasis Type="Italic">4</Emphasis> family goes genomic

Background  

Insertion sequences (ISs) are small, mobile DNA entities able to expand in prokaryotic genomes and trigger important rearrangements. To understand their role in evolution, accurate IS taxonomy is essential. The IS 4 family is composed of ~70 elements and, like some other families, displays extremely elevated levels of internal divergence impeding its classification. The increasing availability of complete genome sequences provides a valuable source for the discovery of additional IS 4 elements. In this study, this genomic database was used to update the structural and functional definition of the IS 4 family.     

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.     

Detection and characterization of a functional insertion sequence, ISVpa2, in Vibrio parahaemolyticus

PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio.     

Insertion sequence-caused large-scale rearrangements in the genome of Escherichia coli

A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 × 10⁻⁴ insertions per genome per generation and the rate of IS homologous recombination is 4.5 × 10⁻⁵ recombinations per genome per generation. These events are mostly contributed by the IS elements IS1, IS2, IS5 and IS186. Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS186 target-sites, 5′-GGGG(N6/N7)CCCC-3′. We also detected 48 long deletions not involving IS elements.     

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.     

Enhancing recombinant protein production with an Escherichia coli host strain lacking insertion sequences

The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23 % of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of ~10^?8/gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (10^?8/gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (~52 % of the total population) 28 h after fermentation began due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant protein production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains.     

Temperature-Induced Change of Chlorophyll a Forms in Phosphatidylcholine Liposomes

Absorption spectra of chlorophyll a in phosphatidylcholine liposomesat different temperatures were analyzed by a curve fitting method.The absorption spectrum was found to be composed of one majorband with a peak at 670–671 nm and minor bands with peaksat 650–652, 662–663 and 684–686 nm. Upon coolingbelow the phase transition temperature of the lipid, the componentabsorbing at 670–671 nm increased significantly at theexpense of the component absorbing at 662–663 nm. No changein the extents of other bands was observed. ¹ CIW-DPB Publication No. 795. ²On leave from the Department of Biology, Faculty of Science,Kanazawa University, Marunouchi, Kanazawa 920, Japan. (Received December 20, 1982; Accepted April 27, 1983)     

The distribution of insertion sequences in the genome of Shigella flexneri strain 2457T

Shigella flexneri, which causes shigellosis in humans, evolved from Escherichia coli. The sequencing of Shigella genomes has revealed that a large number of insertion sequence (IS) elements (over 200 elements) reside in the genome. Although the presence of these elements has been noted previously and summarized, more detailed analyses are required to understand their evolutionary significance. Here, the genome of S. flexneri strain 2457T is used to investigate the spatial distribution of IS copies around the chromosome and the location of elements with respect to genes. It is found that most IS isoforms occur essentially randomly around the genome. Two exceptions are IS91 and IS911, which appear to cluster due to local hopping. The location of IS elements with respect to genes is biased, however, revealing the action of natural selection. The non-coding regions of the genome (no more than 21%) carry disproportionally more IS elements (at least 28%) than the coding regions, implying that selection acts against insertion into genes. Of the genes disrupted by ISs, those involved in signal transduction, intracellular trafficking, and cell motility are most commonly targeted, suggesting selection against genes in these categories.     

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.     

OASIS: an automated program for global investigation of bacterial and archaeal insertion sequences

Insertion sequences (ISs) are simple transposable elements present in most bacterial and archaeal genomes and play an important role in genomic evolution. The recent expansion of sequenced genomes offers the opportunity to study ISs comprehensively, but this requires efficient and accurate tools for IS annotation. We have developed an open-source program called OASIS, or Optimized Annotation System for Insertion Sequences, which automatically annotates ISs within sequenced genomes. OASIS annotations of 1737 bacterial and archaeal genomes offered an unprecedented opportunity to examine IS evolution. At a broad scale, we found that most IS families are quite widespread; however, they are not present randomly across taxa. This may indicate differential loss, barriers to exchange and/or insufficient time to equilibrate across clades. The number of ISs increases with genome length, but there is both tremendous variation and no increase in IS density for genomes >2 Mb. At the finer scale of recently diverged genomes, the proportion of shared IS content falls sharply, suggesting loss and/or emergence of barriers to successful cross-infection occurs rapidly. Surprisingly, even after controlling for 16S rRNA sequence divergence, the same ISs were more likely to be shared between genomes labeled as the same species rather than as different species.     

ISsaga is an ensemble of web-based methods for high throughput identification and semi-automatic annotation of insertion sequences in prokaryotic genomes

Insertion sequences (ISs) play a key role in prokaryotic genome evolution but are seldom well annotated. We describe a web application pipeline, ISsaga (), that provides computational tools and methods for high-quality IS annotation. It uses established ISfinder annotation standards and permits rapid processing of single or multiple prokaryote genomes. ISsaga provides general prediction and annotation tools, information on genome context of individual ISs and a graphical overview of IS distribution around the genome of interest.     

Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor

The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an autonomous basolateral targeting signal that does not mediate rapid endocytosis (Casanova, J. E., G. Apodaca, and K. E. Mostov. Cell. 66:65-75). Alanine-scanning mutagenesis identifies three residues in this region, His656, Arg657, and Val660, that are most essential for basolateral sorting and two residues, Arg655 and Tyr668, that play a lesser role in this process. Progressive truncations suggested that Ser664 and Ile665 might also play a role in basolateral sorting. However, mutation of these residues to Ala or internal deletions of these residues did not affect basolateral sorting, indicating that these residues are probably not required for basolateral sorting. Two-dimensional NMR spectroscopy of a peptide corresponding to the 17-mer signal indicates that the sequence Arg658-Asn-Val-Asp661 has a propensity to adopt a beta-turn in solution. Residues COOH-terminal to the beta-turn (Arg662 to Arg669) seem to take up a nascent helix structure in solution. Substitution of Val660 with Ala destabilizes the turn, while mutation of Arg657 to Ala does not appear to affect the turn structure. Neither mutation detectably altered the stability of the nascent helix in the COOH- terminal portion of the peptide.     

Positive selection on transposase genes of insertion sequences in the Crocosphaera watsonii genome

Insertion sequences (ISs) are mobile elements that are commonly found in bacterial genomes. Here, the structural and functional diversity of these mobile elements in the genome of the cyanobacterium Crocosphaera watsonii WH8501 is analyzed. The number, distribution, and diversity of nucleotide and amino acid stretches with similarity to the transposase gene of this IS family suggested that this genome harbors many functional as well as truncated IS fragments. The selection pressure acting on full-length transposase open reading frames of these ISs suggested (i) the occurrence of positive selection and (ii) the presence of one or more positively selected codons. These results were obtained using three data sets of transposase genes from the same IS family that were collected based on the level of amino acid similarity, the presence of an inverted repeat, and the number of sequences in the data sets. Neither recombination nor ribosomal frameshifting, which may interfere with the selection analyses, appeared to be important forces in the transposase gene family. Some positively selected codons were located in a conserved domain, suggesting that these residues are functionally important. The finding that this type of selection acts on IS-carried genes is intriguing, because although ISs have been associated with the adaptation of the bacterial host to new environments, this has typically been attributed to transposition or transformation, thus involving different genomic locations. Intragenic adaptation of IS-carried genes identified here may constitute a novel mechanism associated with bacterial diversification and adaptation.     

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.     

Experimental analysis of recently transposed insertion sequences in the cyanobacterium Synechocystis sp. PCC 6803.

The genome DNA of the cyanobacterium Synechocystis sp. PCC 6803 carries a number of insertion sequences (Kaneko, T. et al. 1996, DNA Res., 3, 109-136). We analyzed one of the abundant ISs (ISY203 group of IS4 family) in the common three substrains of Synechocystis and found that the four ISs with identical nucleotide sequences were present only in the "Kazusa" strain, whose complete genome sequence had been determined, while absent in ancestral strains (the original strain from Pasteur Culture Collection and its glucose-tolerant derivative). Three of these ISs were found in the genomic sequence as transposase genes of sll1474, sll1780 and slr1635. The fourth was on the plasmid, pSYSM. On the other hand, all three strains had a novel IS (denoted ISY203x), of which the nucleotide sequence was totally identical to the four ISs found only in the Kazusa strain. Since the flanking regions of ISY203x did not match any part of the genome or of the known plasmids of Synechocystis, it is presumably located on a yet uncharacterized plasmid. These suggest that the four ISs in Kazusa strain were recently transposed from ISY203x. Apparently, the transposition inactivated four preexisting genes, of which modified forms are presented as putative genes (sll1473, sll1475, slr1862, slr1863, slr1635 and ssl2982) in the list of the complete genome (CyanoBase: http://www.kazusa.or.jp/cyano/cyano.html). The possible effects of transposition of ISs in Synechocystis are discussed in relation to phenotypic mutations and microevolution.