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.     

Characterization of an insertion sequence (IS891) of novel structure from the cyanobacterium Anabaena sp. strain M-131.

When recombinant plasmids that were transferred to the cyanobacterium Anabaena sp. strain M-131 were transferred back to Escherichia coli, some of the transformants contained inserts. One of the insertion sequences (ISs) was characterized by sequencing. This 1,351-base-pair IS contained an open reading frame that was capable of encoding a peptide of 310 amino acids and had terminal sequences with distinctive structures, but it lacked terminal inverted repeats and did not duplicate target DNA upon insertion. The element bore no significant sequence homology to any sequence stored in the GenBank data base. Restriction analysis of the genomes of Anabaena sp. strain M-131 and Anabaena sp. strain PCC 7120 showed those strains to be closely related. Sequences homologous to the IS element were also present in the DNA of Anabaena strain PCC 7120, but the copy numbers and chromosomal locations of such sequences differed in the two strains. The largest visualized plasmid was 425 kilobases (kb) in M-131 and 410 kb in PCC 7120; at least the former plasmid contained multiple copies of the element, as did a 115-kb plasmid in M-131.     

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.     

Characterization of IS476 and its role in bacterial spot disease of tomato and pepper.

IS476 is an endogenous insertion sequence present in copper-tolerant strains of Xanthomonas campestris pv. vesicatoria. Sequence analysis has revealed that the element is 1,225 base pairs in length, has 26-base-pair inverted repeats, and causes a 4-base-pair target site duplication upon insertion into the avirulence gene avrBs1. Comparison of the full-length sequence with sequences in the National Biomedical Research Foundation and National Institutes of Health data bases showed that one of the predicted IS476 proteins is partially homologous to the putative transposase of IS3 from Escherichia coli, and the inverted repeats of IS476 have significant homology to the inverted repeats of the IS51 insertion sequence of Pseudomonas syringae pv. savastanoi. A transposition assay based on the insertional inactivation of the sacRB locus of Bacillus subtilis was used to demonstrate that one of the three copies of IS476 residing on the 200-kilobase copper plasmid pXVCU1 is capable of transposition in several strains of Xanthomonas campestris. The position of IS476 insertion in several avrBs1 mutants was established and was shown to influence both induction of hypersensitivity and bacterial growth in planta.     

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.     

Nucleotide sequence of Rhizobium meliloti insertion sequence ISRm1: homology to IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens.

Nucleotide sequencing of Rhizobium meliloti insertion sequence ISRm1 showed that it is 1319 nucleotides long and includes 32/31 nucleotide terminal inverted repeats. Analysis of five different insertion sites using sequencing primers complementary to sequences within the left and right ends demonstrated that ISRm1 generates five bp direct repeats at the sites of insertion. Although ISRm1 has shown a target preference for certain short regions (hot spots), there was no apparent similarity in the DNA sequences near the insertion sites. On one strand ISRm1 contains two contiguous open reading frames (ORFs) spanning most of its length. ISRm1 was found to have over 50% sequence homology to insertion sequences IS2 from Escherichia coli and IS426 from Agrobacterium tumefaciens. Their sizes, the sequences of their inverted repeats, and the characteristics of their insertion sites are also comparable, indicating that ISRm1, IS2 and IS426 belong to a class of related insertion sequences. Comparison of the proteins potentially encoded by these insertion sequences showed that the two ORFs found in ISRm1 are also present in IS2 and IS426, suggesting that they may be functional genes.     

Intense transpositional activity of insertion sequences in an ancient obligate endosymbiont

The streamlined genomes of ancient obligate endosymbionts generally lack transposable elements, such as insertion sequences (IS). Yet, the genome of Wolbachia, one of the most abundant bacterial endosymbionts on Earth, is littered with IS. Such a paradox raises the question as to why there are so many ISs in the genome of this ancient endosymbiont. To address this question, we investigated IS transpositional activity in the unculturable Wolbachia by tracking the evolutionary dynamics and history of ISWpi1 elements. We show that 1) ISWpi1 is widespread in Wolbachia, being present in at least 55% of the 40 sampled strains, 2) ISWpi1 copies exhibit virtually identical nucleotide sequences both within and among Wolbachia genomes and possess an intact transposase gene, 3) individual ISWpi1 copies are differentially inserted among Wolbachia genomes, and 4) ISWpi1 occurs at variable copy numbers among Wolbachia genomes. Collectively, our results provide compelling evidence for intense ISWpi1 transpositional activity and frequent ISWpi1 horizontal transmission among strains during recent Wolbachia evolution. Thus, the genomes of ancient obligate endosymbionts can carry high loads of functional and transpositionally active transposable elements. Our results also indicate that Wolbachia genomes have experienced multiple and temporally distinct ISWpi1 invasions during their evolutionary history. Such recurrent exposition to new IS invasions may explain, at least partly, the unusually high density of transposable elements found in the genomes of Wolbachia endosymbionts.     

插入序列共同区元件：细菌中新出现的一种基因捕获系统

摘要：插入序列共同区（Insertion sequence common region,ISCR）元件是一类在结构和功能上与IS91家族相似的特殊插入序列,特点是缺少了末端反向重复序列（Inverted repeats, IRs）,在插入位点不产生直接重复序列,并通过滚环式（Rolling circle, RC）进行转座。ISCR元件作为一种新的基因捕获系统,它可以移动邻近的任何DNA序列,为耐药基因在不同种属细菌间水平传播提供了高效的媒介。世界各地多种革兰氏阴性病原菌中已发现有19种ISCR元件,大部分ISCR元件同时携带了多种耐药基因,提示ISCR有可能会造成细菌多重耐药性的快速传播。本文就ISCR结构特征、类型、移动方式、起源及进化的研究进展进行了综述。     

A family of IS1031 elements in the genome of Acetobacter xylinum: nucleotide sequences and strain distribution

An insertion sequence (here called IS 1031A) from Acetobacter xylinum ATCC 23769 has recently been isolated. This study describes the complete nucleotide sequence of IS 1031A as well as the sequences of two novel iso-IS 1031 elements, IS1031C and IS1031D, from A. xylinum ATCC 23769. The three ISs are all exactly 930 bp long, have imperfect terminal inverted repeats of 24 bp for IS1031A and 21 bp for IS1031C and IS1031D, are flanked by three base pair direct repeats, and contain an open reading frame encoding a putative basic protein of 278 amino acids. Because of nucleotide substitutions, IS1031C and IS1031D differ from IS 1031A by 12.9% while IS1031C differs from IS1031D by only 0.6%. Hybridization analyses of total DNA from nine A. xylinum strains showed that all strains contained IS 1031-like elements varying in copy number from three to at least 16. None of three Acetobacter aceti strains examined contained IS1031-like elements. Taken together, the results suggest that A. xylinum contains a family of IS 1031 elements with considerably diversified nucleotide sequences.     

The inverted repeats of IS<Emphasis Type="Italic">1384</Emphasis>, a newly described insertion sequence from<Emphasis Type="Italic"> Pseudomonas putida</Emphasis> strain H,represent the specific target for integration of IS<Emphasis Type="Italic">1383</Emphasis>

Analysis of a region on plasmid pPGH1 from Pseudomonas putida strain H that is flanked by two copies of IS1383 has revealed an additional element with the typical features of a bacterial insertion sequence. This new IS element, designated IS1384, contains a single ORF of 972 bp, and is flanked by 9-bp inverted repeats. Based on sequence homology and structural characteristics of the putative transposase it encodes, IS1384 belongs to the IS5 subgroup of the IS5 family. Two copies of IS1384 are present on plasmid pPGH1, whereas none could be detected on the chromosome of P. putida strain H. Sequence analysis revealed the presence of two truncated copies of IS1384 on the second plasmid in this strain, pPGH2. The inverted repeats of all IS1384 copies (including the truncated ones) are interrupted by the integration of an IS1383 element. All integrations were found to be site- and orientation-specific. PCR studies and sequence data indicate that IS1383 can form a circular intermediate on excision. In the circular form, the previously described 13-bp inverted repeats of IS1383 are separated by 10 bp that are identical to the 5-bp motif that flanks each side of the element when it is integrated in its target. We provide evidence that these additional nucleotides, although not of inverted symmetry, represent an essential part of the inverted repeats. Furthermore, the data indicate that IS1383 integrated into the inverted repeats of IS1384 by a site-specific recombination rather than a site-specific insertion event.     

Characterization of two insertion sequences, IS701 and IS702, from the cyanobacterium Calothrix species PCC 7601

We describe the characterization of two insertion elements, IS701 and IS702, isolated from Calothrix species PCC 7601. These insertion elements were cloned from spontaneous pigmentation mutants. Both show the characteristics of typical bacterial insertion sequences, i.e. they present long terminal inverted repeats and they duplicate target DNA upon insertion. These elements share no homology with the only other cyanobacterial insertion sequence described so far, IS891. At least 15 copies of IS701 and 9 copies of IS702 were detected by hybridization experiments in the Calothrix 7601 genome. Their occurrence in several cyanobacterial strains is also reported.     

Nucleotide sequence and characterization of a new insertion element, IS240, from Bacillus thuringiensis israelensis

The nucleotide sequence of two repeated sequences (RS) in opposite orientations flanking the 125-kDa toxin gene of Bacillus thuringiensis israelensis (C. Bourgouin et al., J. Bacteriol. 170, 3575-3583, 1988) is reported in this paper. The analysis of these sequences indicates that these two RS display characteristic features of bacterial insertion sequences (IS) and are therefore referred to as IS240. IS240 B is 865 bp long and has two perfect terminal-inverted repeats of 16 bp; IS240 A is 99% identical to IS240 B. A long open reading frame encoding a polypeptide of 235 amino acids spans almost the entire sequence of both IS240 elements. Both the sequence of the inverted repeats and the putative transposases are homologous to IS26 of Proteus vulgaris, IS15-delta of Salmonella panama, IS431 of Staphylococcus aureus, and ISS1 of Streptococcus lactis.     

Isolation and characterization of IS elements repeated in the bacterial chromosome

Shigella sonnei contains repetitive sequences, including an insertion element IS1, which can be isolated as double-stranded DNA fragments by DNA denaturation and renaturation and by treatment with S1 nuclease. In this paper, we describe a method of cloning the IS1 fragments prepared by the S1 nuclease digestion technique into phage M13mp8 RFI DNA. Several clones contained IS1, usually with a few additional bases. We isolated and characterized five other repetitive sequences using this method. One sequence, 1264 base-pairs in length, had terminal inverted repeats and contained two open reading frames. This sequence, called IS600, showed about 44% sequence homology with IS3 and was repeated more than 20 times in the Sh. sonnei chromosome. Another sequence (named IS629, 1310 base-pairs in length), which was repeated six times, was found also to be related to IS3 and thus IS600. Two other sequences (named IS630 and IS640, 1159 and 1092 base-pairs in length, respectively), which were repeated approximately ten times, had characteristic terminal inverted repeats and contained a large open reading frame coding for a protein. The inverted repeat sequences of IS630 were similar to the sequence at one end of IS200, a Salmonella-specific IS element. The fifth sequence, repeated ten times in Sh. sonnei, had about 98% sequence homology with a portion of IS2. The method described here can be applied to the isolation of IS or iso-IS elements present in any other bacterial chromosome.     

Analysis of separate isolates of Bordetella pertussis repeated DNA sequences

Two independent isolates of a Bordetella pertussis repeated DNA unit were sequenced and shown to be an insertion sequence element with five nucleotide differences between the two copies. The sequences were 1053 bp in length with near-perfect terminal inverted repeats of 28 bp, had three open reading frames, and were each flanked by short direct repeats. The two insertion sequences showed considerable homology to two other B. pertussis repeated DNA sequences reported recently: IS481 and a 530 bp repeated DNA unit. The B. pertussis insertion sequence would appear to comprise a group of closely related sequences differing mainly in flanking direct repeats and the terminal inverted repeats. The two isolates reported here, which were from the adenylate cyclase and agglutinogen 2 regions of the genome, were numbered IS48lvl and IS48lv2 respectively.     

The Insertion Sequences of Anabaena sp. Strain PCC 7120 and Their Effects on Its Open Reading Frames

Anabaena sp. strain PCC 7120, widely studied, has 145 annotated transposase genes that are part of transposable elements called insertion sequences (ISs). To determine the entirety of the ISs, we aligned transposase genes and their flanking regions; identified the ISs'' possible terminal inverted repeats, usually flanked by direct repeats; and compared IS-interrupted sequences with homologous sequences. We thereby determined both ends of 87 ISs bearing 110 transposase genes in eight IS families (http://www-is.biotoul.fr/) and in a cluster of unclassified ISs, and of hitherto unknown miniature inverted-repeat transposable elements. Open reading frames were then identified to which ISs contributed and others—some encoding proteins of predictable function, including protein kinases, and restriction endonucleases—that were interrupted by ISs. Anabaena sp. ISs were often more closely related to exogenous than to other endogenous ISs, suggesting that numerous variant ISs were not degraded within PCC 7120 but transferred from without. This observation leads to the expectation that further sequencing projects will extend this and similar analyses. We also propose an adaptive role for poly(A) sequences in ISs.Insertion sequences (ISs) are transposable elements found in prokaryotic and eukaryotic genomes (17). A fully functional bacterial IS comprises one or more transposase genes, ends that are often inverted repeats (IRs), and, between the transposase genes and the ends, sequences termed linkers (32). Diverse bacterial ISs have been classified, and a searchable database of ISs has been constructed (ISfinder [http://www-is.biotoul.fr/]) (28). Miniature inverted-repeat transposable elements (MITEs) and even smaller mobile elements lack their own transposases and are also found in Anabaena spp. (11, 12, 33).Anabaena sp. strain PCC 7120 (also known as Nostoc sp. [25], here denoted Anabaena sp.) is widely used to study the patterned differentiation of dinitrogen-fixing cells called heterocysts. Transposition of ISs in Anabaena sp. has been documented (1, 7-9). We earlier reported, with few details, three genes that are intercepted by ISs in Anabaena sp. (23). We here describe the approach more extensively, organize the ISs of Anabaena sp., and present our efforts to identify Anabaena sp. open reading frames (ORFs) interrupted or contributed to by ISs.     

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.     

Characterization and distribution of two insertion sequences,IS1191 and iso-IS981, in Streptococcus thermophilus: does intergeneric transfer of insertion sequences occur in lactic acid bacteria co-cultures?

A chromosomal repeated sequence from Streptococcus thermophilus was identified as a new insertion sequence (IS), IS1191. This is the first IS element characterized in this species. This 1313 bp element has 28 bp imperfect terminal inverted repeats and is flanked by short direct repeats of 8bp. The single large open reading frame of IS1191 encodes a 391-amino-acid protein which displays homologies with transposases encoded by IS1201 from Lactobacillus helveticus (44.5% amino-acid sequence identity) and by the other ISs of the IS256 family. One of the copies of IS 1191 is inserted into a truncated iso-IS981 element. The nucleotide sequences of two truncated iso-IS981 s from S. thermophilus and the sequence of IS981 element from Lactococcus lactis share more than 99% identity. The distribution of these insertion sequences in L. lactis and S. thermophilus strains suggests that intergeneric transfers occur during co-cultures used in the manufacture of cheese.