IS1397 is active for transposition into the chromosome of Escherichia coli K-12 and inserts specifically into palindromic units of bacterial interspersed mosaic elements

We demonstrate that IS1397, a putative mobile genetic element discovered in natural isolates of Escherichia coli, is active for transposition into the chromosome of E. coli K-12 and inserts specifically into palindromic units, also called repetitive extragenic palindromes, the basic element of bacterial interspersed mosaic elements (BIMEs), which are found in intergenic regions of enterobacteria closely related to E. coli and Salmonella. We could not detect transposition onto a plasmid carrying BIMEs. This unprecedented specificity of insertion into a well-characterized chromosomal intergenic repeated element and its evolutionary implications are discussed.     

Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences

REPs are highly repeated intergenic palindromic sequences often clustered into structures called BIMEs including two individual REPs separated by short linker of variable length. They play a variety of key roles in the cell. REPs also resemble the sub-terminal hairpins of the atypical IS200/605 family of insertion sequences which encode Y1 transposases (TnpA(IS200/IS605)). These belong to the HUH endonuclease family, carry a single catalytic tyrosine (Y) and promote single strand transposition. Recently, a new clade of Y1 transposases (TnpA(REP)) was found associated with REP/BIME in structures called REPtrons. It has been suggested that TnpA(REP) is responsible for REP/BIME proliferation over genomes. We analysed and compared REP distribution and REPtron structure in numerous available E. coli and Shigella strains. Phylogenetic analysis clearly indicated that tnpA(REP) was acquired early in the species radiation and was lost later in some strains. To understand REP/BIME behaviour within the host genome, we also studied E. coli K12 TnpA(REP) activity in vitro and demonstrated that it catalyses cleavage and recombination of BIMEs. While TnpA(REP) shared the same general organization and similar catalytic characteristics with TnpA(IS200/IS605) transposases, it exhibited distinct properties potentially important in the creation of BIME variability and in their amplification. TnpA(REP) may therefore be one of the first examples of transposase domestication in prokaryotes.     

Transposition of IS1397 in the Family Enterobacteriaceae and First Characterization of ISKpn1, a New Insertion Sequence Associated with Klebsiella pneumoniae Palindromic Units

IS1397 and ISKpn1 are IS3 family members which are specifically inserted into the loop of palindromic units (PUs). IS1397 is shown to transpose into PUs with sequences close or identical to the Escherichia coli consensus, even in other enterobacteria (Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Klebsiella oxytoca). Moreover, we show that homologous intergenic regions containing PUs constitute IS1397 transpositional hot spots, despite bacterial interspersed mosaic element structures that differ among the three species. ISKpn1, described here for the first time, is specific for PUs from K. pneumoniae, in which we discovered it. A sequence comparison between the two insertion sequences allowed us to define a motif possibly accounting for their specificity.     

Palindromic unit-independent transposition of IS1397 in Yersinia pestis

Palindromic units (PUs) are intergenic repeated sequences scattered over the chromosomes of Escherichia coli and several other enterobacteria. In the latter, IS1397, an E. coli insertion sequence specific to PUs, transposes into PUs with sequences close to the E. coli consensus. Reasons for this insertion specificity can relate to either a direct recognition of the target (by its sequence or its structure) by the transposase or an interaction between a specific host protein and the PU target DNA sequence. In this study, we show that for Yersinia pestis, a species deprived of PUs, IS1397 can transpose onto its chromosome, with transpositional hot spots. Our results are in favor of a direct recognition of target DNA by IS1397 transposase.     

Les BIMEs: un exemple de séquences répétées chez les entérobactéries

Bacterial interspersed mosaic elements (or BIMEs) are repeated sequences identified in several enterobacterial genomes. BIMEs are a mosaic combination of small sequence motifs. It has been estimated that 500 BIMEs are scattered over the bacterial genome. BIMEs have been identified in several enterobacteria: Escherichia coli, Salmonella typhimurium, Klebsiella sp. and relatives of these bacteria. BIME function is not known, but their interactions with specific proteins (DNA polymerase I, gyrase and integration host factor) suggest that they could be involved in functional organization of bacterial chromosomes. Four other families of interspersed repetitive sequences have been shown to exist in a variety of bacterial genomes. Like BIMEs, these sequences are rather small, contain a region of dyad symmetry, and are found in extragenic locations. Unlike BIMEs, IRU (or ERIC), box C sequences and RSA sequences occur in enterobacteria but also in other Gram-negative bacteria.     

In vivo cleavage of Escherichia coli BIME-2 repeats by DNA gyrase: genetic characterization of the target and identification of the cut site

The Escherichia coli chromosome contains about 300 bacterial interspersed mosaic elements (BIMEs). These elements, located at the 3' end of genes, are composed of three types of alternating repetitive extragenic palindromes (REPs). Based on the type of REP they contain and on their ability to interact with the integration host factor (IHF), BIMEs are subdivided into two families: BIME-1 elements contain an IHF binding site flanked by converging Y and Z1 REPs, whereas BIME-2 elements contain a variable number of alternating Y and Z2 REPs without an IHF site. Although some BIMEs have been implicated in the protection of mRNA against 3' exonucleolytic degradation, the main role of elements belonging to both families remains to be elucidated. In this paper, we used oxolinic acid, a drug that reveals potential sites of DNA gyrase action, to demonstrate that DNA gyrase interacts in vivo with BIME-2 elements. The frequency of cleavage varied from one element to another, and the cleavage pattern observed in elements containing several REPs indicated that DNA gyrase cut DNA every two REPs. A single cleavage site has been identified in the Y REP in six out of seven instances, and the nucleotide sequence of a 44 bp fragment containing the scission point displayed conserved residues at six positions. The lack of one of the conserved residues accounted for the absence of cleavage in most of the Z2 REPs. Our results also showed that cleaved REPs were always associated with another REP, suggesting that a pair of diverging REPs constitutes the target of DNA gyrase. DNA gyrase cleavage at repetitive BIME-2 elements may have consequences for DNA topology and genomic rearrangements.     

Structural and functional diversity among bacterial interspersed mosaic elements (BIMEs)

Palindromic units (PU or REP) were defined as 40-nucleotide DNA sequences which are highly repeated in the genome of several members of the Enterobacteriaceae. They were shown to be a constituent of the bacterial interspersed mosaic element (BIME), in which they are associated with other repetitive sequences. We report here that Escherichia coli PU sequences contain three motifs (Y, Z¹ and Z²), leading to the definition of two BIME families. The BIME-1 family, highly conserved over 145 nucleotides, contains two PUs (motifs Y and Z¹). The BIME-2 family contains a variable number of PUs (motifs Y and Z²). We present evidence, using band shift experiments, that each PU motif binds DNA gyrase with a different affinity. This suggests that the two families are functionally distinct.     

Natural Populations of Escherichia Coli and Salmonella Typhimurium Harbor the Same Classes of Insertion Sequences

Despite their close phylogenetic relationship, Escherichia coli and Salmonella typhimurium were long considered as having distinct classes of transposable elements maintained by either host-related factors or very restricted gene exchange. In this study, genetically diverse collections of E. coli and S. typhimurium (subgroup I) were surveyed for the presence of several classes of insertion sequences by Southern blot analysis and the polymerase chain reaction. A majority of salmonellae contained IS1 or IS3, elements originally recovered from E. coli, while IS200, a Salmonella-specific element, was present in about 20% of the tested strains of E. coli. Based on restriction mapping, the extent of sequence divergence between copies of IS200 from E. coli and S. typhimurium is on the order of that observed in comparisons of chromosomally encoded genes from these taxa. This suggests that copies of IS200 have not been recently transferred between E. coli and S. typhimurium and that the element was present in the common ancestor to both species. IS200 is polymorphic within E. coli but homogeneous among isolates of S. typhimurium, providing evidence that these species might differ in their rates of transfer and turnover of insertion sequences.     

IS186 Insertion at a Hot Spot in the lon Promoter as a Basis for Lon Protease Deficiency of Escherichia coli B: Identification of a Consensus Target Sequence for IS186 Transposition

The radiation sensitivity of Escherichia coli B was first described more than 50 years ago, and the genetic locus responsible for the trait was subsequently identified as lon (encoding Lon protease). We now show that both E. coli B and the first reported E. coli K-12 lon mutant, AB1899, carry IS186 insertions in opposite orientations at a single site in the lon promoter region and that this site represents a natural hot spot for transposition of the insertion sequence (IS) element. Our analysis of deposited sequence data for a number of other IS186 insertion sites permitted the deductions that (i) the consensus target site sequence for IS186 transposition is 5'-(G)(> or =4)(N)(3-6)(C)(> or =4)-3', (ii) the associated host sequence duplication varies within the range of 6 to 12 bp and encompasses the N(3-6) sequence, and (iii) in a majority of instances, at least one end of the duplication is at the G-N (or N-C) junction. IS186-related sequences were absent in closely related bacterium Salmonella enterica serovar Typhimurium, indicating that this IS element is a recent acquisition in the evolutionary history of E. coli.     

Palindromic units are part of a new bacterial interspersed mosaic element (BIME)

Palindromic Units (PU or REP) were defined as DNA sequences of 40 nucleotides highly repeated on the genome of Escherichia coli and other Enterobacteriaceae. PU are found in clusters of up to six occurrences always localized in extragenic regions. By sorting the DNA sequences of the known PU containing regions into different classes, we show here for the first time that, besides the PU themselves, each PU clusters contains a number of other conserved sequence motifs. Seven such motifs were identified with the present list of PU regions. Remarkably, each PU cluster is exclusively composed of a mosaic combination of PU and of these other sequence motifs. We demonstrate directly by hybridization experiments that one of these motifs (called L) is indeed present at a large number of copies on the Escherichia coli chromosome and that its distribution follows the same species specificity as PU sequences themselves. We propose that the mosaic pattern of motif combination in PU clusters reveals a new type of bacterial genetic element which we propose to call BIME for Bacterial Interspersed Mosaic Element. The Escherichia coli genome contains about 500 BIME.     

Is103, a New Insertion Element in Escherichia Coli: Characterization and Distribution in Natural Populations

IS103 is a previously unknown insertion sequence found in Escherichia coli K12. We have sequenced IS103 and find that it is a 1441-bp element that consists of a 1395-bp core flanked by imperfect 23-bp inverted repeats. IS103 causes a 6-bp duplication of the target sequence into which it inserts. There is a single copy of IS103 present in wild-type E. coli K12 strain HfrC. In strain X342 and its descendents there are two additional copies, one of which is located within the bglF gene. IS103 is capable of excising from within bglF and restoring function of that gene. IS103 exhibits 44% sequence identity with IS3, suggesting that the two insertion sequences are probably derived from a common ancestor. We have examined the distribution of IS103 in the chromosomes and plasmids of the ECOR collection of natural isolates of E. coli. IS103 is found in 36 of the 71 strains examined, and it strongly tends to inhabit plasmids rather than chromosomes. Comparison of the observed distribution of IS103 with distributions predicted by nine different models for the regulation of transposition according to copy number and of the effects of copy number on fitness suggest that transposition of IS103 is strongly regulated and that it has only minor effects on fitness. The strong clustering of IS103 within one phylogenetic subgroup of the E. coli population despite its presence on plasmids suggests that plasmids tend to remain within closely related strains and that transfer to distantly related strains is inhibited.     

Identification and sequence of a tet(M) tetracycline resistance determinant homologue in clinical isolates of Escherichia coli

The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host.     

Evidence for convergent evolution of CTX-M-14 ESBL in Escherichia coli and its prevalence

A total of 2440 Escherichia coli strains isolated in 2003 at the Hospital de la Santa Creu i Sant Pau were evaluated for the presence of extended-spectrum beta-lactamases. Two different nucleotide sequences that encode the same beta-lactamase, CTX-M-14, were detected when the bla(CTX-M-14)-genes of 35 E. coli isolates were analysed. Thirty-two of the 35 had the previously described sequence of the bla(CTX-M-14) (AF252622), named bla(CTX-M-14a), and the remaining three isolates showed a nucleotide sequence identical to that of the bla(CTX-M-9) gene except for one nucleotide, named bla(CTX-M-14b). Characterisation of the regions surrounding the bla(CTX-M-14a) showed the ISEcp1 and the IS903 upstream and downstream, respectively, of the bla gene, whereas the regions surrounding the bla(CTX-M-14b) contained the genetic environment described for the bla(CTX-M-9) gene, the In60. Characterisation by hybridisation showed that the bla(CTX-M-14a) was present in IncK plasmids, whereas the bla(CTX-M-14b) was found in the HI2 Inc group. The CTX-M-14 ESBL in E. coli isolates is the result of the convergence of two different genes.     

Promiscuous origin of a chimeric sequence in the Escherichia coli O157:H7 genome

A novel sequence of 2.9 kb in the intergenic region between the mutS and rpoS genes of Escherichia coli O157:H7 and closely related strains replaces a sequence of 6.1 kb in E. coli K-12 strains. At the same locus in Shigella dysenteriae type 1, a sequence identical to that in O157:H7 is bounded by the IS1 insertion sequence element. Extensive polymorphism in the mutS-rpoS chromosomal region is indicative of horizontal transfer events.     

Size and Sequence Polymorphism in the Isocitrate Dehydrogenase Kinase/Phosphatase Gene (Acek) and Flanking Regions in Salmonella Enterica and Escherichia Coli

The sequence of aceK, which codes for the regulatory catalytic enzyme isocitrate dehydrogenase kinase/phosphatase (IDH K/P), and sequences of the 5' flanking region and part or all of the 3' flanking region were determined for 32 strains of Salmonella enterica and Escherichia coli. In E. coli, the aceK gene was 1734 bp long in 13 strains, but in three strains it was 12 bp shorter and the stop codon was TAA rather than TGA. Strains with the shorter aceK lacked an open reading frame (f728) downstream between aceK and iclR that was present, in variable length, in the other strains. Among the 72 ECOR strains, the truncated aceK gene was present in all isolates of the B2 group and half of those of the D group. Other variant conditions included the presence of IS1 elements in two strains and large deletions in two strains. The aceK-aceA intergenic region varied in length from 48 to 280 bp in E. coli, depending largely on the number of repetitive extragenic palindromic (REP) sequences present. Among the ECOR strains, the number of REP elements showed a high degree of phylogenetic association, and sequencing of the region in the ECOR strains permitted partial reconstruction of its evolutionary history. In S. enterica, the normal length of aceK was 1752 bp, but three other length variants, ranging from 1746 to 1785 bp, were represented in five of the 16 strains examined. The flanking intergenic regions showed relatively minor variation in length and sequence. The occurrence of several nonrandom patterns of distribution of polymorphic synonymous nucleotide sites indicated that intragenic recombination of horizontally exchanged DNA has contributed to the generation of allelic diversity at the aceK locus in both species.     

Genetic environments of bla(CTX-M) genes located on conjugative plasmids of Enterobacteriaceae nosocomial isolates collected in Russia within 2003-2007

The study showed that bla(CTX-M) genes were present in the genomes of 71% of cephalosporin resistant Enterobacteriaceae nosocomial isolates (n=833) collected in Russian hospitals within 2003-2007, including 91% of E.coli, 90% of Klebsiella spp., 38% of Enterobacter spp., 31% of Citrobacter spp. (n=9), and 36% of the other Enterobacteriaceae species. The genes belonging to the following subtypes (clusters) were identified: bla(CTX-M-1) (529 bla(CTX-M-15) genes; 25 bla(CTX-M-3) genes; 1 bla(CTX-M-22) gene, 1 bla(CTX-M-23) gene, and 1 bla(CTX-M-34) gene); bla(CTX-M-2) (1 bla(CTX-M-2) gene, and 4 bla(CTX-M-5) genes), and bla(CTX-M-9) (2 bla(CTX-M-9) genes, and 28 bla(CTX-M-14) genes). It was shown that bla(CTX-M) genes were located on high-molecular weight (60-160 bp) conjugative plasmids belonging mainly to the incompatibility groups IncF, IncL/M and IncA/C (bla(CTX-M-15) gene); IncL/M(bla(CTX-M-3) gene); and IncF, IncL/Mand IncI1-ly (CTX-M-14 gene). The gene environments of bla(CTX-M) genes were shown specific for the subtype of the genes. A mobile genetic element ISEcp1 (in some cases deleted or inserted by IS26, IS1, IS10, resTn2, or resTn3 sequences, in direct or reverse position) were detected upstream of bla(CTX-M-3), bla(CTX-M-14), and bla(CTX-M-15) genes. A special characteristic was the sequence between ISEcp1 and bla(CTX-M) gene: 48 bp for bla(CTX-M-15) (except 1 E.coli isolate having such a sequence deleted by 3 bp); 127 bp for bla(CTX-M-3); 42 bp for bla(CTX-M-14). Downstream of bla(CTX-M) and bla(CTX-M-15) genes in the major bacterial isolates orf477 mucA and Delta orf477-Delta mucA sequences were detected respectively. Two isolates had additional Delta orf3 insertion inside of Delta orf477-Delta mucA sequence. Insertion sequence IS903 (intact or deleted) was detected downstream of bla(CTX-M-14) gene. Unlike the others, bla(CTX-M-2) and bla(CTX-M-9) genes were located inside of ISCR1 mobile element, downstream of class 1 integron and orf513 sequence.     

Bacterial interspersed mosaic elements (BIMEs) are present in the genome of Klebsiella

Bacterial interspersed mosaic elements (BIMEs) constitute a family of highly repetitive sequences containing palindromic units (PUs), also called repetitive extragenic palindromes (REPs). BIMEs were originally described in Escherichia coli and Salmonella typhimurium. We show here, by determining the nucleotide sequence of two intergenic regions of Klebsiella pneumoniae, by computer searches, and by hybridization, that sequences with similar characteristics are found in the genome of several Klebsiella species. This reinforces the idea that BIMEs play general and important roles in enterobacteria such as in the organization of the bacterial chromosome.     

IS150: distribution, nucleotide sequence and phylogenetic relationships of a new E. coli insertion element.

Recently we identified the new insertion (IS) sequence IS150 in various strains of Escherichia coli K-12. We have screened other strains of E. coli and Salmonella typhimurium for the presence of homologous sequences. The strains of E. coli K-12 and W tested contain one or more copies of homology to IS150. We have also determined the complete nucleotide sequence of a copy of IS150 inserted into IS1. Comparison of nucleotide and deduced amino acid sequences of IS150, IS2, IS3, IS51, IS600 and IS629 reveals significant homologies suggesting that these elements are members of a family of phylogenetically related insertion sequences.     

A novel IS-like element frequently inserted in a putative virulence regulator in bovine mastitis isolates of Streptococcus dysgalactiae

Streptococcus dysgalactiae, a Lancefield group C streptococcus, is commonly isolated from bovine mastitis. We recently identified a putative regulon in two S. dysgalactiae strains, 8215 and Epi9, consisting of two consecutive genes, dmg and dem, coding for a possible regulatory protein and an M-like protein with fibrinogen- and IgG-binding-properties, respectively. During these studies a short sequence homologous to an IS element was found to be inserted in the dmg gene of strain 8215. The present investigation describes the complete sequence of this IS-like element, named ISSdy1, which consists of 1218 bp and contains two ORFs, flanked by imperfect repeats. The nucleotide sequence of the IS-like element shows 82% identity to the previously reported sequence of IS199 from Streptococcus mutans V403. The deduced amino acid sequences of the ORFs also revealed high homology to transposases from IS elements in Enterococcus faecium, Escherichia coli, and Shigella dysenteriae, all belonging to the IS3 family. We studied the distribution of ISSdy1 in 57 S. dysgalactiae isolates using PCR analysis with specific primers derived from the IS element. Ninety-eight percent of the isolates contained the ISSdy1 element. Surprisingly, in the majority of studied strains a copy of the IS-like element was found to be inserted in the dmg gene, a putative virulence regulator.     

Sequence and characteristics of IS900, an insertion element identified in a human Crohn''s disease isolate of Mycobacterium paratuberculosis.

The complete sequence of an insertion element IS900 in Mycobacterium paratuberculosis is reported. This is the first characterised example of a mycobacterial insertion element. IS900 consists of 1451bp of which 66% is G + C. It lacks terminal inverted and direct repeats, characteristic of Escherichia coli insertion elements but shows a degree of target sequence specificity. A single open reading frame (ORF 1197) coding for 399 amino acids is predicted. This amino acid sequence, and to a lesser extent the nucleotide sequence, show significant homologies to IS110, an insertion element of Streptomyces coelicolor A3(2). It is proposed that IS900, IS110, and similar insertion elements recently identified in disease isolates of Mycobacterium avium are members of a phylogenetically related family. IS900 will provide highly specific markers for the precise identification of Mycobacterium paratuberculosis, useful in defining its relationship to animal and human diseases.