Gene organization and target specificity of the prokaryotic mobile genetic element IS26

Summary The 820-bp mobile genetic element IS26 loses its ability to promote transpositional cointegration (1) by short deletions near the middle of the element causing shifts in both reading frames ORFI (left to right) and ORFII (right to left) and (2) by deletions causing substitutions of the C-terminus of ORFI but not affecting ORFII. The 702-bp ORFI is thus likely to code for the IS26 transposase. An 82-bp long sequence from the left end of IS26 contains a promoter-like structure in front of the start of ORFI at coordinate 64. In appropriately constructed plasmids, this sequence promotes the expression of the galK structural gene. The observation provides additional evidence for the functional relevance of ORFI. Neither the presence nor the absence of an intact IS26 element on the same plasmid affects measurably the degree of the galK gene expression by the IS26 promoter. Sequence comparison of 14 independent integration sites of IS26 and its relatives reveals no striking rules for target selection by the element, and the distrubtion of integration sites of IS26 on small multicopy plasmids is nearly random and independent of the local AT-content.     

Nucleotide sequence of IS26, a new prokaryotic mobile genetic element.

The DNA sequence of a new IS element, the IS26, is 820 bp long and carries 14 bp perfect terminal inverted repeats. Upon integration, IS26 generates an 8 bp duplication of its target sequence. A large open reading frame within IS26 could code for a protein of 234 amino acids. On its reverse strand, IS26 also carries one large open reading frame, 591 bp long, which contains no stop codon within IS26.     

Transposition and targeting of the prokaryotic mobile element IS30 in zebrafish

We provide evidence that a prokaryotic insertion sequence (IS) element is active in a vertebrate system. The transposase of Escherichia coli element IS30 catalyzes both excision and integration in extrachromosomal DNA in zebrafish embryos. The transposase has a pronounced target preference, which is shown to be modified by fusing the enzyme to unrelated DNA binding proteins. Joining the transposase to the cI repressor of phage λ causes transposition primarily into the vicinity of the λ operator in E. coli, and linking to the DNA binding domain of Gli1 also directs the recombination activity of transposase near to the Gli1 binding site in zebrafish. Our results demonstrate the possibility of fusion transposases to acquire novel target specificity in both prokaryotes and eukaryotes.     

A transcriptional terminator sequence in the prokaryotic transposable element IS1

The prokaryotic transposable element IS1 is known to exert a strong polar effect upon integration into an operon. To elucidate this polar effect, we constructed a plasmid which has an IS1 integrated between the 5' half of the tet gene for tetracycline resistance and the cat structural gene for chloramphenicol resistance. The cat gene is expressed by the tet promoter and the presence of IS1 in orientation I, in which the IS1 transposase genes insA and insB are in the same orientation as the cat gene, reduced the cat expression. By introducing deletions or insertions within the IS1 sequence, we were able to map a rho-dependent terminator TIS1A between the insA and insB genes. Translational interruption between these ins genes is important for TIS1A to be an active terminator.     

Functional characterization of a prokaryotic Kir channel

The Kir gene family encodes inward rectifying K+ (Kir) channels that are widespread and critical regulators of excitability in eukaryotic cells. A related gene family (KirBac) has recently been identified in prokaryotes. While a crystal structure of one member, Kir-Bac1.1, has been solved, there has been no functional characterization of any KirBac gene products. Here we present functional characterization of KirBac1.1 reconstituted in liposomes. Utilizing a 86Rb+ uptake assay, we demonstrate that KirBac1.1 generates a K+ -selective permeation path that is inhibited by extraliposomal Ba2+ and Ca2+ ions. In contrast to KcsA (an acid-activated bacterial potassium channel), KirBac1.1 is inhibited by extraliposomal acid (pKa approximately 6). This characterization of KirBac1.1 activity now paves the way for further correlation of structure and function in this model Kir channel.     

A Mycoplasma genetic element resembling prokaryotic insertion sequences

Nucleotide sequence analysis of two Mycoplasma hyopneumoniae-derived copies of a repetitive genetic element revealed structural similarities to typical prokaryotic insertion sequences. This is the first such sequence identified in the class Mollicutes. The element spans approximately 1550bp, with 28bp inverted terminal repeats. Two open reading frames occur within the sequence, one potentially encoding a protein with a size-variant alpha-helical domain containing heptameric leucine periodicity. Hybridization data with several strains from each of two mycoplasma species showed that the repetitive sequence is variably distributed within the M. hyopneumoniae and Mycoplasma hyorhinis chromosomes and indicated that in some cases the repeated sequence is contained within a larger genetic element which may be the result of phage or plasmid insertion.     

IS431, a staphylococcal insertion sequence-like element related to IS26 from Proteus vulgaris

We present the nucleotide sequence of IS431, a new staphylococcal insertion sequence-like element flanking the mercury-resistance determinant of pI524 and associated with the methicillin-resistance determinant. IS431 left is 800 bp long and has a perfect terminal inverted repeat (IR) of 22 bp; IS431 right is 786 bp long and has a terminal IR homologous to the IR of IS431 left except that the terminal 8 bp are absent. Both IRs share a 10-bp homology with the IR of IS26 from Proteus vulgaris. No directly repeated sequences were detected immediately adjacent to the IRs. An open reading frame (ORF) of 675 bp spans most of the IS431 sequence. Its deduced amino acid (aa) sequence shows 40% homology to the 234-aa-long putative transposase coded by ORFI of IS26.     

Genome-wide analysis of mobile genetic element insertion sites

Mobile genetic elements (MGEs) account for a significant fraction of eukaryotic genomes and are implicated in altered gene expression and disease. We present an efficient computational protocol for MGE insertion site analysis. ELAN, the suite of tools described here uses standard techniques to identify different MGEs and their distribution on the genome. One component, DNASCANNER analyses known insertion sites of MGEs for the presence of signals that are based on a combination of local physical and chemical properties. ISF (insertion site finder) is a machine-learning tool that incorporates information derived from DNASCANNER. ISF permits classification of a given DNA sequence as a potential insertion site or not, using a support vector machine. We have studied the genomes of Homo sapiens, Mus musculus, Drosophila melanogaster and Entamoeba histolytica via a protocol whereby DNASCANNER is used to identify a common set of statistically important signals flanking the insertion sites in the various genomes. These are used in ISF for insertion site prediction, and the current accuracy of the tool is over 65%. We find similar signals at gene boundaries and splice sites. Together, these data are suggestive of a common insertion mechanism that operates in a variety of eukaryotes.     

The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts

The roles of restriction-modification (R-M) systems in providing immunity against horizontal gene transfer (HGT) and in stabilizing mobile genetic elements (MGEs) have been much debated. However, few studies have precisely addressed the distribution of these systems in light of HGT, its mechanisms and its vectors. We analyzed the distribution of R-M systems in 2261 prokaryote genomes and found their frequency to be strongly dependent on the presence of MGEs, CRISPR-Cas systems, integrons and natural transformation. Yet R-M systems are rare in plasmids, in prophages and nearly absent from other phages. Their abundance depends on genome size for small genomes where it relates with HGT but saturates at two occurrences per genome. Chromosomal R-M systems might evolve under cycles of purifying and relaxed selection, where sequence conservation depends on the biochemical activity and complexity of the system and total gene loss is frequent. Surprisingly, analysis of 43 pan-genomes suggests that solitary R-M genes rarely arise from the degradation of R-M systems. Solitary genes are transferred by large MGEs, whereas complete systems are more frequently transferred autonomously or in small MGEs. Our results suggest means of testing the roles for R-M systems and their associations with MGEs.     

Landscape of mobile genetic elements and their antibiotic resistance cargo in prokaryotic genomes

Prokaryotic Mobile Genetic Elements (MGEs) such as transposons, integrons, phages and plasmids, play important roles in prokaryotic evolution and in the dispersal of cargo functions like antibiotic resistance. However, each of these MGE types is usually annotated and analysed individually, hampering a global understanding of phylogenetic and environmental patterns of MGE dispersal. We thus developed a computational framework that captures diverse MGE types, their cargos and MGE-mediated horizontal transfer events, using recombinases as ubiquitous MGE marker genes and pangenome information for MGE boundary estimation. Applied to ∼84k genomes with habitat annotation, we mapped 2.8 million MGE-specific recombinases to six operational MGE types, which together contain on average 13% of all the genes in a genome. Transposable elements (TEs) dominated across all taxa (∼1.7 million occurrences), outnumbering phages and phage-like elements (<0.4 million). We recorded numerous MGE-mediated horizontal transfer events across diverse phyla and habitats involving all MGE types, disentangled and quantified the extent of hitchhiking of TEs (17%) and integrons (63%) with other MGE categories, and established TEs as dominant carriers of antibiotic resistance genes. We integrated all these findings into a resource (proMGE.embl.de), which should facilitate future studies on the large mobile part of genomes and its horizontal dispersal.     

Sequence analysis of tyrosine recombinases allows annotation of mobile genetic elements in prokaryotic genomes

Mobile genetic elements (MGEs) sequester and mobilize antibiotic resistance genes across bacterial genomes. Efficient and reliable identification of such elements is necessary to follow resistance spreading. However, automated tools for MGE identification are missing. Tyrosine recombinase (YR) proteins drive MGE mobilization and could provide markers for MGE detection, but they constitute a diverse family also involved in housekeeping functions. Here, we conducted a comprehensive survey of YRs from bacterial, archaeal, and phage genomes and developed a sequence‐based classification system that dissects the characteristics of MGE‐borne YRs. We revealed that MGE‐related YRs evolved from non‐mobile YRs by acquisition of a regulatory arm‐binding domain that is essential for their mobility function. Based on these results, we further identified numerous unknown MGEs. This work provides a resource for comparative analysis and functional annotation of YRs and aids the development of computational tools for MGE annotation. Additionally, we reveal how YRs adapted to drive gene transfer across species and provide a tool to better characterize antibiotic resistance dissemination.     

Characterization of IS999, an unstable genetic element in Mycobacterium avium

An IS3-family insertion element, IS999, was identified in the opportunistic pathogen Mycobacterium avium. The 1347 bp element has 29 bp inverted repeats and two overlapping open reading frames coding for putative transposases. It was detected in the genomes of ten of 12 M. avium isolates examined. Copy numbers ranged from four to 16. IS999 is less stable than IS1245, the most commonly-used marker for typing M. avium isolates. Among 60 colonies picked from a single patient isolate, there were two distinct IS1245 restriction fragment length polymorphism banding patterns compared to eight distinct IS999 patterns (five in one IS1245 group and three in the other). In view of its instability, we asked whether transposition of IS999 might have phenotypic consequences. Nucleotide sequence analysis of insertion sites in four isolates revealed 16 putative structural genes that were variably disrupted by IS999. Insertions into hdhA, a gene that codes for a putative short chain alcohol dehydrogenase, were distributed non-randomly between colony type variants, consistent with phenotypic consequences that exert selective pressure. These observations illustrate the genetic heterogeneity that can exist within populations of M. avium that appear to be homogeneous by IS1245 analysis. IS999 may be a useful marker for tracking, at the sub-strain level, the rapid genetic drift that M. avium isolates undergo in nature and in the laboratory.     

A new mobile genetic element inLactobacillus delbrueckii subsp.bulgaricus

A new IS element (ISL3) was discovered inLactobacillus delbrueckii subsp.bulgaricus during the characterization of the linkage relationships between the two genes important for milk fermentation,β-galactosidase (lacZ) and the cell-wall associated protease (prtP). ISL3 is a 1494 by element, flanked by 38 by imperfect inverted repeats, and generates an 8 by target duplication upon insertion. It contains one open reading frame, encoding a potential polypeptide of 434 amino acids, which shows significant homology (34% identity) to the transposase of theLeuconostoc mesenteroides element IS1165. Molecular analysis of spontaneouslacZ mutants revealed some strains that had sustained deletions of 7 to 30 kb in size, centered on and eliminating the copy of ISL3 next tolacZ. Other deletion endpoints were identified as located immediately adjacent to ISL3. Furthermore, genetic translocations that had occurred via transposition of ISL3 were observed fortuitously in cultures screened for deletion mutants. ISL3 can be found in one to several copies in various strains ofL. delbrueckii. However, it was not present in other dairy lactic acid bacteria tested.     

Detecting motifs and patterns at mobile genetic element insertion site

Mobile genetic elements (MGEs) occupy major proportion of eukaryotic genomes and are present in significant numbers in prokaryote genomes also. Here we report a new method which extracts a motif at the site of insertion of MGE using tools such as DNA SCANNER. The flanking region of the insertion site is extracted and is analyzed in DNA Scanner for physiochemical properties like protein-interaction measures, energy profiles as well as structural parameters. In case significant signals are observed, the most frequently occurring n-mer (5E. histolytica, signals for EhSine1 are found at around 5 bps upstream of insertion and most frequently occurring motif is found to be AAGGT and TCGAA. Signals for Ty3 element in S. cerevisiae are found at 0-3 bps upstream of tRNA, and most frequent motif is GTTCGA (6 bps), GGTTCGA (7 bps) and GGTTCGAT (8 bps). P-element of Drosophila showed remarkable dyad peaks suggesting palindromic site of insertion.     

Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

Background

SXT is an integrating conjugative element (ICE) originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo) and single strand annealing protein (S065, SXT-Bet) encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively.

Results

SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA) molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn²⁺ or Mg²⁺ ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb). When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo.

Conclusions

The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V. cholerae cells, through facilitating homologous DNA recombination events. The results presented here significantly extend our general understanding of the properties and activities of alkaline exonuclease and single strand annealing proteins of viral/bacteriophage origin, and will assist the rational development of bacterial recombineering systems.