Transposase Family Research Articles

Genome Analysis of a Newly Sequenced B. subtilis SRCM117797 and Multiple Public B. subtilis Genomes Unveils Insights into Strain Diversification and Biased Core Gene Distribution.

The bacterium Bacillus subtilis is a widely used study model and industrial workhorse organism that belongs to the group of gram-positive bacteria. In this study, we report the analysis of a newly sequenced complete genome of B. subtilis strain SRCM117797 along with a comparative genomics of a large collection of B. subtilis strain genomes. B. subtilis strain SRCM117797 has 4,255,638bp long chromosome with 43.4% GC content and high coding sequence association with macromolecules, metabolism, and phage genes. Genomic diversity analysis of 232 B. subtilis strains resulted in the identification of eight clusters and three singletons. Of 147 B. subtilis strains included, 89.12% had strain-specific genes, of which 6.75% encoded strain-specific insertion sequence family transposases. Our analysis showed a potential role of strain-specific insertion sequence family transposases in intra-cellular accumulation of strain-specific genes. Furthermore, the chromosomal layout of the core genes was biased: overrepresented on the upper half (closer to the origin of replication) of the chromosome, which may explain the fast-growing characteristics of B. subtilis. Overall, the study provides a complete genome sequence of B. subtilis strain SRCM117797, show an extensive genomic diversity of B. subtilis strains and insights into strain diversification mechanism and non-random chromosomal layout of core genes.

Phenotypic and molecular characterizations of carbapenem-resistant Acinetobacter baumannii isolates collected within the EURECA study

Multi-drug-resistant Acinetobacter baumannii isolates are key pathogens that contribute to the global burden of antimicrobial resistance. This study aimed to investigate the phenotypic and molecular characteristics of carbapenem-resistant A. baumannii (CRAB) isolates from the EURECA clinical trial. In total, 228 CRAB clinical strains were recovered from 29 sites in 10 European countries participating in the EURECA study between May 2016 and November 2018. All strains were reconfirmed centrally for identification and antimicrobial susceptibility testing, and were then subjected to DNA isolation and whole-genome sequencing (WGS), with analysis performed using BacPipe v.1.2.6. K and O typing was performed using KAPTIVE. Overall, 226 (99.1%) strains were confirmed as CRAB isolates. The minimum inhibitory concentration (MIC90) results of imipenem and meropenem were >16 mg/L. WGS showed that the isolates mainly harboured blaOXA-23 (n=153, 67.7%) or blaOXA-72 (n=70, 30.1%). Four blaOXA-72 isolates from Serbia co-harboured blaNDM-1. An IS5 transposase family element, ISAba31, was found upstream of the blaOXA-72 gene harboured on a small (~10-kb) pSE41030-EUR plasmid. The majority of isolates (n=178, 79.1%) belonged to international clone II. Strains belonging to the same sequence type but isolated in different countries or within the same country could be delineated in different clusters by core-genome multi-locus sequence typing (MLST). Whole-genome/core-genome MLST showed high diversity among the isolates, and the most common sequence type was ST2 (n=153, 67.7%). The EURECA A. baumannii strain collection represents a unique, diverse repository of carbapenem-resistant isolates that adds to the existing knowledge of A. baumannii epidemiology and resistance genes harboured by these strains.

MOLECULAR ANALYSIS OF A PUTATIVE EN/SPM-RELATED TRANSPOSON PROTEIN IN BRASSICA JUNCEA

Open reading frame (ORF) of Putative En/Spm transposon in Brassica potentially helps to understand the relationship between various eukaryotic transposable elements. The current study was initially conducted to isolate and analyze the putative En/Spm-related transposon gene from Brassica juncea. PCR products (750 bp) from B. juncea (accession PI 649105 and PI 271442) were cloned, sequenced, and analyzed. Results of BLAST showed identical sequences between two accessions with 100% similarity. The amplified DNA transposon and conserved domain compared to the GenBank database to evaluate the genetic diversity relationships. Sequence of this putative transposon gene from B. juncae was a 98% similar to B. rapa subsp pekinensis at the nucleotide level, and 94% with the En/Spm-related transposon protein of B. oleracea at the amino acid level. Conserved domain architecture was related to transposase_21_pfam0299 and transposase family tnp2 and had a relationship with space outside the cell structure and/or to space outside the plasma membrane.

Resistome, Mobilome and Virulome Analysis of Shewanella algae and Vibrio spp. Strains Isolated in Italian Aquaculture Centers.

Antimicrobial resistance is a major public health concern restricted not only to healthcare settings but also to veterinary and environmental ones. In this study, we analyzed, by whole genome sequencing (WGS) the resistome, mobilome and virulome of 12 multidrug-resistant (MDR) marine strains belonging to Shewanellaceae and Vibrionaceae families collected at aquaculture centers in Italy. The results evidenced the presence of several resistance mechanisms including enzyme and efflux pump systems conferring resistance to beta-lactams, quinolones, tetracyclines, macrolides, polymyxins, chloramphenicol, fosfomycin, erythromycin, detergents and heavy metals. Mobilome analysis did not find circular elements but class I integrons, integrative and conjugative element (ICE) associated modules, prophages and different insertion sequence (IS) family transposases. These mobile genetic elements (MGEs) are usually present in other aquatic bacteria but also in Enterobacteriaceae suggesting their transferability among autochthonous and allochthonous bacteria of the resilient microbiota. Regarding the presence of virulence factors, hemolytic activity was detected both in the Shewanella algae and in Vibrio spp. strains. To conclude, these data indicate the role as a reservoir of resistance and virulence genes in the environment of the aquatic microbiota present in the examined Italian fish farms that potentially might be transferred to bacteria of medical interest.

How mouse RAG recombinase avoids DNA transposition.

The RAG1-RAG2 recombinase (RAG) cleaves DNA to initiate V(D)J recombination. But RAG also belongs to the RNH-type transposase family. To learn how RAG-catalyzed transposition is inhibited in developing lymphocytes, we determined the structure of a DNA strand-transfer complex of mouse RAG at 3.1 Å resolution. The target DNA is a T form (T for transpositional target), which contains two >80° kinks towards the minor groove, only 3 bp apart. RAG2, a late evolutionary addition in V(D)J recombination, appears to enforce the sharp kinks and additional inter-segment twisting in target DNA and thus attenuate unwanted transposition. In contrast to strand-transfer complexes of genuine transposases, where severe kinks occur at the integration sites of target DNA and thus prevent the reverse reaction, the sharp kink with RAG is 1 bp away from the integration site. As a result, RAG efficiently catalyzes the disintegration reaction that restores the RSS (donor) and target DNA.

First report of Burkholderia pseudomallei ST412 and ST734 clones harbouring blaOXA-57 but susceptible to imipenem in India.

Melioidosis caused by Burkholderia pseudomallei has become an important clinical threat, especially in Northern Australia and Southeast Asia. However, the genome information on this pathogen is limited. B. pseudomallei isolates identified from bloodstream infections from inpatients were subjected to whole-genome sequencing by IonTorrent PGM and MinION Oxford Nanopore sequencing technologies. Highly accurate complete genomes of two strains, VB3253 and VB2514, were obtained by a hybrid genome assembly method using both short and long DNA reads. Both isolates carried blaPenI and carbapenemase-encoding blaOXA-57 genes, although the isolates were susceptible to imipenem by E-test method with MIC 1 μg/mL. Multiple IS family transposases specific for all non-fermenting Gram-negative bacteria (NFGNBs)—especially IS3 and IS5, which facilitate mobilization of extended-spectrum β-lactamase (ESBL) and carbapenemase genes—were carried in these genomes. This further adds to the complexity of gene transmission. These IS families were identified only upon hybrid genome assembly and would otherwise be missed.

Transposition of Insertion Sequences was Triggered by Oxidative Stress in Radiation-Resistant Bacterium Deinococcus geothermalis.

During an oxidative stress-response assay on a putative Dps-like gene-disrupted Δdgeo_0257 mutant strain of radiation-resistant bacterium Deinococcus geothermalis, a non-pigmented colony was observed among the normal reddish color colonies. This non-pigmented mutant cell subsequently displayed higher sensitivity to H2O2. While carotenoid has a role in protecting as scavenger of reactive oxygen species the reddish wild-type strain from radiation and oxidative stresses, it is hypothesized that the carotenoid biosynthesis pathway has been disrupted in the mutant D. geothermalis cell. Here, we show that, in the non-pigmented mutant cell of interest, phytoene desaturase (Dgeo_0524, crtI), a key enzyme in carotenoid biosynthesis, was interrupted by transposition of an ISDge7 family member insertion sequence (IS) element. RNA-Seq analysis between wild-type and Δdgeo_0257 mutant strains revealed that the expression level of ISDge5 family transposases, but not ISDge7 family members, were substantially up-regulated in the Δdgeo_0257 mutant strain. We revealed that the non-pigmented strain resulted from the genomic integration of ISDge7 family member IS elements, which were also highly up-regulated, particularly following oxidative stress. The transposition path for both transposases is a replicative mode. When exposed to oxidative stress in the absence of the putative DNA binding protein Dgeo_0257, a reddish D. geothermalis strain became non-pigmented. This transformation was facilitated by transposition of an ISDge7 family IS element into a gene encoding a key enzyme of carotenoid biosynthesis. Further, we present evidence of additional active transposition by the ISDge5 family IS elements, a gene that was up-regulated during the stationary phase regardless of the presence of oxidative stress.

Molecular Epidemiological Screening of the Genome of the Strain Coxiella burnetii NL3262 (Netherlands, 2009) Using Formal Order Analysis

Background . More than 4,000 cases of acute disease in humans were reported during the mass Q fever outbreak in the Netherlands in 2007–2010. The Coxiella burnetii NL3262 strain was isolated during this outbreak from an aborted placenta of a goat and was studied using means of molecular biology and bioinformation techniques. Goals . Approbation of a new bioinformatics approach – formal order analysis – to study the origin of the strains that caused a massive outbreak of Q fever in the Netherlands using the C. burnetii NL3262 strain. Methodology . New tools of the formal order analysis (FOA) «Map of Genes» and «Matrix of Similarity» (available at http://foarlab. org) were used in this work to study the degree of similarity of the genome (chromosome, plasmid) of this strain with the genomes of other strains of C. burnetii. The nucleotide sequences of the chromosomes of 10 C. burnetii strains and 8 plasmids were loaded from GenBank: www.ncbi.nlm.nih.gov/genome. Results . The map of genes data showed that the chromosome of NL3262 strain significantly distanced from the chromosome of other strains by the characteristic of the average remoteness of nucleotides in the chromosome (g) that ranged from 1.448295 (for Dugway 5J108-111) to 1.448865 1.449640 (for CbRSA331). This may be due to the presence of 106 copies of the «transposase family IS110» gene associated with the growth of virulence, while in the chromosomes of other strains their number ranged only from 1 to 48. The similarity matrix showed that 84.9% of C. burnetii NL3262 chromosome components had complete (100%) homology with chromosome components of strain Z3055. The percentage of similar components ranged from 12.06 to 47.14 for chromosomes of other strains. Plasmids of the pQpH1 type of strains NL3262 and RSA 331 contained 50.0% of components with complete homology. For the same type of plasmids of strain RSA 493 and its clones, the index varied from 28.89 to 29.89%, and for plasmids of other types it was from 5.56 to 6.74%. It is shown that the chromosomes of strains NL3262 and Z3055 have the highest percentage of components with complete homology. However, by the g index, chromosomes of these strains are significantly distanced from each other, due to the large number of copies of IS110 in the chromosome of strain NL3262, which caused the formation of 21 collinear blocks. This led to a change in the properties of the Q fever outbreak strains in the Netherlands and the increase in their epidemiological significance, which caused the largest outbreak in the history of the study of this infection. Conclusions . The results of the study, obtained on the basis of the application of formal order analysis, made it possible to make an assumption about the origin of the strains of Q fever in the Netherlands in 2007–2010. It is shown that the leading reason in the reorganization of the C. burnetii genome is the adaptation of the microorganism to a new ecological niche.

Investigation and Comparison of the Flexibility and Dynamics of Tc1/Mariner Transposases

DNA transposons are responsible for transferring genetic information from one location to another and should provide a stable platform for use in human gene therapy. A multi-step process of DNA transposition is catalyzed by a transposase enzyme that should be sufficiently flexible to provide the catalytic site for each step of the reaction. Here we employ a combination of computational (minimal Distance Constraint Model) and experimental (Circular Dichroism spectroscopy) methods to investigate and compare the dynamics, the inter-residue couplings, and the role of DNA binding on the stability and flexibility of two representative members of Tc1/mariner family of transposases, the Sleeping Beauty (SB) and Mos1 transposases. To understand the link between dynamics and function, we further compare results for the original SB transposase and its hyperactive version SB100, which has a 100-fold increase in efficiency. Together, our data provide insights to how these transposases are structured and how they function.

Mos1 Transposase Thermodynamic Stability and Flexibility

DNA transposons are mobile DNA elements that can move (or transpose) from one DNA molecule to another and thereby deliver genetic information into human chromosomes in order to confer a new function or replace a defective gene. This process is catalyzed by a transposase enzyme. The reaction of transposition occurs in several steps, during which two or more transposase enzymes bind to the terminal inverted repeats on the transposon DNA, bring them together to form a synaptic complex, excise the gene flanked by the terminal inverted repeats, and catalyze strand transfer to insert the excised gene at a new location. Thus, transposases must be sufficiently flexible to allow conformational rearrangements of their domains to bind the transposon DNA and to supply a catalytic site during each step of transposition. Here, we investigate the dynamics, thermodynamic stability, and flexibility of Mos1 transposase, a member of the Tc1/mariner family of transposases. We use a computational model called the minimum Distance Constraint Model (mDCM) and the analysis of quantitative stability/flexibility relationships (QSFR). With these tools, we determine the free energy landscape and the flexibility and mechanical coupling of secondary structure elements or residues in Mos1. Our data provide an insight into how Mos1 is structured and how it functions and are applicable to Tc1/mariner transposases in general.

Heteromeric transposase elements: generators of genomic islands across diverse bacteria.

Horizontally acquired genetic information in bacterial chromosomes accumulates in blocks termed genomic islands. Tn7-like transposons form genomic islands at a programmed insertion site in bacterial chromosomes, attTn7. Transposition involves five transposon-encoded genes (tnsABCDE) including an atypical heteromeric transposase. One transposase subunit, TnsB, is from the large family of bacterial transposases, the second, TnsA, is related to endonucleases. A regulator protein, TnsC, functions with different target site selecting proteins to recognize different targets. TnsD directs transposition into attTn7, while TnsE encourages horizontal transmission by targeting mobile plasmids. Recent work suggests that distantly related elements with heteromeric transposases exist with alternate targeting pathways that also facilitate the formation of genomic islands. Tn6230 and related elements can be found at a single position in a gene of unknown function (yhiN) in various bacteria as well as in mobile plasmids. Another group we term Tn6022-like elements form pathogenicity islands in the Acinetobacter baumannii comM gene. We find that Tn6022-like elements also appear to have an uncharacterized mechanism for provoking internal transposition and deletion events that serve as a conduit for evolving new elements. As a group, heteromeric transposase elements utilize diverse target site selection mechanisms adapted to the spread and rearrangement of genomic islands.

ICEA of Mycoplasma agalactiae: a new family of self‐transmissible integrative elements that confers conjugative properties to the recipient strain

Horizontal gene transfer (HGT) is a major force of microbial evolution but was long thought to be marginal in mycoplasmas. In silico detection of exchanged regions and of loci encoding putative Integrative Conjugative Elements (ICE) in several mycoplasma genomes challenged this view, raising the prospect of these simple bacteria being able to conjugate. Using the model pathogen Mycoplasma agalactiae, we demonstrated for the first time that one of these elements, ICEA, is indeed self-transmissible. As a hallmark of conjugative processes, ICEA transfers were DNase resistant and required viable cells. ICEA acquisition conferred ICE-negative strains with the new ability to conjugate, allowing the spread of ICEA. Analysis of transfer-deficient mutants indicated that this process requires an ICEA-encoded lipoprotein of unknown function, CDS14. Formation of a circular extrachromosomal intermediate and the subsequent chromosomal integration of ICEA involved CDS22, an ICEA-encoded product distantly related to the ISLre2 transposase family. Remarkably, ICEA has no specific or no preferential integration site, often resulting in gene disruptions. Occurrence of functional mycoplasma ICE offers these bacteria with a means for HGT, a phenomenon with far-reaching implications given their minute-size genome and the number of species that are pathogenic for a broad host-range.

A proposed mechanism for IS607-family serine transposases

BackgroundThe transposases encoded by the IS607 family of mobile elements are unusual serine recombinases with an inverted domain order and minimal specificity for target DNA.ResultsStructural genomics groups have determined three crystal structures of the catalytic domains of IS607 family transposases. The dimers formed by these catalytic domains are very different from those seen for other serine recombinases and include interactions that usually only occur upon formation of a synaptic tetramer.ConclusionsBased on these structures, we propose a model for how IS607-family transposases could form a synaptic tetramer. The model suggests that, unlike other serine recombinases, these enzymes carry out sequence-specific DNA binding and catalysis in trans: the DNA binding and catalytic domains of each subunit are proposed to interact with different DNA duplexes. The model also suggests an explanation for the minimal target DNA specificity.

Whole-genome sequencing and identification of Morganella morganii KT pathogenicity-related genes.

BackgroundThe opportunistic enterobacterium, Morganella morganii, which can cause bacteraemia, is the ninth most prevalent cause of clinical infections in patients at Changhua Christian Hospital, Taiwan. The KT strain of M. morganii was isolated during postoperative care of a cancer patient with a gallbladder stone who developed sepsis caused by bacteraemia. M. morganii is sometimes encountered in nosocomial settings and has been causally linked to catheter-associated bacteriuria, complex infections of the urinary and/or hepatobiliary tracts, wound infection, and septicaemia. M. morganii infection is associated with a high mortality rate, although most patients respond well to appropriate antibiotic therapy. To obtain insights into the genome biology of M. morganii and the mechanisms underlying its pathogenicity, we used Illumina technology to sequence the genome of the KT strain and compared its sequence with the genome sequences of related bacteria.ResultsThe 3,826,919-bp sequence contained in 58 contigs has a GC content of 51.15% and includes 3,565 protein-coding sequences, 72 tRNA genes, and 10 rRNA genes. The pathogenicity-related genes encode determinants of drug resistance, fimbrial adhesins, an IgA protease, haemolysins, ureases, and insecticidal and apoptotic toxins as well as proteins found in flagellae, the iron acquisition system, a type-3 secretion system (T3SS), and several two-component systems. Comparison with 14 genome sequences from other members of Enterobacteriaceae revealed different degrees of similarity to several systems found in M. morganii. The most striking similarities were found in the IS4 family of transposases, insecticidal toxins, T3SS components, and proteins required for ethanolamine use (eut operon) and cobalamin (vitamin B12) biosynthesis. The eut operon and the gene cluster for cobalamin biosynthesis are not present in the other Proteeae genomes analysed. Moreover, organisation of the 19 genes of the eut operon differs from that found in the other non-Proteeae enterobacterial genomes.ConclusionsThis is the first genome sequence of M. morganii, which is a clinically relevant pathogen. Comparative genome analysis revealed several pathogenicity-related genes and novel genes not found in the genomes of other members of Proteeae. Thus, the genome sequence of M. morganii provides important information concerning virulence and determinants of fitness in this pathogen.

Targeting the Transposase Domain of the DNA Repair Component Metnase to Enhance Chemotherapy

Previous studies have shown that the DNA repair component Metnase (SETMAR) mediates resistance to DNA damaging cancer chemotherapy. Metnase has a nuclease domain that shares homology with the Transposase family. We therefore virtually screened the tertiary Metnase structure against the 550,000 compound ChemDiv library to identify small molecules that might dock in the active site of the transposase nuclease domain of Metnase. We identified eight compounds as possible Metnase inhibitors. Interestingly, among these candidate inhibitors were quinolone antibiotics and HIV integrase inhibitors, which share common structural features. Previous reports have described possible activity of quinolones as antineoplastic agents. Therefore, we chose the quinolone ciprofloxacin for further study, based on its wide clinical availability and low toxicity. We found that ciprofloxacin inhibits the ability of Metnase to cleave DNA and inhibits Metnase-dependent DNA repair. Ciprofloxacin on its own did not induce DNA damage, but it did reduce repair of chemotherapy-induced DNA damage. Ciprofloxacin increased the sensitivity of cancer cell lines and a xenograft tumor model to clinically relevant chemotherapy. These studies provide a mechanism for the previously postulated antineoplastic activity of quinolones, and suggest that ciprofloxacin might be a simple yet effective adjunct to cancer chemotherapy.

Flexibility in MuA transposase family protein structures: functional mapping with scanning mutagenesis and sequence alignment of protein homologues.

MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIβ, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIβ were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.

Development of Hydrolysis Probe-Based Real-Time PCR for Identification of Virulent Gene Targets ofBurkholderia pseudomalleiandB. mallei—A Retrospective Study on Archival Cases of Service Members with Melioidosis and Glanders

Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria responsible for melioidosis and glanders, respectively. Our laboratory developed hydrolysis probe-based real-time polymerase chain reaction assays targeting type three secretion system (TTS) and transposase family protein (TFP) of B. pseudomallei and B. malli, respectively. The assays were validated for target specificity, amplification sensitivity, and reproducibility. A bacterial DNA panel, composed of B. pseudomallei (13 strains), B. mallei (11 strains), Burkholderia species close neighbors (5 strains), and other bacterial species (17 strains), was prepared for specificity testing. Reference DNAs from B. pseudomallei and B. mallei bacterial cultures were used as controls for amplification, limit of detection, and reproducibility testing. The two TaqMan assays, Bp-TTS 1 and Bm-TFP, were optimized and applied in a retrospective study of archived cases from the Armed Forces Institute of Pathology. We tested 10 formalin-fixed paraffin-embedded blocks originally from autopsy specimens of patients who died of melioidosis or glanders during or after overseas tours in 1960s. Polymerase chain reaction results confirmed that DNA samples from formalin-fixed paraffin-embedded blocks of eight patients with melioidosis were positive for Bp-TTS 1 target and two patients with glanders were positive for Bm-TFP target.

Soluble expression, purification and characterization of the full length IS2 Transposase

BackgroundThe two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein.ResultsA soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common to the three dimensional catalytic core motif of the retroviral integrase superfamily.ConclusionsIntractably insoluble transposases, such as the IS2 transposase, prepared by solubilization protocols are often refractory to whole protein structure-function studies. The results described here have validated the use of GFP-tagging and fluorescence-based random mutagenesis in overcoming this limitation at the secondary structure level.

Treponema denticola biofilm-induced expression of a bacteriophage, toxin–antitoxin systems and transposases

Treponema denticola is an oral spirochaete that has been strongly associated with chronic periodontitis. The bacterium exists as part of a dense biofilm (subgingival dental plaque) accreted to the tooth. To determine T. denticola gene products important for persistence as a biofilm we developed a continuous-culture biofilm model and conducted a genome-wide transcriptomic analysis of biofilm and planktonic cells. A total of 126 genes were differentially expressed with a fold change of 1.5 or greater. This analysis identified the upregulation of putative prophage genes in the T. denticola 35405 genome. Intact bacteriophage particles were isolated from T. denticola and circular phage DNA was detected by PCR analysis. This represents the first, to our knowledge, functional bacteriophage isolated from T. denticola, which we have designated varphitd1. In biofilm cells there was also an upregulation of genes encoding several virulence factors, toxin-antitoxin systems and a family of putative transposases. Together, these data indicate that there is a higher potential for genetic mobility in T. denticola when growing as a biofilm and that these systems are important for the biofilm persistence and therefore virulence of this bacterium.

Identification and characterization of repetitive extragenic palindromes (REP)-associated tyrosine transposases: implications for REP evolution and dynamics in bacterial genomes

BackgroundBacterial repetitive extragenic palindromes (REPs) compose a distinct group of genomic repeats. They usually occur in high abundance (>100 copies/genome) and are often arranged in composite repetitive structures - bacterial interspersed mosaic elements (BIMEs). In BIMEs, regularly spaced REPs are present in alternating orientations. BIMEs and REPs have been shown to serve as binding sites for several proteins and suggested to play role in chromosome organization and transcription termination. Their origins are, at present, unknown.ResultsIn this report, we describe a novel class of putative transposases related to IS200/IS605 transposase family and we demonstrate that they are obligately associated with bacterial REPs. Open reading frames coding for these REP-associated tyrosine transposases (RAYTs) are always flanked by two REPs in inverted orientation and thus constitute a unit reminiscent of typical transposable elements. Besides conserved residues involved in catalysis of DNA cleavage, RAYTs carry characteristic structural motifs that are absent in typical IS200/IS605 transposases. DNA sequences flanking rayt genes are in one third of examined cases arranged in modular BIMEs. RAYTs and their flanking REPs apparently coevolve with each other. The rayt genes themselves are subject to rapid evolution, substantially exceeding the substitution rate of neighboring genes. Strong correlation was found between the presence of a particular rayt in a genome and the abundance of its cognate REPs.ConclusionsIn light of our findings, we propose that RAYTs are responsible for establishment of REPs and BIMEs in bacterial genomes, as well as for their exceptional dynamics and species-specifity. Conversely, we suggest that BIMEs are in fact a special type of nonautonomous transposable elements, mobilizable by RAYTs.