Insertion Hotspot Research Articles

Insertion sequences in the CRISPR-Cas system regulate horizontal antimicrobial resistance gene transfer in Shigella strains

Multidrug-resistant (MDR) Shigella strains are an enormous threat to public health. Antimicrobial resistance genes are frequently located on plasmids, phages and integrons, which enter bacterial cells by horizontal gene transfer (HGT). CRISPR-Cas systems are adaptive prokaryotic immune systems in bacteria that confer resistance to foreign genetic material such as phages and other mobile genetic elements. However, this may come at a cost of inhibiting the acquisition of other beneficial genes through HGT. This study investigated how Shigella strains regulate the activity of the CRISPR-Cas system spontaneously when they require an exogenous gene necessary for survival. Insertion sequence (IS) elements were identified in cas genes, such as IS600 in cse2, ISSfl2 in cas6e and IS629 in cse1-cas3. The number of spacers in CRISPR-Cas arrays in strains containing an IS was less than that for strains with no IS. Interestingly, fewer spacers were also found in MDR Shigella isolates. Furthermore, an antimicrobial-resistant strain was constructed by electrotransformation of a resistance plasmid in order to detect changes in the CRISPR-Cas system. It was found that the cse2 gene had a new IS (IS600) in the antimicrobial-resistant strain. Bioinformatics analyses showed that the IS600 insertion hotspot was TGC-GGC in the cse2 gene, and the tertiary structure of the Cse2 protein was different with IS600. IS600 caused a five-order of magnitude decrease in relative expression of the cse2 gene. This study sheds mechanistic light on CRISPR-Cas-mediated HGT of antimicrobial resistance genes in Shigella spp. isolates.

Genomic Islands in Sinorhizobium meliloti Rm1021, Nitrogen-Fixing Symbiont of Alfalfa

In silico analysis of open reading frames of genomic islands of Sinorhizobium meliloti Rm1021 was performed. This strain is a typical representative of soil bacteria forming nitrogen-fixing symbiosis with legume host plants from the alfalfa cross-inoculation group. It was demonstrated that genomic islands had mosaic structure, in which blocks of functional genes, IS-elements and noncoding RNA alternated. Genomic islands contained as well the components of T4SS and T4CP systems, and lacked systems of conjugative mobilization of islands. It was concluded that two of the three islands could be the variants of reduced integrative conjugative elements, and the third island represented a reduced integrated conjugative transposon. Site-specific integration of islands occurred into a 15–31 bp sequence (depending on the island) localized at the 3′-end of the tRNA gene, which is then shifted rightward, while the remaining part of the tRNA gene is completed by a similar sequence that exists in the island. A suggestion on the existence of “speciesspecific insertion hotspots” for genomic islands of root nodule bacteria was put forward.

What matters in chronic Burkholderia cenocepacia infection in cystic fibrosis: Insights from comparative genomics.

Burkholderia cenocepacia causes severe pulmonary infections in cystic fibrosis (CF) patients. Since the bacterium is virtually untreatable by antibiotics, chronic infections persist for years and might develop into fatal septic pneumonia (cepacia syndrome, CS). To devise new strategies to combat chronic B. cenocepacia infections, it is essential to obtain comprehensive knowledge about their pathogenesis. We conducted a comparative genomic analysis of 32 Czech isolates of epidemic clone B. cenocepacia ST32 isolated from various stages of chronic infection in 8 CF patients. High numbers of large-scale deletions were found to occur during chronic infection, affecting preferentially genomic islands and nonessential replicons. Recombination between insertion sequences (IS) was inferred as the mechanism behind deletion formation; the most numerous IS group was specific for the ST32 clone and has undergone transposition burst since its divergence. Genes functionally related to transition metal metabolism were identified as hotspots for deletions and IS insertions. This functional category was also represented among genes where nonsynonymous point mutations and indels occurred parallelly among patients. Another category exhibiting parallel mutations was oxidative stress protection; mutations in catalase KatG resulted in impaired detoxification of hydrogen peroxide. Deep sequencing revealed substantial polymorphism in genes of both categories within the sputum B. cenocepacia ST32 populations, indicating extensive adaptive evolution. Neither oxidative stress response nor transition metal metabolism genes were previously reported to undergo parallel evolution during chronic CF infection. Mutations in katG and copper metabolism genes were overrepresented in patients where chronic infection developed into CS. Among professional phagocytes, macrophages use both hydrogen peroxide and copper for their bactericidal activity; our results thus tentatively point to macrophages as suspects in pathogenesis towards the fatal CS.

Hopping into a hot seat: Role of DNA structural features on IS5-mediated gene activation and inactivation under stress.

Insertion sequence elements (IS elements) are proposed to play major roles in shaping the genetic and phenotypic landscapes of prokaryotic cells. Recent evidence has raised the possibility that environmental stress conditions increase IS hopping into new sites, and often such hopping has the phenotypic effect of relieving the stress. Although stress-induced targeted mutations have been reported for a number of E. coli genes, the glpFK (glycerol utilization) and the cryptic bglGFB (β-glucoside utilization) systems are among the best characterized where the effects of IS insertion-mediated gene activation are well-characterized at the molecular level. In the glpFK system, starvation of cells incapable of utilizing glycerol leads to an IS5 insertion event that activates the glpFK operon, and enables glycerol utilization. In the case of the cryptic bglGFB operon, insertion of IS5 (and other IS elements) into a specific region in the bglG upstream sequence has the effect of activating the operon in both growing cells, and in starving cells. However, a major unanswered question in the glpFK system, the bgl system, as well as other examples, has been why the insertion events are promoted at specific locations, and how the specific stress condition (glycerol starvation for example) can be mechanistically linked to enhanced insertion at a specific locus. In this paper, we show that a specific DNA structural feature (superhelical stress-induced duplex destabilization, SIDD) is associated with “stress-induced” IS5 insertion in the glpFK, bglGFB, flhDC, fucAO and nfsB systems. We propose a speculative mechanistic model that links specific environmental conditions to the unmasking of an insertional hotspot in the glpFK system. We demonstrate that experimentally altering the predicted stability of a SIDD element in the nfsB gene significantly impacts IS5 insertion at its hotspot.

Microbial Signatures Associated with Oropharyngeal and Oral Squamous Cell Carcinomas

The microbiome is fundamentally one of the most unique organs in the human body. Dysbiosis can result in critical inflammatory responses and result in pathogenesis contributing to neoplastic events. We used a pan-pathogen array technology (PathoChip) coupled with next-generation sequencing to establish microbial signatures unique to human oral and oropharyngeal squamous cell carcinomas (OCSCC/OPSCC). Signatures for DNA and RNA viruses including oncogenic viruses, gram positive and negative bacteria, fungi and parasites were detected. Cluster and topological analyses identified 2 distinct groups of microbial signatures related to OCSCCs/OPSCCs. Results were validated by probe capture next generation sequencing; the data from which also provided a comprehensive map of integration sites and chromosomal hotspots for micro-organism genomic insertions. Identification of these microbial signatures and their integration sites may provide biomarkers for OCSCC/OPSCC diagnosis and prognosis as well as novel avenues for study of their potential role in OCSCCs/OPSCCs.

Insertion of Alu elements at a PTEN hotspot in Cowden syndrome.

Cowden syndrome (CS) is an inherited autosomal dominant disorder associated with germline pathogenic variants of the PTEN tumor suppressor gene. Its phenotypical expression is highly variable and the existence of patients with a CS suggestive phenotype without pathogenic PTEN variant may be related to genetic heterogeneity. In order to explore this hypothesis through the detection of potentially deleterious variants enabling us to identify a new candidate gene, we performed whole-exome sequencing (WES) in a series of 22 CS patients without detectable PTEN pathogenic variant using conventional methods for mutation screening. We failed to identify a novel candidate gene, but interestingly in two patients WES revealed the presence of two distinct, previously undescribed Alu insertions with the same break points in exon 5. These insertions were not found in a series of 35 breast carcinomas that showed a loss of PTEN expression without a detectable alteration of this gene. This study reveals the presence of a PTEN Alu insertion hotspot involved in CS, and suggests that undetected PTEN pathogenic variants could contribute to CS.

A hypervariable genomic island identified in clinical and environmental Mycobacterium avium subsp. hominissuis isolates from Germany

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic human pathogen widespread in the environment. Genomic islands (GI)s represent a part of the accessory genome of bacteria and influence virulence, drug-resistance or fitness and trigger bacterial evolution. We previously identified a novel GI in four MAH genomes. Here, we further explored this GI in a larger collection of MAH isolates from Germany (n=41), including 20 clinical and 21 environmental isolates. Based on comparative whole genome analysis, we detected this GI in 39/41 (95.1%) isolates. Although all these GIs integrated in the same insertion hotspot, there is high variability in the genetic structure of this GI: eight different types of GI have been identified, designated A–H (sized 6.2–73.3kb). These GIs were arranged as single GI (23/41, 56.1%), combination of two different GIs (14/41, 34.1%) or combination of three different GIs (2/41, 4.9%) in the insertion hotspot. Moreover, two GI types shared more than 80% sequence identity with sequences of M. canettii, responsible for Tuberculosis. A total of 253 different genes were identified in all GIs, among which the previously documented virulence-related genes mmpL10 and mce. The diversity of the GI and the sequence similarity with other mycobacteria suggests cross-species transfer, involving also highly pathogenic species. Shuffling of potential virulence genes such as mmpL10 via this GI may create new pathogens that can cause future outbreaks.

Hotspot mutations delineating diverse mutational signatures and biological utilities across cancer types.

BackgroundAn important step towards personalizing cancer treatment is to integrate heterogeneous evidences to catalog mutational hotspots that are biologically and therapeutically relevant and thus represent where targeted therapy would likely be beneficial. However, existing methods do not sufficiently delineate varying functionality of individual mutations within the same genes.ResultsWe observed a large discordancy of mutation rates across different mutation subtypes and tumor types, and nominated 702 hotspot mutations in 549 genes in the Catalog of Somatic Mutations in Cancer (COSMIC) by considering context specific mutation characteristics such as genes, cancer types, mutation rates, mutation subtypes and sequence contexts. We observed that hotspot mutations were highly prevalent in Non CpG-island C/G transition and transversion sequence contexts in 10 tumor types, and specific insertion hotspot mutations were enriched in breast cancer and deletion hotspot mutations in colorectal cancer. We found that the hotspot mutations nominated by our approach were significantly more conserved than non-hotspot mutations in the corresponding cancer genes. We also examined the biological significance and pharmacogenomics properties of these hotspot mutations using data in the Cancer Genome Atlas (TCGA) and the Cancer Cell-Line Encyclopedia (CCLE), and found that 53 hotspot mutations are independently associated with diverse functional evidences in 1) mRNA and protein expression, 2) pathway activity, or 3) drug sensitivity and 82 were highly enriched in specific tumor types. We highlighted the distinct functional indications of hotspot mutations under different contexts and nominated novel hotspot mutations such as MAP3K4 A1199 deletion, NR1H2 Q175 insertion, and GATA3 P409 insertion as potential biomarkers or drug targets.ConclusionWe identified a set of hotspot mutations across 17 tumor types by considering the background mutation rate variations among genes, tumor subtypes, mutation subtypes, and sequence contexts. We illustrated the common and distinct mutational signatures of hotspot mutations among different tumor types and investigated their variable functional relevance under different contexts, which could potentially serve as a resource for explicitly selecting targets for diagnosis, drug development, and patient management.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2727-x) contains supplementary material, which is available to authorized users.

Dataset for distribution of INGI/RIME and SLACS CRE transposable elements in Trypanosoma brucei genome.

The current dataset is generated via bio-computational approach by surveying of INGI/RIME and SLACS CRE transposable elements (TEs) in latest update of Trypanosoma brucei genome. The distribution dataset (Supplementary File 1) shows the chromosome wise distribution of INGI/RIME and SLACS CRE transposable elements with the status of their -5′ and -3′ ends, genomic coverage and further elemental description about the completeness on the element. The 5′ upstream flanking sequence of 100 bp was then analyzed to find out possible regions that could act as insertion hotspots. The Fig. 1 represents the ten different motifs found in the 5′ flanking region of the INGI/RIME and SLACS CRE elements. The Supplementary File 2 describes the distribution of these ten motifs in different locations in Trypanosoma brucei genome. These new locations where motifs were found may provide useful information to track the future transposition events of INGI/RIME and SLACS CRE elements in different Trypanosoma species.

Chromosomal distribution and evolution of abundant retrotransposons in plants: gypsy elements in diploid and polyploid Brachiaria forage grasses.

Like other eukaryotes, the nuclear genome of plants consists of DNA with a small proportion of low-copy DNA (genes and regulatory sequences) and very abundant DNA sequence motifs that are repeated thousands up to millions of times in the genomes including transposable elements (TEs) and satellite DNA. Retrotransposons, one class of TEs, are sequences that amplify via an RNA intermediate and reinsert into the genome, are often the major fraction of a genome. Here, we put research on retrotransposons into the larger context of plant repetitive DNA and genome behaviour, showing features of genome evolution in a grass genus, Brachiaria, in relation to other plant species. We show the contrasting amplification of different retroelement fractions across the genome with characteristics for various families and domains. The genus Brachiaria includes both diploid and polyploid species, with similar chromosome types and chromosome basic numbers x = 6, 7, 8 and 9. The polyploids reproduce asexually and are apomictic, but there are also sexual species. Cytogenetic studies and flow cytometry indicate a large variation in DNA content (C-value), chromosome sizes and genome organization. In order to evaluate the role of transposable elements in the genome and karyotype organization of species of Brachiaria, we searched for sequences similar to conserved regions of TEs in RNAseq reads library produced in Brachiaria decumbens. Of the 9649 TE-like contigs, 4454 corresponded to LTR-retrotransposons, and of these, 79.5 % were similar to members of the gypsy superfamily. Sequences of conserved protein domains of gypsy were used to design primers for producing the probes. The probes were used in FISH against chromosomes of accesses of B. decumbens, Brachiaria brizantha, Brachiaria ruziziensis and Brachiaria humidicola. Probes showed hybridization signals predominantly in proximal regions, especially those for retrotransposons of the clades CRM and Athila, while elements of Del and Tat exhibited dispersed signals, in addition to those proximal signals. These results show that the proximal region of Brachiaria chromosomes is a hotspot for retrotransposon insertion, particularly for the gypsy family. The combination of high-throughput sequencing and a chromosome-centric cytogenetic approach allows the abundance, organization and nature of transposable elements to be characterized in unprecedented detail. By their amplification and dispersal, retrotransposons can affect gene expression; they can lead to rapid diversification of chromosomes between species and, hence, are useful for studies of genome evolution and speciation in the Brachiaria genus. Centromeric regions can be identified and mapped, and retrotransposon markers can also assisting breeders in the developing and exploiting interspecific hybrids.

Intron Invasions Trace Algal Speciation and Reveal Nearly Identical Arctic and Antarctic Micromonas Populations.

Spliceosomal introns are a hallmark of eukaryotic genes that are hypothesized to play important roles in genome evolution but have poorly understood origins. Although most introns lack sequence homology to each other, new families of spliceosomal introns that are repeated hundreds of times in individual genomes have recently been discovered in a few organisms. The prevalence and conservation of these introner elements (IEs) or introner-like elements in other taxa, as well as their evolutionary relationships to regular spliceosomal introns, are still unknown. Here, we systematically investigate introns in the widespread marine green alga Micromonas and report new families of IEs, numerous intron presence–absence polymorphisms, and potential intron insertion hot-spots. The new families enabled identification of conserved IE secondary structure features and establishment of a novel general model for repetitive intron proliferation across genomes. Despite shared secondary structure, the IE families from each Micromonas lineage bear no obvious sequence similarity to those in the other lineages, suggesting that their appearance is intimately linked with the process of speciation. Two of the new IE families come from an Arctic culture (Micromonas Clade E2) isolated from a polar region where abundance of this alga is increasing due to climate induced changes. The same two families were detected in metagenomic data from Antarctica—a system where Micromonas has never before been reported. Strikingly high identity between the Arctic isolate and Antarctic coding sequences that flank the IEs suggests connectivity between populations in the two polar systems that we postulate occurs through deep-sea currents. Recovery of Clade E2 sequences in North Atlantic Deep Waters beneath the Gulf Stream supports this hypothesis. Our research illuminates the dynamic relationships between an unusual class of repetitive introns, genome evolution, speciation, and global distribution of this sentinel marine alga.

The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure

The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-222) contains supplementary material, which is available to authorized users.

The rulB gene of plasmid pWW0 is a hotspot for the site‐specific insertion of integron‐like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria

The rulAB operon of Pseudomonas spp. confers fitness traits on the host and has been suggested to be a hotspot for insertion of mobile elements that carry avirulence genes. Here, for the first time, we show that rulB on plasmid pWW0 is a hotspot for the active site-specific integration of related integron-like elements (ILEs) found in six environmental pseudomonads (strains FH1–FH6). Integration into rulB on pWW0 occurred at position 6488 generating a 3 bp direct repeat. ILEs from FH1 and FH5 were 9403 bp in length and contained eight open reading frames (ORFs), while the ILE from FH4 was 16 233 bp in length and contained 16 ORFs. In all three ILEs, the first 5.1 kb (containing ORFs 1–4) were structurally conserved and contained three predicted site-specific recombinases/integrases and a tetR homologue. Downstream of these resided ORFs of the ‘variable side’ with structural and sequence similarity to those encoding survival traits on the fitness enhancing plasmid pGRT1 (ILEFH1 and ILEFH5) and the NR-II virulence region of genomic island PAGI-5 (ILEFH4). Collectively, these ILEs share features with the previously described type III protein secretion system effector ILEs and are considered important to host survival and transfer of fitness enhancing and (a)virulence genes between bacteria.

Denaturing Gradient Gel Electrophoresis (DGGE) as a Powerful Novel Alternative for Differentiation of Epizootic ISA Virus Variants

Infectious Salmon Anemia is a devastating disease critically affecting world-wide salmon production. Chile has been particularly stricken by this disease which in all cases has been directly related with its causative agent, a novel orthomyxovirus which presents specific and distinctive infective features. Among these, two molecular markers have been directly associated with pathogenicity in two of the eight RNA sub genomic coding units of the virus: an insertion hot spot region present in viral segment 5 and a Highly Polymorphic Region (HPR) located in viral segment 6. Here we report the successful adaptation of a PCR-dependent denaturing gel electrophoresis technique (DGGE), which enables differentiation of selected reported HPR epizootic variants detected in Chile. At the same time, the technique allows us to distinguish one nucleotide differences in sequences associated with the intriguing, and still not well-understood, insertion events which tend to occur on RNA Segment 5. Thus, the versatility of the technique opens new opportunities for improved understanding of the complex biology of all ISA variants as well as possible applications to other highly variable pathogens.

Structural characteristics of genomic islands associated with GMP synthases as integration hotspot among sequenced microbial genomes

tRNA, tmRNA and some small RNA genes are recognized as general integration hotspots of genomic islands (GIs). The GMP synthase gene (guaA) has been firstly identified as one insertion hotspot of foreign DNA fragments. Thirty four islands integrated into the guaA genes were identified in the 987 completely sequenced archaeal and bacterial genomes. These alien islands were widely distributed within the host strains belonging to Proteobacteria, Firmicutes and Actinobacteria. The analysis of structural characteristics of these GIs is important for further determination of the island mobility and transference into suitable hosts. The putative functional integrases encoded by guaA-associated islands were mainly composed of phage P4 integrases, and followed by phage PhiLC3 integrases. Interestingly, island-encoding AlpA is close to P4 integrase and is deduced to be the positive transcriptional regulatory factor of P4 integrase while the XRE protein is close to PhiLC3 integrase and may be the negative transcriptional regulatory factor of PhiLC3 integrase. An 8-bp consensus sequence (5′-GAGTGGGA-3′) within the direct repeats of these GIs is the cutting site of the P4 integrases encoding by guaA-associated islands, in which the third nucleotide (G) is the key site. The large-scale investigation of the content of GMP synthase gene hotspots may be useful to find important functional islands within members of many key bacterial species and to transfer useful islands into more suitable hosts.

A large scale comparative genomic analysis reveals insertion sites for newly acquired genomic islands in bacterial genomes

BackgroundBacterial virulence enhancement and drug resistance are major threats to public health worldwide. Interestingly, newly acquired genomic islands (GIs) from horizontal transfer between different bacteria strains were found in Vibrio cholerae, Streptococcus suis, and Mycobacterium tuberculosis, which caused outbreak of epidemic diseases in recently years.ResultsUsing a large-scale comparative genomic analysis of 1088 complete genomes from all available bacteria (1009) and Archaea (79), we found that newly acquired GIs are often anchored around switch sites of GC-skew (sGCS). After calculating correlations between relative genomic distances of genomic islands to sGCSs and the evolutionary distances of the genomic islands themselves, we found that newly acquired genomic islands are closer to sGCSs than the old ones, indicating that regions around sGCSs are hotspots for genomic island insertion.ConclusionsBased on our results, we believe that genomic regions near sGCSs are hotspots for horizontal transfer of genomic islands, which may significantly affect key properties of epidemic disease-causing pathogens, such as virulence and adaption to new environments.

Exceptional lability of a genomic complex in rice and its close relatives revealed by interspecific and intraspecific comparison and population analysis

BackgroundExtensive DNA rearrangement of genic colinearity, as revealed by comparison of orthologous genomic regions, has been shown to be a general concept describing evolutionary dynamics of plant genomes. However, the nature, timing, lineages and adaptation of local genomic rearrangement in closely related species (e.g., within a genus) and haplotype variation of genomic rearrangement within populations have not been well documented.ResultsWe previously identified a hotspot for genic rearrangement and transposon accumulation in the Orp region of Asian rice (Oryza sativa, AA) by comparison with its orthologous region in sorghum. Here, we report the comparative analysis of this region with its orthologous regions in the wild progenitor species (O. nivara, AA) of Asian rice and African rice (O. glaberrima) using the BB genome Oryza species (O. punctata) as an outgroup, and investigation of transposon insertion sites and a segmental inversion event in the AA genomes at the population level. We found that Orp region was primarily and recently expanded in the Asian rice species O. sativa and O. nivara. LTR-retrotransposons shared by the three AA-genomic regions have been fixed in all the 94 varieties that represent different populations of the AA-genome species/subspecies, indicating their adaptive role in genome differentiation. However, LTR-retrotransposons unique to either O. nivara or O. sativa regions exhibited dramatic haplotype variation regarding their presence or absence between or within populations/subpopulations.ConclusionsThe LTR-retrotransposon insertion hotspot in the Orp region was formed recently, independently and concurrently in different AA-genome species, and that the genic rearrangements detected in different species appear to be differentially triggered by transposable elements. This region is located near the end of the short arm of chromosome 8 and contains a high proportion of LTR-retrotransposons similar to observed in the centromeric region of this same chromosome, and thus may represent a genomic region that has recently switched from euchromatic to heterochromatic states. The haplotype variation of LTR-retrotransposon insertions within this region reveals substantial admixture among various subpopulations as established by molecular markers at the whole genome level, and can be used to develop retrotransposon junction markers for simple and rapid classification of O. sativa germplasm.

Expansion of the known Klebsiella pneumoniae species gene pool by characterization of novel alien DNA islands integrated into tmRNA gene sites

Klebsiella pneumoniae is an important bacterial pathogen of man that is commonly associated with opportunistic and hospital-associated infections. Increasing levels of multiple-antibiotic resistance associated with this species pose a major emerging clinical problem. This organism also occurs naturally in other diverse environments, including the soil. Consistent with its varied lifestyle and membership of the Enterobacteriaceae family, K. pneumoniae genomes exhibit highly plastic architecture comprising a core genome backbone interspersed with numerous and varied alien genomic islands. In this study the size of the presently known K. pneumoniae pan-genome gene pool was estimated through analysis of complete sequences of three chromosomes and 31 plasmids belonging to K. pneumoniae strains. In addition, using a PCR-based strategy the genomic content of eight tRNA/ tmRNA gene sites that serve as DNA insertion hotspots were investigated in 28 diverse environmental and clinical strains of K. pneumoniae. Sequencing and characterization of five newly identified horizontally-acquired tmRNA-associated islands further expanded the archived K. pneumoniae gene pool to a total of 7648 unique gene members. Large-scale investigation of the content of tRNA/ tmRNA hotspots will be useful to identify and/or survey accessory sequences dispersed amongst hundreds to thousands of members of many key bacterial species.

Different Genetic Elements Carrying the tet (W) Gene in Two Human Clinical Isolates of Streptococcus suis

The genetic support for tet(W), an emerging tetracycline resistance determinant, was studied in two strains of Streptococcus suis, SsCA and SsUD, both isolated in Italy from patients with meningitis. Two completely different tet(W)-carrying genetic elements, sharing only a tet(W)-containing segment barely larger than the gene, were found in the two strains. The one from strain SsCA was nontransferable, and aside from an erm(B)-containing insertion, it closely resembled a genomic island recently described in an S. suis Chinese human isolate in sequence, organization, and chromosomal location. The tet(W)-carrying genetic element from strain SsUD was transferable (at a low frequency) and, though apparently noninducible following mitomycin C treatment, displayed a typical phage organization and was named ΦSsUD.1. Its full sequence was determined (60,711 bp), the highest BLASTN score being Streptococcus pyogenes Φm46.1. ΦSsUD.1 exhibited a unique combination of antibiotic and heavy metal resistance genes. Besides tet(W), it contained a MAS (macrolide-aminoglycoside-streptothricin) fragment with an erm(B) gene having a deleted leader peptide and a cadC/cadA cadmium efflux cassette. The MAS fragment closely resembled the one recently described in pneumococcal transposons Tn6003 and Tn1545. These resistance genes found in the ΦSsUD.1 phage scaffold differed from, but were in the same position as, cargo genes carried by other streptococcal phages. The chromosome integration site of ΦSsUD.1 was at the 3' end of a conserved tRNA uracil methyltransferase (rum) gene. This site, known to be an insertional hot spot for mobile elements in S. pyogenes, might play a similar role in S. suis.

High-frequency of P{lacW} double local reinsertions in chromosomal region 100C of Drosophila melanogaster are amenable to a homing-like mechanism.

Herein we present results concerning the transposit ion pattern of the P{lacW} artificial mobile element in the male germline of Drosophila melanogaster . Using the power of inverse PCR followed by sequencing strategy to analyze a collection of 55 transgenic lines, we recovered 18 independent secondary reinsertions of the P{lacW} gammaCop057302 parental transposon affecting gammaCop gene located in 100C. Bioinformatics investigation revealed that 8 out of them reside within or very close to the starting element, repre senting extreme cases of secondary local reinsertio ns (equivalent to double local reinsertions). Similar molecular phenomena were previously reported for a PZ artificial element transposing in the male germlin e. Mapping of some secondary reinsertions obtained in our experiment revealed two putative insertional hot-spots located in the 3' half of P{lacW} , namely in the mini-white allele and in the adjacent pBR322 fragment. The relative high frequency of double lo cal reinsertions resemble homing, a phenomenon consisting in the preferential inserti on of transgenic transposons harboring fragments from a D. melanogaster gene into or nearby the genomic copy of the cloned gene. Such biases in the pattern of transposition of artificial mobile eleme nts may be products of a molecular behavior with st ill unknown biological significance.