Types Of Insertion Sequences Research Articles

Genome Plasticity by Insertion Sequences Learned From a Case of Radiation-Resistant Bacterium Deinococcus geothermalis.

The genome of the radiation-resistant bacterium Deinococcus geothermalis contains 19 types of insertion sequences (ISs), including 93 total transposases (Tpases) in 73 full-length ISs from the main chromosome and 2 mega plasmids. In this study, 68 ISs from the D. geothermalis genome were extracted to implicate the earlier genome before its mutation by transposition of ISs. The total size of eliminated ISs from genome was 78.85 kb. From these in silico corrections of mutation by the ISs, we have become aware of some bioinformatics factualness as follows: (1) can reassemble the disrupted genes if the exact IS region was eliminated, (2) can configure the schematic clustering of major DDE type Tpases, (3) can determine IS integration order across multiple hot spots, and (4) can compare genetic relativeness by the partial synteny analysis between D. geothermalis and Deinococcus strain S9. Recently, we found that several IS elements actively transferred to other genomic sites under hydrogen peroxide-induced oxidative stress conditions, resulting in the inactivation of functional genes. Therefore, the single species genome’s mobilome study provides significant support to define bacterial genome plasticity and molecular evolution from past and present progressive transposition events.

Short communication: Whole-genome sequence analysis of 4 fecal blaCMY-2-producing Escherichia coli isolates from Holstein dairy calves

This study was carried out to determine the antimicrobial resistance (AMR) genes and mobile genetic elements of 4 fecal blaCMY-2-producing Escherichia coli isolated from Holstein dairy calves on the same farm using whole-genome sequencing. Genomic analysis revealed that 3 of the 4 isolates shared similar genetic features, including sequence type (ST), serotype, plasmid characteristics, insertion ST, and virulence genes. In addition to genes encoding for complex multidrug resistance efflux systems, all 4 isolates were carriers of genes conferring resistance to β-lactams (blaCMY-2, blaTEM-1B), tetracyclines (tetA, tetB, tetD), aminoglycosides [aadA1, aph(3")-lb, aph(6)-ld], sulfonamides (sul2), and trimethoprim (dfrA1). We also detected 4 incompatibility plasmid groups: Inc.F, Inc.N, Inc.I, and Inc.Q. A novel ST showing a new purA and mdh allelic combination was found. The 4 isolates were likely enterotoxigenic pathotypes of E. coli, based on serotype and presence of the plasmid Inc.FII(pCoo). This study provides information for comparative genomic analysis of AMR genes and mobile genetic elements. This analysis could give some explanation to the multidrug resistance characteristics of bacteria colonizing the intestinal tract of dairy calves in the first few weeks of life.

Metagenome analysis of antibiotic resistance genes in fecal microbiota of chickens

Numerous environmental reservoirs contribute to the problem of acquired antibiotic resistance of human pathogens. One environmental reservoir of particular importance is the intestinal bacteria of poultry. Antibiotics are often used to prevent sickness and improve production in chicken farms. Hence, the intestinal microbial community of chickens as a reservoir for antibiotic resistance genes (ARGs) has received increasing attention. Therefore, the aim of this study was to investigate the occurrence, diversity, and abundance of ARGs and mobile genetic elements (MGEs), and the distribution of plasmids in layer and broiler feces using Illumina high-throughput sequencing. Metagenomic analysis showed that Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were predominate in chicken feces. At the genus level, Escherichia coli may be predominant in chicken feces with a relative abundance of plasmids. Sequencing reads revealed differences in the distribution of plasmids in microbial communities between layer and broiler feces. Genes coding for antibiotic resistance were identified in both communities. BLAST analysis against the Antibiotic Resistance Genes Database (ARDB) further revealed that layer and broiler feces contained various ARGs with high abundances, among which vancomycin (van) was the most abundant, accounting for >36% of all reads of ARGs. Beta-lactam resistance genes (bl) were highly enriched in the broiler feces, among which bl2e_cbla was the most abundant, but was absent in layer feces. Over 20 types of insertion sequences were detected in each fecal sample. These results highlight the prevalence of ARGs, plasmids, and MGEs in chicken feces, which warrants further studies to better elucidate the risk to public health.

ISCR2 is associated with the dissemination of multiple resistance genes among Vibrio spp. and Pseudoalteromonas spp. isolated from farmed fish.

58 multiresistant strains representing diverse genera were isolated from farmed fish in an aquaculture facility. Resistant rates of strains harboring ISCR2, an insertion sequence type element, were higher than those in which this element was absent. Full genome sequencing of a Vibrio isolate containing ISCR2 confirmed that it is associated with multiple resistance genes, many of which are of clinical relevance. We describe the structural variation within ISCR2, and its distribution throughout multiple diverse aquatic genera, including Vibrio, Shewenalla, Pseudoalteromonas and Psychrobacter, suggesting the potential role of ISCR2 in disseminating antibiotic resistance. We also observe, and experimentally verify, a novel macrolide resistance gene that is also associated with ISCR2.

Distribution of IS629 and stx genotypes among enterohemorrhagic Escherichia coli O157 isolates in Yamaguchi Prefecture, Japan, 2004-2013.

Patterns of insertion sequence (IS)629, norV genotype, and Shiga toxin (Stx) genotype distribution were investigated amongst 203 enterohemorrhagic Escherichia coli O157 isolates collected in Yamaguchi Prefecture, Japan, between 2004 and 2013. A total of 114 IS629 patterns were identified; these were divided into eight IS groups (A–H). Ninety isolates carried an intact norV gene, whereas 113 isolates carried a norV with a 204-bp deletion. Other than one isolate from IS group G, all isolates with an intact norV belonged to groups A–F, whereas isolates with a mutant norV belonged to IS groups G and H. Seven stx genotypes were identified, and of those, stx1a/stx2a was predominant (n=105), followed by stx2c (n=32) and stx2a (n=27). The stx1a/stx2a genotype was associated with the mutant norV isolates, whereas isolates with an intact norV had the stx2c genotype. Therefore, certain combinations of IS type and stx genotype appear to be more frequent among O157 clades which may be useful for detection of predominant subtypes in the interest of public health.

Metagenomic Profiling of Antibiotic Resistance Genes and Mobile Genetic Elements in a Tannery Wastewater Treatment Plant

Antibiotics are often used to prevent sickness and improve production in animal agriculture, and the residues in animal bodies may enter tannery wastewater during leather production. This study aimed to use Illumina high-throughput sequencing to investigate the occurrence, diversity and abundance of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in aerobic and anaerobic sludge of a full-scale tannery wastewater treatment plant (WWTP). Metagenomic analysis showed that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria dominated in the WWTP, but the relative abundance of archaea in anaerobic sludge was higher than in aerobic sludge. Sequencing reads from aerobic and anaerobic sludge revealed differences in the abundance of functional genes between both microbial communities. Genes coding for antibiotic resistance were identified in both communities. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) further revealed that aerobic and anaerobic sludge contained various ARGs with high abundance, among which sulfonamide resistance gene sul1 had the highest abundance, occupying over 20% of the total ARGs reads. Tetracycline resistance genes (tet) were highly rich in the anaerobic sludge, among which tet33 had the highest abundance, but was absent in aerobic sludge. Over 70 types of insertion sequences were detected in each sludge sample, and class 1 integrase genes were prevalent in the WWTP. The results highlighted prevalence of ARGs and MGEs in tannery WWTPs, which may deserve more public health concerns.

Miniature Transposable Sequences Are Frequently Mobilized in the Bacterial Plant Pathogen Pseudomonas syringae pv. phaseolicola

Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.6×10−5 and 1.1×10−6, depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria.

Origin and Examination of a Leafhopper Facultative Endosymbiont

Eukaryotes engage in intimate interactions with microbes that range in age and type of association. Although many conspicuous examples of ancient insect associates are studied (e.g., Buchnera aphidicola), fewer examples of younger associations are known. Here, we further characterize a recently evolved bacterial endosymbiont of the leafhopper Euscelidius variegatus (Hemiptera, Cicadellidae), called BEV. We found that BEV, continuously maintained in E. variegatus hosts at UC Berkeley since 1984, is vertically transmitted with high fidelity. Unlike many vertically transmitted, ancient endosymbioses, the BEV–E. variegatus association is not obligate for either partner, and BEV can be cultivated axenically. Sufficient BEV colonies were grown and harvested to estimate its genome size and provide a partial survey of the genome sequence. The BEV chromosome is about 3.8 Mbp, and there is evidence for an extrachromosomal element roughly 53 kb in size (e.g., prophage or plasmid). We sequenced 438 kb of unique short-insert clones, representing about 12% of the BEV genome. Nearly half of the gene fragments were similar to mobile DNA, including 15 distinct types of insertion sequences (IS). Analyses revealed that BEV not only shares virulence genes with plant pathogens, but also is closely related to the plant pathogenic genera Dickeya, Pectobacterium, and Brenneria. However, the slightly reduced genome size, abundance of mobile DNA, fastidious growth in culture, and efficient vertical transmission suggest that symbiosis with E. variegatus has had a significant impact on genome evolution in BEV.Electronic supplementary materialThe online version of this article (doi:10.1007/s00284-011-9893-5) contains supplementary material, which is available to authorized users.

Insertion Sequences as Highly Resolutive Genomic Markers for Sequence Type 1 Legionella pneumophila Paris

The causative agent of legionellosis, Legionella pneumophila, colonizes all natural and human-made water networks, thus constituting the source of contaminated aerosols responsible for airborne human infections. Efficient control of infections, especially during epidemics, necessitates the fastest and most resolutive identification possible of the bacterial source for subsequent disinfection of reservoirs. We thus compared recognized typing approaches for Legionella with a method based on characterization of insertion sequence (IS) content. A total of 86 clinical or environmental isolates of L. pneumophila, including 84 Paris isolates, sampled from 25 clinical investigations in France between 2001 and 2007, were obtained from the Legionella National Reference Center. All strains were typed by monoclonal antibody subgrouping, sequence-based typing, pulsed-field gel electrophoresis, and restriction fragment length polymorphism based on the presence or absence of IS elements. We identified six different types of IS elements in L. pneumophila Paris and used them as genomic markers in hybridization experiments. One IS type, ISLpn11, revealed a high discriminatory power. Simpson's index of discrimination, calculated from the distribution of IS elements, was higher than that obtained with the other typing methods used for L. pneumophila Paris. Moreover, specific ISLpn11 copies were found only in strains isolated from particular cities. In more than half of the cases, each clinical isolate had an ISLpn11 profile that was recovered in at least one environmental isolate from the same geographical location, suggesting that our method could identify the infection source. Phylogenetic analysis suggests a clonal expansion for the L. pneumophila Paris strain.

Rapid Detection of a Point Mutation in the parC Gene Associated with Decreased Susceptibility to Fluoroquinolones in Mycoplasma bovis

Comparison of the quinolone resistance-determining regions (QRDRs) in 42 Mycoplasma bovis clinical isolates revealed amino acid substitutions at both GyrA (position 83) and ParC (position 84) in 10/11 enrofloxacin-resistant strains. The mutation present in the parC QRDR was discriminative for enrofloxacin resistance by parC PCR-restriction fragment length polymorphism. Comparison of molecular profiles by insertion sequence typing suggests that the currently prevalent enrofloxacin-resistant M. bovis strain evolved by selection under field conditions from one of the susceptible strains.

Spontaneous transposition of IS 1096 or IS Msm3 leads to glycopeptidolipid overproduction and affects surface properties in Mycobacterium smegmatis

Natural modification of the colony appearance is a phenomenon that has not been fully understood in mycobacteria. Here, we show that Mycobacterium smegmatis ATCC607 displays a low-frequency spontaneous morphological variation that correlates with the acquisition of a panel of new phenotypes, such as aggregation, biofilm formation and sliding motility. These variants produce larger amounts of glycopeptidolipid (GPL), a cell-surface component, than did the wild-type strain. This conversion results from the transposition of two types of insertion sequences, IS1096 and ISMsm3, into two loci. One locus is the promoter region of the mps operon, the GPL biosynthesis gene cluster, leading to the overexpression of these genes. The other locus is the lsr2 gene, which encodes a small basic histone-like protein that likely plays a regulatory role at the mps promoter and also controls pigment production. This study demonstrates that insertion sequence mobility play a crucial role in the acquisition of new phenotypes.

Genetic and Phenotypic Variability among Salmonella enterica Serovar Typhimurium Isolates from California Dairy Cattle and Humans

Fifty-six human and 24 adult dairy cattle isolates of Salmonella enterica serovar Typhimurium from a single county in California were compared using ribotyping, insertion sequence typing (IS200), pulsed-field gel electrophoresis, plasmid typing, phage typing, and antimicrobial resistance testing. The majority of the isolates fell into one of two groups which were phage types DT104 and DT193. Combining the information from all typing methods, a total of 45 different "clusters" were defined, with 35 of those including only a single isolate. The library of isolates had a high degree of variability, but antibiotic resistance and plasmid typing each defined single clusters in which human or bovine isolates predominated (chi2, P < 0.05).

Molecular Typing Reveals a Unique Clone of Salmonella enterica Serotype Typhi among Indian Strains

Molecular Typing Reveals a Unique Clone of <i>Salmonella enterica</i> Serotype Typhi among Indian Strains

Insertion sequence typing of Mycobacterium tuberculosis: characterization of a widespread subtype with a single copy of IS6110

DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.

Genotypic diversity of an indigenous Rhizobium meliloti field population assessed by plasmid profiles, DNA fingerprinting, and insertion sequence typing

One hundred and twenty-five Rhizobium meliloti isolates nodulating field-grown Medicago sativa cv. Europe were evaluated for plasmid content and for symbiotic effectiveness with this host. Relative to the strain used commercially in France (RCR2011), isolates showed a comparable level of symbiotic effectiveness, although two isolates were poorly effective. Twenty-four distinct plasmid profiles were observed among these isolates, with a plasmid number varying between one and five per isolate. All isolates but one harboured a plasmid equivalent in size to that of the R. meliloti 41 megaplasmid. Sizes of the other plasmids varied from 45 to &gt;600 kb. The major plasmid group (representing 30% of the whole population) consisted of isolates carrying a megaplasmid plus a large plasmid with an average size of 230 kb. To assess the validity of plasmid grouping, 32 isolates representative of 12 plasmid groups were further characterized on the basis of their genomic DNA restriction patterns and their insertion sequence (IS) fingerprints, using the indigenous insertion sequence ISRm1 as a probe. The DNA restriction patterns and IS fingerprints were highly distinctive among the 32 isolates examined since 20 and 16 types, respectively, were observed. These two characteristics are strongly correlated with the plasmid profiles of the strains, confirming the validity of plasmid grouping in assessing diversity of natural populations of R. meliloti. Plasmid profile analysis, although less discriminating than DNA restriction patterns or IS fingerprinting, is the method of choice when many isolates need to be typed. Key words: Rhizobium meliloti, ecology, diversity, field population, symbiotic effectiveness.

The Interplay of Communication Device Output Mode and Interaction Style between Nonspeaking Persons and their Speaking Partners

This study sought to determine how augmentative communication device output modes differentially affected various aspects of interactions between nonspeaking persons (NSPs) and their speaking partners (SPs). It was hypothesized that when an electronic output display (EOD) was added to a communication board, the semipermanent display of information would lessen the dyad's need to adopt specialized turn and message formulation conventions, permitting the NSP to construct more complex messages with fewer communication breakdowns. A series of 10 interactional teaching tasks were recorded for two adult male nonhandicapped dyads performing under the two output conditions (+/- EOD). Interaction transcripts were analyzed with regard to quantitative differences within and between dyads with respect to turn taking, message formulation, propositional content, and several types of insertion sequences (guessing, confirmation queries, message reformulations). With the exception of message reformulation, changes due to output mode were nonexistent or inconsistent for the variables measured within and across dyads. The addition of the EOD significantly lowered the rate of message reformulation and the total number of reformulation-related turns. Results are discussed with regard to research and clinical implications for augmentative communication.

Bombyx mori 28S ribosomal genes contain insertion elements similar to the Type I and II elements of Drosophila melanogaster.

We have examined the 28S ribosomal genes of the silkmoth, Bombyx mori, for the presence of insertion sequences. Two types of insertion sequences were found, each approximately 5 kb in length, which do not share sequence homology. Comparison of the nucleotide sequences of the junction regions with the uninserted gene reveals that one type of insertion has resulted in a 14 bp duplication of the 28S coding region at the insertion site. The location of this insertion and the 14 bp duplication are identical to that found in the Type I ribosomal insertion element of Drosophila melanogaster. The second type of insertion element is located at a site corresponding to approximately 75 bp upstream of the first type. The location of this insertion, the variability detected at its 5' junction, and a short region of sequence homology at its 3' junction suggest that it is related to the Type II element of D. melanogaster. This is the first example of a Type II-like rDNA insertion outside of sibling species of D. melanogaster, and the first example of a Type I-like rDNA insertion outside of the higher Diptera.