DO PHAGES EFFICIENTLY SHUTTLE TRANSPOSABLE ELEMENTS AMONG PROKARYOTES?

The distribution, dynamics, and evolution of insertion sequences (IS), the most frequent class of prokaryotic transposable elements, are conditioned by their ability to horizontally transfer between cells. IS horizontal transfer (HT) requires shuttling by other mobile genetic elements. It is widely assumed in the literature that these vectors are phages and plasmids. By examining the relative abundance of IS in 454 plasmid and 446 phage genomes, we found that IS are very frequent in plasmids but, surprisingly, very rare in phages. Our results indicate that IS rarity in phages reflects very strong and efficient postinsertional purifying selection, mainly caused by a higher density of deleterious insertion sites in phages compared to plasmids. As they do not tolerate IS insertions, we conclude that phages may be rather poor vectors of IS HT in prokaryotes, in sharp contrast with the conventional view.     

Novel insertion sequence IS1380 from Acetobacter pasteurianus is involved in loss of ethanol-oxidizing ability.

Acetobacter pasteurianus NCI1380, a thermophilic strain isolated from the surface culture of acetic acid fermentation, showed genetic instability to produce at high frequency spontaneous mutants which were deficient in ethanol oxidation because of the loss of alcohol dehydrogenase activity. Southern hybridization experiments with the cloned alcohol dehydrogenase-cytochrome c gene cluster as the probe showed insertion of an unknown DNA fragment into a specific position in the cytochrome c gene in most of the mutant strains. Cloning and sequencing analyses revealed that the inserted sequence was 1,665 bp in length and had a terminal inverted repeat of 15 bp. In addition, this inserted sequence was found to generate a 4-bp duplication at the inserted site upon transposition. The target site specificity was not very strict, but a TCGA sequence appeared to be preferentially used. The inserted sequence contains two long open reading frames of 461 and 222 amino acids which are overlapped and encoded by different strands. Although these open reading frames showed no homology to any protein registered in the DNA data bases, the longer open reading frame contained many basic amino acids (87 of 461), as was observed with transposases of so-called insertion sequence (IS) elements. All of these characteristics are typical of IS elements, and the sequence was named IS1380. The copy number of IS1380 in a cell of A. pasteurianus NCI1380 was estimated to be about 100. Several strains of acetic acid bacteria also contained IS1380 at high copy numbers. These results suggest that IS1380 is associated with the genetic loss of ethanol-oxidizing ability as well as the genetic instability of acetic acid bacteria in general.     

Understanding Genetic Factors in Idiopathic Scoliosis, a Complex Disease of Childhood

Idiopathic scoliosis (AIS) is the most common pediatric spinal deformity, affecting ~3% of children worldwide. AIS significantly impacts national health in the U. S. alone, creating disfigurement and disability for over 10% of patients and costing billions of dollars annually for treatment. Despite many investigations, the underlying etiology of IS is poorly understood. Twin studies and observations of familial aggregation reveal significant genetic contributions to IS. Several features of the disease including potentially strong genetic effects, the early onset of disease, and standardized diagnostic criteria make IS ideal for genomic approaches to finding risk factors. Here we comprehensively review the genetic contributions to IS and compare those findings to other well-described complex diseases such as Crohn’s disease, type 1 diabetes, psoriasis, and rheumatoid arthritis. We also summarize candidate gene studies and evaluate them in the context of possible disease aetiology. Finally, we provide study designs that apply emerging genomic technologies to this disease. Existing genetic data provide testable hypotheses regarding IS etiology, and also provide proof of principle for applying high-density genome-wide methods to finding susceptibility genes and disease modifiers.     

Isopropylbenzene catabolic pathway in Pseudomonas putida RE204: nucleotide sequence analysis of the ipb operon and neighboring DNA from pRE4

Pseudomonas putida RE204 employs a set of plasmid-specified enzymes in the catabolism of isopropylbenzene (cumene) and related alkylbenzenes. A 21,768 bp segment of the plasmid pRE4, whose sequence is discussed here, includes the ipb (isopropylbenzene catabolic) operon as well as associated genetic elements. The ipb operon, ipbAaAbAcAdBCEGFHD, encodes enzymes catalyzing the conversion of isopropylbenzene to isobutyrate, pyruvate, and acetyl-coenzyme A as well as an outer membrane protein (IpbH) of uncertain function. These gene products are 75 to 91% identical to those encoded by other isopropylbenzene catabolic operons and are somewhat less similar to analogous proteins of related pathways for the catabolism of mono-substituted benzenes. Upstream of ipbAa, ipbR encodes a positive regulatory protein which has about 56% identity to XylS regulatory proteins of TOL (xylene/toluate) catabolic plasmids. This similarity and that of the DNA sequence in the proposed ipb operator-promoter region (ipbOP) to the same region of the xyl meta operon (xylOmPm) suggest that, although the IpbR and XylS regulatory proteins recognize very different inducers, their interactions with DNA to activate gene expression are similar. Upstream of ipbR is an 1196 bp insertion sequence, IS1543, related to IS52 and IS1406. Separating ipbR from ipbAa are 3 additional tightly clustered IS elements. These are IS1544, related to IS1543, IS52, and other members of the IS5 family; IS1545, related to IS1240; and IS1546, related to IS1491. Encompassing the ipb catabolic genes and the other genetic elements and separated from each other by 18,492 bp, are two identical, directly repeated 1007 bp DNA segments. Homologous recombination between these segments appears to be responsible for the occasional deletion of the intervening DNA from pRE4.     

Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease and may contribute to the onset and development of Crohn's disease in humans. Rapid detection of Map is fundamental because of its reported isolation from pasteurized milk and its potential for transmission through environmental sources. In this study, we developed two independent real-time quantitative PCR assays targeting the IS900 genetic insertion sequence and the F57 sequence, which proved capable of detecting and quantifying Map DNA. Validation and standardization of the developed methods were performed by evaluating diagnostic trueness, precision, and accuracy of the techniques. Specificity of the IS900 and F57 methods was verified in both in silico and experimental studies. The assays were found to be very accurate and precise with high repeatability and reproducibility. Moreover, the two real-time assays were very specific for Map, discriminating most of mycobacterial and nonmycobacterial species.     

Large-Scale Genomic Analysis Suggests a Neutral Punctuated Dynamics of Transposable Elements in Bacterial Genomes

Insertion sequences (IS) are the simplest and most abundant form of transposable DNA found in bacterial genomes. When present in multiple copies, it is thought that they can promote genomic plasticity and genetic exchange, thus being a major force of evolutionary change. The main processes that determine IS content in genomes are, though, a matter of debate. In this work, we take advantage of the large amount of genomic data currently available and study the abundance distributions of 33 IS families in 1811 bacterial chromosomes. This allows us to test simple models of IS dynamics and estimate their key parameters by means of a maximum likelihood approach. We evaluate the roles played by duplication, lateral gene transfer, deletion and purifying selection. We find that the observed IS abundances are compatible with a neutral scenario where IS proliferation is controlled by deletions instead of purifying selection. Even if there may be some cases driven by selection, neutral behavior dominates over large evolutionary scales. According to this view, IS and hosts tend to coexist in a dynamic equilibrium state for most of the time. Our approach also allows for a detection of recent IS expansions, and supports the hypothesis that rapid expansions constitute transient events—punctuations—during which the state of coexistence of IS and host becomes perturbated.     

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.     

Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence

Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identified distinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans and M. marinum, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS2404 and IS2606, have recently been reported in M. ulcerans, and significantly, these elements are not present in M. marinum. Hybridization of the AseI restriction fragments from M. ulcerans with IS2404 and IS2606 indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently diverged from M. marinum by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of M. tuberculosis, where species diversity is being driven mainly by the activity of mobile DNA elements.     

F-statistics of clonal diploids structured in numerous demes

Abstract The expected apportionment of genetic diversity in diploid clonal organisms structured in numerous subpopulations is explored. Under the specific assumptions considered, corresponding, for instance, to clonal pathogens infecting a large number of hosts, the co-ancestry between individuals within subpopulations is the only nontrivial quantity. Thus the population structure can be fully described either by F(ST) or F(IS), as F(ST) = -F(IS)/(1 - F(IS)). We show that, for most of the parameter space considered, including simulations where equilibrium is not reached and/or where homoplasy is high, the number of effective migrants is most accurately estimated as Nm = -(1 + F(IS))/4F(IS). We further propose a criterion to test for the absence of cryptic sexual reproduction based on the F-statistics F(IS) and F(ST), which is applied to three previously published empirical data sets.     

Association of the genetic polymorphisms in immunoinflammatory microRNAs with risk of ischemic stroke and subtypes in an Iranian population

Stroke is one of the most common type of cerebrovascular disease threatening human health and life with high mortality, disability, and morbidity. Ischemic stroke (IS) is determined to be a complex disease containing a group of heterogeneous disorders with various environmental and genetic risk factors. This study evaluated the polymorphisms of microRNAs involved in inflammatory routes leading to stroke in an Iranian population. This study evaluated the associations of hsa-mir-608 C/G rs4919510, hsa-mir-499 A/G rs3746444, and hsa-mir-145 C/T rs190323149 polymorphisms in precursor miRNAs with the risk of IS. These microRNA polymorphisms were analyzed in 470 patients with IS and 489 control subjects. The TOAST criteria was applied for IS subtypes classification. The frequency of the allele G of hsa-mir-499/rs3746444 A/G revealed significant association with IS in comparison with controls ( p < 0.0001, OR = 1.838, 95% CI = 1.406–2.401). Increased IS risks were associated with hsa-mir-499/ rs3746444 A/G genotypes in diverse genetic model (homozygote comparison: p = 0.004, OR = 2.136, 95% CI = 1.269–3.597; heterozygote comparison: p = 0.029, OR = 1.373, 95% CI = 1.033–1.825). Statistical analysis in IS subtypes showed that cardio-embolic patients compared with other subtypes (large artery atherosclerosis and lacunar) had higher frequency of G allele (LAA vs. CEI, p = 0.017; LAC vs. CEI, p = 0.009), AG genotype (LAA vs. CEI, p = 0.016; LAC vs. CEI, p = 0.013). Nevertheless, this study did not find any association between the alleles and genotypes of mir-608 C/G rs4919510 SNP and IS, respectively ( p > 0.05). The current investigation provided verification that hsa-mir-499 rs3746444 A/G polymorphism may be associated with a significantly increased risk of IS in an Iranian population.     

Relaxed natural selection alone does not permit transposable element expansion within 4,000 generations in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Insertion sequences (ISs) are transposable genetic elements in bacterial genomes. IS elements are common among bacteria but are generally rare within free-living species, probably because of the negative fitness effects they have on their hosts. Conversely, ISs frequently proliferate in intracellular symbionts and pathogens that recently transitioned from a free-living lifestyle. IS elements can profoundly influence the genomic evolution of their bacterial hosts, although it is unknown why they often expand in intracellular bacteria. We designed a laboratory evolution experiment with Escherichia coli K-12 to test the hypotheses that IS elements often expand in intracellular bacteria because of relaxed natural selection due to (1) their generally small effective population sizes (N _e) and thus enhanced genetic drift, and (2) their nutrient rich environment, which makes many biosynthetic genes unnecessary and thus selectively neutral territory for IS insertion. We propagated 12 populations under four experimental conditions: large N _e versus small N _e, and nutrient rich medium versus minimal medium. We found that relaxed selection over 4,000 generations was not sufficient to permit IS element expansion in any experimental population, thus leading us to hypothesize that IS expansion in intracellular symbionts may often be spurred by enhanced transposition rates, possibly due to environmental stress, coupled with relaxed natural selection.     

Characterization of an insertion sequence element associated with genetically diverse plant pathogenic Streptomyces spp

Streptomycetes are common soil inhabitants, yet few described species are plant pathogens. While the pathogenicity mechanisms remain unclear, previous work identified a gene, nec1, which encodes a putative pathogenicity or virulence factor. nec1 and a neighboring transposase pseudogene, ORFtnp, are conserved among unrelated plant pathogens and absent from nonpathogens. The atypical GC content of nec1 suggests that it was acquired through horizontal transfer events. Our investigation of the genetic organization of regions adjacent to the 3' end of nec1 in Streptomyces scabies 84.34 identified a new insertion sequence (IS) element, IS1629, with homology to other IS elements from prokaryotic animal pathogens. IS1629 is 1,462 bp with 26-bp terminal inverted repeats and encodes a putative 431-amino-acid (aa) transposase. Transposition of IS1629 generates a 10-bp target site duplication. A 77-nucleotide (nt) sequence encompassing the start codon and upstream region of the transposase was identified which could function in the posttranscritpional regulation of transposase synthesis. A functional copy of IS1629 from S. turgidiscabies 94.09 (Hi-C-13) was selected in the transposon trap pCZA126, through its insertion into the lambda cI857 repressor. IS1629 is present in multiple copies in some S. scabies strains and is present in all S. acidiscabies and S. turgidiscabies strains examined. A second copy of IS1629 was identified between ORFtnp and nec1 in S. acidiscabies strains. The diversity of IS1629 hybridization profiles was greatest within S. scabies. IS1629 was absent from the 27 nonpathogenic Streptomyces strains tested. The genetic organization and nucleotide sequence of the nec1-IS1629 region was conserved and identical among representatives of S. acidiscabies and S. turgidiscabies. These findings support our current model for the unidirectional transfer of the ORFtnp-nec1-IS1629 locus from IS1629-containing S. scabies (type II) to S. acidiscabies and S. turgidiscabies.     

Single nucleotide polymorphisms and disease gene mapping

Single nucleotide polymorphisms are the most important and basic form of variation in the genome, and they are responsible for genetic effects that produce susceptibility to most autoimmune diseases. The rapid development of databases containing very large numbers of single nucleotide polymorphisms, and the characterization of haplotypes and patterns of linkage disequilibrium throughout the genome, provide a unique opportunity to advance association strategies in common disease rapidly over the next few years. Only the careful use of these strategies and a clear understanding of their statistical limits will allow novel genetic determinants for many of the common autoimmune diseases to be determined.     

Individual diet specialisation in sparrows is driven by phenotypic plasticity in traits related to trade‐offs in animal performance

Individual diet specialisation (IS) is frequent in many animal taxa and affects population and community dynamics. The niche variation hypothesis (NVH) predicts that broader population niches should exhibit greater IS than populations with narrower niches, and most studies that examine the ecological factors driving IS focus on intraspecific competition. We show that phenotypic plasticity of traits associated with functional trade‐offs is an important, but unrecognised mechanism that promotes and maintains IS. We measured nitrogen isotope (δ¹⁵N) and digestive enzyme plasticity in four populations of sparrows (Zonotrichia capensis) to explore the relationship between IS and digestive plasticity. Our results show that phenotypic plasticity associated with functional trade‐offs is related in a nonlinear fashion with the degree of IS and positively with population niche width. These findings are opposite to the NVH and suggest that among individual differences in diet can be maintained via acclimatisation and not necessarily require a genetic component.     

新型抗生素抗性基因传播元件ISCR及其生态风险

抗生素抗性基因(antibiotic resistance genes, ARGs)作为一种新型的环境污染物,成为多个学科关注的焦点.其在不同环境介质中的扩散和传播具有极大的环境危害性,对人类健康造成严重威胁.插入序列共同区(insertion sequence common region, ISCR),是一种新发现的抗性基因传播元件,因其特殊的遗传结构,能够通过滚环复制及同源重组等机制移动邻近的任何DNA序列,是ARGs在不同DNA分子或不同种属细菌间水平传播的高效媒介.目前世界上发现了27种ISCR元件.大量间接证据表明,ISCR可能与许多耐药基因的移动和扩散有关,特别是多重耐药性(multiple drug resistance, MDR)形成与传播.因此,ISCR很可能是抗生素抗性基因在环境中扩散传播的关键因子.本文就ARGs水平传播、ISCR结构特征、ISCR种类及其相关ARGs及其研究方法等进行综述,并揭示ISCR元件可能的生态风险,提出了今后的研究重点,以期为今后深入开展相关研究打下基础.     

Linkage disequilibrium in natural populations of Trypanosoma cruzi (flagellate), the agent of Chagas' disease

We have studied linkage disequilibrium in natural populations of Trypanosoma cruzi, the agent of Chagas' disease, by analyzing (i) a set of 524 stocks from the whole geographical range of the parasite, characterized at four gene loci coding for enzymes; (ii) a subsample of 121 stocks characterized at 12 enzyme loci; and (iii) a subset of 386 stocks from six locations in Bolivia, characterized by four enzyme loci. Our results show that the linkage disequilibrium reaches the maximum possible value, given the observed allelic frequencies, for almost all the locus pairs. This result is most consistent with the hypothesis that genetic recombination is absent or very rare in T. cruzi natural populations. Partition of the linkage disequilibrium variance for the six Bolivian populations shows that both inter- and intrapopulation components are substantial and that the relationships among the components are D2IS less than D2ST, and D'2IS less than D'2ST. These inequalities are interpreted as the result of an interplay between genetic drift, rare or absent mating, and clonal selection in generating linkage disequilibrium in T. cruzi populations.     

Natural transposon mutagenesis of clinical isolates of Mycobacterium tuberculosis: how many genes does a pathogen need?

Transposable elements can affect an organism's fitness through the insertional inactivation of genes and can therefore be used to identify genes that are nonessential for growth in vitro or in animal models. However, these models may not adequately represent the genetic requirements during chains of human infection. We have therefore conducted a genome-wide survey of transposon mutations in Mycobacterium tuberculosis isolates from cases of human infection, identifying the precise, base-specific insertion sites of the naturally occurring transposable element IS6110. Of 294 distinct insertions mapped to the strain H37Rv genome, 180 were intragenic, affecting 100 open reading frames. The number of genes carrying IS6110 in clinical isolates, and hence apparently not essential for infection and transmission, is very much lower than the estimates of nonessential genes derived from in vitro studies. This suggests that most genes in M. tuberculosis play a significant role in human infection chains. IS6110 insertions were underrepresented in genes associated with virulence, information pathways, lipid metabolism, and membrane proteins but overrepresented in multicopy genes of the PPE family, genes of unknown function, and intergenic sequences. Population genomic analysis of isolates recovered from an organism's natural habitat is an important tool for determining the significance of genes or classes of genes in the natural biology of an organism.     

Distribution and activity of IS elements in Streptococcus mutans

Insertion sequence (IS) elements are widely distributed selfmobilizing genetic elements that can affect genetic changes by integrating into the chromosome, mediating genetic rearrangements and facilitating horizontal gene transfer. Three members of the IS3 family have been identified in Streptococcus mutans: IS199 was discovered by its transposition while ISSmu1 and ISSmu2 were predicted from the genome sequence of S. mutans UA159. Sixty-eight strains of S. mutans were screened by PCR for carriage of the IS elements IS199, ISSmu1 and ISSmu2. Twenty-seven (30%) of the strains were positive for IS199, 33 (49%) were positive for ISSmu1 and 51 (75%) carried ISSmu2. All three IS were found in 11 strains, two were found in various combinations in 31, one was found in 16, while only 10 strains had none of the three IS for which we screened. ISSmu1 was demonstrated to be capable of transposition at a low frequency but no transposition of ISSmu2 was observed.     

Insertion sequence inversions mediated by ectopic recombination between terminal inverted repeats

Transposable elements are widely distributed and diverse in both eukaryotes and prokaryotes, as exemplified by DNA transposons. As a result, they represent a considerable source of genomic variation, for example through ectopic (i.e. non-allelic homologous) recombination events between transposable element copies, resulting in genomic rearrangements. Ectopic recombination may also take place between homologous sequences located within transposable element sequences. DNA transposons are typically bounded by terminal inverted repeats (TIRs). Ectopic recombination between TIRs is expected to result in DNA transposon inversions. However, such inversions have barely been documented. In this study, we report natural inversions of the most common prokaryotic DNA transposons: insertion sequences (IS). We identified natural TIR-TIR recombination-mediated inversions in 9% of IS insertion loci investigated in Wolbachia bacteria, which suggests that recombination between IS TIRs may be a quite common, albeit largely overlooked, source of genomic diversity in bacteria. We suggest that inversions may impede IS survival and proliferation in the host genome by altering transpositional activity. They may also alter genomic instability by modulating the outcome of ectopic recombination events between IS copies in various orientations. This study represents the first report of TIR-TIR recombination within bacterial IS elements and it thereby uncovers a novel mechanism of structural variation for this class of prokaryotic transposable elements.