Molecular evidence for independent occurrence of IS6110 insertions at the same sites of the genome of Mycobacterium tuberculosis in different clinical isolates

Several characteristics of Mycobacterium tuberculosis (e.g., conserved genome and low growth rate) have severely restricted the study of the microorganism. The discovery of IS6110 raised hopes of overcoming these obstacles. However, our knowledge of this IS element is relatively limited; even its two basic characteristics (transposition mechanism and target site selection) are far from well understood. In this study, IS6110 insertions in ipl loci (iplA and iplB) in two collections of clinical isolates of M. tuberculosis from different geographic locations, one from Scotland and the other from Thailand, were investigated. Five different IS6110 insertions in the loci were identified: ipl-4::IS6110, ipl-5::IS6110, ipl-11::IS6110, ipl-12::IS6110, and ipl-13::IS6110. An attempt to establish the phylogenetic relationship of the isolates containing these insertions was unsuccessful, suggesting that some of these insertions may have arisen from more than one event. This possibility is further supported by the observation that IS6110 copies existed in the same site but with different orientations in different isolates, and the insertion site of ipl-1::IS6110 harbored IS6110 copies in both iplA and iplB in different strains. All these suggest the independent occurrence of IS6110 insertions at the same sites of the genome of M. tuberculosis in different clinical isolates. The implications of this finding are discussed.     

Evolutionary relationships among strains of Mycobacterium tuberculosis with few copies of IS6110

Molecular typing of Mycobacterium tuberculosis by using IS6110 shows low discrimination when there are fewer than five copies of the insertion sequence. Using a collection of such isolates from a study of the epidemiology of tuberculosis in London, we have shown a substantial degree of congruence between IS6110 patterns and both spoligotype and PGRS type. This indicates that the IS6110 types mainly represent distinct families of strains rather than arising through the convergent insertion of IS6110 into favored positions. This is supported by identification of the genomic sites of the insertion of IS6110 in these strains. The combined data enable identification of the putative evolutionary relationships of these strains, comprising three lineages broadly associated with patients born in South Asia (India and Pakistan), Africa, and Europe, respectively. These lineages appear to be quite distinct from M. tuberculosis isolates with multiple copies of IS6110.     

Analysis of sequence diversity among IS6110 sequence of Mycobacterium tuberculosis: possible implications for PCR based detection

The IS6110 belongs to the family of insertion sequences (IS) of the IS3 category. This insertion sequence was reported to be specific for Mycobacterium tuberculosis complex and hence is extensively exploited for laboratory detection of the agent of tuberculosis and for epidemiological investigations based on polymerase chain reaction. IS6110 is 1361-bp long and within this sequence different regions have been utilized as targets in the identification of M. tuberculosis by PCR. However, the results are not always consistent, specific and sensitive. In recent years, a few clinical investigations raised concerns over IS6110 specificity and sensitivity in the diagnosis of tuberculosis due to false-positive (homology with other target DNA besides M. tuberculosis) or false negative (due to absence of copies of IS6110) results with IS6110 specific primers. To unravel the variations in IS6110 sequences, an insilico analysis of IS6110 sequence of different strains of M. tuberculosis was carried out. Our results of comparative analysis of IS6110 insertion sequences of M. tuberculosis complex suggests that, IS6110 insertion sequences harbored variations in its sequence, which is evident from the phylogenetic analysis. Importantly, IS6110 sequence has divergence within the copies of same strain and formed different clusters. A list of IS6110 specific primers used in various clinical investigation of tuberculosis was obtained from the literature and their performance scrutinized. Our study emphasizes the need to develop PCR assays (multiplex format) targeting more than one region of the genome of M. tuberculosis.     

A genomic library-based amplification approach (GL-PCR) for the mapping of multiple IS6110 insertion sites and strain differentiation of Mycobacterium tuberculosis

Evidence suggests that insertion of the IS6110 element is not without consequence to the biology of Mycobacterium tuberculosis complex strains. Thus, mapping of multiple IS6110 insertion sites in the genome of biomedically relevant clinical isolates would result in a better understanding of the role of this mobile element, particularly with regard to transmission, adaptability and virulence. In the present paper, we describe a versatile strategy, referred to as GL-PCR, that amplifies IS6110-flanking sequences based on the construction of a genomic library. M. tuberculosis chromosomal DNA is fully digested with HincII and then ligated into a plasmid vector between T7 and T3 promoter sequences. The ligation reaction product is transformed into Escherichia coli and selective PCR amplification targeting both 5' and 3' IS6110-flanking sequences are performed on the plasmid library DNA. For this purpose, four separate PCR reactions are performed, each combining an outward primer specific for one IS6110 end with either T7 or T3 primer. Determination of the nucleotide sequence of the PCR products generated from a single ligation reaction allowed mapping of 21 out of the 24 IS6110 copies of two 12 banded M. tuberculosis strains, yielding an overall sensitivity of 87,5%. Furthermore, by simply comparing the migration pattern of GL-PCR-generated products, the strategy proved to be as valuable as IS6110 RFLP for molecular typing of M. tuberculosis complex strains. Importantly, GL-PCR was able to discriminate between strains differing by a single IS6110 band.     

Mechanisms regulating the copy numbers of six LTR retrotransposons in the genome of Drosophila melanogaster

There has been debate over the mechanisms that control the copy number of transposable elements in the genome of Drosophila melanogaster. Target sites in D. melanogaster populations are occupied at low frequencies, suggesting that there is some form of selection acting against transposable elements. Three main theories have been proposed to explain how selection acts against transposable elements: insertions of a copy of a transposable element are selected against; chromosomal rearrangements caused by ectopic exchange between element copies are selected against; or the process of transposition itself is selected against. The three theories give different predictions for the pattern of transposable element insertions in the chromosomes of D. melanogaster. We analysed the abundance of six LTR (long terminal repeat) retrotransposons on the X and fourth chromosomes of multiple strains of D. melanogaster, which we compare with the predictions of each theory. The data suggest that no one theory can account for the insertion patterns of all six retrotransposons. Comparing our results with earlier work using these transposable element families, we find a significant correlation between studies in the particular model of copy number regulation supported by the proportion of elements on the X for the different transposable element families. This suggests that different retrotransposon families are regulated by different mechanisms.     

IS6110-mediated deletion polymorphism in the direct repeat region of clinical isolates of Mycobacterium tuberculosis

This study investigates the phenomenon of IS6110-mediated deletion polymorphism in the direct repeat (DR) region of the genome of Mycobacterium tuberculosis. Clinical isolates and their putative predecessors were compared using a combination of DR region restriction fragment length polymorphism, IS6110 DNA fingerprinting, spoligotyping, and DNA sequencing, which allowed the mapping of chromosome structure and deletion junctions. The data suggest that adjacently situated IS6110 elements mediate genome deletion. However, in contrast to previous reports, deletions appear to be mediated by inversely oriented IS6110 elements. This suggests that these events may occur via mechanisms other than RecA-mediated homologous recombination. The results underscore the important role of IS6110-associated deletion hypervariability in driving M. tuberculosis genome evolution.     

结核分枝杆菌中插入序列的研究

用人型复合分枝杆菌的特异插入序列IS6110和IS1081制 备探针,对5种限制性内切酶消化的结核分枝杆菌DNA进行杂交。结果表明,经PvuⅡ酶消化 的结核分枝杆菌DNA,用IS6110制备的探针进行杂交呈现高度多态性,说明IS6110对于人型 复合分枝杆菌分型研究和结核病流行病学研究具有很大价值。用IS6110制备的317bp探针对4 6株人结核分枝杆菌分离株多态性分析研究证实,这些菌株呈现高度DNA多态性,而且所含拷 贝数也极为不同,一般含7～18个拷贝。     

Population Genetics and Molecular Evolution of DNA Sequences in Transposable Elements. I. A Simulation Framework

A population genetic simulation framework is developed to understand the behavior and molecular evolution of DNA sequences of transposable elements. Our model incorporates random transposition and excision of transposable element (TE) copies, two modes of selection against TEs, and degeneration of transpositional activity by point mutations. We first investigated the relationships between the behavior of the copy number of TEs and these parameters. Our results show that when selection is weak, the genome can maintain a relatively large number of TEs, but most of them are less active. In contrast, with strong selection, the genome can maintain only a limited number of TEs but the proportion of active copies is large. In such a case, there could be substantial fluctuations of the copy number over generations. We also explored how DNA sequences of TEs evolve through the simulations. In general, active copies form clusters around the original sequence, while less active copies have long branches specific to themselves, exhibiting a star-shaped phylogeny. It is demonstrated that the phylogeny of TE sequences could be informative to understand the dynamics of TE evolution.     

Multiple copies of IS10 in the Enterobacter cloacae MD36 chromosome.

Repetitive sequences were isolated and characterized as double-stranded DNA fragments by treatment with S1 nuclease after denaturation and renaturation of the total DNA of Enterobacter cloacae MD36. One repetitive sequence was identical to the nucleotide sequence of IS10-right (IS10R), which is the active element in the plasmid-associated transposon Tn10. Unexpectedly, 15 copies of IS10R were found in the chromosomal DNA of E. cloacae MD36. One copy of the central region of Tn10 was found in the total DNA of E. cloacae MD36. IS10Rs in restriction fragments isolated from the E. cloacae MD36 total DNA showed 9-bp duplications adjacent to the terminal sequences that are characteristic of Tn10 transposition. This result suggests that many copies of IS10R in E. cloacae MD36 are due to transposition of IS10R alone, not due to transposition of Tn10 or to DNA rearrangement. I also found nine copies of IS10 in Shigella sonnei HH109, two and four copies in two different natural isolates of Escherichia coli, and two copies in E. coli K-12 strain JM109 from the 60 bacterial strains that were examined. All dam sites in the IS10s in E. cloacae MD36 and S. sonnei HH109 were methylated. Tn10 and IS10 transpose by a mechanism in which the element is excised from the donor site and inserted into the new target site without significant replication of the transposing segment; thus, the copy numbers of the elements in the cell are thought to be unchanged in most circumstances. Accumulation of IS10 copies in E. cloacae MD36 has interesting evolutionary implications.     

Cotranslational control of DNA transposition: a window of opportunity

Transposable elements are important in genome dynamics and evolution. Bacterial insertion sequences (IS) constitute a major group in number and impact. Understanding their role in shaping genomes requires knowledge of how their transposition activity is regulated and interfaced with the host cell. One IS regulatory phenomenon is a preference of their transposases (Tpases) for action on the element from which they are expressed (cis) rather than on other copies of the same element (trans). Using IS911, we show in vivo that activity in cis was ~200 fold higher than in trans. We also demonstrate that a translational frameshifting pause signal influences cis preference presumably by facilitating sequential folding and cotranslational binding of the Tpase. In vitro, IS911 Tpase bound IS ends during translation but not after complete translation. Cotranslational binding of nascent Tpase permits tight control of IS proliferation providing a mechanistic explanation for cis regulation of transposition involving an unexpected partner, the ribosome.     

Size matters: non-LTR retrotransposable elements and ectopic recombination in Drosophila

The Drosophila melanogaster genome contains approximately 100 distinct families of transposable elements (TEs). In the euchromatic part of the genome, each family is present in a small number of copies (5-150 copies), with individual copies of TEs often present at very low frequencies in populations. This pattern is likely to reflect a balance between the inflow of TEs by transposition and the removal of TEs by natural selection. The nature of natural selection acting against TEs remains controversial. We provide evidence that selection against chromosome abnormalities caused by ectopic recombination limits the spread of some TEs. We also demonstrate for the first time that some TE families in the Drosophila euchromatin appear to be only marginally affected by purifying selection and contain many copies at high population frequencies. We argue that TEs in these families attain high population frequencies and even reach fixation as a result of low family-wide transposition rates leading to low TE copy numbers and consequently reduced strength of selection acting on individual TE copies. Fixation of TEs in these families should provide an upward pressure on the size of intergenic sequences counterbalancing rapid DNA loss through small deletions. Copy-number-dependent selection on TE families caused by ectopic recombination may also promote diversity among TEs in the Drosophila genome.     

Characterization of in vitro constructed IS30-flanked transposons

In order to facilitate functional studies on the mobile genetic element IS30, a resident of the Escherichia coli chromosome, transposon structures with two copies of IS30 flanking the chloramphenicol-resistance gene cat were constructed in vitro. Transposons containing IS30 as direct repeats (Tn2700 and Tn2702) transpose from multicopy plasmids into the genome of phage P1-15, thus giving rise to special transduction for cat with frequencies between 10(-5) and 10(-8)/plaque-forming unit. In contrast, transposon structures with IS30 in inverted repeat (Tn2701 and Tn2703) showed no detectable (less than 10(-9] transposition activity in vivo. By restriction analysis, two insertion sites of Tn2700 and Tn2702 on the phage P1-15 genome were indistinguishable from those observed earlier with a single copy of the IS30 element. These two insertion sites were used several times independently by Tn2700 and Tn2702. This confirms the non-random target selection by the element and it indicates that transposition of Tn2700 and Tn2702 follows the same rules as that of IS30.     

Stimulation of transposition of the Mycobacterium tuberculosis insertion sequence IS6110 by exposure to a microaerobic environment

The Mycobacterium tuberculosis-specific insertion sequence IS6110/986 has been widely used as a probe because of the multiple polymorphism observed among different strains. To investigate transposition of IS6110, a series of artificially constructed composite transposons containing IS6110 and a kanamycin resistance marker were constructed. The composite transposons were inserted into a conditionally replicating, thermosensitive, Escherichia coli-mycobacterial shuttle vector and introduced into M. smegmatis mc2155. Lawns of transformants were grown at the permissive temperature on kanamycin-supplemented agar and subsequently prevented from further growth by shifting to the non-permissive temperature. Under normal atmospheric conditions, kanamycin-resistant papillae appeared after only about 5-6 weeks of incubation. However, these events were not associated with transposon mobilization. In contrast, lawns that were exposed to a 48 h microaerobic shock generated kanamycin-resistant papillae after only 6-14 days. These events were generated by conservative transposition of the IS6110 composite transposon into the M. smegmatis chromosome, with loss of the shuttle vector. In common with other IS3 family elements, transposition of IS6110 is thought to be controlled by translational frameshifting. However, we were unable to detect any significant frameshifting within the putative frameshifting site of IS6110, and the level of frameshifting was not affected by microaerobic incubation. The finding that transposition of IS6110 is stimulated by incubation at reduced oxygen tensions may be relevant to transposition of IS6110 in M. tuberculosis harboured within TB lesions.     

Mapping of IS6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticity

Southern hybridization was used in combination with IS6110 insertion-locus-specific probes in a comparative study to determine the structure of chromosomal domains flanking IS6110 elements in clinical isolates of Mycobacterium tuberculosis. The resulting restriction fragment length polymorphism (RFLP) data demonstrated three mutational mechanisms responsible for the polymorphisms observed: IS6110 insertion, chromosomal mutation and deletion. The frequency of IS6110 insertion within many of the chromosomal regions demonstrates that preferential integration regions are common in M. tuberculosis. Mapping the IS6110 insertion positions and chromosomal deletions in relation to the M. tuberculosis H37Rv and M. bovis BCG genome sequences reveals numerous disruptions of predicted open reading frames (ORFs). A phylogenetic tree, based on the mutational data, showed a number of independently evolving lineages of M. tuberculosis, while analysis of the mutational events occurring at each branch point suggests both divergent and convergent evolution. A significant positive correlation was demonstrated between the mutation rate and the frequency of occurrence of different isolates in families of strains, suggesting that evolution may impact on strain 'fitness' or that strain proliferation may increase the chance of mutation. We conclude that the genome of clinical isolates of M. tuberculosis continues to evolve.     

Nucleotide sequence and characterization of a repetitive DNA element from the genome of Bordetella pertussis with characteristics of an insertion sequence

A repeating element of DNA has been isolated and sequenced from the genome of Bordetella pertussis. Restriction map analysis of this element shows single internal ClaI, SphI, BstEII and SalI sites. Over 40 DNA fragments are seen in ClaI digests of B. pertussis genomic DNA to which the repetitive DNA sequence hybridizes. Sequence analysis of the repeat reveals that it has properties consistent with bacterial insertion sequence (IS) elements. These properties include its length of 1053 bp, multiple copy number and presence of 28 bp of near-perfect inverted repeats at its termini. Unlike most IS elements, the presence of this element in the B. pertussis genome is not associated with a short duplication in the target DNA sequence. This repeating element is not found in the genomes of B. parapertussis or B. bronchiseptica. Analysis of a DNA fragment adjacent to one copy of the repetitive DNA sequence has identified a different repeating element which is found in nine copies in B. parapertussis and four copies in B. pertussis, suggesting that there may be other repeating DNA elements in the different Bordetella species. Computer analysis of the B. pertussis repetitive DNA element has revealed no significant nucleotide homology between it and any other bacterial transposable elements, suggesting that this repetitive sequence is specific for B. pertussis.     

Estimating change rates of genetic markers using serial samples: applications to the transposon IS6110 in Mycobacterium tuberculosis

In infectious disease epidemiology, it is useful to know how quickly genetic markers of pathogenic agents evolve while inside hosts. We propose a modular framework with which these genotype change rates can be estimated. The estimation scheme requires a model of the underlying process of genetic change, a detection scheme that filters this process into observable quantities, and a monitoring scheme that describes the timing of observations. We study a linear "birth-shift-death" model for change in transposable element genotypes, obtaining maximum-likelihood estimators for various detection and monitoring schemes. The method is applied to serial genotypes of the transposon IS6110 in Mycobacterium tuberculosis. The estimated birth rate of 0.0161 (events per copy of the transposon per year) and death rate of 0.0108 are both significantly larger than the estimated shift rate of 0.0018. The sum of these estimates, which corresponds to a "half-life" of 2.4 years for a typical strain that has 10 copies of the element, substantially exceeds a previous estimate of 0.0135 total changes per copy per year. We consider experimental design issues that enable the precision of estimates to be improved. We also discuss extensions to other markers and implications for molecular epidemiology.     

新型Taq Man-MGB探针在结核分枝杆菌实时PCR检测中的应用

为建立一种比现有方法敏感、准确性高、重复性好的结核分枝杆菌DNA定性定量检测方法 ,以TaqMan探针技术为基础 ,运用TaqMan MGB探针 ,实时检测临床标本中的结核分枝杆菌DNA .用来自临床标本的DNA及克隆于载体的IS6 1 1 0序列检测所建立方法的有效性 .结果显示 ,所建立方法的最低检测限度为 1个基因拷贝 反应 ,在每反应 1 0 0 ～ 1 0 8拷贝范围内 ,Ct 值同DNA量的对数呈线性关系 .同一模板不同时间或同一时间不同管内扩增 ,所得Ct 值恒定 .用该方法检测 37例结核分枝杆菌培养阳性的痰液标本 ,敏感度为 1 0 0 % ;用该方法检测 1 6例TB系列阴性参考品 ,特异性为1 0 0 % .结果表明 ,所建立的方法是用于结核分枝杆菌定性定量检测较理想的方法     

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.     

Immunity to repeated transposition of the insertion sequence IS21

The ability of pBR325 derivatives carrying a copy of IS21-element to accept the second copy of this element from plasmid pRP19.6, a temperature-sensitive for replication mutant of RPI containing the duplicated IS21 was studied. It was shown that the frequency of IS21 transposition into plasmids pBR32S::IS21 differing by localization IS21 was lower by two orders of magnitude as compared to that of pBR325. The restriction endonuclease analysis revealed that the insertion of the second copy of IS21 resulted in the formation of pBR325 derivatives carrying the tandem repeated copies of IS21. It was also shown that the plasmids pBR325::IS21 were capable of increasing the frequency of pRP19.6 insertion into the bacterial chromosome from 3-9 to 200-300 times depending on IS21 localization. On the basis of the results obtained and literature data the possible mechanism of the transposition immunity is discussed.     

A branching-process model for the evolution of transposable elements incorporating selection

We have formulated a very general mathematical model to analyze the evolution of transposable genetic elements in prokaryotic populations. Transposable genetic elements are DNA sequences able to replicate and insert copies of themselves at new locations in the genome. This work characterizes the equilibrium distribution of copy number under the influence of copy number-dependent selection, transposition and deletion. Our principal results concern the equilibrium distribution of copy number in response to various selective regimes. For particular transposition patterns (e.g. unregulated transposition or copy number-dependent transposition), equilibrium distributions are calculated numerically for a variety of specific selection patterns. Selection is quantified through specification of the expected number of offspring for individuals of each type, which is generally a non-increasing function of copy number, in accord with the usual evolutionary speculations.