Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains.

Gene probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.     

Restriction Fragment Length Polymorphism Analysis of Epidemiologically Related Mycobacterium tuberculosis Isolates

Restriction fragment length polymorphism (RFLP) analysis of a large number of Japanese isolates of Mycobacterium tuberculosis, containing isolates from small outbreaks of M. tuberculosis infection, and clinical isolates of M. bovis BCG, was carried out using a DNA probe derived from the insertion sequence IS986. Clinical isolates of M. tuberculosis had a high degree of RFLP. The occurrences of the IS element varied from 1 to 19, the majority of isolates having 8 to 15 copies. Very similar fingerprints, however, were seen among strains isolated in the Kanto district. In particular, 3 strains were of the same pattern with or without an additional band. Similarity of the banding patterns of strains islated in the same district was observed in other areas. Six groups of strains, each group arising from a suspected common source of infection, were analyzed. Of these, 5 showed identical fingerprints within each group, but one showed different fingerprints. RFLP patterns of three strains isolated from individuals with lymphadenitis developed about two months after BCG vaccination, and one strain isolated from a bladder cancer patient with BCG instillation therapy were identical to those of BCG-Tokyo which had been used for the vaccination and therapy. These results confirm that RFLP analysis using IS986 is a suitable tool for epidemiology of tuberculosis.     

IS150: distribution, nucleotide sequence and phylogenetic relationships of a new E. coli insertion element.

Recently we identified the new insertion (IS) sequence IS150 in various strains of Escherichia coli K-12. We have screened other strains of E. coli and Salmonella typhimurium for the presence of homologous sequences. The strains of E. coli K-12 and W tested contain one or more copies of homology to IS150. We have also determined the complete nucleotide sequence of a copy of IS150 inserted into IS1. Comparison of nucleotide and deduced amino acid sequences of IS150, IS2, IS3, IS51, IS600 and IS629 reveals significant homologies suggesting that these elements are members of a family of phylogenetically related insertion sequences.     

The presence of insertion elements<Emphasis Type="Italic">IS</Emphasis>861 and<Emphasis Type="Italic">IS</Emphasis>1548 in group B streptococci

The presence of insertion elements (IS) IS861 and IS1548 in the collection of 211 Streptococcus agalactiae strains isolated from pregnant women and dairy cows was assayed. IS861 was found in 67 human strains (59%) and 36 bovine strains (37%), IS1548 in 13 human strains (12%) and 16 bovine strains (16%). Two combinations, IS861+ IS1548- and IS861- IS1548-, were widely distributed in both human and bovine strains. The copy number and the restriction fragment length polymorphism (RFLP) of the two IS were determined in human group B streptococcus (GBS) strains. A minimum of 8 copies of IS1548 were detected in GBS strains while the copy number of IS861 varied from 1 to 9. The number of different hybridizing patterns with IS861 and IS1548 probes was 9 and 6, respectively. These hybridization patterns were divided into several clusters. All strains with IS were also clustered according to pulsed field-gel electrophoresis (PFGE) patterns. A correlation was found between the results of PFGE- and IS-based clustering.     

Identification of a novel insertion sequence element in Streptococcus agalactiae. bspeller@imib.rwth-aachen.de

Gain and loss of bacterial pathogenicity is often associated with mobile genetic elements. A novel insertion sequence (IS) element designated ISSa4 was identified in Streptococcus agalactiae (group B streptococci). The 963bp IS element is flanked by 25bp perfect inverted repeats and led to the duplication of a 9bp target sequence at the insertion site. ISSa4 contains one open reading frame coding for a putative transposase of 287 aa and exhibits closest similarities to insertion elements of the IS982 family which has previously not been identified in streptococci. Analysis of different S. agalactiae strains showed that the copy number of ISSa4 in S. agalactiae varies significantly between strains. The S. agalactiae strain with the highest copy number of ISSa4 was nonhemolytic and harbored one copy inserted in cylB, which encodes the membrane-spanning domain of the putative hemolysin transporter (Spellerberg et al., 1999. Identification of genetic determinants for the hemolytic activity of Streptococcus agalactiae by ISS1 transposition. J. Bacteriol. 181, 3212-3219). Determination of the distribution of ISSa4 in different S. agalactiae strains revealed that ISSa4 could be detected only in strains isolated after 1996, which might indicate a recent acquisition of this novel insertion element by S. agalactiae.     

Is103, a New Insertion Element in Escherichia Coli: Characterization and Distribution in Natural Populations

IS103 is a previously unknown insertion sequence found in Escherichia coli K12. We have sequenced IS103 and find that it is a 1441-bp element that consists of a 1395-bp core flanked by imperfect 23-bp inverted repeats. IS103 causes a 6-bp duplication of the target sequence into which it inserts. There is a single copy of IS103 present in wild-type E. coli K12 strain HfrC. In strain X342 and its descendents there are two additional copies, one of which is located within the bglF gene. IS103 is capable of excising from within bglF and restoring function of that gene. IS103 exhibits 44% sequence identity with IS3, suggesting that the two insertion sequences are probably derived from a common ancestor. We have examined the distribution of IS103 in the chromosomes and plasmids of the ECOR collection of natural isolates of E. coli. IS103 is found in 36 of the 71 strains examined, and it strongly tends to inhabit plasmids rather than chromosomes. Comparison of the observed distribution of IS103 with distributions predicted by nine different models for the regulation of transposition according to copy number and of the effects of copy number on fitness suggest that transposition of IS103 is strongly regulated and that it has only minor effects on fitness. The strong clustering of IS103 within one phylogenetic subgroup of the E. coli population despite its presence on plasmids suggests that plasmids tend to remain within closely related strains and that transfer to distantly related strains is inhibited.     

Characterization of a Mycobacterium bovis BCG insertion sequence related to the IS21 family

The structure and distribution of a Mycobacterium bovis BCG insertion element of the IS21 family were investigated. Several IS21-like elements found in mycobacterial genomes were separated in four types, following their nucleic acid similarities. The M. bovis BCG IS21 element is highly similar to IS1533 (class I), 70% similar to IS1534 (class II), 52% similar to IS1532 (class III) of Mycobacterium tuberculosis, and 54% similar to both an Mycobacterium avium serovar 2 and an M. avium silvaticum IS (class IV). The M. bovis BCG IS21 element of the class I appears to be present in a single copy in the genome of M. bovis BCG, M. bovis, M. tuberculosis and Mycobacterium africanum and to be absent from all other tested species of the Corynebacteria-Mycobacteria-Nocardia group.     

中国、日本和菲律宾水稻白叶枯病菌的母株与单细胞系的遗传与致病性差异的分析

以J3、IS1112、IS1113和ERIC为引物对17组(56株)来自中国、日本和菲律宾的水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)代表菌株和它们的单细胞系进行IS-PCR和Rep-PCR扩增以及田间致病性鉴定,研究母株和单细胞系之间的遗传和致病性变异.结果表明:1)母株与单细胞系属于同一分子谱型的比率依次为:52.9%(J3)、23.5%(IS1112)、29.4%(IS1113)和35.3%(ERIC); 2)综合4个引物的PCR扩增结果,采用UPGMA聚类,母株与其全部单细胞系相似率在90%以上的组群占29.4%,差异率达到30%～41%的组群占参试组群的52.9%;以带位相似率80%为簇界,参试菌可以划分为15簇,来自中国、日本和菲律宾的9个组的母株和单细胞系聚合在同一簇中,占参试菌的52.9%,说明三国病菌在遗传上存在高度相关性;三国病菌都存在簇群差异,以菲律宾茵系变异最大,如Pxo79和Pxo112母株和单细胞系各分在不同的3簇中,Pxo79、Pxo86、Pxo99和Pxo112等菌系的母株和单细胞系之间的差异率达到了41%;结果证明:母株是"由遗传上有不同程度差异的细胞"组成的混合体.3)田问致病性测定表明,母株与其单细胞系毒力不同的有15组,占88.2%,说明母株是由毒力不同的单细胞系组成的混合体,但病原菌的分子谱型以及遗传分簇与致病型之间没有相关性.     

Diversity of mammary tumor viral genes within the genus Mus, the species Mus musculus, and the strain C3H.

Proviral sequences complementary to the C3H mouse mammary tumor virus RNA genome are present in the DNA of early occurring mammary tumors of C3H/HeN mice and are absent from apparently normal C3H/HeN tissues; these sequences are non-germ line transmitted in C3H/HeN mice and have been termed tumor-associated sequences; (W. Drohan et al., J. Virol. 21:986-995, 1977). We report here that tumor-associated sequences are present in the DNA of spontaneous mammary tumors that occur early in the life of several inbred, high-tumor-incidence mouse strains but are absent in mammary tumors that occur later in life in low- and moderate-tumor-incidence strains. These sequences are also absent in apparently normal organs tested from numerous laboratory mouse strains, feral mice, Mus musculus subspecies, and other Mus species. Sequences represented in tumor-associated sequence RNA, however, are present as endogenous provirus in GR mice (at approximately four copies per haploid genome) and in two of five substrains of C3H mice tested (at approximately one copy per haploid genome). The two substrains of C3H mice positive for endogenous tumor-associated sequence provirus were recently (circa 1930) separated from the negative substrains of C3H mice. The results may be explained by the unlikely chance segregation of proviral sequences or by the recent integration of viral genes (within the last few decades). Whereas radioactively labeled mouse mammary tumor virus 60-70S RNA or complementary DNA detected mouse mammary tumor virus-related proviral information in all laboratory mouse strains, feral mice, subspecies of M. musculus, and other species of Mus, the use of tumor-associated sequence RNA clearly revealed the genetic diversity that may exist between different colonies or substrains of "inbred" laboratory mice commonly used in cancer research.     

Analysis of transposable elements inserted in the genomes of bacteriophages Mu and P1.

We have examined the genomes of the temperate bacteriophages Mu and P1 and some of their insertion mutants for hybridization with the prokaryotic transposable elements IS1 and IS2. We used the DNA blotting-hybridization technique in which denatured DNA fragments are transferred to nitrocellulose paper directly from agarose gels and hybridized to 32P-labeled probe DNA. The 800 base pair insertion in an X mutant of Mu was found to hybridize with IS1. The chloramphenicol resistance transposon, Tn9, in Mu X cam mutants was found to be located at or close to the sites of IS1 insertion in X mutants; Tn9 also hybridized with IS1. The restriction endonuclease BalI cleaved IS1 once; it cleaved Tn9 in all Mu X cam mutants twice to release a fragment of about 1700 base pairs. These results support the conclusion that Tn9 contains one copy of IS1 at each end. In the P1cam isolate, from which Tn9 was transposed to Mu, BalI made a third cut in Tn9 giving rise to fragments of about 850 base pairs. The data further suggested that Tn9 is present in tandem copies in the P1cam isolate we examined. P1 itself was found to harbor IS1. The two P1 strains tested had a common fragment containing IS1; one strain had an additional copy of IS1. The IS1 element common to the P1 strains was shown to be the site of the Tn9 insertion in the P1cam isolate examined. No hybridization between IS2 and any of the Mu and P1 strains could be detected.     

Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis.

DNA from 1008 strains of Mycobacterium avium subspecies paratuberculosis, digested by restriction endonucleases PstI and BstEII, was hybridised with a standard IS900 probe prepared by PCR and labelled non-radioactively by ECL. DNA fingerprints were scanned by CCD camera and analysed using the software Gel Compar (Applied Maths, Kortrijk, Belgium). Thirteen restriction fragment length polymorphism (RFLP) (PstI) types were detected, which where designated as A, B, C, D, E, F, G, H, I, J, K, L and M in accordance with the study of Pavlik et al. (1995) [Pavlik, I., Bejckova, L., Pavlas, M., Rozsypalova, V., Koskova, S., 1995. Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities. Vet. Microbiol. 45, 311-318]. Twenty RFLP (BstEII) types were detected and designated as C1-3, C5, C7-20, S1 and I1 in accordance with the study by Collins et al. 1990 [Collins, D.M., Gabric, D.M., de Lisle, G.W., 1990. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 28, 1591-1596]. A combination of both RFLP (PstI) and RFLP (BstEII) results revealed a total of 28 different RFLP types. All the RFLP types and detailed protocols are available at Intemet web site WWW...: http:/ /www.vri.cz/wwwrflptext.htm.     

Distribution of IS5 in bacteria

Four different strains of Escherichia coli and several other bacteria were examined by Southern analysis for the presence of the insertion element IS5 and IS5-like sequences. Variations in the copy number, degree of homology and restriction pattern of the IS5-like sequence were found among the different organisms. The number and distribution of IS5 sequences do not appear to correlate with the evolutionary relationship of the bacteria in which they occur.     

Genetic diversity of a natural population of Sinorhizobium meliloti, detected during analysis of a cryptic plasmid and ISRm2011-2 fingerprints]

Fifty-six natural strains of alfalfa nodule bacteria were isolated from samples of the soil under wild legume and alfalfa in two different field sites of Irkutsk oblast. Based on the results of analysis of plasmid profile, 11 different types of strains were detected, and 43 types were identified based on the results of hybridization with the insertion sequence element ISRm2011-2. Significant differences were found in the plasmid profile and IS fingerprints between strains isolated from the soil under alfalfa and the soil under legume. In contrast, strains growing at some distance from each other differed only in the IS fingerprints. From a comparison of results obtained in the assessment of plasmid profile and in analysis of IS fingerprints with results of RFLP analysis in strains, the conclusion about the transference of cryptic plasmids between strains and genetic rearrangements in strains of this population was drawn.     

The Haemophilus influenzae capsulation gene cluster: a compound transposon

The population of capsulate Haemophilus influenzae is divided into two phylogenetic divisions. Here we show that in division I strains the capsulation (cap) gene cluster lies between direct repeats of a novel insertion sequence (IS)-like element, IS1016. cap has apparently been mobilized in the chromosome as a compound transposon by IS1016, and the repeats have provided a molecular substrate for reversible cap gene amplification, with augmentation of capsule production, through unequal homologous recombination. Such amplification has occurred in serotype b strains, but in these a large direct repeat of cap genes has become fixed in the population. We have found a 1.2 kb deletion at one end of this duplicated capb locus, removing most of one copy of the polysaccharide export gene bexA. We have shown that this makes capsulation dependent on preservation of the direct repeat structure in order to avoid recombination-mediated loss of the other copy of bexA. Type b strains with this cap configuration are disseminated worldwide and currently cause nearly all invasive Haemophilus infections, leading us to speculate that the 1.2 kb deletion occurred in an ancestral type b strain and conferred significant biological advantage.     

Genetic Diversity of a Natural Population of Sinorhizobium melilotiRevealed in Analysis of Cryptic Plasmids and ISRm2011-2 Fingerprints

Fifty-six natural strains of alfalfa nodule bacteria were isolated from samples of the soil under wild legume and alfalfa in two different field sites of Irkutsk oblast. Based on the results of analysis of plasmid profile, 11 different types of strains were detected, and 43 types were identified based on the results of hybridization with the insertion sequence element ISRm2011-2. Significant differences were found in the plasmid profile and IS fingerprints between strains isolated from the soil under alfalfa and the soil under legume. In contrast, strains growing at some distance from each other differed only in the IS fingerprints. From a comparison of results obtained in the assessment of plasmid profile and in analysis of IS fingerprints with results of RFLP analysis in strains, the conclusion about the transference of cryptic plasmids between strains and genetic rearrangements in strains of this population was drawn.     

Organization of lin genes and IS6100 among different strains of hexachlorocyclohexane-degrading Sphingomonas paucimobilis: evidence for horizontal gene transfer

The organization of lin genes and IS6100 was studied in three strains of Sphingomonas paucimobilis (B90A, Sp+, and UT26) which degraded hexachlorocyclohexane (HCH) isomers but which had been isolated at different geographical locations. DNA-DNA hybridization data revealed that most of the lin genes in these strains were associated with IS6100, an insertion sequence classified in the IS6 family and initially found in Mycobacterium fortuitum. Eleven, six, and five copies of IS6100 were detected in B90A, Sp+, and UT26, respectively. IS6100 elements in B90A were sequenced from five, one, and one regions of the genomes of B90A, Sp+, and UT26, respectively, and were found to be identical. DNA-DNA hybridization and DNA sequencing of cosmid clones also revealed that S. paucimobilis B90A contains three and two copies of linX and linA, respectively, compared to only one copy of these genes in strains Sp+ and UT26. Although the copy number and the sequence of the remaining genes of the HCH degradative pathway (linB, linC, linD, and linE) were nearly the same in all strains, there were striking differences in the organization of the linA genes as a result of replacement of portions of DNA sequences by IS6100, which gave them a strange mosaic configuration. Spontaneous deletion of linD and linE from B90A and of linA from Sp+ occurred and was associated either with deletion of a copy of IS6100 or changes in IS6100 profiles. The evidence gathered in this study, coupled with the observation that the G+C contents of the linA genes are lower than that of the remaining DNA sequence of S. paucimobilis, strongly suggests that all these strains acquired the linA gene through horizontal gene transfer mediated by IS6100. The association of IS6100 with the rest of the lin genes further suggests that IS6100 played a role in shaping the current lin gene organization.     

A family of IS1031 elements in the genome of Acetobacter xylinum: nucleotide sequences and strain distribution

An insertion sequence (here called IS 1031A) from Acetobacter xylinum ATCC 23769 has recently been isolated. This study describes the complete nucleotide sequence of IS 1031A as well as the sequences of two novel iso-IS 1031 elements, IS1031C and IS1031D, from A. xylinum ATCC 23769. The three ISs are all exactly 930 bp long, have imperfect terminal inverted repeats of 24 bp for IS1031A and 21 bp for IS1031C and IS1031D, are flanked by three base pair direct repeats, and contain an open reading frame encoding a putative basic protein of 278 amino acids. Because of nucleotide substitutions, IS1031C and IS1031D differ from IS 1031A by 12.9% while IS1031C differs from IS1031D by only 0.6%. Hybridization analyses of total DNA from nine A. xylinum strains showed that all strains contained IS 1031-like elements varying in copy number from three to at least 16. None of three Acetobacter aceti strains examined contained IS1031-like elements. Taken together, the results suggest that A. xylinum contains a family of IS 1031 elements with considerably diversified nucleotide sequences.     

Further mapping of IS2 and IS3 in the lac-purE region of the Escherichia coli K-12 genome: structure of the F-prime ORF203.

The sequence organization of the F-prime ORF203 was determined by heteroduplex analysis. This large, type II F-prime (Scaife, 1967) contains lac, proC, and purE genes derived from the W1485 subline of Escherichia coli K-12. The IS3 and IS2 elements previously found in the lac-proC-purE region derived from the 58-161 subline (Hu et al., 1975) are also present in the same locations in the bacterial deoxyribonucleic acid (DNA) from the W1485 subline. Recombination between the IS2 region of F and an IS2 element located between lac and proC on the bacterial DNA apparently led to the formation of the perental Hfr, OR21. IS2 is thus directly repeated, with one copy of each element appearing at each of the two junctions between F and the bacterial sequences on ORF203. The F plasmid is found together with ORF203 in the plasmid DNA, and this probably forms from ORF203 by recombination between the directly repeated IS2 elements. ORF203 appears to have been excised from the Hfr chromosome by recombination between the IS3 sequence alpha3beta3 located counterclockwise of lac and the directly repeated IS3 sequence alpha4beta4 located clockwise of purE.     

wbeT sequence typing and IS1004 profiling of Vibrio cholerae isolates

Aims: To investigate the molecular basis for serotype variation in Vibrio cholerae O1 and the genetic relatedness amongst different serotypes isolated from 2004 to 2008 in Iran. Methods and Results: Despite the presence of all three serotypes of V. cholerae O1 (Ogawa, Inaba and Hikojima) in Iran in the last decade, the Inaba strains have been the dominated serotype. Sequence analysis of wbeT determined only a single substitution of G for A at position 295 in all Inaba strains resulting in a replacement of serine to proline. No difference was found in the copy numbers and profile of IS1004 between the classical and El Tor V. cholerae O1 strains, supporting the clonality amongst the isolates obtained over 5 years in Iran. In addition, Southern blots of HpaII‐digested chromosomal DNAs of our Ogawa and Inaba isolates showed the presence of an incomplete copy of IS1004 for all isolates. Conclusions: IS1004 profiling can be a reliable method for analysis of clonal dissemination of V. cholerae. The results indicated that specific point mutation at a particular position within the wbeT of V. cholerae O1 strains in Iran may occur which, in turn, may result in serotype switching. Significance and Impact of the Study: Understanding the molecular basis for serotype conversion of V. cholerae and their genetic relatedness could give insights for the incoming cholera epidemic prediction and control.