Demonstration of IS<Emphasis Type="Italic">711</Emphasis> transposition in <Emphasis Type="Italic">Brucella ovis</Emphasis> and <Emphasis Type="Italic">Brucella pinnipedialis</Emphasis>

Background  

The Brucella genome contains an insertion sequence (IS) element called IS711 or IS6501, which is specific to the genus. The copy number of IS711 varies in the genome of the different Brucella species, ranging from 7 in B. abortus, B. melitensis and B. suis to more than 30 in B. ovis and in Brucella strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS711, but the occurrence of this element with a high copy number in some species, and the isolation of Brucella strains with "ectopic" copies of IS711 suggested that this IS could still transpose.     

Identification and characterization of IS1381, a new insertion sequence in Streptococcus pneumoniae.

A new insertion sequence (IS1381) was identified in the genome of Streptococcus pneumoniae R6 as an 846-bp segment containing 20-bp terminal inverted repeats and flanked by 7-bp direct repeats. The three sequenced copies of this element have two overlapping open reading frame (ORF) genes named orfA and orfB. However, significant variations between individual copies were found, suggesting that inactivating mutations have occurred in an original single ORF. Accordingly, the consensus IS1381 element derived from the comparison of the three available copies should contain a single ORF sufficient to encode a basic protein of 267 amino acids which exhibited high similarity to the putative transposases of ISL2 from Lactobacillus helveticus and of IS702 from the cyanobacterium Calothrix sp. strain PCC 7601. A minimum of five to seven copies were detected by hybridization experiments in the R6 genome. In remarkable contrast with the two previously reported pneumococcal insertion sequences, several copies of IS1381 have been detected in all of the clinical isolates tested so far. Interestingly, Streptococcus oralis NCTC 11427 (type strain), a close relative of pneumococcus, does not contain this element, but its occurrence in the type strain of Streptococcus mitis (NCTC 12261) suggests that this species has exchanged DNA with S. pneumoniae directly or through an intermediate species.     

Three new loci of insertion element IS1112 in Chinese strains of Xanthomonas oryzae pv. oryzae

Insertion sequence IS1112 is a repetitive element with a relatively high number of copies in Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice (Oryza sativa L.). Three new loci of IS1112 were identified in seven Chinese strains of Xoo using a single oligonucleotide primer J3; 5'-GCTCAGGTCAGGTGGCCTGG-3' by insertion-sequence-based polymerase chain reaction (IS-PCR). Among the three new loci of IS1112, two were located in the open-reading frame region of genes fhuA and cirA, which encode TonB-dependent receptors, and the third in ISXo2, another type of insertion sequence in Xoo genome. Three variants of IS1112 were identified in those three loci based on their sequence similarities: two were identical to IS1112a and IS1112b, reported in strain PXO86 from the Philippines, while the third was a new member of IS1112, defined as IS1112d. Inserting IS1112 in gene fhuA caused three bases, GGT, to be duplicated at the target site, but inserting it in gene cirA did not cause any duplication in the target site. The diversity of IS1112 sequence and insertion loci in Xoo genome and their potential effects are discussed.     

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.     

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.     

An Acetobacter xylinum insertion sequence element associated with inactivation of cellulose production.

An insertion sequence (IS) element, IS1031, caused insertions associated with spontaneous cellulose deficient (Cel-) mutants of Acetobacter xylinum ATCC 23769. The element was discovered during hybridization analysis of DNAs from Cel- mutants of A. xylinum ATCC 23769 with pAXC145, an indigenous plasmid from a Cel- mutant of A. xylinum NRCC 17005. An IS element, IS1031B, apparently identical to IS1031, was identified on pAXC145. IS1031 is about 950 bp. DNA sequencing showed that the two elements had identical termini with inverted repeats of 24 bp containing two mismatches and that they generated 3-bp target sequence duplications. The A. xylinum ATCC 23769 wild type carries seven copies of IS1031. Southern hybridization showed that 8 of 17 independently isolated spontaneous Cel- mutants of ATCC 23769 contained insertions of an element homologous to IS1031. Most insertions were in unique sites, indicating low insertion specificity. Significantly, two insertions were 0.5 kb upstream of a recently identified cellulose synthase gene. Attempts to isolate spontaneous cellulose-producing revertants of these two Cel- insertion mutants by selection in static cultures were unsuccessful. Instead, pseudorevertants that made waxlike films in the liquid-air interface were obtained. The two pseudorevertants carried new insertions of an IS1031-like element in nonidentical sites of the genome without excision of the previous insertions. Taken together, these results suggest that indigenous IS elements contribute to genetic instability in A. xylinum. The elements might also be useful as genetic tools in this organism and related species.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.     

IS200: a Salmonella-specific insertion sequence

A new IS element (IS200) has been identified in Salmonella. The sequence was identified as an IS element by the following criteria: its insertion caused the mutation hisD984; six copies of the sequence are present in strain LT2 of S. typhimurium; and transposition of the sequence has been observed on several occasions. IS200 is found in almost all Salmonella species examined but is absent from most other enteric bacteria. The specificity of this element for Salmonella (and the absence of IS1-IS4 from Salmonella) suggest that transfer of insertion sequences between bacterial groups may be less extensive than is commonly believed. Alternatively, the distribution may suggest that these elements play a selectively important role in bacteria.     

Characterization of IS1001, an insertion sequence element of Bordetella parapertussis.

By analysis of repetitive DNA in Bordetella parapertussis, an insertion sequence element, designated IS1001, was identified. Sequence analysis revealed that IS1001 comprised 1,306 bp and contained inverted repeats at its termini. Furthermore, several open reading frames that may code for transposition functions were identified. The largest open reading frame coded for a protein comprising 406 amino acid residues and showed homology to TnpA, which is encoded by an insertion sequence element (IS1096) found in Mycobacterium smegmatis. Examination of flanking sequences revealed that insertion of IS1001 occurs preferentially in stretches of T's or A's and results in a duplication of target sequences of 6 to 8 bases. IS1001 was found in about 20 copies in 10 B. parapertussis strains analyzed. No restriction fragment length polymorphism was observed in B. parapertussis when IS1001 was used as a probe. An insertion sequence element similar or identical to IS1001 was found in B. bronchiseptica strains isolated from pigs and a rabbit. In these strains, about five copies of the IS1001-like element were present at different positions in the bacterial chromosome. Neither B. pertussis nor B. bronchiseptica strains isolated from humans and dogs contained an IS1001-like element. Therefore, IS1001 may be used as a specific probe for the detection of B. parapertussis in human clinical samples.     

Characterization of two insertion sequences, IS701 and IS702, from the cyanobacterium Calothrix species PCC 7601

We describe the characterization of two insertion elements, IS701 and IS702, isolated from Calothrix species PCC 7601. These insertion elements were cloned from spontaneous pigmentation mutants. Both show the characteristics of typical bacterial insertion sequences, i.e. they present long terminal inverted repeats and they duplicate target DNA upon insertion. These elements share no homology with the only other cyanobacterial insertion sequence described so far, IS891. At least 15 copies of IS701 and 9 copies of IS702 were detected by hybridization experiments in the Calothrix 7601 genome. Their occurrence in several cyanobacterial strains is also reported.     

Analysis of separate isolates of Bordetella pertussis repeated DNA sequences

Two independent isolates of a Bordetella pertussis repeated DNA unit were sequenced and shown to be an insertion sequence element with five nucleotide differences between the two copies. The sequences were 1053 bp in length with near-perfect terminal inverted repeats of 28 bp, had three open reading frames, and were each flanked by short direct repeats. The two insertion sequences showed considerable homology to two other B. pertussis repeated DNA sequences reported recently: IS481 and a 530 bp repeated DNA unit. The B. pertussis insertion sequence would appear to comprise a group of closely related sequences differing mainly in flanking direct repeats and the terminal inverted repeats. The two isolates reported here, which were from the adenylate cyclase and agglutinogen 2 regions of the genome, were numbered IS48lvl and IS48lv2 respectively.     

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.     

Characterization and distribution of IS8301 in the radioresistant bacterium Deinococcus radiodurans

The insertion sequence element IS8301 isolated from the radiation resistant bacterium Deinococcus radiodurans strain KD8301 was characterized. IS8301 is comprised of 1,736-bp, lacks terminal inverted repeats and does not duplicate target DNA upon its insertion. The amino acid sequence homology of two open reading frames encoded in IS8301 indicates that this insertion sequence element belongs to the IS200/IS605 group. There were seven loci completely identical with the IS8301 sequence in the published D. radiodurans R(1) genome sequence. The genome distribution profiles of IS8301 in strain KD8301 as well as in the three different laboratory isolates (KR(1), MR(1), and R(1)) of wild-type D. radiodurans were investigated using genomic hybridization analysis. At least 21 strong hybridization signals were detected in strain KD8301 while only one hybridization signal was detected in strain KR(1), the parent strain of KD8301. In strain MR1, a different wild-type isolate, six strong hybridization signals were detected. In spite of the identification of seven copies of IS8301 in the published D. radiodurans R(1) genome sequence, only one hybridization signal was detected in strain R(1) purchased from American Type Culture Collection. Using inverse PCR and sequencing analyses, total 13 different insertion loci of IS8301 in the D. radiodurans genome were identified. Sequence comparison of the flanking region of insertion sites indicated that the sequence 5'-TTGAT-3' preceded the left end of IS8301 in all cases.     

Classification of Brucella strains isolated from marine mammals by infrequent restriction site-PCR and development of specific PCR identification tests

Brucella strains have been isolated since the 1990s from a wide variety of marine mammals and represent potential zoonotic pathogens. They have distinctive phenotypic and molecular characteristics from the terrestrial mammal Brucella species, and two new species names have been previously proposed based on DNA polymorphism at the omp2 locus and their preferential host, i.e. Brucella cetaceae for cetacean isolates and Brucella pinnipediae for pinniped isolates. The results presented in this study on characterization of these strains by infrequent restriction site-PCR (IRS-PCR), taking into account the higher number of IS711 elements in their genome compared to terrestrial mammal Brucella species, supports this classification. The nucleotide sequences of specific DNA fragments detected by IRS-PCR were determined and used to develop PCR identification tests for either B. cetaceae or B. pinnipediae.     

Osteological and molecular identification of Brucellosis in ancient Butrint, Albania

Ancient skeletal remains can harbor unique information about past civilizations at both the morphological and molecular levels. For instance, a number of diseases manifest in bone, some of which have been confirmed through DNA analysis, verifying their presence in ancient populations. In this study, anthropological analysis of skeletal remains from the ancient Albanian city of Butrint identified individuals with severe circular lytic lesions on their thoracic and lumbar vertebrae. Differential diagnosis suggested that the lesions resulted from pathologies known to affect these skeletal regions, such as tuberculosis (TB) or brucellosis. Relevant bones of two adolescent males from the 10th to 13th century AD that displayed the lesions, along with unaffected individuals, were collected in the field. Genetic screening of the skeletal samples for TB was repeatedly negative, thus additional testing for Brucella spp.-bacteria of livestock and the causative agent of brucellosis in humans-was conducted. Two Brucella DNA markers, the IS6501 insertion element and Bcsp31 gene, amplified from the affected vertebrae and/or ribs, whereas all unaffected individuals and control samples were negative. Subsequent DNA sequencing confirmed the presence of the brucellar IS6501 insertion element. On the basis of the skeletal lesions, negative tests for TB, and positive Brucella findings, we report a confirmed occurrence of brucellosis in archaeologically recovered human bone. These findings suggest that brucellosis has been endemic to the area since at least the Middle Ages.     

Genome sequence of Lactobacillus helveticus, an organism distinguished by selective gene loss and insertion sequence element expansion

Mobile genetic elements are major contributing factors to the generation of genetic diversity in prokaryotic organisms. For example, insertion sequence (IS) elements have been shown to specifically contribute to niche adaptation by promoting a variety of genetic rearrangements. The complete genome sequence of the cheese culture Lactobacillus helveticus DPC 4571 was determined and revealed significant conservation compared to three nondairy gut lactobacilli. Despite originating from significantly different environments, 65 to 75% of the genes were conserved between the commensal and dairy lactobacilli, which allowed key niche-specific gene sets to be described. However, the primary distinguishing feature was 213 IS elements in the DPC 4571 genome, 10 times more than for the other lactobacilli. Moreover, genome alignments revealed an unprecedented level of genome stability between these four Lactobacillus species, considering the number of IS elements in the L. helveticus genome. Comparative analysis also indicated that the IS elements were not the primary agents of niche adaptation for the L. helveticus genome. A clear bias toward the loss of genes reported to be important for gut colonization was observed for the cheese culture, but there was no clear evidence of IS-associated gene deletion and decay for the majority of genes lost. Furthermore, an extraordinary level of sequence diversity exists between copies of certain IS elements in the DPC 4571 genome, indicating they may represent an ancient component of the L. helveticus genome. These data suggest a special unobtrusive relationship between the DPC 4571 genome and its mobile DNA complement.     

Characterization of IS476 and its role in bacterial spot disease of tomato and pepper.

IS476 is an endogenous insertion sequence present in copper-tolerant strains of Xanthomonas campestris pv. vesicatoria. Sequence analysis has revealed that the element is 1,225 base pairs in length, has 26-base-pair inverted repeats, and causes a 4-base-pair target site duplication upon insertion into the avirulence gene avrBs1. Comparison of the full-length sequence with sequences in the National Biomedical Research Foundation and National Institutes of Health data bases showed that one of the predicted IS476 proteins is partially homologous to the putative transposase of IS3 from Escherichia coli, and the inverted repeats of IS476 have significant homology to the inverted repeats of the IS51 insertion sequence of Pseudomonas syringae pv. savastanoi. A transposition assay based on the insertional inactivation of the sacRB locus of Bacillus subtilis was used to demonstrate that one of the three copies of IS476 residing on the 200-kilobase copper plasmid pXVCU1 is capable of transposition in several strains of Xanthomonas campestris. The position of IS476 insertion in several avrBs1 mutants was established and was shown to influence both induction of hypersensitivity and bacterial growth in planta.     

IS861, a group B streptococcal insertion sequence related to IS150 and IS3 of Escherichia coli.

A 1,442-base-pair (bp) insertion sequence (IS861) was identified in the type III group B streptococcal (GBS) strain COH-1. It is flanked by 26-bp imperfect inverted repeats and contains two open reading frames, 1 and 2, encoding 141- and 277-amino-acid proteins, respectively. A 3-bp target sequence, ACA, is duplicated and flanks each inverted repeat. IS861 shares greater than 30% homology with IS3 and IS150 of Escherichia coli, primarily in the region of their putative transposases. Northern (RNA) analysis revealed that RNA is actively transcribed in vivo by IS861 and 17- and 36-kilodalton proteins were synthesized in E. coli maxicell assays. Multiple copies of IS861 were observed throughout the chromosome of COH-1, and one of the copies is located near genes involved in GBS capsule synthesis. IS861 is the first insertion sequence identified in GBS. Its role in GBS and the significance of its relationship to the phylogenetically similar insertion sequences typified by IS150 and IS3 of E. coli are unknown.     

Cloning and sequence of IS1000, a putative insertion sequence from Thermus thermophilus HB8

The complete nucleotide sequences of two copies of a putative insertion sequence IS1000 from Thermus thermophilus HB8 are presented. IS1000 is 1196 base pairs long, contains a long open reading frame which could code for a protein of 317 amino acids, and has imperfect terminal inverted repeats of 6 base pairs (confirmed by the terminal sequencing of 4.5 copies of IS1000), but does not cause a target site duplication. There are at least 6 copies of IS1000 in the genome of T. thermophilus HB8. A search of the GEN-EMBL data base revealed that the putative 317 amino acid protein had significant homology with open reading frames in the transposable elements IS110 of Streptomyces coelicolor and IS492 of Pseudomonas atlantica.