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PubMed1999

Excision of IS492 requires flanking target sequences and results in circle formation in Pseudoalteromonas atlantica.

The gram-negative marine bacterium Pseudoalteromonas atlantica produces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492 at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production in P. atlantica. Examination of IS492 transposition in P. atlantica by using a PCR-based assay revealed a circular form of IS492 that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492 circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492 insertion sites on the P. atlantica chromosome that we identified by using inverse PCR. IS492 circle formation correlated with precise excision of IS492 from the P. atlantica eps target sequence when introduced into Escherichia coli on a plasmid. Deletion analyses of the flanking host sequences at the eps insertion site for IS492 demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492 and circle formation in E. coli. Excision of IS492 in E. coli also depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492 is suggested by the creation of a new strong promoter for expression of mooV by the joining of the ends of the insertion sequence element at the circle junction.

Base Sequence↗
PubMed1991

Activation of an endogenous retrovirus enhancer by insertion into a heterologous context.

An enhancer element is located in the U3 portion of exogenous avian retrovirus long terminal repeats (LTRs). A similar element has not been detected in the LTRs of ev-1 and ev-2, two avian endogenous viruses (evs) that normally are not expressed in vivo. Experiments were initiated to determine whether minor nucleotide differences in the U3 region of a previously untested ev that is ubiquitously expressed in vivo (ev-3) might confer enhancer function on the LTR of this provirus. This question was addressed by inserting U3 regions from ev-3 and from ev-1 and/or ev-2 both upstream of the herpesvirus thymidine kinase gene promoter and in place of the major enhancer domains of the Rous sarcoma virus LTR and determining their relative effects on transcription. U3 regions from all evs tested were unable to enhance transcription from the thymidine kinase gene promoter, indicating that nucleotide differences in the ev U3 regions do not affect their relative enhancer function and therefore are unlikely to play a role in their differential expression in vivo. Unexpectedly, however, all ev U3 regions were able to augment transcription in an orientation-independent manner in the ev-Rous sarcoma virus hybrid LTRs. Further experiments conducted to determine why this enhancer activity is not detectable in intact ev LTRs demonstrated that it was not due to removal of repressor sequences in the ev fragments used that might normally be present in intact ev LTRs. The lack of detectable enhancer activity in intact ev LTRs also was not explained by a defect in ev promoters that makes them unresponsive to enhancers in cis. These experiments therefore identify sequences that, although unable to function detectably as enhancers in their natural context, can function efficiently in a heterologous context. Data are discussed in terms of the modularity of enhancer elements and possible interactions between enhancers and promoter-specific sequences.

Avian Sarcoma Viruses↗
PubMed1991

Transposon mutagenesis in Proteus mirabilis.

A technique of transposon mutagenesis involving the use of Tn5 on a suicide plasmid was developed for Proteus mirabilis. Analysis of the resulting exconjugants indicated that Tn5 transposed in P. mirabilis at a frequency of ca. 4.5 x 10(-6) per recipient cell. The resulting mutants were stable and retained the transposon-encoded antibiotic resistance when incubated for several generations under nonselective conditions. The frequency of auxotrophic mutants in the population, as well as DNA-DNA hybridizaiton to transposon sequences, confirmed that the insertion of the transposon was random and the Proteus chromosome did not contain significant insertional hot spots of transposition. Approximately 35% of the mutants analyzed possessed plasmid-acquired ampicillin resistance, although no extrachromosomal plasmid DNA was found. In these mutants, insertion of the Tn5 element and a part or all of the plasmid had occurred. Application of this technique to the study of swarmer cell differentiation in P. mirabilis is discussed.

DNA Transposable Elements↗
PubMedDate not supplied

Variation of beta-galactosidase expression from Mudlac elements during the development of Escherichia coli colonies.

Bacterial colonies reveal the action of systems for multicellular regulation of genetic activity during growth and development. Colonies produced on agar indicator medium by Escherichia coli strains carrying the transposable genetic fusion element Mudlac displayed organized patterns of differential beta-galactosidase expression. One feature of these patterns was the presence of phenotypically distinct concentric rings containing cells that were not genetically distinct. A second feature of the patterns was the appearance of sectorial populations with novel phenotypes, frequently displaying coincident variation in several characters, such as enzyme activity, multicellular aggregation and rate of spread over the agar substrate. Subcloning analysis of sectors with more expansive growth phenotypes revealed that they contained bacteria expressing novel developmental sequences on more than one kind of medium. These novel developmental sequences could be transmitted to progeny bacteria but were reversible during growth in liquid medium. More stable clonal variation in patterns of beta-galactosidase expression arose during storage in liquid medium. Most of these changes correlated with transpositions or rearrangements of Mudlac sequences. Some changes in beta-galactosidase expression involved interactions between Mudlac elements and unlinked Mu derivatives. These results revealed the operation of novel control systems regulating beta-galactosidase expression from the lacZ sequences in a chromosomal Mudlac element and demonstrated at least two different kinds of clonal variation events which affected pattern formation in bacterial colonies.

DNA Transposable Elements↗
PubMed1996

A region of a Sym plasmid of Rhizobium leguminosarum biovar phaseoli has similarity to prokaryotic insertion sequences and to eukaryotic integrases.

Near the nod and nif genes of the Sym plasmid pRP2JI of Rhizobium leguminosarum biovar phaseoli are three open reading frames whose deduced polypeptide products have similarities to those of genes in bacterial insertion sequences. The similarity of one of these ORFs was significantly greater to that of the integrase region of pol proteins of eukaryotic retroviruses and transposable elements in animals and plants than it was to the transposases of prokaryotic insertion sequences. In the noncoding region of the IS-like element, there was a sequence similar to that which had been identified close to nod genes in Azorhizobium caulinodans.

Amino Acid Sequence↗
PubMed1984

Escherichia coli alpha-haemolysin synthesis and export genes are flanked by a direct repetition of IS91-like elements.

A revised physical map of the alpha-haemolysin plasmid pHly152 has been constructed. The known position of the hly genes in the restriction map of pHly152 allowed us to locate in it a direct repeat of IS elements flanking the hly genes of pHly152. These elements are IS92L, which is a derivative of the previously characterised element IS91 (1.85 kb) by insertion of a sequence of 1.2 kb, and IS92R, an element related to IS91 by a deletion of 0.7 kb and substitution of a 0.2 kb sequence of IS91 by a 1.2 kb heterologous sequence. IS92L is, in turn, flanked by an inverted repetition of sequences of 1.4 kb. These and previously published data strongly suggest that the hly genes spread at some time in evolution by means of the recombinational activity of IS91-like elements.

Bacterial Proteins↗
PubMed1995

Repeated DNA sequences in mycobacteria.

In tuberculosis, it is often important to establish the source of infection and to determine whether disease is due to a new strain of Mycobacterium tuberculosis or to relapse. To cope with the resurgence of tuberculosis and atypical mycobacterioses in AIDS patients, on the one hand, and to overcome the limitations of classical bacteriological procedures on the other, the development of rapid, sensitive, and reliable diagnostic and epidemiologic tools is highly desirable. Molecular typing methods are often based on repeated genes such as those for rRNA. Ribotyping is of limited use with pathogenic mycobacteria, as the slow-growers possess a single rRNA operon, while the fast-growers have two. This problem has been overcome by the discovery and study of repeated DNA elements in mycobacterial genomes, as these provide an alternative pathway for diagnostic and epidemiological investigations.

Base Sequence↗
PubMed1983

An insertion mutation associated with constitutive expression of repressible acid phosphatase in Saccharomyces cerevisiae.

The PHO83 mutation in Saccharomyces cerevisiae, which had been detected on the basis of constitutive production of repressible acid phosphatase and mapped at the end of the PHO5 locus, was analysed by Southern hybridization with cloned DNA fragments of the PHO5 gene as probe. It was shown that this mutant has a DNA insertion of about 6 kilobase pairs, probably in the 5'-noncoding region of the PHO5 gene. Production of repressible acid phosphatase by the PHO83 mutant is partially independent of the function of the PHO2 and PHO4 genes, the positive regulatory genes whose functions are indispensable for PHO5 expression. PHO83 mutants are constitutive in a and alpha cells, either haploid or diploid, but not in non-mating cells, MATa/MAT alpha or a certain sterile mutation. These observations strongly suggest that the PHO83 mutation is caused by insertion of a Ty element in the 5'-noncoding region of the PHO5 gene.

Acid Phosphatase↗
PubMed1996

A group II intron in a conjugative transposon from the gram-positive bacterium, Clostridium difficile.

We have been studying the conjugative transposon Tn5397, originally isolated from the Gram-positive pathogen Clostridium difficile. Physical analysis of this transposon demonstrated that it contained a group II intron. This is the first report of an intron in a conjugative transposon and the first report of a group II intron in Gram-positive bacteria. The intron interrupted a gene in Tn5397 that is almost identical to orf14 from Tn916. DNA hybridisation analysis showed that elements related to Tn5397, containing the group II intron, were present in five other C. difficile strains from different geographical locations suggesting that the element is likely to be widely distributed.

Cloning, Molecular↗
PubMed1997

Gene transfer, gentamicin resistance and enterococci.

Enterococci are versatile pathogens by virtue of their ability to exhibit low-level intrinsic resistance to clinically useful antibiotics and their tolerance to adverse environmental conditions. In the last 20 years these pathogens have become progressively more difficult to treat because of their aptitude for acquiring antibiotic-resistance genes. Of increasing concern is the rapid dissemination of the AAC6'-APH2" bi-functional aminoglycoside modifying enzyme. This enzyme confers high-level resistance to gentamicin and all other related aminoglycosides with the exception of streptomycin. The gene conferring this phenotype has been associated with both narrow and broad host range plasmids, and has recently been found on conjugative transposons. The nature of these conjugative elements raises the possibility of the resistance gene spreading to other pathogenic bacteria.

Anti-Bacterial Agents↗
PubMed1999

IS1626, a new IS900-related Mycobacterium avium insertion sequence.

An insertion sequence designated IS1626 was isolated and characterized from a Mycobacterium avium clinical strain. IS1626 was detected by high-stringency hybridization with the pMB22/S12 probe from IS900 of Mycobacterium paratuberculosis. IS1626 is 1418 bp in size and has a G+C content of 65 mol%. It has neither terminal inverted repeats nor flanking direct repeats. Analysis of three IS1626 insertion sites in the M. avium strain and the corresponding potential insertion sites in two IS1626-free M. avium strains indicated a consensus sequence of CATGCN(4-5)TCCTN(2)G for IS1626 insertion. In the three clones examined, IS1626 has the same orientation with respect to this target site. IS1626 has two major ORFs. ORF1179 encodes a predicted protein of 393 amino acids. ORF930, on the complementary strand of ORF1179, encodes a protein of 310 amino acids. The Shine-Dalgarno sequence for ORF930 is partially located in the flanking region, similar to other IS900-related elements. Analysis of the comparable features of insertion sequences and their variable occurrence in related organisms is useful for studying the evolution of these elements and their hosts.

Amino Acid Sequence↗
PubMed2003

CACTA transposons in Triticeae. A diverse family of high-copy repetitive elements.

In comparison with retrotransposons, which comprise the majority of the Triticeae genomes, very few class 2 transposons have been described in these genomes. Based on the recent discovery of a local accumulation of CACTA elements at the Glu-A3 loci in the two wheat species Triticum monococcum and Triticum durum, we performed a database search for additional such elements in Triticeae spp. A combination of BLAST search and dot-plot analysis of publicly available Triticeae sequences led to the identification of 41 CACTA elements. Only seven of them encode a protein similar to known transposases, whereas the other 34 are considered to be deletion derivatives. A detailed characterization of the identified elements allowed a further classification into seven subgroups. The major subgroup, designated the "Caspar " family, was shown by hybridization to be present in at least 3,000 copies in the T. monococcum genome. The close association of numerous CACTA elements with genes and the identification of several similar elements in sorghum (Sorghum bicolor) and rice (Oryza sativa) led to the conclusion that CACTA elements contribute significantly to genome size and to organization and evolution of grass genomes.

DNA Transposable Elements↗
PubMed2005

Genetic diversity in the lactose operons of Lactobacillus helveticus strains and its relationship to the role of these strains as commercial starter cultures.

Two novel insertion sequence elements, ISLhe1 and ISLhe15, were located upstream of the genes encoding the beta-galactosidase enzyme in Lactobacillus helveticus commercial starter strains. Strains with the IS982 family element, ISLhe1, demonstrated reduced beta-galactosidase activity compared to the L. helveticus type strain, whereas strains with the ISLhe15 element expressed beta-galactosidase in the absence of lactose.

Base Sequence↗
PubMed1987

Complete nucleotide sequence of insertion element IS4351 from Bacteroides fragilis.

The nucleotide sequence and genetic analyses of one of the directly repeated sequences flanking the macrolide-lincosamide-streptogramin B drug resistance determinant, ermF, from the Bacteroides fragilis R plasmid, pBF4, suggested that this region is an insertion sequence (IS) element. This 1,155-base-pair element contained partially matched (20 of 25 base pairs) terminal-inverted repeats, overlapping, anti-parallel open reading frames, and nine promoterlike sequences, including three that were oriented outward. Analysis of this sequence revealed no significant nucleotide homology to 13 other known IS elements. Inasmuch as Southern blot hybridization analysis detected homologous sequences in chromosomal DNA and its G+C content (42 mol%) was similar to that of B. fragilis, the data suggested that this element is of Bacteroides origin. Transposition promoted by this element was demonstrated in recA E. coli. Recombinants were recovered by selecting for the activation of a promoterless chloramphenicol resistance gene on the plasmid pDH5110 and were characterized by restriction endonuclease mapping and Southern blot hybridization. We propose that this IS element be designated IS4351.

Bacteroides fragilis↗
PubMed1990

A cryptic 65-kilobase-pair transposonlike element isolated from Bacteroides uniformis has homology with Bacteroides conjugal tetracycline resistance elements.

A 65-kilobase-pair element, XBU4422, which has some transposonlike characteristics but carries no known antibiotic resistance genes, has been isolated from Bacteroides uniformis 0061. XBU4422 was trapped on Bacteroides-Escherichia coli shuttle vectors during experiments in which one of the conjugal Bacteroides tetracycline resistance (Tcr) elements was being used to mobilize the shuttle vectors to Bacteroides recipients. Results of Southern hybridization experiments showed that XBU4422 is normally integrated in the B. uniformis 0061 chromosome and is found only in some strains. Insertion of XBU4422 in the shuttle vectors was site specific and orientation specific. Nonmobilizable vectors that had acquired XBU4422 became transmissible and could be transferred to Bacteroides or E. coli recipients. In B. uniformis transconjugants, the XBU4422 insertion in the vectors was usually intact, but XBU4422 was always lost in matings with E. coli, Bacteroides thetaiotaomicron, or B. ovatus. The loss of XBU4422 did not visibly alter the vector; in the case of E. coli, the loss of the insertion appeared to be RecA dependent. Although XBU4422 carried no antibiotic resistances, it shared regions of homology with six conjugal Bacteroides Tcr elements; this homology was strongest with the ends of XBU4422. Using a strain of B. thetaiotaomicron that contains no XBU4422-hybridizing sequences, we showed that the ends of XBU4422 were probably reacting with the ends of the Tcr elements. These results provide the first direct evidence that the Tcr elements, like XBU4422, are integrated in the chromosome and that insertion of the least some Tcr elements, such as TcrEmr DOT, is relatively site specific.

Bacteroides↗
PubMed1992

TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization.

We describe a set of elements based on the transposon TnphoA for making transcriptional fusions to the lacZ gene and for making translational fusions to the phoA or lacZ structural gene. Each element can be switched, one for another, by homologous recombination, thereby allowing testing for transcription, translation, or cell surface localization determinants at the same site within a gene. We describe three kinds of elements for making each fusion type. Two kinds are transposition proficient (Tnp+): one encodes kanamycin resistance, and the other encodes tetracycline resistance. The third kind is transposition defective (Tnp-) and encodes kanamycin resistance. In addition, we describe one Tnp- element that has no reporter gene and encodes chloramphenicol resistance; this element is used primarily as a tool to aid in switching fusions. Switching is efficient because each element has in common 254 bp of DNA at the phoA end and 187 bp (or more) of DNA at the IS50R end of TnphoA, and switching is straightforward because individual elements encode different drug resistances. Thus, switched recombinants can be selected as drug-resistant transductants, and they can be recognized as ones that have lost the parental drug resistance and fusion phenotype. Further, switching Tnp+ elements to Tnp- elements reduces problems due to transposition that can arise in P1 crosses or cloning experiments. Some TnphoA and TnphoA' elements cause polar mutations, while others provide an outward promoter for downstream transcription. This feature is especially useful in the determination of operon structures. Strategies for the use of TnphoA and TnphoA' elements in gene analysis are also described.

Amino Acid Sequence↗
PubMed1998

Identification and characterization of IS1411, a new insertion sequence which causes transcriptional activation of the phenol degradation genes in Pseudomonas putida.

A new insertion sequence (IS element), IS1411, was identified downstream of the phenol degradation genes pheBA that originated from plasmid DNA of Pseudomonas sp. strain EST1001. According to sequence analysis, IS1411 belongs to a new family of IS elements that has recently been named the ISL3 family (J. Mahillon and M. Chandler, Microbiol. Mol. Biol. Rev. 62:725-774, 1998). IS1411 generates 8-bp duplication of the target DNA and carries 24-bp inverted repeats (IRs), highly homologous to the IRs of other IS elements belonging to this family. IS1411 was discovered as a result of insertional activation of promoterless pheBA genes in Pseudomonas putida due to the presence of outward-directed promoters at the left end of IS1411. Both promoters located on the IS element have sequences that are similar to the consensus sequence of Escherichia coli sigma70. IS1411 can produce IS circles, and the circle formation is enhanced when two copies of the element are present in the same plasmid.

Amino Acid Sequence↗