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PubMed2002

Biodegradation of atrazine and related s-triazine compounds: from enzymes to field studies.

s-Triazine ring compounds are common industrial chemicals: pesticides, resin intermediates, dyes, and explosives. The fate of these compounds in the environment is directly correlated with the ability of microbes to metabolize them. Microbes metabolize melamine and the triazine herbicides such as atrazine via enzyme-catalyzed hydrolysis reactions. Hydrolytic removal of substituents on the s-triazine ring is catalyzed by enzymes from the amidohydrolase superfamily and yields cyanuric acid as an intermediate. Cyanuric acid is hydrolytically processed to yield 3 mol each of ammonia and carbon dioxide. In those cases studied, the genes underlying the hydrolytic reactions are localized to large catabolic plasmids. One such plasmid, pADP-1 from Pseudomonas sp. ADP, has been completely sequenced and contains the genes for atrazine catabolism. Insertion sequence elements play a role in constructing different atrazine catabolic plasmids in different bacteria. Atrazine chlorohydrolase has been purified to homogeneity from two sources. Recombinant Escherichia coli strains expressing atrazine chlorohydrolase have been constructed and chemically cross-linked to generate catalytic particles used for atrazine remediation in soil. The method was used for cleaning up a spill of 1,000 pounds of atrazine to attain a level of herbicide acceptable to regulatory agencies.

Atrazine↗
PubMed1984

Nucleotide sequence of the transposable element IS15.

We have determined the complete nucleotide sequence of the right (R) copy of the insertion sequence IS15 which flanks, in direct orientation, the composite transposon Tn1525. IS15-R, which is capable of independent transposition, is 1648 bp long and has short (14 bp) perfect inverted repeats at its termini. Analysis of the nucleotide sequence indicates that IS15-R results from the transposition, in direct orientation, of a smaller (820 bp long) IS, designated IS15-delta, into itself. This integration event is accompanied by the duplication of 8 bp in the target DNA. IS15-delta possesses two large overlapping open reading frames (ORF) located on opposite strands. Because of this particular structure, IS15 possesses four large ORFs which, due to the integration event, exhibit some differences with those of the parental IS15-delta.

Base Sequence↗
PubMed1996

Identification of a native Dichelobacter nodosus plasmid and implications for the evolution of the vap regions.

Studies on the role of various virulence factors of the ovine pathogen, Dichelobacter nodosus, have suffered from the absence of a mechanism for the introduction of DNA into this organism. As an initial step in the development of genetic methods, we have identified and cloned a native 10-kb plasmid, pJIR896, from a clinical isolate. This plasmid was found to be a circular form of vap region 1/3 that is found in the reference strain, A198. However, pJIR896 lacked the duplicated region present in the A198 sequence and instead contained a 1.7-kb putative insertion sequence, IS1253, which shared similarity to a number of unusual IS elements. A model is proposed for the evolution of vap region 1/3 which involves the integration of a plasmid, such as pJIR896, and subsequent rearrangements resulting from the deletion or transposition of IS1253.

Base Sequence↗
PubMed1996

Human erythrocyte dematin and protein 4.2 (pallidin) are ATP binding proteins.

Dematin and protein 4.2 are peripheral membrane proteins associated with the cytoplasmic surface of the human erythrocyte plasma membrane. Isoforms of dematin and protein 4.2 exist in many nonerythroid cells. In solution, dematin is a trimeric protein containing two subunits of 48 kDa and one subunit of 52 kDa. Recent determination of the primary structure of the 52 kDa subunit of dematin showed that it contains an additional 22-amino acid sequence in the headpiece domain. An alignment of the 22-amino acid insertion sequence revealed that the 52 kDa subunit of dematin shares a novel 11-amino acid motif with protein 4.2. In this communication, we report that the conserved 11-amino acid motif in dematin52 and protein 4.2 contains a nucleotide binding P-loop. Direct binding of ATP is demonstrated to the glutathione S-transferase fusion proteins containing corresponding segments of dematin52 and protein 4.2 as well as to purified protein 4.2. The binding of ATP to the recombinant domains of dematin52 and protein 4.2 is specific, saturable, and of high affinity. The nucleotide specificity of the P-loop is restricted to ATP since no detectable binding was observed with GTP. These results show that the 11-amino acid motif provides an ATP binding site in dematin52 and protein 4.2. Although the functional significance of ATP binding is not yet clear, our findings open new perspectives for the function of dematin and protein 4.2 in vivo.

Adenosine Triphosphate↗
PubMed1989

Compilation of DNA sequences of Escherichia coli.

We have compiled the DNA sequence data for E. coli K12 available from the GENBANK and EMBO databases and over a period of several years independently from the literature. We have introduced all available genetic map data and have arranged the sequences accordingly. As far as possible the overlaps are deleted and a total of 940,449 individual bp is found to be determined till the beginning of 1989. This corresponds to a total of 19.92% of the entire E. coli chromosome consisting of about 4,720 kbp. This number may actually be higher by some extra 2% derived from the sequence of lysogenic bacteriophage lambda and the various insertion sequences. This compilation may be available in machine readable form from one of the international databanks in some future.

Base Sequence↗
PubMed2005

Improved diagnosis of mycobacterial infections in formalin-fixed and paraffin-embedded sections with nested polymerase chain reaction.

Traditional histological diagnosis of mycobacterial infection in formalin-fixed and paraffin-embedded (FFPE) tissues is insensitive and poorly specific. To improve this, we developed nested polymerase chain reaction (PCR) protocols for detecting a Mycobacterium genus-specific 65-kDa heat shock protein (HSP65) sequence and the M. tuberculosis complex-specific insertion sequence IS6110 in FFPE sections. Protocols were optimized on tissues from 20 patients with a final clinical diagnosis of mycobacterial infection. Amplicons were controlled by sequencing and restriction endonuclease digestion. PCR could detect as few as three mycobacterial genomes per reaction. Assays showed 100% sensitivity and specificity for both M. tuberculosis complex and M. avium complex infection. Paraffin blocks from a second group of 26 patients with histological evidence of necrotizing granulomas of unknown etiology were then analyzed as a surrogate group to test the assay under conditions similar to those applying during routine diagnosis. Twenty-three of these blocks contained amplifiable DNA; nine were positive for M. tuberculosis complex DNA and four for other types of mycobacterial DNA. Furthermore, digestion of HSP65 amplicons with NarI could distinguish M. tuberculosis from M. avium complex. In conclusion, our nested PCR assays can be used as reliable tools for the detection of mycobacterial infections in FFPE tissues. The assays are simple and rapid to perform and show improved sensitivity and specificity compared to previously reported protocols.

DNA, Bacterial↗
PubMed2003

Molecular analysis of incHI1 antimicrobial resistance plasmids from Salmonella serovar Typhi strains associated with typhoid fever.

The first outbreak of multidrug-resistant (MDR) typhoid fever in Vietnam was in 1993, and by 1995 nearly 90% of cases were MDR. Plasmid HCM1, sequenced in full, is an incHI1 plasmid from Salmonella enterica serovar Typhi strain CT18, isolated in Vietnam in 1993. Restriction analysis shows that pHCM1 shares a restriction fragment length polymorphism (RFLP) pattern with plasmids isolated from the first outbreak and 10 of 17 MDR plasmids isolated from sporadic cases occurring at the same time in Vietnam. A core region of pHCM1 has significant DNA sequence similarity to plasmid R27, isolated in 1961 from S. enterica in the United Kingdom. There are five regions of DNA in pHCM1 which are not present in R27. Two of these are putative acquisition regions; the largest is 34.955 kbp in length and includes sequences of several antibiotic resistance genes and several insertion sequences. The borders of this region are defined by two identical IS10 left elements, associated with an inversion of DNA or with a truncated Tn10 element. The second, smaller region is 14.751 kbp and carries a trimethoprim resistance gene dfr14A cassette associated with a class 1 integrase. In 1993 to 1994, restriction analysis revealed some variations in the structures of Salmonella serovar Typhi MDR plasmids which were mapped to the two putative acquisition regions and three smaller variable regions. In 1996 a single RFLP type, RFLP7, was found to carry the dfrA7 and sul-1 genes, which were not present on R27 or pHCM1. This plasmid type appears to have a selective advantage over other plasmids with the same resistance phenotype.

Conjugation, Genetic↗
PubMed1995

Genetic Diversity of Xanthomonas oryzae pv. oryzae in Asia.

Restriction fragment length polymorphism and virulence analyses were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae, the rice bacterial blight pathogen, from several rice-growing countries in Asia. Two DNA sequences from X. oryzae pv. oryzae, IS1112, an insertion sequence, and avrXa10, a member of a family of avirulence genes, were used as probes to analyze the genomes of 308 strains of X. oryzae pv. oryzae collected from China, India, Indonesia, Korea, Malaysia, Nepal, and the Philippines. On the basis of the consensus of three clustering statistics, the collection formed five clusters. Genetic distances within the five clusters ranged from 0.16 to 0.51, and distances between clusters ranged from 0.48 to 0.64. Three of the five clusters consisted of strains from a single country. Strains within two clusters, however, were found in more than one country, suggesting patterns of movement of the pathogen. The pathotype of X. oryzae pv. oryzae was determined for 226 strains by inoculating five rice differential cultivars. More than one pathotype was associated with each cluster; however, some pathotypes were associated with only one cluster. Most strains from South Asia (Nepal and India) were virulent to cultivars containing the bacterial blight resistance gene xa-5, while most strains from other countries were avirulent to xa-5. The regional differentiation of clusters of X. oryzae pv. oryzae in Asia and the association of some pathotypes of X. oryzae pv. oryzae with single clusters suggested that strategies that target regional resistance breeding and gene deployment are feasible.

Journal Article↗
PubMed2002

Identification and broad dissemination of the CTX-M-14 beta-lactamase in different Escherichia coli strains in the northwest area of Spain.

During the course of a molecular epidemiology study of mechanisms of antibiotic resistance in the area served by our hospital (516,000 inhabitants), we isolated the gene encoding CTX-M-14 beta-lactamase. Thirty clinical strains (27 Escherichia coli and 3 Klebsiella pneumoniae isolates) with a phenotype of extended-spectrum beta-lactamase were collected from January to October 2001 and studied for the presence of the CTX-M-14 beta- lactamase gene. By isoelectric point determination, PCR, and nucleotide sequencing, we detected the presence of this gene in 17 E. coli strains belonging to 15 different genotypes (REP-PCR) causing infections in 17 different patients. Epidemiological studies based on medical records did not suggest any relationship between the patients infected with these E. coli strains and, interestingly, 7 of 30 patients harboring strains with extended-spectrum beta-lactamases never had contact with the hospital environment before the clinical E. coli isolation. Conjugation experiments revealed that this gene was plasmid mediated in the 17 E. coli strains, and plasmid restriction fragment length polymorphisms showed 9 different patterns in the 17 E. coli strains. By PCR, the sequence of the tnpA transposase gene of the insert sequence ISEcp-1 was detected in all the plasmids harboring the CTX-M-14 gene. These results strongly suggest that plasmid dissemination between different E. coli strains in addition to a mobile element (transposon) around the beta-lactamase gene may be involved in the spreading of the CTX-M-14 gene. This study reinforces the hypothesis that the epidemiology of the prevalence of the beta-lactamase genes is changing and should alert the medical community to the increase in the emergence of the CTX-M beta-lactamases worldwide.

Adolescent↗
PubMed1990

Recombination between irradiated shuttle vector DNA and chromosomal DNA in African green monkey kidney cells.

An autonomously replicating shuttle vector was used to investigate enhancement of plasmid-chromosome recombination in mammalian host cells by gamma irradiation and UV light. Sequences homologous to the shuttle vector were stably inserted into the genome of African green monkey kidney cells to act as the target substrate for these recombination events. The shuttle vector molecules were irradiated at various doses before transfection into the mammalian host cells that contained the stable insertions. The homologous transfer of the bacterial ampicillin resistance gene from the inserted sequences to replace a mutant ampicillin sensitivity gene on the shuttle vector was identified by the recovery of ampicillin-resistant plasmids after Hirt extraction and transformation into Escherichia coli host cells. Gamma irradiation increased homologous shuttle vector-chromosome recombination, whereas UV light did not increase the frequency of recombinant plasmids detected. Introducing specific double-strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was increased by UV irradiation of the plasmid but did not change with time. The ampicillin-resistant recombinant plasmid molecules analyzed appeared to rise mostly from nonconservative exchanges that involved both homologous and possibly nonhomologous interactions with the host chromosome. The observation that similar recombinant structures were obtained from all the plasmid treatments and host cells used suggests a common mechanism for plasmid-chromosome recombination in these mammalian cells.

Animals↗
PubMed1983

Movable DNA sequences.

Movable sequences described previously in the bacterial genome as insertion sequences and transposons seem to be present in a wide range of forms in all organisms. Structure, mechanism of transposition and regulatory function of these sequences are discussed.

Animals↗
PubMed1993

Identification and characterization of IS1138, a transposable element from Mycoplasma pulmonis that belongs to the IS3 family.

Insertion sequence (IS) elements are mobile genetic elements found in prokaryotes. We have identified a repetitive element from Mycoplasma pulmonis, a murine pathogen, that is similar to eubacterial IS elements. By subcloning a single strain of M. pulmonis, we isolated a variant clone in which the IS element had undergone an apparent transposition event. The nucleotide sequences of the element, designated IS1138, and the target site into which it inserted were determined. IS1138 consists of 1288 bp with 18 bp perfect terminal inverted repeats. Sequence analysis of the target site before and after insertion of IS1138 identified a 3 bp duplication of target DNA flanking the element. The predicted amino acids encoded by the major open reading frame of IS1138 share significant similarity with the transposases of the IS3 family. Southern hybridization analysis indicates that repetitive sequences similar to IS1138 are present in most, if not all, strains of M. pulmonis, but IS1138-like sequences were not detected in other mycoplasmal species.

Amino Acid Sequence↗
PubMed1991

Insertion of cellular sequences in the genome of bovine viral diarrhea virus.

The genomic sequences of four pestiviruses, two BVDV strains (Osloss and NADL, both of which are cytopathogenic) and two HCV strains, were analyzed. Comparative studies revealed the presence of small insertions of cellular sequences in the genomes of both BVDV strains; the insertions are located in a region coding for a nonstructural protein. Such insertions are not present in the HCV sequences. The insertion identified in BVDV Osloss encodes a complete ubiquitin-like element. The sequence inserted in the BVDV NADL genome shows no homology to a ubiquitin gene but is almost identical with another bovine mRNA sequence. Molecular characterization of a BVDV "pair", isolated from an animal with mucosal disease, led to the detection of a ubiquitin-like sequence in the genome of the cytopathogenic strain, but not of the noncytopathogenic strain. It is proposed that recombination between viral and cellular RNA leads to formation of cpBVDV genomes. This hypothesis has direct implications for the pathogenesis of mucosal disease.

Amino Acid Sequence↗
PubMed1996

Mobilization of vancomycin resistance by transposon-mediated fusion of a VanA plasmid with an Enterococcus faecium sex pheromone-response plasmid.

A striking feature of recent outbreaks of vancomycin-resistant (VmR) enterococci is the apparent horizontal dissemination of resistance determinants. The plasmids pHKK702 and pHKK703 from Enterococcus faecium clinical isolate R7 have been implicated in the conjugal transfer of VmR. pHKK702 is a 41-kb plasmid that contains an element indistinguishable from the glycopeptide-resistance transposon Tn1546. pHKK703 is an approx. 55-kb putative sex pheromone-response plasmid that is required for conjugative mobilization of pHKK702. During experiments in which strain R7 was used as a donor, a highly conjugative VmR transconjugant was isolated that formed constitutive cellular aggregates. Restriction analyses and DNA hybridizations revealed that the transconjugant harbored a single plasmid of approx. 92 kb and this plasmid (pHKK701) was composed of DNA from both pHKK702 and pHKK703. Results from DNA sequence analyses showed that a 39-kb composite transposon (Tn5506) from pHKK702 had inserted into pHKK703. The left end of Tn5506 contained a single insertion sequence (IS) element, IS1216V2, whereas the right end was composed of a tandem IS structure consisting of the novel 1065-bp IS1252 nested within an IS1216V1 element. Transposition of Tn5506 from pHKK702 to pHKK703 created an 8-bp target sequence duplication at the site of insertion and interrupted an ORF (ORFX) that was 91% identical to that of prgX, a gene proposed to negatively regulate sex pheromone response of the E.faecalis plasmid, pCF10. We propose that the interruption of ORFX by Tn5506 led to the constitutive cellular aggregation phenotype and thereby enhanced the efficiency with which VmR was transferred. Similar IS1216V-mediated transposition events may contribute to the horizontal spread of glycopeptide resistance among enterococci in nature.

Amino Acid Sequence↗
PubMed2000

Insertion sites for recombinant vaccinia virus construction: effects on expression of a foreign protein.

The expression of antigens or other molecules from recombinant vaccinia viruses requires the insertion of coding sequence at specific sites in the viral genome. Here we investigate the influence of two different sites on the level of protein expressed during a viral infection. The level of immune response in mice to vaccinia virus-expressed murine interleukin 2 (IL-2) or IL-4 varied depending on whether the coding sequence was inserted into the vaccinia virus thymidine kinase (tk) gene or into the HindIII F fragment of the viral genome where herpes simplex virus (HSV) tk was used as a selectable marker. In each case the intensity of the response was greater when the relevant gene was expressed from the HindIII F insertion site. In order to quantify these differences a series of recombinant viruses expressing luciferase was constructed. Luciferase activity from coding sequence inserted into the HindIII F fragment was significantly higher than that from the tk gene insertion, provided HSV tk(+) constructs were compared. Insertion of a marker gene (HSV tk) into the HindIII F site with disruption of the F7L open reading frame led to a reduced level of luciferase expressed from the tk insert, despite more than 45 kb of intervening sequence. In mice, luciferase expression was higher from the HindIII F inserted gene than from the tk insert in both lungs and ovaries.

Animals↗
PubMed1997

Conservation of IS6110 sequence in strains of Mycobacterium tuberculosis with single and multiple copies.

The insertion sequence IS6110/IS986 is used extensively for detecting and typing Mycobacterium tuberculosis. In order to appreciate the evolutionary and epidemiological significance of differences in the fingerprint pattern, it is necessary to understand the factors influencing transposition frequency. Although most strains have many copies of this element, some have only one or two copies; it has been suggested that this apparent transpositional defect arises from the minor differences in the sequence of the element from different strains. In contrast, we have found that the sequence is identical in high and low copy number strains, indicating that external factors, such as the presence of adjacent promoters, must be responsible for differences in copy number.

Base Sequence↗