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An insertion sequence, IS22, in Pseudomonas aeruginosa PAO was identified by its genetic properties. It occurred as a single copy in the chromosome, as shown by Southern hybridization. A restriction map of it was constructed.
A constitutively expressed beta-lactamase gene from a clinical isolate of Stenotrophomonas maltophilia, J675Ia, has been cloned. Its DNA sequence is almost identical to that of bla(TEM2) (one nucleotide change) and the expressed enzyme is a Bush type 2a penicillinase with an amino acid sequence identical to that of TEM-2. The bla(TEM) gene was present within a novel Tn1/Tn3-type transposon in the genome of isolate J675Ia and the transposon was able to mobilize bla(TEM) on to the broad host-range conjugative plasmid, R388. When transferred to an Escherichia coli recipient, R388::Tn conferred high-level ampicillin resistance. This represents the first identification of a TEM beta-lactamase in S. maltophilia and the first evidence that this important clinical pathogen is able to act as a reservoir for mobile beta-lactamase genes in the hospital environment.
A large set of endogenous small RNAs of predominantly 21 to 24 nucleotides was identified in Arabidopsis. These small RNAs resembled micro-RNAs from animals and were similar in size to small interfering RNAs that accumulated during RNA silencing triggered by multiple types of inducers. Among the 125 sequences identified, the vast majority (90%) arose from intergenic regions, although small RNAs corresponding to predicted protein-coding genes, transposon-like sequences, and a structural RNA gene also were identified. Evidence consistent with the derivation of small RNAs of both polarities, and from highly base-paired precursors, was obtained through the identification and analysis of clusters of small RNA loci. The accumulation of specific small RNAs was regulated developmentally. We propose that Arabidopsis small RNAs participate in a wide range of post-transcriptional and epigenetic events.
Thioredoxin reductases (TRR) serve critical roles in maintaining cellular redox states. Two isoforms of TRR have been identified in mammals: both contain a penultimate selenocysteine residue that is essential for catalytic activity. A search of the genome of the invertebrate, Caenorhabditis elegans, reveals a gene highly homologous to mammalian TRR, with a TGA selenocysteine codon at the corresponding position. A selenocysteyl-tRNA was identified in this organism several years ago, but no selenoproteins have been identified experimentally. Herein we report the first identification of a C. elegans selenoprotein. By (75)Se labeling of C. elegans, one major band was identified, which migrated with the predicted mobility of the C. elegans TRR homologue. Western analysis with an antibody against human TRR provides strong evidence for identification of the C. elegans selenoprotein as a member of the TRR family. The 3'-untranslated region of this gene contains a selenocysteine insertion sequence (SECIS) element that deviates at one position from the previously invariant consensus "AUGA." Nonetheless, this element functions to direct selenocysteine incorporation in mammalian cells, suggesting conservation of the factors recognizing SECIS elements from worm to man.
The genome of the diploid hemiascomycetous yeast Candida tropicalis, an opportunistic human pathogen and an important organism for industrial applications, was explored by the analysis of 2541 Random Sequenced Tags (RSTs) covering about 20% of its genome. Comparison of these sequences with Saccharomyces cerevisiae and other species permitted the identification and the analysis of a total of more than 1000 novel genetic elements of C. tropicalis. Moreover, the present study confirms that in C. tropicalis, the rare CUG codon is read as a serine and not a leucine. The sequences have been deposited at EMBL with the accession numbers AL438875-AL441602.
Group II introns are an abundant class of self-splicing RNAs, found primarily in the organelles of plants and lower eukaryotes and in bacteria. The first bacterial group II intron identified to be functional for splicing in vivo was the Ll.ltrB intron of Lactococcus lactis. It has served as an excellent model for the study of group II intron structure and function. Taking advantage of the tools of bacterial genetics and biochemical methodologies, details of Ll.ltrB splicing and homing reactions have been elucidated and are similar to those of fungal group II introns. This review provides a summary of these results. Of particular interest is the potential use of Ll.ltrB as an agent for targeted gene disruption. In addition, the development of a genetic system to analyze Ll.ltrB splicing promises to provide new insight into group II intron structure and function. Identification and analysis of group II introns in other bacterial species is a continuing process, and a discussion of published reports on these introns is provided here. Limited functional data is available for most of these introns, but sequence analysis points out several common themes, most notably that bacterial group II introns are almost always carried on mobile genetic elements.
The rice disease resistance gene Xa21, which encodes a receptor-like kinase, is a member of a multigene family. Based on comparisons of genomic sequences of seven family members, seventeen transposon-like elements were identified in the 5' and 3' flanking regions and introns of these genes. Sequence characterization revealed that these elements are diverse, showing similarity to maize Ds, CACTA and miniature inverted repeat-like elements, as well as novel elements. Only two elements were located in presumed coding regions, indicating that integration of transposable elements at the Xa21 disease resistance locus occurred preferentially in noncoding regions.
Sphingomonas paucimobilis var. EPA505 utilizes fluoranthene (FLA), naphthalene (NAP), and phenanthrene (PHE) as sole carbon sources for energy and growth. A genetic library of EPA505 was constructed using mini-Tn5 promoter reporter genes encoding for tetracycline resistance (tc(p-)) or luminescence (luxAB(p-)). Out of 2250 Tn5 mutants, ten were deficient in utilization of FLA, NAP, and/or PHE as sole carbon sources. Three classes of Tn5 mutants were defined: classI (nap(-)phe(-)fla(-)), classII (nap(-)phe(-)), and classIII (fla(-)). Four of five mutants in classI did not express dioxygenase function, whereas one classI mutant and all classII and classIII mutants retained dioxygenase activity. In Tn5 tc(p-) classI mutants 200 and 394 (dioxygenase negative) and classII mutant 132 (dioxygenase positive), promoter reporter was expressed when induced with FLA, NAP, PHE, other polycyclic aromatic hydrocarbons (PAHs), and several proposed PAH-derived catabolites. The Tn5 tc(p-) derived classIII mutant 104 was induced only with PAHs and not with PAH-derived catabolites. DNA sequence analysis of cloned regions of classI mutant 200 revealed that Tn5 inserted into a gene that shared (96%) DNA sequence homology with 2,3-dihydroxybiphenyl 1,2-dioxygenase that is designated pbhA. Nucleotide sequences downstream of pbhA shared (84%) homology to a Rieske-type ferredoxin subunit gene of a multicomponent dioxygenase designated pbhB. The Tn5 tc(p-) in classII mutant 132 occurred within sequences that shared (74%) homology with a trans-o-hydroxybenzylidene-pyruvate hydratase-aldolase gene (pbhC). Sequence analysis of the region proximal to this gene revealed a putative promoter that contained a binding site for a LysR transcriptional activator. In classIII mutant 104, the Tn5 tc(p-) resided within a region that shared 94% nucleotide homology to that of a pyruvate phosphate dikinase gene known to be involved in cellular uptake of glucose. The FLA-specific catabolic gene disrupted in mutant 104 was designated phbD. Functional and sequence analyses of promoter probe mutants allowed identification of four genes necessary for the utilization of PAHs that are controlled by at least two promoters that are affected by a wide range of aromatic compounds.
The avoidance response to repellent odorants in Drosophila melanogaster, a response essential for survival, provides an advantageous model for studies on the genetic architecture of behavior. Transposon tagging in a highly inbred strain of flies in combination with a rapid and simple statistical behavioral assay enables the identification of not only large phenotypic effects, but also small aberrations from wild-type avoidance behavior. The recent completion of the sequence of the Drosophila genome facilitates the molecular characterization of transposon-tagged genes and correlation between gene expression and behavior in smell-impaired (smi) mutant lines. Quantitative genetic analyses of a collection of smi lines in a coisogenic background revealed an extensive network of epistatic interactions among genes that shape the olfactory avoidance response. The identification and functional characterization of proteins encoded by smi genes that form part of the olfactory subgenome and correlation of polymorphisms in these genes with variation in odor-guided behavior in natural populations will advance our understanding of the genetic architecture of chemosensory behavior.
Pseudomonas syringae pv. tomato PT23.2 produces the chlorosis-inducing phytotoxin coronatine. Thirty-eight chlorosis-defective mutants of PT23.2 were previously generated by using the transposon Tn5. Five mutants contained Tn5 insertions in the indigenous plasmid pPT23A; the remaining 33 mutants either were missing pPT23A (29 mutants) or contained deletions in this plasmid (4 mutants). These results suggested that pPT23A was involved in coronatine production in strain PT23.2. This plasmid was introduced into P. syringae pv. syringae PS61, which does not produce coronatine. A bioassay for coronatine suggested that PS61(pPT23A) transconjugants were able to make this phytotoxin. In a chemical analysis, organic acids were isolated from PT23.2, PS61, and the transconjugant PS61(pPT23A); these were derivatized to their methyl esters and analyzed by gas chromatography. The derivatized organic acids extracted from PT23.2 and PS61(pPT23A) contained peaks that corresponded to coronafacic acid, coronafacoylvaline, and coronatine, but these were absent in the extracts from the wild-type strain PS61. The identification of these components was confirmed by combined gas chromatography-mass spectrophotometry. Therefore, the acquisition of pPT23A by PS61 resulted in biosynthesis of coronafacic acid, coronafacoylvaline, and coronatine, clearly demonstrating the involvement of pPT23A in coronatine production in P. syringae pv. tomato.
Pituitary-derived trophic hormones regulate cell-type-specific expression of VL30 retrotransposons in tissues that are engaged in steroidogenesis. We show that adrenocorticotropic hormone and forskolin induced VL30 transcription in the steroidogenic adrenal cell line Y1 and that the transcriptional activation was cell type- and protein kinase A-dependent. Three novel cAMP-responsive elements (CREs), within the VL30 long terminal repeat, were identified and shown to activate transcription synergistically when templates bearing multiple sites were compared with templates bearing a single site. This type of regulation was evident only in forskolin-treated cells, and the response elements were found to be inactive as mediators of constitutive transcription. In vitro binding analyses indicated that a consensus CRE and the nonconsensus VL30 CREs differ with respect to binding affinity and specificity to a number of nuclear factors that were identified to be related to proteins within the CREB, Jun, and C/EBP families of transcription factors. The relatively low affinity and/or a restricted binding specificity of the VL30 CREs made it possible to detect forskolin-induced binding of CREB- and Jun-related proteins to these sequences. We suggest that cAMP-induced transcription, specific for steroidogenic cells, can be mediated by a novel type of nonconsensus CREs and that the mechanism for this type of gene regulation is distinct from that mediated through a consensus CRE. We also report the identification of a novel factor, distinct from previously characterized CRE-binding proteins, that constitutively binds to the identified CREs.
Bacillus cereus strains ATCC 10987 and ATCC 14579 harbor an approximately 155-bp repeated element, bcr1, which is conserved in B. cereus, B. anthracis, B. thuringiensis, and B. mycoides but not in B. subtilis and B. licheniformis. In this study, we show by Southern blot hybridizations that bcr1 is present in all 54 B. cereus group strains tested but absent in 11 Bacillus strains outside the group, suggesting that bcr1 may be specific and ubiquitous to the B. cereus group. By comparative analysis of the complete genome sequences of B. cereus ATCC 10987, B. cereus ATCC 14579, and B. anthracis Ames, we show that bcr1 is exclusively present in the chromosome but absent from large plasmids carried by these strains and that the numbers of full-length bcr1 repeats for these strains are 79, 54, and 12, respectively. Numerous copies of partial bcr1 elements are also present in the three genomes (91, 128, and 53, respectively). Furthermore, the genomic localization of bcr1 is not conserved between strains with respect to chromosomal position or organization of gene neighbors, as only six full-length bcr1 loci are common to at least two of the three strains. However, the intergenic sequence surrounding a specific bcr1 repeat in one of the three strains is generally strongly conserved in the other two, even in loci where bcr1 is found exclusively in one strain. This finding indicates that bcr1 either has evolved by differential deletion from a very high number of repeats in a common ancestor to the B. cereus group or is moving around the chromosome. The identification of bcr1 repeats interrupting genes in B. cereus ATCC 10987 and ATCC 14579 and the presence of a flanking TTTAT motif in each end show that bcr1 exhibits features characteristic of a mobile element.
We report a rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species - Bacteroides caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis and B. vulgatus. These 10 species were first divided into three subgroups by multiplex PCR-G, followed by three multiplex PCR assays with three species-specific primer mixtures for identification to the species level. The primers were designed from nucleotide sequences of the 16S rRNA, the 16S-23S rRNA intergenic spacer region and part of the 23S rRNA gene. The established two-step multiplex PCR identification scheme was applied to the identification of 155 clinical isolates of the B. fragilis group that were previously identified to the species level by phenotypic tests. The new scheme was more accurate than phenotypic identification, which was accurate only 84.5% of the time. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of the B. fragilis group species. This will permit more accurate assessment of the role of various B. fragilis group members in infections and of the degree of antimicrobial resistance in each of the group members.
Mycobacterium marinum, a causative agent of fish tuberculosis, is one of the most closely related Mycobacterium species (outside the M. tuberculosis complex) to M. tuberculosis, the etiologic agent of human tuberculosis. Signature-tagged mutagenesis was used to identify genes of M. marinum required for in vivo survival in a goldfish model of mycobacterial pathogenesis. Screening the first 1008 M. marinum mutants led to the identification of 40 putative virulence mutants. DNA sequence analysis of these 40 mutants identified transposon insertions in 35 unique loci. Twenty-eight out of 33 (85%) loci encoding putative virulence genes have homologous genes in M. tuberculosis.
The vanA gene cluster is carried as a part of Tn1546-like elements. The genetic diversity in Tn1546-like elements has been documented previously. The differences described thus far have included the integration of insertion sequence (IS) elements IS1216V, IS1251, IS1476, and IS1542. Among these, IS1216V has been reported to be widespread in VanA enterococci of diverse geographic areas, whereas IS1542 and IS1476 have been reported only in the United Kingdom and Canada, respectively. We investigated the distribution of ISs among 20 vanA-containing Enterococcus faecium isolates from human patients in nine different university hospitals in Korea. Pulsed-field gel electrophoresis (PFGE) was performed to identify the clonality of the isolates. Moreover, PCR amplification of the internal regions of Tn1546 was performed for structural analysis of the van gene, and both DNA strands of the PCR amplicons were directly sequenced by the dideoxy termination method. The PFGE patterns revealed a high degree of clonal diversity. Structural analyses of the van gene detected IS1542 and IS1216V in the genomes of all 20 isolates, whereas it did not detect IS1476 or IS1251 in the genomes of any of the isolates. In addition, IS19 was detected in the vanS-vanH intergenic region of one isolate. These data indicate that identification of the IS within a vanA gene cluster could be a useful tool in epidemiological investigations. In addition, the distribution of ISs associated with Tn1546-like elements among the Korean isolates is therefore similar to that among European vancomycin-resistant enterococci.
In the traditional study of tuberculosis epidemiology, information about social contact of persons and patient's illness history used to be an only relevant basis for elucidating transmission of tuberculosis infection. Therefore, it was very difficult to give a clear conclusion of whether isolates from different patients derived from a common source of infection or not. Recently, the subspecies typing of M. tuberculosis strains has become possible, based on the visualization of multiple loci of an insertion sequence (IS6110) that is a relatively stable gene fragment existing in a specific region of the genome. The variability of the number of copies and locations of this IS6110 in a genome is the basis that enables this technique to be used for the above purpose, which is a unique tool applicable to the analysis of M. tuberculosis. Generally, this technique, i.e., restriction fragment length polymorphism (RFLP) analysis, depends on the diversity of pattern of any polymorphic marker found in a genome of a strain. Among various markers so far developed and examined, IS6110 has been proved most appropriate for the purpose of typing strains of M. tuberculosis complex, especially in such circumstances as in Japan where isolated strains' RFLP patterns are similar each with others so that finer subtyping is needed. In this lecture, I would like to review the following topics based on the world literature of molecular epidemiology and the findings of our own that we have achieved during 1992 through 2001 in our Institute; 1) typing of the isolates for the identification of the infection source, 2) pathogenesis of tuberculosis under low incidence situation, 3) predominance of certain genotypes endemic in an area, 4) cross-contamination of isolates in the laboratory, 5) the stability of IS6110 patterns, 6) phylogeny of M. tuberculosis complex, and 7) differentiation between M. tuberculosis and M. bovis BCG.
We have identified genes required for competence of Neisseria meningitidis, a naturally transformable human pathogen. Although not comprehensive, our analysis identified competence-defective mutants with transposon insertions in genes not previously implicated in this process in Neisseria.
In order to set up a sensitive and reliable detection method to monitor environmentally released genetically engineered microorganisms (GEMs) a 72-bp, double-stranded DNA fragment has been built by annealing and ligating four synthetic oligonucleotides. Binding sites for two 20-mer oligonucleotides are situated inside the DNA fragment, flanking the centre. Into the central part of the construction a 30-nucleotide identification sequence has been fitted. Thanks to the presence of the two oligonucleotide binding sites, the synthetic construction ("number-plate") can be submitted to enzymatic amplification using the polymerase chain reaction (PCR), thus enabling the identification system to take advantage of the outstanding sensitivity of this technique. When released into a freshwater microcosm, cells of Pseudomonas putida carrying a "number-plated" chromosome could be easily and rapidly detected merely by submitting boiled cell sediments to PCR amplification.