Cloning, sequencing, and transcriptional analysis of the recA gene of Pseudomonas cepacia

Nakazawa, Takahiro; Kimoto, Mitsuaki; Abe, Mitsuko

doi:10.1016/0378-1119(90)90471-3

Cited by 20 publications

(13 citation statements)

References 21 publications

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“…This putative promoter overlaps the Cheo box which we have shown here to be the binding site for LexA. Such an arrangement of overlapping promoter and LexA binding site has been described for several gram-negative bacterial recA genes (16,39,60).…”

Section: Discussionsupporting

confidence: 74%

Determination of DNA sequences required for regulated Mycobacterium tuberculosis RecA expression in response to DNA-damaging agents suggests that two modes of regulation exist

1997

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The recA gene of Mycobacterium tuberculosis has previously been cloned and sequenced (E. O. Davis, S. G. Sedgwick, and M. J. Colston, J. Bacteriol. 173:5653-5662, 1991). In this study, the expression of this gene was shown to be inducible in response to various DNA-damaging agents by using a transcriptional fusion to the reporter gene encoding chloramphenicol acetyltransferase. A segment of DNA around 300 bp upstream of the coding region was shown to be required for expression. However, primer extension analysis indicated that the transcriptional start sites were 47 and 93 bp upstream of the translation initiation codon. Sequence motifs with homology to two families of Escherichia coli promoters but also with significant differences were located near these proposed transcription start sites. The differences from the E. coli consensus patterns would explain the previously described lack of expression of the M. tuberculosis recA gene from its own promoter in E. coli. In addition, the M. tuberculosis LexA protein was shown to bind specifically to a sequence, GAAC-N4-GTTC, overlapping one of these putative promoters and homologous to the Bacillus subtilis Cheo box involved in the regulation of SOS genes. The region of DNA 300 bp upstream of the recA gene was shown not to contain a promoter, suggesting that it functions as an upstream activator sequence.

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Section: Discussionsupporting

confidence: 74%

Determination of DNA sequences required for regulated Mycobacterium tuberculosis RecA expression in response to DNA-damaging agents suggests that two modes of regulation exist

1997

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“…The partial ORF was determined to encode 654 amino acids, and amino acids 37 to 654 exhibited significant similarity with the amino acids of RecG proteins from N. meningitidis (56% identity), P. aeruginosa (57% iden- The amino acid sequence encoded by this ORF was found to be 100% identical to the sequence of the RecA protein from several strains of Burkholderia vietnamiensis, and it exhibited 97 to 98% identity with RecA proteins from multiple strains of B. cepacia. The sequence upstream of the recA start codon was very similar to that previously described for Pseudomonas cepacia (32). A putative SOS box (a palindromic sequence that binds the LexA repressor protein of the SOS response system) was identified 100 bp upstream of the start codon, overlapping with a potential Ϫ10 promoter consensus sequence ( Fig.…”

Section: Resultssupporting

confidence: 56%

Requirement of DNA Repair Mechanisms for Survival of Burkholderia cepacia G4 upon Degradation of Trichloroethylene

Yeager

Bottomley²,

Arp

2001

Appl Environ Microbiol

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A Tn5-based mutagenesis strategy was used to generate a collection of trichloroethylene (TCE)-sensitive (TCS) mutants in order to identify repair systems or protective mechanisms that shield Burkholderia cepacia G4 from the toxic effects associated with TCE oxidation. Single Tn5 insertion sites were mapped within open reading frames putatively encoding enzymes involved in DNA repair (UvrB, RuvB, RecA, and RecG) in 7 of the 11 TCS strains obtained (4 of the TCS strains had a single Tn5 insertion within a uvrB homolog). The data revealed that the uvrB-disrupted strains were exceptionally susceptible to killing by TCE oxidation, followed by the recA strain, while the ruvB and recG strains were just slightly more sensitive to TCE than the wild type. The uvrB and recA strains were also extremely sensitive to UV light and, to a lesser extent, to exposure to mitomycin C and H 2 O 2 . The data from this study establishes that there is a link between DNA repair and the ability of B. cepacia G4 cells to survive following TCE transformation. A possible role for nucleotide excision repair and recombination repair activities in TCE-damaged cells is discussed.Trichloroethylene (TCE), a suspected human carcinogen (17), has been used extensively as a metal degreaser, fumigant, and solvent for dry cleaning and in other commercial applications. Because of its widespread use and persistence, TCE is one the most commonly detected organic pollutants at hazardous waste sites and in municipal groundwater supplies in the United States (24, 48). Although it has not been demonstrated that microorganisms can utilize TCE as a growth-supporting substrate under aerobic conditions, a number of bacteria are able to degrade TCE cometabolically; in this process nonspecific oxygenases catalyze the initial transformation (1,8,44).The practicality of utilizing bacteria to degrade TCE via aerobic cometabolism has been questioned, however, due to the cytotoxicity that is almost universally associated with this process. Loss of TCE-degradative activity is often observed with whole cells during TCE transformation (34,36,43,45,49), and each of the TCE-degrading enzymes that have been purified to homogeneity and examined to date (toluene dioxygenase, toluene 2-monooxygenase, and soluble methane monooxygenase) exhibits turnover-dependent inactivation upon TCE oxidation (12,22,33). Additionally, TCE degradation can result in injuries that adversely affect more basic cellular functions, such as general respiratory activity and cell viability (4,16,43,49). Although the exact nature of the destructive species remains unknown, it has been proposed that acyl chlorides, generated from hydrolysis or rearrangement of TCE epoxide (monooxygenase-catalyzed reactions) or TCEdioxetane (dioxygenase-catalyzed reactions), cause damage by alkylating various cellular constituents (12,22,33,43,46). Since cellular toxicity can potentially limit the sustainability of TCE biodegradation under aerobic conditions, a concerted effort has been directed towards identifying strains t...

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“…Such elements have been implicated in the recruitment of foreign genes for novel catabolic functions (15,43,54,60). Although there is considerable information about the biochemical activities of P. cepacia (2,3,11,25,26,35,40,45,51) and although certain genes and IS elements have been isolated and characterized (5,7,10,15,16,33,36,42,54,59,61), little is known about the overall organization of genes or the genomic distribution of IS elements. To gain such information we undertook the construction of a macrorestriction map of the genome of P. cepacia 17616 by using recently developed techniques for the manipulation of large DNA fragments (8,17,19,(46)(47)(48) To test the phenotypes of auxotrophic strains, 0.5% potassium phthalate or mannitol was substituted for the yeast extract and the medium was supplemented with 50 jig of each required amino acid per ml.…”

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confidence: 99%

Multiple replicons constituting the genome of Pseudomonas cepacia 17616

1994

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Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction fragments containing rare restriction enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 megabases (Mb). Inclusion of the 170-kb cryptic plasmid present in this strain gave an overall estimate of genome size of 7 Mb. Other Southern hybridization experiments indicated that the three large replicons contained rRNA genes as well as insertion sequence elements identified previously in this strain. The distribution of Swal, PacI, and Pmel sites on the three replicons was determined. A derivative of TnS-751 carrying a SwaI site was used to inactivate and map genes on the 2.5-and 3.4-Mb replicons. Mutants were isolated in which the 2.5-and 0.9-Mb replicons had been reduced in size to 1.8 and 0.65 Mb, respectively. The loss of DNA from the 2.5-Mb replicon was associated with lysine auxotrophy, P-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources. DNA fragments corresponding in size to randomly linearized forms of the different replicons were detected in unrestricted DNA by pulsed-field gel electrophoresis. The results provide a framework for further genetic analysis of strain 17616 and for evaluation of the genomic complexities of other P. cepacia isolates.Pseudomonas cepacia has attracted attention because of its extraordinary biodegradative abilities and its potential as an agent of bioremediation (2,13,22,25,35,44,45,51,56). The genome of this bacterium contains a large number of insertion sequences (ISs) (25, 27), which were identified on the basis of their abilities to promote genomic rearrangements (4, 12) and to activate the expression of neighboring genes (15,43,60). Such elements have been implicated in the recruitment of foreign genes for novel catabolic functions (15,43,54,60). Although there is considerable information about the biochemical activities of P. cepacia (2,3,11,25,26,35,40,45,51) and although certain genes and IS elements have been isolated and characterized (5,7,10,15,16,33,36,42,54,59,61), little is known about the overall organization of genes or the genomic distribution of IS elements. To gain such information we undertook the construction of a macrorestriction map of the genome of P. cepacia 17616 by using recently developed techniques for the manipulation of large DNA fragments (8,17,19,(46)(47)(48) To test the phenotypes of auxotrophic strains, 0.5% potassium phthalate or mannitol was substituted for the yeast extract and the medium was supplemented with 50 jig of each required amino acid per ml. For growth in liquid medium, 10-to 20-ml cultures were shaken in 125-ml flasks. For growth on plates, the medium was supplemented with 2% (wt/vol) agar. Preparation of genomic DNA for pulsed-field gel electrophoresis (PFGE). Agarose plugs containing intact chromosomal DNA were prepared essentially as described by Smith and Cantor (47). Approximatel...

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Cloning, sequencing, and transcriptional analysis of the recA gene of Pseudomonas cepacia

Cited by 20 publications

References 21 publications

Determination of DNA sequences required for regulated Mycobacterium tuberculosis RecA expression in response to DNA-damaging agents suggests that two modes of regulation exist

Determination of DNA sequences required for regulated Mycobacterium tuberculosis RecA expression in response to DNA-damaging agents suggests that two modes of regulation exist

Requirement of DNA Repair Mechanisms for Survival of Burkholderia cepacia G4 upon Degradation of Trichloroethylene

Multiple replicons constituting the genome of Pseudomonas cepacia 17616

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