Transcriptional and translational analyses of recA mutant alleles in Pseudomonas aeruginosa

Horn, Joseph M.; Ohman, Dennis E.

doi:10.1128/jb.170.4.1637-1650.1988

Cited by 27 publications

(40 citation statements)

References 55 publications

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“…The conjugative helper plasmid pRK2013 (Figurski & Helinski, 1979) was used in triparental matings to transfer recombinant plasmids to P. aeruginosa strains FRDl (Ohman & Chakrabarty, 1981), FRD261 algB5O (Goldberg & Ohman, 1984), FRD284 recA7 (Ohman et af., 1985), PAOl (Holloway et al, 1979), and PD03 recA7 (Horn & Ohman, 1988). Samples (0.2 ml) of stationary-phase L broth cultures of E. coli HBlOl (containing a recombinant plasmid), HBlOl(pRK2013), and P. aeruginosa were mixed, collected on a membrane filter (25 mm diam., 0.45 pm pore size; Millipore), and incubated cell side up on the surface of an L agar plate for 6 to 24 h at 37 "C. Cells were resuspended in saline (5ml) and spread onto MM agar supplemented with the appropriate amino acids and tetracycline to select for transconjugants of the P. aeruginosa recipient.…”

Section: Methodsmentioning

confidence: 99%

Precise excision and instability of the transposon Tn5 in Pseudomonas aeruginosa

Goldberg

Won²,

Ohman³

1990

Journal of General Microbiology

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Excision of the transposon Tn5 from sites of insertion in plasmid DNA was shown to occur at high frequency in Pseudomonas aeruginosa Plasmids with Tn5 insertions conjugated poorly into P. aeruginosa and adversely affected growth compared to the respective parental plasmids. The kanamycin-resistance phenotype of Tn5 was expressed poorly in P. aeruginosa and kanamycin-sensitive strains were common during the manipulation of the P. aeruginosa transconjugants. Examination of plasmid DNA isolated from kanamycin-sensitive P. aeruginosa transconjugants revealed excision of Tn5 sequences. A plasmid containing a selectable marker (mercury resistance) inactivated by a Tn5 insertion was constructed, and Tn5 excised precisely, permitting the expression of the mercury-resistance marker at high frequency in P. aeruginosa and at the expected low frequency (lo-') in Escherichia cofi. The recombinational mechanism that promotes frequent Tn5 excision in P. aeruginosa operated in the absence of the P. ueruginosa recA gene product. Fragments of Tn5 were also examined for excision and instability in P. ueruginosa. A plasmid containing the terminal 485 bp of inverted repeat sequences from Tn5, but lacking the transposase or kanamycin-resistance genes, also showed precise excision of Tn5 DNA at high frequency ( in P. ueruginosa. Unlike plasmids containing a complete Tn5 insertion, this plasmid transferred to P. aeruginosa at normal frequencies and growth of the host was not severely impaired. In contrast, plasmids containing either IS50 element transferred to P. aeruginosa at greatly reduced frequencies, and transconjugants containing the IS50R element (which contains the active transposase gene) were small and especially difficult to maintain. P. aeruginosa transconjugants harbouring a plasmid containing only the DNA between the IS50 elements (which included the kanamycin-resistance gene) were of normal size and stably maintained. These observations suggest that frequent and precise excision of Tn5 in P. aeruginosa required the long inverted repeat sequences at the termini of Tn5. The adverse effects conferred by IS50 on the transconjugant formation and growth of P. aeruginosa were apparently not required to promote Tn5 excision.

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Section: Methodsmentioning

confidence: 99%

Precise excision and instability of the transposon Tn5 in Pseudomonas aeruginosa

Goldberg

Won²,

Ohman³

1990

Journal of General Microbiology

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“…In P. aeruginosa, UV-dependent prophage activation is also regulated by the RecA protein Wokjohn , and expression of the recA gene itself is autoregulated and induced by DNAdamaging agents (Horn & Ohman, 1988;. Furthermore, the E. coli and P.…”

Section: Discussionmentioning

confidence: 99%

“…aeruginosa has been observed to be relatively sensitive to (Kung & Lee, 1973;Krishnapillai, 1975;Kokjohn & Miller, 1985;McBeth, 1989) and non-mutable by (Lehrbach et al, 1979;McBeth, 1989) UV radiation. Mutations in various loci, including recA (Kokjohn & Miller, 1987Horn & Ohman, 1988), which increase the UV sensitivity of this species have been isolated (Krishnapillai, 1975).…”

Section: Introductionmentioning

confidence: 99%

Inducible UV repair potential of Pseudomonas aeruginosa PAO

1990

Journal of General Microbiology

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Pseudomonas aeruginosa P A 0 lacks UV-inducible Weigle reactivation and Weigle mutagenesis of UV-damaged bacteriophages. This lack of UV-inducible, error-prone DNA repair appears to be due to the absence of efficiently expressed umuDC-like genes in this species. When the P. mruginosa recA gene is introduced into a recA(Def) mutant of Escherichia coli K12, the P. aeruginosa recA gene product is capable of mediating UV-induced mutagenesis, indicating that it could participate in a red-lexA-like regulatory network and function in inducible DNA repair pathways if such existed in P. aevuginosa. The presence of the IncP9, UV-resistance plasmid R2 in RecA+ strains of P. aeruginosa P A 0 allows UV-inducible, mutagenic DNA repair of UV-irradiated bacteriophages. R2 also greatly stimulates the ability of UV radiation to induce mutagenesis of the bacterial chromosome. When R2 is introduced into P. aeruginosa strains containing either the recA908 or recAlO2 mutation, plasmid-mediated UV resistance and Weigle reactivation are not observed. These observations suggest that the increased protection afforded to P. aeruginosa by R2 is derived from a RecA-mediated, DNA-damage-inducible, error-prone DNA repair system which complements the lack of a chromosomally encoded umuDC-like operon.

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“…RecA protein is mechanistically involved in both recombinational repair and induced mutagenesis. Similar roles have been postulated for the product of the P. aeruginosa recA gene (10,17). Like their E. coli counterparts, P. aeruginosa recA mutants are deficient in recombination and extremely sensitive to UV irradiation and other DNA-damaging agents (4,8).…”

mentioning

confidence: 88%

The CAM-OCT plasmid enhances UV responses of Pseudomonas aeruginosa recA mutants

McBeth

1990

J Bacteriol

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The effect of the CAM-OCT plasmid on responses to UV irradiation of Pseudomonas aeruginosa recA mutants was characterized. Mutant alleles examined included rec-1, rec-2, and recA7::Tn501. The plasmid substantially enhanced both survival and mutagenesis of RecA- cells after treatment with UV light. Survival of the RecA-(CAM-OCT) cells after UV irradiation was intermediate between that seen in the wild-type P. aeruginosa PAO1 and the increased survival seen in PAO1(CAM-OCT) cells. Mutability was quantitated by the reversion to carbenicillin resistance of strains carrying a bla(Am) mutation on a derivative of plasmid RP1. UV-induced mutagenesis of CAM-OCT carrying recA mutants occurred at levels comparable to that seen in PAO1(CAM-OCT). The ability of CAM-OCT plasmid to suppress the recombination deficiency in recA mutants was tested by assaying for bacteriophage F116L-generalized transduction of a Tn7 insertion in the alkane utilization genes of CAM-OCT. Transduction of the Tn7 insertion was not detected in RecA-(CAM-OCT) strains but was easily seen in PAO1(CAM-OCT), indicating that the plasmid does not encode a recA analog. The results indicate that the CAM-OCT UV response genes are expressed in RecA- cells, which differs from results seen with other UV response-enhancing plasmids. The results suggest that CAM-OCT either encodes several UV responses genes itself or induces chromosomal UV response genes by an alternate mechanism.

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Transcriptional and translational analyses of recA mutant alleles in Pseudomonas aeruginosa

Cited by 27 publications

References 55 publications

Precise excision and instability of the transposon Tn5 in Pseudomonas aeruginosa

Precise excision and instability of the transposon Tn5 in Pseudomonas aeruginosa

Inducible UV repair potential of Pseudomonas aeruginosa PAO

The CAM-OCT plasmid enhances UV responses of Pseudomonas aeruginosa recA mutants

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