Evidence that an Additional Mutation Is Required To Tolerate Insertional Inactivation of the<i>Streptomyces lividans recA</i>Gene

Vierling, Silke; Weber, Tilmann; Wohlleben, Wolfgang; Muth, Günther

doi:10.1128/jb.183.14.4374-4381.2001

Cited by 18 publications

(22 citation statements)

References 43 publications

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“…To our knowledge, the only other recA gene for which a disruption was achieved only under special circumstances is that of Streptomyces lividans (Muth et al, 1997), in which viable mutant strains were found to lack only short extensions of the RecA C-terminus, and displayed residual RecA activity. Recent studies in S. lividans suggested that an additional mutation (resulting in a sporulation defect) is required to tolerate total recA deficency, although its role remains obscure (Vierling et al, 2001). Although the observed inability to inactivate recA2 demands further investigation, our present data indicate that this locus might be required for sustaining cell viability in B. megaterium.…”

Section: Expression Of Reca1 and Reca2 Is Damageinduciblementioning

confidence: 60%

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

2005

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Isolation and subsequent knockout of a recA-homologous gene in Bacillus megaterium DSM 319 resulted in a mutant displaying increased sensitivity to mitomycin C. However, this mutant did not exhibit UV hypersensitivity, a finding which eventually led to identification of a second functional recA gene. Evidence for recA duplicates was also obtained for two other B. megaterium strains. In agreement with potential DinR boxes located within their promoter regions, expression of both genes (recA1 and recA2) was found to be damage-inducible. Transcription from the recA2 promoter was significantly higher than that of recA1. Since a recA2 knockout could not be achieved, functional complementation studies were performed in Escherichia coli. Heterologous expression in a RecA null mutant resulted in increased survival after UV irradiation and mitomycin C treatment, proving both recA gene products to be functional in DNA repair. Thus, there is evidence for an SOS-like pathway in B. megaterium that differs from that of Bacillus subtilis.

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Section: Expression Of Reca1 and Reca2 Is Damageinduciblementioning

confidence: 60%

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

2005

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“…In fact, ϳ10% of E. coli recA mutants are anucleate, and an additional fraction show signs of chromosomal degradation (35,49). By contrast, Streptomyces lividans recA mutants are not viable at all unless additional compensatory mutations are selected that suppress the lethal effect of the absence of RecA (41). In T. thermophilus recA mutants, there are also defects in chromosome condensation and asymmetric partitioning, but this could represent only a minor fraction (ϳ10 to 15%) of the cells and cannot by itself explain the dramatic decrease in viability observed (Fig.…”

Section: Discussionmentioning

confidence: 99%

Temperature-Dependent Hypermutational Phenotype inrecAMutants ofThermus thermophilusHB27

et al. 2003

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The recA gene from Thermus thermophilus HB27 was cloned and engineered to obtain insertion (recA::kat) and deletion (⌬recA) derivatives. Transcription of recA in this extreme thermophile was induced by mitomycin C, leading to the synthesis of a monocistronic mRNA. This DNA damage-mediated induction was dependent on the integrity of recA. In addition to UV sensitivity, the recA mutants of T. thermophilus showed severe pleiotropic defects, ranging from irregular nucleoid condensation and segregation to a dramatic reduction in viability during culture. An increase in the frequency of both carotenoidless and auxotrophic mutants within surviving cells of the ⌬recA strain indicated a high mutation rate. As RecA is not required for plasmid transformation, we have used the ␣-lacZ gene fragment and the ampicillin resistance gene from Escherichia coli as passenger reporters to confirm such high mutation rates. Our data support the idea that the absence of RecA results in a hypermutational phenotype in T. thermophilus. Furthermore, a direct relationship is deduced between the growth temperature and mutation rate, which finally has a deleterious effect on cell survival in the absence of RecA.RecA is a multifunctional protein that plays key roles in several cellular processes, such as recombination and DNA repair in Bacteria (10, 23). Accordingly, the sequences of RecA proteins from different origins are well conserved, allowing the construction of robust phylogenetic trees (8). Homologues to the RecA protein have also been described in Eucarya and Archaea (17, 32).The functions of RecA have been analyzed in great detail in Escherichia coli, where different mutants are available (42). RecA controls the expression of the SOS regulon through its inducible coprotease activity. This activity is acquired when RecA binds to single-stranded DNA regions originated by the arrest of DNA replication or as a consequence of DNA damage. After a conformational change, activated RecA promotes autocleavage of the SOS repressor LexA, which in the E. coli LexA protein occurs at the Ala 84 -Gly 85 peptide bond. This allows access by the RNA polymerase to at least 30 gene promoters, including those of the recA and lexA genes (42). The final goal of the overexpression of the SOS genes is to permit the cell to grow by repairing most of the damage and also by translesion DNA synthesis using the specialized DNA polymerase V. This DNA polymerase requires the help of RecA and single-stranded DNA-binding proteins to act (28, 37). The translesion synthesis mechanism is responsible for the hypermutational effect that the induction of the SOS system has in E. coli and other bacteria. In fact, recA mutants of E. coli are defective in DNA damage-mediated mutagenesis and show a low spontaneous mutation rate (42).RecA is also required for homologous recombination in normal cells (30). Therefore, and despite their extreme UV sensitivity, recA mutants of E. coli are commonly used as hosts for genetic engineering, since the stability of plasmid constructs is rel...

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“…The acquisition of a rare compensatory mutation enabling the spirochete to overcome recA inactivation as a necessary precondition for isolation of a recA null mutant cannot be ruled out. Such a compensatory mutation has been suggested for tolerance of a recA null mutation in Streptomyces lividans (57).…”

Section: Discussionmentioning

confidence: 99%

Borrelia burgdorferi vlsE Antigenic Variation Is Not Mediated by RecA

Liveris¹,

Mulay

Sandigursky³

et al. 2008

Infect Immun

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RecA is a key protein linking genetic recombination to DNA replication and repair in bacteria. Previous functional characterization of Borrelia burgdorferi RecA indicated that the protein is mainly involved in genetic recombination rather than DNA repair. Genetic recombination may play a role in B. burgdorferi persistence by generation of antigenic variation. We report here the isolation of a recA null mutant in an infectious B. burgdorferi strain. Comparison of the in vitro growth characteristics of the mutant with those of the wild-type strain under various conditions showed no significant differences. While the RecA mutant was moderately more sensitive to UV irradiation and mitomycin C than the wild-type strain, the lack of RecA abolished allelic exchange in the mutant. Absence of RecA did not affect the ability of the mutant to infect mice. However, the RecA mutant was attenuated for joint infection in competitive-infection assays with the wild-type strain. vlsE sequence variation in mice was observed in both wild-type and RecA mutant spirochetes, indicating that the mechanism of antigenic variation is not homologous genetic recombination.

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Evidence that an Additional Mutation Is Required To Tolerate Insertional Inactivation of theStreptomyces lividans recAGene

Cited by 18 publications

References 43 publications

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

Temperature-Dependent Hypermutational Phenotype inrecAMutants ofThermus thermophilusHB27

Borrelia burgdorferi vlsE Antigenic Variation Is Not Mediated by RecA

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