Transcriptional and Mutational Analyses of theStreptomyces lividans recXGene and Its Interference with RecA Activity

Abstract

The role of the 20,922-Da RecX protein and its interference with RecA activity were analyzed in Streptomyces lividans. The recX gene is located 220 bp downstream of recA. Transcriptional analysis by reverse transcriptase PCR demonstrated that recX and recA constitute an operon. While recA was transcribed at a basal level even under noninducing conditions, a recA-recX cotranscript was only detectable after induction of recA following DNA damage. The recA-recX cotranscript was less abundant than the recA transcript alone. The recX gene was inactivated by gene replacement. The resulting mutant had a clearly diminished colony size, but was not impaired in recombination activity, genetic instability, and resistance against UV irradiation. Expression of an extra copy of the S. lividans recA gene under control of the thiostrepton-inducible tipA promoter was lethal to the recX mutant, demonstrating that RecX is required to overcome the toxic effects of recA overexpression. Since inactivation of the recX gene did not influence transcription of recA, the putative function of the RecX protein might be the downregulation of RecA activity by interaction with the RecA protein or filament.

Differential cross-complementation patterns of Escherichia coli and Neisseria gonorrhoeae RecA proteins

“…Work in Pseudomonas aeruginosa, Mycobacterium smegmatis and Streptomyces lividans has demonstrated that overexpression of recA from a plasmid construct is deleterious or lethal in the absence of recX (Papavinasasundaram et al, 1997 ;Sano, 1993 ;Vierling et al, 2000), leading to the hypothesis that RecX acts by downregulating expression of recA or activity of the RecA protein. However, this hypothesis has not been rigorously tested, and a recX mutation of S. lividans does not affect any RecA-mediated processes or RecA levels (Vierling et al, 2000). Gc also contains a recX homologue ; however, it is not located downstream of recA.…”

“…In mycobacteria and Streptomyces lividans the genes are cotranscribed (Papavinasasundaram et al, 1997 ;Vierling et al, 2000). Further work has led to the hypothesis that RecX acts by downregulating RecA activity (Sano, 1993 ;Papavinasasundaram et al, 1998 ;Vierling et al, 2000), though this hypothesis has not been directly tested, and a recX mutation in S. lividans does not affect any RecA-mediated processes or RecA expression levels. E. coli contains a RecX homologue located downstream of recA, designated oraA (Zaitsev et al, 1994).…”

“…2) (Sano, 1993 ;Vierling et al, 2000). We therefore wanted to determine whether RecA protein levels were influenced by the presence of recX and whether the level of …”

Analysis of the Role of recA in Phenotypic Switching of Pseudomonas tolaasii

“…This argues against a direct role of recX in pheN duplication-related phenotypic switching from 1116S to 1116R forms in P. tolaasii. Although the precise role of recX in recA-mediated recombination is still under investigation, the ability of recX to reduce the toxicity of recA overexpression has been demonstrated in P. aeruginosa (18), Streptococcus lividans (20), and Mycobacterium smegmatis (16).…”

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

“…The genetic instability of S. lividans strains was measured as the ratio of chloramphenicol-sensitive colonies, as described by Vierling et al (36).…”