Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities

Abstract

Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy-and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA ؉ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDC Ec ). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDC Ec proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants.The SOS regulon, which plays an important role in bacterial responses to DNA damage, has been most extensively studied in Escherichia coli (41). This regulon is composed of more than 20 genes (see reference 12) and is involved in several physiological responses such as DNA repair and mutagenesis, whose expression is transcriptionally regulated by LexA and RecA proteins. LexA repressor binds to similar operators of most SOS genes, including recA and lexA. Treatments that cause DNA damage or stalled DNA replication lead to the activation of the RecA coprotease which facilitates the autocatalytic cleavage of LexA and the subsequent derepression of SOScontrolled genes (see reference 13). The umuDC operon, as well as the analogous mucAB operon, is induced as part of the SOS response, and its products are required for most UV and chemical mutagenesis in E. coli. The UmuD protein is processed to the mutagenically active form UmuDЈ by activated RecA through a mechanism similar to that of the cleavage of LexA (2, 22, 34). The mechanism of SOS mutagenesis is not yet clarified, but biochemical evidence suggests that the UmuCUmuDЈ complex and RecA help DNA polymerase III holoenzyme to facilitate error-prone translesion DNA synthesis (29).The closely related species Salmonella typhimurium has a SOS regulatory system which resembles that of E. coli (37). Several SOS loci such as recA (28), uvrB (31), uvrD (25), sulA (4), and several other din (damage-inducible) loci...

Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica

“…Our microarray analyses show a moderate induction of the SOS response, exemplified by the genes recA , umuCD , uvrABY , sulA and mutH . Induction of the SOS response would normally be linked to a cessation of bacterial replication caused by the SulA protein (Clerch et al ., 1996). However, the repressor protein for sulA , encoded by lexA , showed a similar induction of expression, suggesting a balancing role for LexA in the activation and suppression of the SOS response.…”

Identification of Salmonella functions critical for bacterial cell division within eukaryotic cells

“…As intracellular bacteria are exposed to DNA‐damaging agents (Schlosser‐Silverman et al ., 2000), we examined if the arrest in the division process could be directed by SulA, the classic SOS gene product inhibiting cell division (Hill et al ., 1997). We transduced the sulA46::Spc insertion (Clerch et al ., 1996) into the SL1344 strain. Upon infection of MelJuSo cells with the resulting strain TH101, we still observed filamentous bacteria (Fig.…”

Phenotypes of lexA Mutations in Salmonella enterica : Evidence for a Lethal lexA Null Phenotype Due to the Fels-2 Prophage

“…A strain carrying a lexA insertion mutation after base 11 of the coding sequence (lexA40::Kan; Fig. 1B) was constructed in vitro and kindly provided to us by Llagostera and Garriga (15). This strain (TT17653; ϭ UA1685) also carries a constructed sulA46::Spc (spectinomycin resistance) insertion that was rendered essential by the lexA defect and could not be removed by transduction.…”

“…The insertion mutations lexA40::Kan (Fig. 1B) and sulA46::Spc were constructed and provided by Montserrat Llagostera and Xavier Garriga (15). The reported structures were confirmed by sequencing.…”