Allele replacement in Escherichia coli by use of a selectable marker for resistance to spectinomycin: replacement of the lexA gene

Abstract

We replaced the Escherichia coli lexA gene by a segment of DNA coding for resistance to spectinomycin and streptomycin. The use of this segment expands the range of selectable markers usable for allele replacement. The availability of this null lexA mutation will facilitate genetic analysis of lexA and the SOS regulon.The Escherichia coli LexA protein controls the expression of the SOS regulon (11,17). Many In order to overcome these problems, we have substituted another selectable marker, resistance to spectinomycin (Spcr), for the lexA gene and have introduced it into the chromosome by homologous recombination (18). The new allele of lexA, which we term lexA300(Del) [where "(Del)" extends the nomenclature of reference 11 and signifies a deletion], was made from plasmid pJWL197 (Fig. 1A), in which a DNA segment coding for resistance to spectinomycin and streptomycin was flanked by homology with E. coli sequences lying on both sides of lexA. Plasmid DNA was linearized by digestion with Sacl and PstI and introduced into strain KP520 (a sulA100::TnS derivative [3] of strain JC9387 recB21 recC22 sbcB15 [16]) as described previously (18), and then selection for resistance to 30 jig of spectinomycin per ml was carried out. Since this level of drug gave heterogenous colony morphology, 20 ,ug/ml was used in all subsequent selections. Because strain KP520 and most other strains used in work on the SOS system carry an rpsL allele conferring resistance to streptomycin (Strr), simultaneous selection for Strr was not possible. In a later step, the lexA300 marker was transduced into other recipient strains by P1 transduction and selection for Spcr.Several lines of evidence indicated that lexA300(Del) completely replaces the resident lexA sequences (Fig. 1B). * Corresponding author.First, recipients carrying lexA7J::TnS became sensitive to kanamycin upon transduction to Spcr, indicating tight linkage between this marker and lexA71. Second, Southern analysis of DNA from Spcr transductants of recipients carrying lexA+ or lexA71::TnS (data not shown) showed that the substitution altered the size of restriction fragments in the region spanning lexA in a manner consistent with the maps in Fig. 1B and that flanking regions were unaltered. Analysis of four of the initial Spcr transformants of strain KP520 also showed that the chromosomal lexA sequences were deleted and that the omega fragment was present on fragments of the predicted size. Third, lexA300 inactivated the LexA repressor function, as judged by several tests using sulA::lacZ operon fusions. Levels of P-galactosidase were identical to those observed in strains carrying lexA7J::Tn5.When strain JL1478, a derivative of JL1436 (L. Lin and J. W. Little, manuscript in preparation) carrying lexA3(Ind-) on the chromosome and expressing low levels of f3-galactosidase, was transduced to Spcr, it also displayed a constitutive level of enzyme. In addition, when a lexA7J: :TnS strain and a lexA300 derivative were transformed with a multicopy plasmid, pJWL147, carrying a lacP-lexA+ ...

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

“…The original sulA lexA mutant shows no growth defect. This behavior is similar to that of E. coli lexA null mutants, whose lethal phenotype is corrected by a sulA mutation (22,26).…”

Overexpression of MutS impairs DNA mismatch repair and causes cell division defect inE.coli

“…This suggests that MutS binding to DNA contributes to the DNA DSBs and cell elongation phenotype. To confirm whether DNA DSBs induced SOS response, using sulA::lacZ fusions 42,43 , we measured SOS response and found that arabinose induced MutS-overexpression elicited significantly higher SOS response than uninduced levels of MutS-overexpression (Fig.6I).…”

A phenotype for enigmatic DNA polymerase II: A pivotal role for pol II in replication restart in UV-irradiated Escherichia coli

“…The presence of the ⌬(araD-polB)::⍀ allele was selected on LB agar plates containing 20 g͞ml spectinomycin and was confirmed by PCR analysis (see below). Use of higher concentrations of spectinomycin were avoided because they resulted in colonies with different size morphology consistent with the acquisition of additional chromosomal mutations in rpsE (25). To simplify notation, we refer to the ⌬(araD-polB)::⍀ allele as ⌬polB.…”