Transcriptional and translational analyses of recA mutant alleles in Pseudomonas aeruginosa

Abstract

Recombinant plasmids containing the recA gene from Pseudomonas aeruginosa were used in complementation, transcriptional, and translational studies to examine the nature of rec-102 and rec-2, mutations which confer a recA-like mutant phenotype on P. aeruginosa PAO strains. For comparison, recA7::Tn501 mutants of strain PAO were constructed by gene replacement. The rec-2 and rec-102 alleles were shown to be recA alleles; plasmids containing the recA gene complemented the three rec mutant strains for defects associated with recA mutation. Northern blot analyses indicated that the recA gene in P. aeruginosa was transcribed as two distinct mRNAs of approximately 1.2 and 1.4 kilobases (kb). A plasmid encoding both transcripts of recA complemented all defects associated with the three recA mutations rec-2, rec-102, and recA7. However, a 2.4-kb subclone (pJH13) encoding only the smaller transcript of the recA gene was expressed differently in the three recA allele backgrounds and served as a tool to distinguish the nature of the rec-2 and rec-102 mutations in recA. A minicell analysis showed that a plasmid expressing both of the recA gene transcripts or one that expressed only the smaller transcript both produced the same 42-kilodalton recA protein. A chloramphenicol acetyltransferase gene fusion in the 3' end of the recA transcript showed that the recA gene of P. aeruginosa was induced following treatment with a DNA-damaging agent (methyl methanesulfonate). The recA7 mutant constructed here showed no recA-related transcript or protein under inducing conditions, and pJH13 in this host produced only low levels of the smaller recA transcript and low levels of recA protein. The rec-2 mutant produced a detectable transcript but no recA protein following induction. The presence of low levels of activated recA protein encoded by pJH13 in the rec-2 mutant resulted in wild-type transcriptional levels of chromosomally encoded recA, but no recA protein was detectable. Thus, the rec-2 allele of recA was normal with respect to induction of mRNA, but these transcripts were defective in either translation or synthesis of a stable protein. The rec-102 mutant also produced a detectable transcript and no recA protein following induction, but having pJH13 in the cell to produce low levels of activated recA protein resulted in overproduction of chromosomally encoded recA transcripts and active recA protein. Thus, the recA defect in the rec-102 mutant is apparently in the interaction between recA and a lexA-like repressor.