Cohabitation of two different lexA regulons in Pseudomonas putida

Abstract

In contrast to the vast majority of the members of the domain Bacteria, several Pseudomonas and Xanthomonas species have two lexA genes, whose products have been shown to recognize different LexA binding motifs, making them an interesting target for studying the interplay between cohabiting LexA regulons in a single species. Here we report an analysis of the genetic composition of the two LexA regulons of Pseudomonas putida KT2440 performed with a genomic microarray. The data obtained indicate that one of the two LexA proteins (LexA1) seems to be in control of the conventional Escherichia coli-like SOS response, while the other LexA protein (LexA2) regulates only its own transcriptional unit, which includes the imuA, imuB, and dnaE2 genes, and a gene (PP_3901) from a resident P. putida prophage. Furthermore, PP_3901 is also regulated by LexA1 and is required for DNA damage-mediated induction of several P. putida resident prophage genes. In silico searches suggested that this marked asymmetry in regulon contents also occurs in other Pseudomonas species with two lexA genes, and the implications of this asymmetry in the evolution of the SOS network are discussed.

FIG. 1.

Schematic diagram showing P. putida mitomycin C-induced transcriptional units (TUs) and their relationship to either LexA1 or LexA2 protein regulation. All genes shown had an induction factor higher than 2.0-fold in microarray data compared with either mitomycin C-treated or untreated wild-type expression cells. Data obtained by microarray analysis were confirmed for all the genes using quantitative RT-PCR (see Materials and Methods). Filled and open circles indicate the presence of LexA1 and LexA2 binding sites, respectively, in the corresponding promoter region. For the genes identified in the genome annotation, the gene designations are shown. Open reading frames without designations are indicated by their locus names. Genes belonging to P. putida genomic island 79 (from position 4363000 to position 4428000 of P. putida genome sequence and annotated as a bacteriophage [39]) are indicated by dark gray arrows. Connected arrows indicate polycistronic transcriptional units.

FIG. 2.

(A) Structural arrangement of the P. putida PP_3901 gene promoter. LexA1 and LexA2 binding sites and their positions with respect to the ATG translational starting codon are indicated. The bent arrows indicate the positions of the oligonucleotides used to obtain the PP_3901 promoter probe for EMSA experiments. (B) EMSA of the P. putida PP_3901 promoter in the absence or in the presence of either LexA1 or LexA2 purified protein. To determine the specificity of binding of both proteins, a 300-fold molar excess of either nonlabeled lexA1 or lexA2 promoter was used as a specific or nonspecific competitor fragment depending on the purified LexA protein that was used in each case.

FIG. 3.

Relative expression levels of the PP_3901 gene in the P. putida wild-type (Wt) strain and in lexA1 and lexA2 mutant derivatives of this strain. The expression level was measured by quantitative RT-PCR and is the ratio of the relative mRNA concentration of the PP_3901 gene in mitomycin C-treated (+) and untreated (−) cells to the relative PP_3901 mRNA concentration in the untreated P. putida wild-type strain. Relative mRNA concentrations were normalized to P. putida trpA gene expression. In each case, the mean value from three independent experiments (each performed in triplicate) is shown.

FIG. 4.

Relative expression levels of PP_3873 and PP_3894 prophage genes in the P. putida wild-type (Wt) strain and PP_3901, lexA1, and lexA2 mutant derivatives of this strain measured by quantitative RT-PCR. The relative expression level is the ratio of the relative mRNA concentration of either PP_3873 (light gray columns) or PP_3894 (dark gray columns) genes in mitomycin C-treated (+) and untreated (−) cells to the relative mRNA concentration of the gene in the untreated isogenic P. putida wild-type strain. The relative mRNA concentration of each gene was normalized to P. putida trpA gene expression. In each case, the mean value from three independent experiments (each performed in triplicate) is shown.