Control of expression of divergent Pseudomonas putida put promoters for proline catabolism

Abstract

Pseudomonas putida KT2440 uses proline as the sole C and N source. Utilization of this amino acid involves its uptake, which is mediated by the PutP protein, and its conversion into glutamate, mediated by the PutA protein. Sequence analysis revealed that the putA and putP genes are transcribed divergently. Expression from the putP and putA genes was analyzed at the mRNA level in different host backgrounds in the absence and presence of proline. Expression from the put promoters was induced by proline. The transcription initiation points of the putP and putA genes were precisely mapped via primer extension, and sequence analysis of the upstream DNA region showed well-separated promoters for these two genes. The PutA protein acts as a repressor of put gene expression in P. putida because expression from the put promoters is constitutive in a host background with a knockout putA gene. This regulatory activity is independent of the catabolic activity of PutA, because we show that a point mutation (Glu896-->Lys) that prevents catalytic activity allowed the protein to retain its regulatory activity. Expression from the put promoters in the presence of proline in a putA-proficient background requires a positive regulatory protein, still unidentified, whose expression seems to be sigma(54) dependent because the put genes were not expressed in a sigma(54)-deficient background. Expression of the putA and putP genes was equally high in the presence of proline in sigma(38)- and ihf-deficient P. putida backgrounds.

FIG. 1

DNA sequence of the intergenic region between the putA and putP genes. The ATG start codon of the genes is boxed; the transcription initiation point of each gene is marked by an asterisk, and the −10 and −35 regions of each promoter are underlined.

FIG. 2

Expression of the putA and putP genes of P. putida KT2440 under different growth conditions. mRNA was prepared as described previously (20). Cells were grown in different media as follows. Lane 1, M8 minimal medium with 20 mM proline; lane 2, M9 minimal medium with 20 mM proline; lane 3, M9 with 20 mM proline and succinate; lane 4, M9 with succinate. Primer extension analysis was done as described in Materials and Methods with a primer complementary to putA or putP mRNA.

FIG. 3

Expression from the putA and putP promoters in P. putida KT2442, the PutA-deficient derivative S14D2, and the PutA Glu896→Lys mutant. Cells were grown in the absence (−) or presence (+) of proline. The strains were P. putida KT2442 (lanes 1 and 2), P. putida S14D2 (lanes 3 and 4), and P. putida KT2442-Pro21 (lanes 5 and 6). Other conditions are as described in the legend for Fig. 2.

FIG. 4

Expression from the putA and putP gene promoters in different host backgrounds. P. putida cells were grown in the presence (+) or absence (−) of proline. The strains used were the wild type (lane 1), IHF-deficient mutant (lane 2), ς⁵⁴-deficient mutant (lane 3), ς³⁸-deficient mutant (lane 4), and ptsN-deficient mutant (lane 5). Other conditions are as described in the legend for Fig. 2.