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Crc (catabolite repression control) protein of Pseudomonas aeruginosa has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in Pseudomonas putida. The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the P. putida crc gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of the bkd operon, were isolated and identified as crc and vacB (rnr) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels.

The effect of growth in 2xYT medium on catabolite repression control in Pseudomonas putida has been investigated using the bkd operon, encoding branched-chain keto acid dehydrogenase. Crc (catabolite repression control protein) was shown to be responsible for repression of bkd operon transcription in 2xYT. BkdR levels were elevated in a P. putida crc mutant, but bkdR transcript levels were the same in both wild type and crc mutant. This suggests that the mechanism of catabolite repression control in rich media by Crc involves posttranscriptional regulation of the bkdR message.

Reinvestigation of the transcriptional start site of the bkd operon of Pseudomonas putida revealed that the transcriptional start site was located 86 nucleotides upstream of the translational start. There was a sigma 70 binding site 10 bp upstream of the transcriptional start site. The dissociation constants for BkdR, the transcriptional activator of the bkd operon, were 3.1 x 10(-7) M in the absence of L-valine and 8.9 x 10(-8) M in the presence of L-valine. Binding of BkdR to substrate DNA in the absence of L-valine imposed a bend angle of 92 degrees in the DNA. In the presence of L-valine, the angle was 76 degrees. BkdR did not bind to either of the two fragments of substrate DNA resulting from digestion with AgeI. Because AgeI attacks between three potential BkdR binding sites, this suggests that binding of BkdR is cooperative. P. putida JS110 and JS112, mutant strains which do not express any of the components of branched-chain keto acid dehydrogenase, were found to contain missense mutations in bkdR resulting in R40Q and T22I changes in the putative helix-turn-helix of BkdR. Addition of glucose to the medium repressed expression of lacZ from a chromosomal bkdR-lacZ fusion, suggesting that catabolite repression of the bkd operon was the result of reduced expression of bkdR. These data are used to present a model for the role of BkdR in transcriptional control of the bkd operon.