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.Pseudomonads play an important role in nature because of their ability to metabolize natural and manufactured organic chemicals. Many of these compounds are environmental pollutants, such as benzene, toluene, xylene, ethylbenzene, styrene, and chlorobenzoates (18), and their removal has been named bioremediation. Although the enzymic pathways responsible for degradation of these pollutants may be effective when the target compound is the sole growth-supporting substrate, in nature these compounds are present as mixtures, and some substrates may be degraded preferentially. Catabolite repression control refers to the ability of an organism to preferentially metabolize one carbon source over another when both are present in the growth medium. Because of the importance of pseudomonads to bioremediation efforts, understanding the control of catabolite repression is important so that more efficient, genetically modified organisms can be utilized in the removal of these environmental pollutants.The molecular mechanisms of catabolite repression control have been extensively characterized in enteric bacteria, where glucose is the preferred carbon source. In these organisms, enzymes of the phosphoenolpyruvate-dependent phosphotransferase system mediate catabolite repression control by regulation of cyclic AMP (cAMP) concentration via adenylate cyclase activity (22). The strongest repressing substrates in Pseudomonas spp. are acetate, tricarboxylic acid cycle intermediates, and glucose (4, 10, 26). Unlike Escherichia coli, in Pseudomonas species adenylate cyclase activity, cAMP phosphodiesterase activity, and cAMP pools do not fluctuate with carbon source, nor does the addition of cAMP relieve repression of catabolite responsive pathways (21, 25). In addition, only one phosphotransferase system (fructose) has been identified in Pseudomonas (5), suggesting that PTS components are not involved in catabolite repression control in pseudomo...

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.The molecular mechanisms of catabolite repression have been well described in enteric bacteria, where enzymes of the phosphoenolpyruvate phosphotransferase system mediate catabolite repression control by regulation of cAMP concentration via adenylate cyclase activity (19). However, a similar mechanism does not appear to be present in Pseudomonas because adenylate cyclase activity and cAMP pools do not fluctuate with carbon source, nor does addition of cAMP relieve repression of catabolite responsive pathways (12, 18). The only protein thus far shown to be involved in catabolite repression in Pseudomonas is Crc of P. aeruginosa, but a function has not been identified (13). However, Crc does not appear to bind DNA (13), suggesting that it is not simply a DNA-binding negative regulator.Crc is involved in catabolite repression of P. putida branched-chain keto acid dehydrogenase (BCKAD), glucose-5-phosphate dehydrogenase, and amidase by glucose and succinate in synthetic media (11). BCKAD is encoded by the four structural genes of the bkd operon, which is positively regulated by BkdR (15). BkdR is a homologue of Lrp (leucineresponsive protein), which is a global transcriptional regulator in Escherichia coli (4). However, pseudomonads and enteric bacteria live in complex media in nature and not in chemically defined media. Expression of lrp is downregulated in nutritionally rich media (6), which suggested that this might also be the case with bkdR. In this report, the effect of 2xYT medium on the expression of bkdR in wild type and in a crc mutant of P. putida was studied to determine if catabolite repression control of the bkd operon might be accomplished by controlling the level of BkdR in the cell.Crc downregulates BCKAD activity in 2xYT. The wild-type strains of P. putida and P. aeruginosa, their crc mutants, and the complemented mutants (11) were grown to an A 660 of ϳ0.6 in 100 ml of 2xYT plus 0.3% valine and 0.1% isoleucine (wt/vol) and then harvested; cell extracts were then prepared as described earlier (16). P. putida JS394 had five-to sixfold higher activity than either PpG2 or JS394 (pJRS196) ( Table 1), and a similar result was obtained when BCKAD activity of PAO8020 was compared to the activities of PAO1 and PAO8020 (pPZ352). These results demonstrate that Crc is involved in catabolite repression control of BCKAD activity by 2xYT in both P. putida and P. aeruginosa. However, the BCKAD activities of the crc were much lower than that ...

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 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 ؋ 10 ؊7 M in the absence of L-valine and 8.9 ؋ 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؇ in the DNA. In the presence of L-valine, the angle was 76؇. 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.The branched-chain keto acid dehydrogenase complex of Pseudomonas putida is the second enzyme in the pathway for the metabolism of branched-chain amino acids. There are three components in the complex, E1, the dehydrogenase, E2, the transacylase, and E3, the lipoamide dehydrogenase. The E1 component of P. putida branched-chain keto acid dehydrogenase has been purified and shown to be a heterotetramer (10). The E3 component is a specific lipoamide dehydrogenase, , which has been crystallized and whose structure has been solved (19). The four polypeptides constituting branched-chain keto acid dehydrogenase are encoded by the bkd operon (2-4), which is expressed as a polycistronic message.Branched-chain keto acid dehydrogenase of P. putida is induced during growth on branched-chain amino or keto acids (18). Expression is also under catabolite repression control by glucose and succinate (28). Expression of the bkd operon is positively regulated by BkdR, which is encoded by a structural gene which is divergently transcribed from the bkd operon (17). BkdR is a homolog of Lrp, the leucine-responsive protein of Escherichia coli (5). Lrp is a global regulator in E. coli, but the low copy number of BkdR suggests that its main purpose in P. putida is regulation of the bkd operon (16). The region to which BkdR binds was identified by DNase I protection studies (16) as a large segment of DNA between bkdR and bkdA1, the latter being the first gene of the bkd operon (Fig. 1). BkdR is a homotetramer (14, 16), and three tetramers of BkdR bind to its substrate DNA (12). Millimolar L-branched-chain amino acids cause a conformational change in BkdR (14), and the fact that L-b...

Active E l component of Pseudomonas putida branched-chain-oxoacid dehydrogenase was purified from F1 putida strains carrying pJRS84 which contains bkdR (encoding the transcriptional activator) and bkdAl and hkdA2 (encoding the a and 1) subunits). Expression was inducible, however, 4 5 , 39-and 37-kDa proteins were produced instead of the expected 45-kDa and 37-kDa proteins. The 45-kDa protein was identified as Ela and the 37-kDa and 39-kDa proteins were identified as separate translational products of bkdA2 by their N-terminal sequences. The N-terminal amino acid of the 39-kDa protein was leucine instead of methionine. The 4 5 , 39-and 37-kDa proteins were also produced in wild-type f?putida. Translation of bkdA1 and hkdA2 from an Escherichia coli expression plasmid produced only 45-kDa and 39-kDa proteins, with N-terminal methionine on the 39-kDa protein. The insertion of guanine residues 5' to the first ATG of bkdA2 did not affect expression of El/? in I? putida including the N-terminal leucine which appears to eliminate the possibility of ribosome jumping. The Z-average molecular mass of the E l component was determined by sedimentation equilibrium to be 172 -C 9 kDa compared to a calculated value of 166 kDa for the heterotetramer and a Stokes radius of 5.1 nm. E l u Ser313, which is homologous to the phosphorylated residue of rat liver E l u , was converted to alanine resulting in about a twofold increase in K,n, but no change in KcL,r. S315A and S319A mutations had no effect on K,, or K,,, indicating that these residues do not play a major part in catalysis of Eln,P,.Keywords: branched-chain-oxoacid dehydrogenase ; dihydrolipoamide dehydrogenase ; multienzyme complexes ; protein engineering ; protein expression.Branched-chain-oxoacid dehydrogenase is a multienzyme complex with a structure similar to pyruvate and 2-oxoglutarate dehydrogenases. These complexes play important roles in energy metabolism of aerobic bacteria and most eukaryotes. Mutations affecting these complexes lead to serious and frequently fatal diseases in man [I, 21. Oxoacid dehydrogenase complexes consist of three components, E l , which catalyzes decarboxylation and dehydrogenation of the oxoacid, E2, which catalyzes transacylation of the acyl group from E2 to coenzyme A and E3, which catalyzes the oxidation of the lipoyl residue of E2 and allows the reaction to recycle 131. E3 is dihydrolipoamide dehydrogenase 141 and generally there is a single dimeric dihydrolipoamide dehydrogenase in each species for pyruvate, 2-oxoglutarate, and branched-chain-oxoacid dehydrogenases and glycine decarboxylase. However, Pseudomonas putidu has specific dihydrolipoamide dehydrogenases, Lpd-glc [5] [17] contain amino acid sequences similar to the phosphorylation sites, the bacterial complexes are not phosphorylated. The activity of F1 putida branched-chain-oxoacid dehydrogenase however, is stimulated by all three L-branched chain amino acids [23].It was established several years ago that the subunit stoichiometry of the E l component of bovine pyruvate d...

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