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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 (leucine-responsive 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 A₆₆₀ of ~0.6 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 obtained in minimal media (11), indicating that something in addition to Crc is involved in catabolite repression control in synthetic medium.

Crc reduces the level of BCKAD in 2xYT. BCKAD is a multienzyme complex with three components. The E1 component of P. putida BCKAD is an heterotetramer (10), the structure of which has just recently been determined (1). The E2 component is a transacylase (2), and the E3 component is a specific lipoamide dehydrogenase (3). In mammalian cells, BCKAD activity is regulated by a posttranslational modification: E1 contains two phosphorylation sites, and the phosphorylation state regulates activity of the complex (17). Although P. putida E1 is not phosphorylated (10), it is possible that catabolite repression of P. putida BCKAD activity could be the result of some other kind of posttranslational modification of BCKAD or could be the result of reduction in transcription of the bkd operon. To distinguish between these two possibilities first, Western blots with anti-E1 serum (10) were employed. P. putida PpG2, JS394, and JS394(pJRS196), and P. aeruginosa strains PAO1, PAO8020, and PAO8020(pJRS196) were grown to an A₆₆₀ of ~0.6 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). Five micrograms of protein was loaded onto a sodium dodecyl sulfate (SDS)-8.5% polyacrylamide gel electrophoresis (PAGE) gel, blotted to Hybond-enhanced chemiluminescence (ECL) membrane, and treated with anti-E1 serum. As seen in Fig. 1, E1 protein levels were greatly increased in the crc mutants P. putida JS394 and P. aeruginosa PAO8020 compared to the other four strains. The increase in E1 reflected the increased BCKAD activities found in these extracts (Table 1). Therefore, repression of BCKAD activity by Crc is due to a reduction in the amount of BCKAD. P. putida PpG2 grown in glucose minimal medium had no BCKAD activity, nor could E1 be detected by Western blots.

View larger version (36K): [in this window] [in a new window]   FIG. 1.   Western blot with anti-E1 serum (10) of crc mutants grown in 2xYT plus valine-isoleucine. The extracts in lanes 1 to 6 were from cultures grown in 2xYT plus 0.3% valine and 0.1% isoleucine (wt/vol); the extract in lane 7 was from a culture grown in glucose minimal medium. Each lane contained 5 µg of protein. Lane 1, P. putida PpG2; lane 2, P. putida JS394; lane 3, P. putida JS394(pJRS196); lane 4, P. aeruginosa PAO1; lane 5, P. aeruginosa PAO8020; lane 6, P. aeruginosa PA8020(pJRS196); lane 7, P. putida PpG2 grown in glucose. All cultures were grown to an A660 of between 0.6 and 0.8, and cell extracts were prepared as described before (16). Electrophoresis was done in an SDS-8.5% PAGE gel. Western blots were screened by using the ECL-Western blotting analysis system (Amersham Pharmacia Biotech) with Hybond-ECL nitrocellulose membranes according to the manufacturer's instructions. To determine whether the ECL detection method was quantitative, increasing amounts of PpG2 grown in valine-isoleucine-lactate medium were loaded on a gel and used for Western blotting with anti-E1 serum. The blot was scanned with a Molecular Dynamics densitometer, and pixel values versus micrograms of protein were graphed and shown to be a linear plot (data not shown).

Crc regulates the level of BkdR produced in 2xYT. To characterize the role of BkdR in catabolite repression of BCKAD activity, Western blots with anti-BkdR serum were used to measure BkdR levels in wild type and the crc mutant of P. putida in grown in 2xYT plus valine-isoleucine and valine-isoleucine synthetic media. Then, 200 µg of cell extracts from P. putida PpG2, JS394, and JS394(pJRS196) grown in 2xYT plus 0.3% valine-0.1% isoleucine, along with P. putida PpG2 grown in 0.3% valine-0.1% isoleucine synthetic medium (16) alone or with 40 mM succinate, were loaded on an SDS-12% PAGE gel, blotted to Hybond-P membrane, and treated with anti-BkdR serum. As seen in Fig. 2, P. putida JS394 grown in 2xYT plus valine-isoleucine had higher levels of BkdR than either PpG2 or JS394(pJRS196) grown under the same conditions. This result demonstrates that Crc plays a major role controlling the level of BkdR in 2xYT and suggests that relief of catabolite repression control in crc mutants is due to a higher level of BkdR. BkdR levels of P. putida PpG2 were much higher in minimal media than in 2xYT (lanes 4 to 5 of Fig. 2), corresponding to the higher BCKAD activity in minimal medium (11). Also, the amount of BkdR was not repressed when succinate was added to the inducing medium, suggesting a different kind of control in minimal media. No BkdR was detected in P. putida JS386 which contains a bkdR-lacZ translational fusion (14).

View larger version (9K): [in this window] [in a new window]   FIG. 2.   Levels of BkdR in wild type and in the crc mutant of P. putida. These cultures were grown and harvested as in Fig. 1, and 200 µg of each cell extract was loaded onto an SDS-12% PAGE gel, blotted to Hybond-P membrane, and treated with anti-BkdR (14). More protein was used than in the experiment of Fig. 1 because of the low copy number of BkdR per cell. Hybond-polyvinylidene difluoride (PVDF) membranes (Amersham) were used in this experiment because PVDF has a better binding capacity for low-molecular-weight proteins. Proteins were separated by electrophoresis in an SDS-12% PAGE gel. Lane 1, P. putida grown in 2xYT plus 0.3% valine-0.1% isoleucine; lane 2, P. putida JS394 grown in 2xYT plus 0.3% valine-0.1% isoleucine; lane 3, P. putida JS394(pJRS196) grown in 2xYT plus 0.3% valine-0.1% isoleucine; lane 4, P. putida PpG2 grown in 0.3% valine-0.1% isoleucine synthetic medium; lane 5, P. putida PpG2 grown in 0.3% valine-0.1% isoleucine plus 40 mM succinate; lane 6, P. putida JS386 grown in 2xYT plus 0.3% valine-0.1% isoleucine; lane 7, 20 ng of purified BkdR.

Repression of BCKAD activity by 2xYT involves posttranscriptional regulation of bkdR expression. Since both BCKAD and BkdR levels are elevated in the JS394 grown in 2xYT supplemented with valine and isoleucine (Fig. 1 and 2), mRNA levels of bkdR and bkdA1, which encodes E1, were of interest in determining the mechanism of action of Crc in repression by 2xYT. P. putida PpG2, JS394, and JS394(pJRS196) were grown in 2xYT plus valine-isoleucine medium. P. putida JS382 (15), a mutant with a deletion in bkdR, which does not produce bkd operon mRNA, was grown under the same conditions for use as a negative control. At mid-log phase, total RNA was purified from each culture. Five micrograms total RNA was transferred to a GeneScreen Plus membrane by means of a vacuum suction HYBRI-SLOT Filtration Manifold (Life Technologies). The membrane was prehybridized for 1 h at 42°C and then hybridized overnight with 0.5 µCi of bkdA1 or bkdR mRNA probes at the same temperature. After a washing at 65°C, the membrane was exposed to a Phosphor Screen (Molecular Dynamics) overnight. The membrane was stripped by boiling for 30 min in 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) plus 1% SDS and then prehybridized and hybridized as described above with 0.3 µCi of 5'-end-labeled 16S RNA probe. The adjusted pixel value for each mRNA was obtained by normalizing each slot pixel value to its 16S RNA value and then averaging the duplicates and subtracting the average of the normalized P. putida JS382 pixel values. After hybridization and a washing, the membrane was exposed to a Phosphor Screen for 1 to 2 h.