John R. Sokatch

Crc Is Involved in Catabolite Repression Control of the bkd Operons of Pseudomonas putida and Pseudomonas aeruginosa

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.

Catabolite Repression Control by Crc in 2xYT Medium Is Mediated by Posttranscriptional Regulation of bkdR Expression in Pseudomonas putida

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.

Oxidation of glycine by Pseudomonas putida requires a specific lipoamide dehydrogenase

Pseudomonas putida produces two lipoamide dehydrogenases with molecular weights of 49,000 and 56,000 designated LPD-val and LPD-glc, respectively. LPD-val is required for oxidation of valine, since it is specifically utilized as the E3 component of branched-chain keto acid dehydrogenase. Since glycine oxidation by bacteria and mammals also requires lipoamide dehydrogenase, we desired to determine which lipoamide dehydrogenase would be used by the P. putida glycine oxidation system. When grown in a medium with glycine as the sole nitrogen source, P. putida produced a single lipoamide dehydrogenase with a molecular weight of 56,000 and which reacted with antiserum to LPD-glc. The partially purified glycine oxidation system from P. putida was stimulated by LPD-glc but not by LPD-val and was inhibited by anti-LPD-glc, but not by anti-LPD-val. It was not possible to detect LPD-val in extracts of cells grown in glucose-glycine medium by the use of anti-LPD-val. LPD-glc was five times as active as LPD-val in catalyzing the oxidation of purified protein H, the heat-stable, lipoic acid-containing protein of the glycine oxidation system. These results indicate that LPD-glc is specifically utilized for glycine oxidation in P. putida.

Relationship of lipoamide dehydrogenases from Pseudomonas putida to other FAD-linked dehydrogenases.

Pseudomonas putida produces two lipoamide dehydrogenases, LPD‐glc and LPD‐val. LPD‐val is specifically required as the lipoamide dehydrogenase of branched‐chain keto acid dehydrogenase and LPD‐glc fulfills all other requirements for lipoamide dehydrogenase. Both proteins are dimers with one FAD per subunit. LPD‐glc has an absorption maximum at 455 nm, but LPD‐val has a maximum at 460 nm. Comparison of amino acid compositions revealed that LPD‐glc was more closely related to Escherichia coli and pig heart lipoamide dehydrogenase than to LPD‐val. LPD‐val did not appear to be closely related to any of the proteins compared with the possible exception of mercuric reductase.

Construction of chromosomal recA mutants of Pseudomonas putida PpG2.

The recA gene of Pseudomonas putida PpG2 was cloned by complementation of the recA mutations of Escherichia coli strains DH5 alpha and HB101. The nucleotide sequence of the DNA fragment was determined and shown to contain recA and a downstream partial open reading frame. Two mutants of P. putida PpG2, strains JS387 and JS388, were constructed by insertional inactivation of recA with a tetracycline-resistance gene in both orientations. Both mutants acquired sensitivity to methyl methanesulfonate (MMS) and both failed to undergo homologous recombination. While the recA mutation of P. putida JS388 was complemented in trans by recA of P. putida, the JS387 mutant was difficult to transform and transformants exhibited varying degrees of sensitivity to MMS. Therefore, P. putida JS388 can be used as a carrier of recombinant plasmids, but JS387 is not a suitable host for this purpose.

Isolation of two lipoamide dehydrogenases from Pseudomonasputida grown on valine

Abstract Pseudomonas putida PPG2 grown on valine as the principal carbon and energy source produces two species of lipoamide dehydrogenase, molecular weights 56k and 49k. Pseudomonas putida grown on glucose as the carbon source produces only the 56k lipoamide dehydrogenase. The 56k proteins from P. putida grown on glucose and valine were not separated by SDS-Page and may be the same proteins. Pyruvate dehydrogenase of mutant JS94 lacking lipoamide dehydrogenase was restored by the 56k protein but not by the 49k protein. Branched chain keto acid dehydrogenase was partially restored by the 49k protein, but not the 56k protein.

[30] - Purification of Branched-Chain Keto Acid Dehydrogenase Regulator from Pseudomonas putida

BkdR can be isolated in nearly pure form as a tetramer by this procedure, which involves hyperexpressing bkdR from a plasmid, purification by chromatography on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, and dialysis to precipitate BkdR. BkdR is relatively insoluble in aqueous buffers but can be kept in solution in buffer with 50% (v/v) glycerol and 0.2 M NaCl. Cultures of E. coli DH5 alpha (pJRS119) should be maintained at 30 degrees to promote plasmid stability. Because BkdR is prone to form intermolecular disulfide bonds, buffers for SDS-PAGE should contain fresh 0.5% (v/v) 2-mercaptoethanol.