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Transcriptional analysis of the promoter region of the Pseudomonas putida branched-chain keto acid dehydrogenase operon.

ABSTRACT: Branched-chain keto acid dehydrogenase is a multienzyme complex produced by Pseudomonas putida when it is grown in a minimal medium containing branched-chain amino acids. A 1.87-kilobase (kb) DNA fragment was cloned and sequenced which contained 0.24 kb of the E1 alpha structural gene and 1.6 kb of upstream DNA. There were 854 base pairs (bp) of noncoding DNA upstream of bkdA1, the first gene of the bkd operon, and 592 bp between the transcriptional and translational starts. The G + C content of the noncoding region was 56.7% compared with 65.2% for all the structural genes of the operon. A partial open reading frame was found on the strand opposite that of the bkd operon beginning at base 774. When the bkd promoter was cloned into the promoter probe vector pKT240, streptomycin resistance was obtained in P. putida but not Escherichia coli with the promoter in both orientations, which indicates either that the bkd promoter is bidirectional or that there are two promoters in this region. A series of ordered deletions on both sides of the proposed site of the start of transcription revealed that almost 700 bp upstream of the start of translation were required for expression. Streptomycin resistance was also obtained in an rpoN mutant of P. putida KT2440 containing constructs with the intact bkd promoter, indicating that the bkd operon does not require the rpoN sigma factor for expression. Another construct containing the bkd promoter, bkdA1, and bkdA2 in pKT240 was used to transform P. putida JS113, a mutant which was unable to produce the E1 subunits of the branched-chain keto acid dehydrogenase. In this case, very high inducible expression of the bkd operon was obtained.

SUBMITTER: Madhusudhan KT

PROVIDER: S-EPMC526879 | biostudies-other | 1990 Oct

REPOSITORIES: biostudies-other

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Transcriptional analysis of the promoter region of the Pseudomonas putida branched-chain keto acid dehydrogenase operon.

Madhusudhan K T KT Huang G G Burns G G Sokatch J R JR

Journal of bacteriology 19901001 10

Branched-chain keto acid dehydrogenase is a multienzyme complex produced by Pseudomonas putida when it is grown in a minimal medium containing branched-chain amino acids. A 1.87-kilobase (kb) DNA fragment was cloned and sequenced which contained 0.24 kb of the E1 alpha structural gene and 1.6 kb of upstream DNA. There were 854 base pairs (bp) of noncoding DNA upstream of bkdA1, the first gene of the bkd operon, and 592 bp between the transcriptional and translational starts. The G + C content of ...[more]

PMID: 2211503

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Project description:Pseudomonas putida is a metabolically robust soil bacterium that employs a diverse set of pathways to utilize a wide range of nutrients. The versatility of this microorganism contributes to both its environmental ubiquity and its rising popularity as a bioengineering chassis. In P. putida, the newly named dbu locus encodes a transcriptional regulator (DbuR), D-amino acid oxidase (DbuA), Rid2 protein (DbuB), and a putative transporter (DbuC). Current annotation implicates this locus in the utilization of D-arginine. However, data obtained in this study showed that genes in the dbu locus are not required for D-arginine utilization, but, rather, this locus is involved in the catabolism of multiple D-branched-chain amino acids (D-BCAA). The oxidase DbuA was required for catabolism of each D-BCAA and D-phenylalanine, while the requirements for DbuC and DbuB were less stringent. The functional characterization of the dbu locus contributes to our understanding of the metabolic network of P. putida and proposes divergence in function between proteins annotated as D-arginine oxidases across the Pseudomonas genus.IMPORTANCEPseudomonas putida is a non-pathogenic bacterium that is broadly utilized as a host for bioengineering and bioremediation efforts. The popularity of P. putida as a chassis for such efforts is attributable to its physiological versatility and ability to metabolize a wide variety of compounds. Pathways for L-amino acid metabolism in this microbe have been rather well studied, primarily because of their relevance to efforts in foundational physiology research, as well as the commercial production of economically pertinent compounds. However, comparatively little is known about the metabolism of D-amino acids despite evidence showing the ability of P. putida to metabolize these enantiomers. In this work, we characterize the D-BCAA catabolic pathway of P. putida and its integration with the essential L-BCAA biosynthetic pathway. This work expands our understanding of the metabolic network of Pseudomonas putida, which has potential applications in efforts to model and engineer the metabolic network of this organism.

Project description:A novel mutation, causing a phenotype we named frogleg because its most obvious characteristic is a severe splaying of the hind limbs, arose spontaneously in a colony of Sprague-Dawley rats. Frogleg is a complex phenotype that includes abnormalities in hind limb function, reduced brain weight with dilated ventricles and infertility. Using micro-satellite markers spanning the entire rat genome, the mutation was mapped to a region of rat chromosome 1 between D1Rat131 and D1Rat287. Analysis of whole genome sequencing data within the linkage interval, identified a missense mutation in the branched-chain alpha-keto dehydrogenase kinase (Bckdk) gene. The protein encoded by Bckdk is an integral part of an enzyme complex located in the mitochondrial matrix of many tissues which regulates the levels of the branched-chain amino acids (BCAAs), leucine, isoleucine and valine. BCAAs are essential amino acids (not synthesized by the body), and circulating levels must be tightly regulated; levels that are too high or too low are both deleterious. BCKDK phosphorylates Ser293 of the E1α subunit of the BCKDH protein, which catalyzes the rate-limiting step in the catabolism of the BCAAs, inhibiting BCKDH and thereby, limiting breakdown of the BCAAs. In contrast, when Ser293 is not phosphorylated, BCKDH activity is unchecked and the levels of the BCAAs will decrease dramatically. The mutation is located within the kinase domain of Bckdk and is predicted to be damaging. Consistent with this, we show that in rats homozygous for the mutation, phosphorylation of BCKDH in the brain is markedly decreased relative to wild type or heterozygous littermates. Further, circulating levels of the BCAAs are reduced by 70-80% in animals homozygous for the mutation. The frogleg phenotype shares important characteristics with a previously described Bckdk knockout mouse and with human subjects with Bckdk mutations. In addition, we report novel data regarding peripheral neuropathy of the hind limbs.

Dataset Information

Transcriptional analysis of the promoter region of the Pseudomonas putida branched-chain keto acid dehydrogenase operon.

Publications

Transcriptional analysis of the promoter region of the Pseudomonas putida branched-chain keto acid dehydrogenase operon.

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