We cloped the structural genes for the individual subunits of the branched-chain keto acid dehydrogenase multienzyme complex on a 7.8-kilobase EcoRI-SstI restriction fragment of Pseudomonas putida chromosomal DNA by cloning into the broad-host-range vector pKT230. A direct selection system for growth on valine-isoleucine agar was achieved by complementation of P. putida branched-chain keto acid dehydrogenawe mutants. The recombinant plasmid, pSS1-1, increased expression of branched-chain ketQ acid dehydrogenase up to five times in wild-type P. putida. The complex was expressed cQnstitutively in P. putida(pSSl-l) but was inducible in Escherichia coli HB101(pSS1-1) by high valine. E. coli minicells tra'nsformed with pSS1-1 produced three polypeptides which did not match the fopr polypeptides of the purified complex. To resolve this problem, we inserted P. putida DNA from pSS1-i into pUC18 And pUC19. The pUC-derived plasmids were used as DNA templates in an E. coli transcription-translation system. Four polypeptides were produced from the pUC18-derived plasmid which had the correct molecular weights, showing that the structural genes had been cloned. Since only weak bands were produced with the pUC19-derived plasmid, the direction of transcription was established. The locations and order of all the structural genes of branched-chain keto acid dehydrogenase were located by restriction enzyme mapping.Branched-chain keto acid dehydrogenase is an enzyme common to the catabolism of valine, leucine, and isoleucine. The enzyme has been purified from mammals (25, 26) and from Pseudomonas putida (31), Pseudomonas aeruginosa (21), and Bacillus subtilis (17). In each species, it is a multienzyme complex composed of three functional subunits: El, the dehydrogenase-decarboxylase; E2, the transacylase; and E3, lipoamide dehydrogenase. Purified branched-chain keto acid dehydrogena'se from mammnals is composed of four polypeptides, the E-1 subunit being composed of two dissimilar proteins. The E2 and E3 subunits of the Pseudomonas complexes have been identified, but it was not clear whether El consisted of one or two polypeptides. P. putida and P. aeruginosa are unusual in that they possess two functionally and structurally distinct lipoamide dehydrogenases, 29). LPD-Val is the specific E3 subunit of branched-chain keto acid dehydrogenase. Mutations affecting subunits of branched-chain keto acid dehydrogenase including LPD-Val map by conjugation at a single location on the P. putida chromosomes, suggesting that the structural genes are linked (37). LPD-Glc is the E3 subunit of 2-ketoglutarate and probably pyruvate dehydrogenase and is the L-factor of glycine decarboxylase in P.putida (28,29). In Escherichia coli there is a single lipoamide dehydrogenase which functions as the E3 subunit of the pyruvate and 2-ketoglutarate dehydrogrnases (11).There is some evidence which suggests that branchedchain keto acid dehydrogenase evolved from pyruvate dehydrogenase. Lowe et al. (17) have isolated a dual-purpose keto acid dehydrogenase fro...

(Reçu le 26 juin 1989; accepté le 14 janvier 1991) Résumé -L'étude porte sur l'influence du polymorphisme génétique de la~-lactoglobuline (~-Lg) et de la x-caséine (K-Cn) sur la composition du lait et ses aptitudes à la coagulation et à la transformation fromagère. Quatre-vingt-trois fabrications de type Camembert au lait cru ont été réalisées au laboratoire à partir du lait de 230 vaches Pie Noire. Huit combinaisons génétiques de la~-Lg et de la K-Cn ont été étudiées; les mesures ont porté sur la composition physico-chimique des laits de fabrication, caillés et sérums, les paramètres d'aptitude à la coagulation mesurés à l'aide d'un Formagraph et les bilans et pourcentages de récupération des matières lors des fabrications. L'influence positive du génotype BB et de la~-Lg est mise en évidence essentiellement sur le taux de caséines du lait (+ 4,7% par rapport au génotype AA) et sur la rétention des matières azotées totales dans le caillé (+ 2,9%). Le génotype BB de la K-Cn exerce des effets bénéfiques plus marqués, notamment sur le temps de coagulation des laits et la fermeté maximale des gels (respectivement -24% et +37% par rapport à l'homozygote AA), expliqués notamment par une composition protéique plus favorable. Il en résulte une meilleure récupération des constituants du lait dans les caillés BB en K-Cn. Le génotype AB occupe une position intermédiaire. L'analyse factorielle discriminante souligne le caractère additif de l'interaction entre les variants gé-nétiques de la~-Lg et de la K-Cn. Un classement de qualité fromagère est proposé, le double homozygote BB en~-Lg/BB en K-Cn occupant la place la plus favorable. variant génétique / x-casèlne 1~-Iactoglobuline 1 aptitude fromagère Summary -Influence of genetic variants of~-Iactoglobulin and x-caseln on milk composition and cheese-making capacity.

Branched-chain keto acid dehydrogenase, an enzyme in the common pathway of branched-chain amino acid catabolism of Pseudomonas putida, is a multienzyme complex which catalyzes the oxidative decarboxylation of branched-chain keto acids. The objective of the present study was to isolate strains with mutations of this and other keto acid dehydrogenases and to map the location of the mutations on the chromosome of P. putida. Several strains with mutations of branched-chain keto acid dehydrogenase, two pyruvate and two 2-ketoglutarate dehydrogenase, were isolated, and the defective subunits were identified by biochemical analysis. By using a recombinant XYL-K plasmid to mediate conjugation, these mutations were mapped in relation to a series of auxotrophic and other catabolic mutations. The last time of entry recorded was at approximately 35 min, and the data were consistent with a single point of entry. Branched-chain keto acid dehydrogenase mutations affecting El, El plus E2, and E3 subunits mapped at approximately 35 min. One other strain affected in the common pathway was deficient in branched-chain amino acid transaminase, and the mutation was mapped at 16 min. The mutations in the two pyruvate dehydrogenase mutants, one deficient in El and the other deficient in El plus E2, mapped at 22 minutes. The 2-ketoglutarate dehydrogenase mutation affecting the El subunit mapped at 12 minutes. A 2-ketoglutarate dehydrogenase mutant deficient in E3 was isolated, but the mutation proved too leaky to map. Branched-chain keto acid dehydrogenase of Pseudomonas putida is a multienzyme complex in the common pathway of branched-chain amino acid metabolism. The complex is composed of three subunits: El, the dehydrogenase, E2, the * Corresponding author. sex factor (12) to mediate conjugation. No genes in keto acid metabolism have previously been mapped in P. putida, and this is the first report to map genes encoding branched-chain keto acid dehydrogenase in any organism. MATERIALS AND METHODS Organisms and growth conditions. Both P. putida PpG2 and the donor for the interrupted matings, strain AC143 (12), were obtained from I. C. Gunsalus. E. coli NECO 100, used for transposon mutagenesis, was obtained from David Gibson. The mutants and their genotypes are listed in Table 12 PpG2 Wild type I. C.