Short communication

A method for isolating constitutive mutants for carbohydrate-catabolizing enzymes

Strains of $\text{E}\text{.}\text{coli}$ with fusions between the $\text{lac}$ structural genes and the promoter region of the fumarate reductase system were constructed from a parental strain deleted in the native $\text{lac}$ operon. Like fumarate reductase in wild-type cells, β-galactosidase in these fusion strains is inducible by fumarate, but only under anaerobic conditions. From one of these strains, three classes of mutants altered in the expression of the hybrid operon were isolated. By anaerobic selection for growth on lactose in the absence of fumarate, mutants that synthesize β-galactosidase constitutively both aerobically and anaerobically were obtained. By aerobic selection for growth on lactose in the presence of fumarate, mutants that are inducible in the enzyme both aerobically and anaerobically and mutants that are inducible in the enzyme only aerobically were obtained. The regulatory behaviors of the mutants studied suggest that substrate and respiratory control of the expression of the fumarate reductase complex are mechanistically connected.

Wild type Escherichia coli K-12 cannot grow on xylitol and we have been unsuccessful in isolating a mutant directly which had acquired this new growth ability. However, a mutant had been selected previously for growth on L-1,2-propanediol as the sole source of carbon and energy. This mutant constitutively synthesized a propanediol dehydrogenase. Recently, we have found that this dehydrogenase fortuitously converted xylitol to D-xylose which could normally be metabolized by E. coli K-12. In addition, it was also discovered that the D-xylose permease fortuitously transported xylitol into the cell. A second mutant was thus isolated from the L-1,2-propanediol-growing mutant that was constitutive for enzymes of the D-xylose pathway. This mutant could indeed grow on xylitol as the sole source of carbon and energy, by utilizing the enzymes normally involved in D-xylose and L-1,2-propanediol metabolism.

We performed experiments in search of a plating medium which would differentiate lactose constitutive mutants from wild-type cells of lactic streptococci and of a broth medium that would enrich for lactose constitutive mutants. No selective medium was applicable for selection of lactose constitutive mutants from Group N streptococci. Prolonged growth in a chemostat with lactose as the limiting growth factor, however, enriched for these mutants from Streptococcus lactis C2. The mutants synthesized phospho-β-galactosidase constitutively with an activity 1.5 to 4 times that of the parent culture. The slow growth of S. lactis C2 and the acquisition of ability to grow on lactobionic acid by the mutants confirmed their constitutive nature. In comparison to S. lactis C2, the constitutive mutant M10 grew rapidly in milk whereas the lag phase was 45 min with the parent strain when the inoculum was low. During this study we observed a change in the specific activity of phospho-β-galactosidase in S. lactis C2 under different growth conditions. Stage of growth markedly influenced enzyme activity. In addition, in complex broth lactose utilizing enzymes were repressed by glucose. However, in a semi-synthetic medium containing glucose, the phospho-β-galactosidase activity remained essentially constant for glucose concentrations ranging from .5 to 10 mg per ml. S. lactis C2 did not exhibit a glucose-lactose growth diauxie and. appeared to possess a high basal amount of phospho-β-galactosidase activity.

Oleate uptake by Escherichia coli showed saturation kinetics with a K_m of 34 μm and an activation energy of 6.25 kcal/mole indicating that the rate limiting step in oleate uptake involves an enzyme-catalyzed step. The rate of oleate uptake was decreased by the respiratory poisons, arsenate and 4-pentenoate, which apparently is activated to pentenoyl CoA, thus reducing the intracellular concentration of free intracellular CoA. These data indicated that oleate uptake is dependent on cellular ATP and CoA. During short pulses with [1-¹⁴C]oleate, most of the radioactivity which was taken up was released as ¹⁴C0₂; cells accumulated radioactivity in phospholipids and compounds with the chromatographic mobility of Krebs cycle intermediates. Neither free fatty acid nor oleyl CoA were detectable in the cells. The results support the hypothesis that long-chain fatty acids are translocated by the long-chain fatty acyl CoA synthetase and that uptake is the rate limiting step in the utilization of exogenous fatty acid.

A strain of Pseudomonas putida was found to oxidize L-lysine by an inducible pathway through δ-aminovalerate, and D-lysine via Δ¹-piperideine-2-carboxylate and pipecolic acid. Each pathway is selectively induced by L- or D-lysine and the appropriate intermediates. Lysine racemase is inadequate to permit growth on L-lysine after a block in the L-pathway, but is sufficient to permit cross induction of D-lysine-related enzymes.