Decreased riboflavin formation in mutants of Aerobacter (Enterobacter) aerogenes deficient in the butanediol pathway.

Riboflavin concentration increased linearly for more than 60 h in wild type cultures, whereas in three mutants deficient in the formation of acetoin and 2,3-butanediol the production ceased at the end of exponential growth.     

Physiological and biochemical role of the butanediol pathway in Aerobacter (Enterobacter) aerogenes.

Aerobacter (Enterobacter) aerogenes wild type and three mutants deficient in the formation of acetoin and 2,3-butanediol were grown in a glucose minimal medium. Culture densities, pH, and diacetyl, acetoin, and 2,3-butanediol levels were recorded. The pH in wild-type cultures dropped from 7.0 to 5.8, remained constant while acetoin and 2,3-butanediol were formed, and increased to pH 6.5 after exhaustion of the carbon source. More 2,3-butanediol than acetoin was formed initially, but after glucose exhaustion reoxidation to acetoin occurred. The three mutants differed from the wild type in yielding acid cultures (pH below 4.5). The wild type and one of the mutants were grown exponentially under aerobic and anaerobic conditions with the pH fixed at 7.0, 5.8, and 5.0, respectively. Growth rates decreased with decreasing pH values. Aerobically, this effect was weak, and the two strains were affected to the same degree. Under anaerobic conditions, the growth rates were markedly inhibited at a low pH, and the mutant was slightly more affected than the wild type. Levels of alcohol dehydrogenase were low under all conditions, indicating that the enzyme plays no role during exponential growth. The levels of diacetyl (acetoin) reductase, lactate dehydrogenase, and phosphotransacetylase were independent of the pH during aerobic growth of the two strains. Under anaerobic conditions, the formation of diacetyl (acetoin) reductase was pH dependent, with much higher levels of the enzyme at pH 5.0 than at pH 7.0. Lactate dehydrogenase and phosphotransacetylase revealed the same pattern of pH-dependent formation in the mutant, but not in the wild type.     

The influence of minerals on the formation of 2,3 butanediol by Aerobacter aerogenes

Summary The presence of a small concentration of nickel or zinc ions in the cultures ofAerobacter aerogenes increases the formation of 2,3 butanediol in the cultures. The presence of 1 per cent of milk increases the formation of 2,3 butanediol in the cultures and decreases the consumption of sugar. Copper ions inhibit the formation of 2,3 butanediol.The combination of zinc and nickel ions decrease the formation of 2,3 butanediol in the cultures ofAerobacter aerogenes and even with addition of milk this combination is not beneficial to the formation of the diol. Nickel in presence of milk increases the formation of 2,3 butanediol.     

The formation of phenol in the degradation ofp-hydroxybenzoic acid byKlebsiella aerogenes (Aerobacter aerogenes)

After growth ofK. aerogenes in chemically defined media consisting of mineral salts andp-hydroxybenzoate with or without glucose, phenol was found in the culture fluid at concentrations inhibiting further growth. Bacteria adapted to mineral salts medium containingp-hydroxybenzoate as sole source of carbon and energy produced small but isolable quantities of 3,4-dihydroxybenzoic acid and catechol and oxidized these substances as rapidly asp-hydroxybenzoate. Bacteria adapted to mineral salts medium containing glucose as sole carbon and energy source did not oxidizep-hydroxybenzoate, 3,4-dihydroxybenzoate or catechol. Bacteria adapted to glucose medium or top-hydroxybenzoate medium did not oxidize or utilize phenol as sole carbon and energy source. A metabolic pathway forp-hydroxybenzoate degradation is proposed and the formation of phenol is attributed to a side reaction.     

Regulation of the L-arabinose catabolic pathway in Aerobacter aerogenes

Three enzymes of the l-arabinose catabolic pathway in Aerobacter aerogenes, l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, are specifically induced in the presence of l-arabinose. Mutants constitutive for kinase activity are also constitutive for the isomerase and 4-epimerase activities, suggesting that these three enzymes are coordinately controlled in A. aerogenes. l-Ribulokinase activity can still be induced in the presence of l-arabinose in an isomerase-deficient strain of A. aerogenes. Since l-arabinose is not converted to l-ribulose in such a strain, it appears that l-arabinose must be the inducer of l-ribulokinase, as well as the coordinately controlled isomerase and 4-epimerase. As in the metabolism of l-arabinose, growth of A. aerogenes on l-arabitol also requires a 4-epimerase for the conversion of l-ribulose-5-phosphate to d-xylulose-5-phosphate. However, loss of ability to metabolize l-arabinose, due to a deficiency in 4-epimerase synthesis in the presence of l-arabinose, does not affect growth on l-arabitol. In addition, synthesis of the 4-epimerase associated with l-arabitol metabolism is not accompanied by l-arabinose isomerase or l-ribulokinase synthesis. These results suggest either the existence of two different l-ribulose-5-phosphate 4-epimerases in A. aerogenes, or of two different regulatory mechanisms for the control of the same epimerase.     

Effect of Sodium on the Transport and Utilization of Citric Acid by Aerobacter (Enterobacter) aerogenes

Sodium inhibited citrate uptake by two of the four strains of Aerobacter (Enterobacter) aerogenes used in these studies, had no effect on one strain, and stimulated citrate uptake by one strain. Two of the four strains grew well anaerobically on citrate in the presence of Na(+), one grew poorly, and one grew not at all either in the presence or absence of Na(+). Na(+) stimulated the aerobic growth of one strain on citrate, increased the total growth but not the rate of growth of one strain, and prolonged the lag phase but not the rate of growth or total growth of two strains. The experimental data reported herein, therefore, indicate that there are appreciable physiological differences among strains of A. aerogenes.     

Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene

Microbial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid production.     

The oxidative degradation of benzoate and catechol byKlebsiella aerogenes (Aerobacter aerogenes)

Klebsiella aerogenes NCTC 418 adapted to mineral salts medium with benzoate as sole source of carbon and energy oxidized catechol without a lag and as rapidly as benzoate. It also oxidizedcis-cis-muconate, (+)-muconolactone and -ketoadipate without a lag but less rapidly. For each mol of benzoate, catechol,cis-cis-muconate, (+)-muconolactone or -ketoadipate oxidized by fresh, intact benzoate-adapted bacteria, 6.0, 5.0, 4.0, 4.0 and 4.0 mol of O₂ respectively were taken up. Incubation of cell-free extracts of ultrasonically disrupted benzoateadapted bacteria with catechol in the presence of EDTA affordedcis-cis-muconate. Incubation of heat-treated cell-free extracts withcis-cis-muconate yielded (+)-muconolactone. Incubation of cell-free extracts with catechol,cis-cis-muconate, or (+)-muconolactone gave -ketoadipate. Cell-free extracts of the organism adapted to mineral salts medium containingp-hydroxybenzoate as sole source of carbon and energy also converted catechol to -ketoadipate. Strains adapted to glucose, benzoate orp-hydroxybenzoate did not contain detectable amounts of catechol-2,3-oxygenase. The above observations are consistent with the following pathway inK. aerogenes: benzoate (orp-hydroxybenzoate) catecholcis-cis-muconate(+)-muconolactone-ketoadipate.The author wishes to thank Professor M. W. Partridge for chemical advice, and Miss E. S. Smalley and Mr. P. J. Wragg for technical assistance.     

Metabolism of D-mannose in Aerobacter aerogenes: evidence for a cyclic pathway

Kamel, M. Y. (Michigan State University, East Lansing), and R. L. Anderson. Metabolism of d-mannose in Aerobacter aerogenes: evidence for a cyclic pathway. J. Bacteriol. 92:1689-1697. 1966.-Evidence is presented which suggests a cyclic pathway for the constitutive utilization of d-mannose in extracts of Aerobacter aerogenes PRL-R3. d-Mannose is phosphorylated with d-glucose-6-phosphate to yield d-mannose-6-phosphate and d-glucose. d-Glucose-6-phosphate may be regenerated by isomerization of d-mannose-6-phosphate through d-fructose-6-phosphate, or by phosphorylation of d-glucose with adenosine-5'-triphosphate. The pathway involves the participation of four constitutive enzymes: d-glucose-6-phosphate isomerase, d-mannose-6-phosphate isomerase, a stereospecific d-glucokinase, and a phosphotransferase which phosphorylates d-mannose with d-glucose-6-phosphate, acetyl phosphate, or carbamyl phosphate. The absence of d-mannokinase (adenosine-5'-triphosphate:d-mannose phosphotransferase) activity in extracts of this organism suggests that the pathway may be of functional significance. Also, the pathway accounts for an apparent 2-epimerization of d-mannose to d-glucose that was observed in extracts.     

Phosphatase synthesis in Klebsiella (Aerobacter) aerogenes growing in continuous culture

1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (K_m 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO₄³⁻-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F⁻, PO₄³⁻) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined.