The OCT plasmid encodes D-lysine membrane transport and catabolic enzymes in Pseudomonas putida.

Pseudomonas putida (oleovorans) (Pp(OCT)) cured of its OCT plasmid (Pp) no longer grows on D-lysine. Conjugation of PpTrp- with three different methionine auxotrophs carrying the OCT plasmid resulted in PpTrp- (OCT) organisms that grew on D-lysine. Three early D-lysine catabolic enzymes encoded by the OCT plasmid are a lysine racemase, the proposed conversion of D-lysine to delta 1-piperidine-2-carboxylate (P2C), for which we provide evidence, and P2C reductase which converts P2C to pipecolate. In Pp the rate of active D-lysine transport is reduced by 30 to 50%. We consider this to reflect a loss of the gene for the D-lysine carrier while the remaining activity is due to a chromosomally coded L-lysine or D-ornithine carrier or both. The membrane carriers and catabolic enzymes for D- and L-lysine in P. putida P2 and for L-lysine in Pp(OCT) are chromosomally coded.

The ethanol sensitivity of calcium taken up by a depolarization-dependent process in mouse strains DBA and C57BL.

In vitro effects of ethanol on calcium taken up by synaptosomes were examined in two strains of mice, C57BL and DBA, that exhibit marked differences in alcohol sensitivity and preference. There were no significant strain differences in basal or depolarization-dependent synaptosomal calcium levels. Ethanol did not reduce the basal calcium level but instead reduced depolarization-dependent calcium levels with the same potency in both strains. These results do not support a role for changes of calcium levels as the basis for differences in ethanol sensitivity in these mouse strains.

Naphthalene association and uptake in Pseudomonas putida.

Two methods for bacterial membrane transport, filtration and flow dialysis, were used to study cellular association of Pseudomonas putida with naphthalene. It is not technically possible to determine the exact cellular or vesicular location of the naphthalene, and because it is hydrophobic, it could be at the membrane(s) rather than inside the cells. As an index of naphthalene having crossed the inner membrane we used the intracellular formation of its first catabolite. An energized membrane or ATP was not essential for association or movement into the cell. Evidence for a nonspecific association and a movement into cells by simple diffusion are the lack of saturation of association, an absence of inhibition of association by protein inhibitors and structural analogs, and the passage of naphthalene through cell membranes in the presence of iodoacetamide. Specific naphthalene metabolism gene expression was not required for association.

Age differences in the oxidation of and neural response to ethanol in mice.

Male mice (C57BL/6J) from three age groups, 4, 14, and 26 months, were tested to determine their oxidation rate of intragastrically infused ethanol and the response of the centrally mediated jaw-jerk reflex to ethanol challenge. There were no age-related differences in the rate of oxidation of ethanol. However, age differences in ethanol-induced decrement of the jaw-jerk amplitude were significant. The 26 month animals were the most affected, followed by the 4 month group. The middle aged mice (14 month) showed the greatest resistance to the depression induced by ethanol. The results were evaluated with regard to the current hypotheses.

Oxidation of alcohol in free-moving mice from high and low preference strains.

Free-moving mice from the high-alcohol preference C57BL/6J strain and low-preference DBA/2J strain were slowly fed [2-14C]ethanol intragastrically until anesthesia was achieved. Behavior was monitored in a Plexiglas metabolic chamber while 14CO2 was simultaneously trapped to determine the rate of ethanol metabolism. Average time to the loss of the righting reflex in the DBA/2J was 21.9 min and 27.9 min for the C57BL/6J strain (p less than 0.005). Elimination of 14CO2 was slightly higher (n.s.) in the DBA/2J strain for the entire monitoring period. Infusion of ethanol via the tail vein yielded identical results indicating that the slower elimination rate in the C57BL/6J strain could not be the result of slower absorption across the gut wall. Infusion via the tail vein with radioactive sodium bicarbonate indicated that the DBA/2J strain has a higher rate of CO2 expiration (n.s.). Consequently, the higher rate of 14CO2 expiration from ethanol oxidation may not reflect a higher rate of metabolism. These results are discussed in terms of the apparent differences between these strains in neural sensitivity to ethanol.

A quantitative analysis of ethanol and acetaldehyde expired by inbred mouse strains.

Expired ethanol and acetaldehyde were measured after an oral injection of ethanol in C57BL/6J and DBA/2J mouse strains by a combination of several techniques in a sequence involving a method for trapping expired radioactive compounds, separation of compounds by gas chromatography, isolation of radioactive ethanol and acetaldehyde, and their quantitative analysis by liquid scintillation spectrophotometry. With the specific activities used in evaluation of the technique (0.1 Ci/mole, acetaldehyde; 1.1 Ci/mol, ethanol) the lower limit of sensitivity using 500 microliters from a 10 ml trap is 955 pmoles for acetaldehyde and 101 pmoles for ethanol. However, in the animal experiments, injected ethanol has a specific activity of 1.1 Ci/mol which would make the specific activity of expired metabolically formed acetaldehyde the same. This results in a lower limit of sensitivity for acetaldehyde of 80 pmoles. The two strains were monitored for 80 min following an oral injection of 3.8 g/Kg of (2-14C) ethanol. Comparing the two strains on the expiration of each compound the curves were identical.

Transport of C5 dicarboxylate compounds by Pseudomonas putida.

Induced glutarate and 2-oxoglutarate uptake and transport by Pseudomonas putida were investigated in whole cells and membrane vesicles, respectively. Uptake of 2-oxoglutarate, but not glutarate, was against a concentration gradient to 1.7-fold greater than the initial extracellular concentration. Membrane vesicles transported 2-oxoglutarate and glutarate against gradients to intramembrane concentrations fivefold greater than the initial extravesicle concentrations. The rates of transport of both compounds were greatest in the presence of the artificial electron donor system phenazine methosulfate-ascorbate. Malate and D-lactate were the only naturally occurring compounds that served as electron donors. Uptake and transport were inhibited by KCN, NaN3, and 2,2-dinitrophenol. Kinetic parameters of transport were: glutarate, apparent Km--1.22 mM, Vmax--400 nmol/min per mg of membrane protein; 2-oxoglutarate, apparent Km--131 microM, Vmax--255 nmol/min per mg of membrane protein. Studies of competitive inhibition indicated a common system for transport of five C5 dicarboxylate compounds. The apparent Km and Ki values with 2-oxoglutarate as a substrate placed the substrate affinity for transport in the order 2-oxoglutarate greater than glutarate greater than D-2-hydroxyglutarate and L-2-hydroxyglutarate greater than glutaconate.

Butanediols: selection, open field activity, and NAD reduction by liver extracts in inbred mouse strains.

Mice from the high-ethanol preferring C57BL strain and low-ethanol preferring DBA strain were tested for their preference for butanediols. The C57BL strain showed a significantly higher preference for a 10% (v/v) solution of 1,3-butanediol than the DBA strain. The C57BL strain also showed a significantly greater consumption of 1,2- and 2,3-butanediol, but the separation between strains was much smaller than with 1,3-butanediol. Both strains uniformly avoided 1,4-butanediol. Tolerance for 1,3-butanediol was tested in an open-field monitor at 3 doses. At the lowest dose the DBA strain was hyperactive and the C57BL were unaffected. At the highest dose both strains were equally depressed. The specific activity of NAD reduction on incubation of liver extracts with 1,3-butanediol and ethanol as substrates was higher with both compounds in extracts from the C57BL strain.

Physiological basis for preferential uptake of D-alpha-aminoadipate over the L-isomer by Alcaligenes denitrificans.

Alcaligenes denitrificans, pre-incubated with D-alpha-aminoadipate and assayed for L-isomer uptake without removal of extracellular D-isomer, exhibits a reduced rate of uptake and a reduced level at which steady state is achieved. During D- or L-isomer uptake, intracellular alpha-aminoadipate is exclusively the L-configuration. These data are consistent with an intracellular, mediated reduction in L-isomer uptake as the physiological basis for preferential D-alpha-aminoadipate uptake by A. denitrificans growing on racemic alpha-aminoadipate. Translocated D-alpha-aminoadipate is rapidly metabolized to form an L-isomer pool which subsequently reduces the rate of L-isomer uptake and the level at which steady state occurs resulting in a preferred D-isomer uptake. Competitive inhibition of L-alpha-aminoadipate uptake by the D-isomer or a difference in the maximum rates of uptate uptake is an inducible process expressed only in the presence of that compound and while uptake of L-alpha-animoadipate is also inducible there is a low rate of constitutive uptake. While L-alpha-aminoadipate uptake occurs against a concentration gradient, uptake of the D-isomer is not against a gradient. D- and L-isomer uptake are active processes since both are inhibited by azide, cyanide and 2,4-dinitrophenol.

Transport of octanoate by Pseudomonas oleovorans.

The properties of a system for octanoate transport in Pseudomonas oleovarans are described. Transport is inducible and energy dependent, shows saturation kinetics, and concentrates against a gradient. Optimal transport is at pH 6.0 and 28 C. Apparent K(m) and V(max) values are, respectively, 7.0 muM octanoate and 0.68 nmol of octanoate transported per min per mg (dry mass) of cells. Fatty acids from C(7) to C(12) are competitive inhibitors, whereas alkanes, alkenes, and esters of the same carbon chain lengths show no inhibition. The K(i) values for heptanoate, nonanoate, decanoate, undecanoate, and dodecanoate are 17, 3.4, 3.2, 1.2, and 2.4 muM, respectively. The molecular specificity of this transport system is a linear hydrocarbon chain of no less than 6 to at least 11 carbon atoms and a carboxyl group.

Induction specificity and catabolite repression of the early enzymes in camphor degradation by Pseudomonas putida.

The ability of bornane and substituted bornanes to induce the early enzymes for d(+)-camphor degradation and control of these enzymes by catabolite repression were studied in a strain of a Pseudomonas putida. Bornane and 20 substituted bornane compounds showed induction. Of these 21 compounds, bornane and 8 of the substituted bornanes provided induction without supporting growth. Oxygen, but not nitrogen, enhanced the inductive potency of the unsubstituted bornane ring. All bornanedione isomers caused induction, and those with substituents on each of the three consecutive carbon atoms, including the methyl group at the bridgehead carbon, showed induction without supporting growth. Although it was not possible to obtain experimental data for a case of absolute gratuitous induction by compounds not supporting growth, indirect evidence in support of gratuitous induction is presented. It is proposed that the ability of P. putida to tolerate the unusually high degree of possible gratuitous induction observed for camphor catabolism may be related to the infrequent occurrence of bicyclic ring structures in nature. Survival of an organism with a broad specificity for gratuitous induction is discussed. Glucose and succinate, but not glutamate, produced catabolite repression of the early camphor-degrading enzymes. Pathway enzymes differ in their degree of sensitivity to succinate-provoked catabolite repression. The ability of a compound to produce catabolite repression is not, however, directly related to the duration of the lag period (diauxic lag) between growth on camphor and growth on the repressing compound.