Energization of Comamonas testosteroni ATCC 17454 for indicating toxic effects of chlorophenoxy herbicides.

The toxicity of chlorophenoxy herbicides to a bacterium, strongly related to the well-known species Delftia (formerly Comamonas) acidovorans that are able to detoxify these xenobiotics, was investigated. The oxidation of n-hexanol via alcohol dehydrogenases, coupled with the generation of ATP by electron transport phosphorylation (ETP), was used as an indicator for energy-toxic effects on the growth of Comamonas testosteroni ATCC 17454. Uncoupling--reductions in ATP synthesis accompanied by increased respiration--was found to be induced by 1 mM of the classic uncoupler 2,4-dinitrophenol (2,4-DNP) at pH 7.0 and 8.0. At pH 5.4 and 6.0, the ATP synthesis and respiration were strongly inhibited by both 2,4-DNP and the chlorophenoxy herbicides tested. In contrast, 5 mM of 2,4-dichlorophenoxyacetic acid (2,4-D) and of 2-(2,4-dichlorophenoxy)-propanoic acid (2,4-DCPP) were required for detectable uncoupling effects--reduction of the P/O ratios by about 30%--at pH 7.0. These chemicals may have less uncoupling power because the concentration of their protonated (undissociated) forms (pKa values 2.7 and 3.0) is an order of magnitude lower than that of 2,4-DNP (pKa = 4.0) at this pH value. Strong uncoupling accompanied by increased respiration, like that induced by 1 mM 2,4-DNP, was also caused by 5 mM 4-(2,4-dichlorophenoxy)-butyric acid (2,4-DCPB), which correlates with its high pKa value of 4.6. The order of toxicity of the chlorophenoxy herbicides (2,4-D < 2,4-DCPP < 2,4-DCPB) to the ETP, which correlates well with the lipophilicity of their undissociated forms (log P 2.7 < 3.4 < 3.5, respectively), was confirmed by measuring their capacity to inhibit the growth of Comamonas testosteroni ATCC 17454. The results show that energization via alcohol dehydrogenases can be used as an indicator for investigating energy-toxic effects of organics on the ETP and growth of chlorophenoxy herbicide-detoxifying bacteria.

Suitability of the trans/cis ratio of unsaturated fatty acids in Pseudomonas putida NCTC 10936 as an indicator of the acute toxicity of chemicals.

This study explored the suitability of using the trans/cis ratio of unsaturated fatty acids as an indicator of the acute toxicity of membrane active hazardous chemicals. The conversion of cis into trans fatty acids in Pseudomonas putida NCTC 10936 in response to 4-chlorophenol and temperature changes was compared with the results from another kind of toxicity test using the same organism, based on the sensitivity of its xylose oxidation-driven ATP synthesis to uncoupling. The response of both indicators is believed to be largely due to changes in the fluidity of the cytoplasmic membrane. However, the electron transport phosphorylation reacted faster and more sensitively to the fluidizing effect of 4-chlorophenol than the isomerization of unsaturated fatty acids. Therefore, measuring the trans/cis ratio does not provide as good early warning signals of acute toxicity as monitoring the response of the electron transport phosphorylation. If used as an indicator of chemostress, with Pseudomonas species as test organisms, the ratio should only be used in conjunction with other parameters reflecting the energetic state of the cells.

Regulation of poly(beta-hydroxybutyrate) synthesis in Methylobacterium rhodesianum MB 126 growing on methanol or fructose.

The intracellular concentration of CoA metabolites and nucleotides was determined in batch cultures of Methylobacterium rhodesianum grown on methanol and shifted to growth on fructose. The intracellular concentration of CoA decreased from a high value of 0.6 nmol/mg poly(beta-hydroxybutyrate)-free bacterial dry mass during growth on methanol to a low value of 0.03 nmol/mg poly(beta-hydroxybutyrate)-free bacterial dry mass after a shift to fructose as a carbon source. The levels of NADH, NADPH, and acetyl-CoA were also lower. Under these conditions, acetyl-CoA was metabolized by both citrate synthase and beta-ketothiolase, and poly(beta-hydroxybutyrate) synthesis and growth occurred simultaneously during growth on fructose. Moreover, the level of ATP was approximately 50% lower during growth on fructose, supporting the hypothesis of a bottleneck in the energy supply during the growth of M. rhodesianum with fructose.

The toxicity of substituted phenolic compounds to a detoxifying and an acetic acid bacterium.

In the detoxifying bacterium Acinetobacter calcoaceticus 69-V and in the acetic acid bacterium Acetobacter methanolicus MB 58, glucose and xylose are oxidized, respectively, via PQQ-dependent membrane-bound dehydrogenases, which are linked to the respiratory chain in a manner enabling energy conservation via electron transport phosphorylation (ETP) in the cytoplasmic membrane. Neither the glucose and gluconic acid nor the xylose and xylonic acid are metabolized. Therefore, measurements of sugar oxidation-driven ATP syntheses ought not to be disturbed by ATP drainage caused by anabolic processes. Studying the effect of substituted phenolic compounds on these energization processes reveals that their toxicity increases with an increasing degree of chlorination and that A. calcoaceticus 69-V is more stable than A. methanolicus MB 58 against chlorinated phenols. On the other hand, A. methanolicus MB 58 is more stable against 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D), especially in the acidic pH range, in which the sensitivity of ATP synthesis to the uncouplers is higher than that of respiration. The toxicity caused by protonophoric activities ought to be barely detectable by respiratory and dehydrogenase tests. The luminescence system of Photobacterium phosphoreum tested in the luminescent bacteria test was much more sensitive. This test system should be used as a screening tool and the effects measured must be confirmed by toxicity tests evaluating the stability of bacteria themselves involved in processes of detoxification as well as the production of toxic metabolites, monitored with respect to their velocity and efficiency.

The glucose dehydrogenase-mediated energization of Acinetobacter calcoaceticus as a tool for evaluating its susceptibility to, and defence against, hazardous chemicals.

Cells of Acinetobacter calcoaceticus 69-V could be energized by glucose oxidation after the growth on acetate, ethanol, hexanol and benzoate. The velocities of glucose oxidation-driven ATP syntheses were relatively constant in the range from pH 5.4 to 7.5. With decreasing pH values (7.0, 6.0, 5.4) ATP synthesis was inhibited more strongly by the action of 2,4-dinitrophenol and at the same pH value glucose oxidation was nearly unimpaired or inhibited more weakly. This finding is expressed by a decrease of the P/O ratios, indicating the uncoupling of the electron-transport phosphorylation by 2,4-dinitrophenol. The sensitivity towards this uncoupling effect was higher in ethanol-grown cells of Acinetobacter calcoaceticus 69-V than in hexanol- or acetate-grown cells. This increase in sensitivity was accompanied by a decrease of the ratio of saturated (mainly C16:0) to unsaturated (C16:1, C18:1) fatty acids in ethanol-grown cells compared with hexanol-grown ones. The knowledge of such differences in the susceptibility and its molecular background, e.g. possible substrate-induced changes of the fatty acid composition of the cytoplasmic membranes, should help elucidate mechanisms of poisoning by membrane-active hazardous chemicals and develop defence strategies.

Detoxification of formaldehyde by acetic acid bacteria.

Formaldehyde resistance of methylotrophic and non-methylotrophic Acetobacter strains was investigated. A facultatively methylotrophic Acetobacter methanolicus (MB58) gets rid of free formaldehyde by assimilating it. Heterotrophically growing cells tolerate 12 mM free formaldehyde. Non-methylotrophic but methanol oxidizing Acetobacter pasteurianus strains possess the same level of formaldehyde resistance. Formaldehyde resistance can be drastically lowered down to 4 mM by blocking the formate dehydrogenase by means of hypophosphite. Acetobacter spp. Martin 1 and LMG 76.10 are not able to oxidize methanol or formaldehyde via formate to CO2 and possess a significantly lower formaldehyde resistance (4 mM). Hence high formaldehyde resistance of the Acetobacter spp. investigated is based above all on a properly operating linear dissimilatory sequence. The dissimilatory RuMP cycle can hardly help detoxify formaldehyde.

Completion of the amino acid sequence of the alpha 1 chain of human basement membrane collagen (type IV) reveals 21 non-triplet interruptions located within the collagenous domain.

The cDNA and protein sequences of the N-terminal half of human basement membrane collagen (type IV) have been determined. Overlapping cDNA clones were constructed by repeated primer extension with synthetic oligonucleotides. They cover 2953 bp, beginning at the 5' end of the corresponding mRNA. At the protein level, the sequence of the cyanogen bromide peptide CB6 adjacent to the 7S domain has been additionally elucidated. The data presented here complete the protein sequence and nearly the entire cDNA sequence of the human alpha 1(IV) chain. The amino-terminal half of the alpha 1(IV) chain contains 8 cysteine residues involved in intramolecular and intermolecular cross-links. The entire triple-helical domain of alpha 1(IV) is interrupted by 21 non-triplet regions.

Structure of human-basement-membrane (type IV) collagen. Complete amino-acid sequence of a 914-residue-long pepsin fragment from the alpha 1(IV) chain.

The complete amino acid sequence of the 914-residue-long pepsin fragment alpha 1 (IV)95 from the alpha 1 chain of human placental basement membrane (type IV) collagen is presented. This sequence contains 12 interruptions of the collagenous triplet sequence Gly-Xaa-Yaa which varied in length from 1 to 11 residues. The distribution of amino acids between the Xaa and Yaa position was similar to that found in interstitial collagens but the extent of proline and lysine hydroxylation differed. Computer comparisons of the alpha 1 (IV)95 sequence with those of the interstitial collagen chains did not reveal any homology, whereas a comparison with the partial sequences of mouse tumor and bovine lens capsule alpha 1 (IV) showed an approximately 85% identity. The unique sequence characteristics of type IV collagen are discussed in relation to its macromolecular structure and to the interstitial collagens.

The binding of bongkrekate to mitochondria.

The binding of bongkrekate to mitochondrial membrane was investigated using [3H]bongkrekate. These measurements were designed to examine the previously derived reorienting site mechanism which implies that bongkrekate binds to the single carrier site only from the inner face of the mitochondrial membrane. The binding studies confirm pH-dependent accumulation of [3H]bongkrekate inside the mitochondria which superimposes on to binding of carrier sites. By breaking the membrane with Lubrol or sonication, binding to the carrier sites can be titrated and Kd approximately equal to 5 X 10(-8) M is determined. ADP or ATP increases the amount of binding but does not change the Kd. Reciprocally bongkrekate increases ADP binding in those sections of a titration curve where bongkrekate binding is increased by ADP. [35S]Atractylate is displaced by [3H]bongkrekate at a 1:1 molar ratio. This displacement is dependent on ADP concentration with the Km = 0.5 X 10(-6) M. The earlier described isomer, isobongkrekate, also binds specifically to the carrier sites. From competition with bongkrekate a ratio KisoBKAd/KBKAd = 0.10 is determined. [35S]Carboxyatractylate displaces most of [3H]isobongkrekate but only little of [3H]bongkrekate. The rate of displacement is more than 10-times faster for isobongkrekate than for bongkrekate. The displacement is dependent on ADP with a Km = 5 X 10(-6) M. All these results are fully consistent with the single site reorienting mechanism. In no instant do bongkrekate and atractylate as well as ADP or ATP bind simultaneously to the carrier.

[Regulation of PEP-carboxylase of the facultative methylotrop Acetobacter sp. MB 58]. / Regulation der PEP-Carboxylase des fakultativ methylotrophen Acetobacter sp. MB 58.

Acetobacter sp. MB 58 assimilates methanol via the fructose-1,6-bisphosphate variant of the hexulose phosphate pathway. Glyceraldehyde-3-phosphate originates as net product of an assimilation loop involving the regeneration of the C1-acceptor ribulose-5-phosphate and must be available for the de novo synthesis of the C1-acceptor as well as for the oxidative glycolysis. It is made probable in a regulatory model that this is accomplished via alternating anabolic and catabolic phases which are controlled by concerted action of PEP-carboxylase and pyruvate kinase. Whereas Ac-CoA is a crucial effector and alpha-ketoglutarate and aspartate are inhibitors for the PEP-carboxylase, the pyruvate kinase is assumed to be regulated by energy charge.

Dihydroxyacetone kinase of methanol-assimilating yeast. I. Regulation of dihydroxyacetone kinase from Candida methylica in situ.

In order to investigate the control behaviour of dihydroxyacetone kinase of methanol-grown Candida methylica under nearly physiological conditions kinetic and regulatory studies were carried out in situ. Yeast cells were made permeable to substrate and reaction products by treatment with Triton X-100. Normal Michaelis-Menten kinetics resulted in dependence upon the dihydroxyacetone concentration, both at the pH optimum of 7.6 and near the physiological pH-value of 6.5. The Km obtained for dihydroxyacetone was 0.02 mM, independent of the pH-value. The plots of dihydroxyacetone kinase activity as a function of the ATP concentration revealed complex kinetic characteristics with plateau regions. The maximum reaction rate was reached only after a lag time both at pH 7.6 and concentrations of ATP higher than 5 mM and at pH 6.5 and concentrations of ATP higher than 1.25 mM. Among a great number of tested metabolites no inhibitors of dihydroxyacetone kinase were found. Dihydroxyacetone kinase activity depending upon energy charge according to Atkinson exhibited curves of the U-type. These results and further data concerning the regulation of other enzymes obtained with C. methylica and other yeasts were the basis to propose a preliminary overall model of fine control of the carbon and energy metabolism of methanol-utilizing yeasts.

Isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekate-protein complex.

1. The isolation of the ADP/ATP translocator from beef heart mitochondria as the bongkrekateprotein complex is described, using hydroxyapatite chromatography and gel filtration in Triton X-100 solution. 2. The inhibitor is bound to the protein prior to solubilization with detergent for protection against denaturation. Only the intact bongkrekate-protein passes easily through the hydroxyapatite column. Bongkrekate shileds the protein in contrast to carboxyatractylate only partially against proteinases present in the crude extract. 3. The isolated bongkrekate protein shows the same molecular weights in dodecylsulfate and Triton X-100, the same amino acid composition and the same isoelectric point as the earlier isolated carboxyatractylate-protein complex. It differs by its higher sensitivity against trypsin and thermolysin. 4. The identity of both proteins is demonstrated by interconversion of the bongkrekate-protein into the carboxyatractylate-protein. The process requires the catalysis by ADP or ATP, the natural substrates of the protein. 5. The formation of the extractable [3H]bongkrekate-protein complex in mitochondria requires the presence of ADP or ATP. 6. These data, the immunological studies presented earlier, and the differences in the reactivity of -SH groups of the isolated bongkrekate and carboxyatractylate complexes (to be published) indicate that both proteins represent different conformational states of the translocator protein (m-state and c-state).