Search

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.

Monitoring biotechnological processes involves acquiring information about key metabolic events and, ideally, single cell states should be determined to obtain comprehensive data on the physiological status of the surveyed population. In this paper, growth stages of the strain Acinetobacter calcoaceticus 69-V were characterised at the single cell level using flow cytometry. Four methods for analysing bacterial cellular characteristics by fluorescence were compared with respect to their sensitivity to changes in the physiological states induced by changing micro-environmental conditions. DNA analysis was confirmed to be highly informative with regard to the multiplication activity of the population. Measuring the membrane potential related fluorescence intensity (MPRFI) and the rRNA content were found to be useful for describing high-active cell states. A method for the measurement of the fluidity related fluorescence intensity (FRFI) was developed, since it allowed changes in the fluidity of the bacterial membrane to be detected, and thereby provided a valuable means of tracking adaptation of the population to micro-environmental deviations from optimal growth conditions.

In the detoxifying bacteriumAcinetobacter calcoaceticus69-V and in the acetic acid bacteriumAcetobacter methanolicusMB 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 thatA. calcoaceticus69-V is more stable thanA. methanolicusMB 58 against chlorinated phenols. On the other hand,A. methanolicusMB 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 ofPhotobacterium phosphoreumtested 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.

A method for the determination and quantification of intracellular nucleotides and dinucleotides from bacterial biomass is described. The cellular metabolism was stopped and nucleotides were extracted by rapidly transferring suspension into potassium hydroxide to give a final concentration of 0.05 M. The respective (di)nucleotides were proven to be stable under these conditions. Detection of the nucleotides was carried out after neutralization of the extracts by ion-pair reversed-phase HPLC using a biphasic linear gradient. The complete separation of all the compounds considered was achieved. The chromatographic patterns of (di)nucleotides of cellular extracts correlated well with the profile of a standard solution. The concentration of the respective (di)nucleotides and their ratio to each other, expressed for instance as the energy charge, corresponds to the metabolic state of the population and is thus an indication that the intracellular level of the respective (di)nucleotides was properly fixed. The present method is suitable for determining the intracellular concentration of (di)nucleotides in one run.

Changes of membrane-potential-related fluorescence intensity (MPRFI) in Acinetobacter calcoaceticus 69-V were measured by flow cytometry. For that purpose the cells were stained with the dye 3,3′ -dihexyloxacarbocyanine iodide (diOC6(3)), which is bound to the inner side of the cytoplasmic membrane bilayer. The MPRFI is actually an expression of the viability of bacterial cells. This was shown by the sensitivity to the ionophores gramicidin and valinomycin.The MPRFI depends on the growth phase (acetate as carbon and energy source) and increases by the addition of glucose, which is oxidized and provides biologically useful energy as shown by the synthesis of ATP. A decrease in the MPRFI and in the synthesis of ATP can be provoked by the pH-dependent action of the uncoupler 2,4-DNP.The flow cytometric detection of MPRFI is suitable for the rapid detection of growth-phase-dependent variations of the membrane potential and for the appraisal of the nature of injury to bacterial membranes after the action of membrane-active xenobiotics.