Verwertung von Trimethylammoniumverbindungen durch Acinetobacter calcoaceticus

Zusammenfassung Die Verwertung von Carnitin und Carnitinderivaten (O-Acylcarnitine, Carnitincarboxyl-derivate) und strukturverwandten Trimethylammoniumverbindungen (Betaine und Stickstoffbasen) durch Acinetobacter calcoaceticus wurde anhand des Wachstums und des quantitativen Nachweises der Metabolite untersucht. Der Stamm wuchs auf l-Carnitin, l-O-Acylcarnitinen und -Butyrobetain als jeweils einziger C-Quelle. Der Verbrauch dieser Verbindungen und das Wachstum korrelierten mit der Spaltung der C-N-Bindung und mit dem gebildeten Trimethylamin. d-Carnitin wurde metabolisiert, wenn als zusätzliche C-Quelle l-Carnitin im Nährmedium vorhanden war, oder wenn die Bakterien mit l-oder dl-Carnitin vorinkubiert worden waren. Mit d-Carnitin als einziger C-Quelle wuchsen die Bakterien jedoch nicht. Die Bakterien oxidierten Cholin zu Glycinbetain in Gegenwart einer zusätzlichen C-Quelle, Glycinbetain selbst wurde nicht assimiliert. In Hinsicht auf den Abbau quaternärer Stickstoffverbindungen besitzt Acinetobacter calcoaceticus im Vergleich zu anderen Carnitin-verwertenden Bakterienarten einen für ihn charakteristischen Stoffwechselweg.

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Utilization of trimethylammonium-compounds by Acinetobacter calcoaceticus

The utilization of carnitine and carnitine derivatives (O-acylcarnitines, carnitine carboxylderivatives) and structure-related trimethylammonium-compounds (betaines and nitrogen-bases) by Acinetobacter calcoaceticus was studied by means of the control of growth and the quantitative detection of metabolites. The strain grew only on l-carnitine, l-O-acylcarnitines, and -butyrobetaine as the sole carbon sources. The utilization of these compounds and the growth correlated with the cleavage of the C-N bond and thereby with the formation of trimethylamine. d-Carnitine was metabolized, if an additional carbon source, like l-carnitine, was present in the incubation mixture, or if the bacteria were preincubated with l-or dl-carnitine, but no growth was observed on d-carnitine as the sole carbon source. The bacteria oxidized choline to glycinebetaine in the presence of additional carbon sources, glycinebetaine itself was not assimilated. With regard to the catabolism of quaternary nitrogen compounds Acinetobacter calcoaceticus shows a different pathway in comparison with other bacterial species metabolizing carnitine.

     

Relationship between phage resistance and emulsan production,interaction of phages with the cell-surface of Acinetobacter calcoaceticus RAG-1

The hydrocarbon-degrading strain Acinetobacter calcoaceticus RAG-1 produces an extracellular emulsifying agent capable of forming stable oil-in-water emulsions. The bioemulsifier, termed emulsan, is a polyanionic heteropolysaccharide (M.W. 10⁶) composed mainly of N-acyl D-galactosamine and an N-acyl hexosamine uronic acid. In order to probe the interaction of emulsan with the cell surface prior to its release into the growth medium, two new virulent bacteriophages for A. calcoaceticus RAG-1 were isolated from sewage and the properties of phage resistant mutants were studied. The two phages, ap-2 and ap-3, were differentiated on the basis of plaque morphology, electron microscopy and buoyant density. Isolated mutants of A. calcoaceticus RAG-1 which were resistant to one of the two phages retained sensitivity to the other phage. Resistance to phage ap-3 was accompanied by a severe drop in emulsan production. Independently isolated derivatives of A. calcoaceticus RAG-1 with a defect in emulsan production also turned out to be resistant towards phage ap-3. Antibodies prepared against purified emulsan specifically inhibited phage ap-3 adsorption to the cell surface of the parental strain.     

Enzymologie der n-alkanoxidation bei acinetobacter

Enzymology of n-alkane degradation was studied by using different Acinetobactersp¨? Cell-free extracts of A. calcoaceticus69/V oxidize the n-alkane to the corresponding fatty acid via the intermediately formed n-alkanol Soluble and particulate components are necessary for activity. Independently, a rubredoxin and a NADH-dependent rubredoxin reductase were isolated from this strain. Rubredoxin is induced by n-alkanes. The occurence of a cytochrome P-450 was observed only in other strains of Acinetobacter. A. calcoaceticus69/V contains a particulate pyridine nucleotide-independent and a soluble NADP-dependent alcohol dehydrogenase; the particulate aldehyde dehydrogenase is also NADP-dependent. The particulate dehydrogenases are inducible. The results concerning the regulation of enzymes of the citrate and glyoxylate cycle as well as of the malic enzyme indicate that the corresponding fatty acids formed from the alkanes are metabolized via β-oxidation.     

Inducibility of cytochrome P-450 in Acinetobacter calcoaceticus by n-alkanes

Summary Cytochrome P-450, detectable in n-hexadecane-grown cells of Acinetobacter calcoaceticus, is not found during growth on complex media, sugars, or various metabolic indermediates, such as mono- or dicarboxylic acids. Cytochrome P-450 formation is observed after shifting cells from a non-hydrocarbon medium to a minimal medium with n-hexadecane as the sole source of carbon, or after addition of n-hexadecane to cultures growing on a non-inducing carbon source. The content increases with time. Induction of cytochrome P-450 is inhibited by streptomycin, chloramphenicol, and rifampicin. Besides n-hexane, other n-alkanes from n-hexane up to n-hexadecane are inducers, whereas cetyl alcohol or palmitic acid are not. The results indicate that the occurrence of cytochrome P-450 in n-alkane-induced cells reflects a de novo protein synthesis. Regulation seems not to be governed by catabolite repression but by the presence of an inducer molecule, which is either an n-alkane or a very similar molecule.     

Assimilation of γ-butyrobetaine,and d-and l-carnitine by resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida

The metabolic pattern of utilization of [1,2,3,4-¹⁴C, methyl-³H] -butyrobetaine and d-and l-[1-¹⁴C, methyl-³H]carnitine has been examined with variously grown resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida. Ps. putida grown on d, l-carnitine as the sole source of carbon, degraded only l-carnitine with stoichiometric accumulation of glycinebetaine. Alternatively, when grown on -butyrobetaine, Ps. putida rapidly metabolized -butyrobetaine, and to a lesser but significant extent, both d-and l-carnitine with equivalent formation of trimethylamine and degradation of the betaine carbon skeleton. Ac. calcoaceticus grown on either d,l-carnitine or -butyrobetaine, effectively utilized all three betaines at nearly the same rates. Disappearance of each of these quarternary ammonium compounds was accompanied by stoichiometric formation of trimethylamine and degradation of the carbon backbone. Utilization of the betaines and corresponding formation of trimethylamine by resting cell suspensions of appropriately grown Ac. calcoaceticus and Ps. putida, was essentially abolished under conditions of anaerobiosis and severely impaired in the presence of sodium cyanide, sodium azide, 2,4-dinitrophenol or 2,2-bipyridine. The results of the present investigations with resting cell suspensions of both Ac. calcoaceticus and Ps. putida do not support an earlier suggestion that -butyrobetaine degradation in these organisms proceeds by its prior hydroxylation to l-carnitine. Indeed, disrupted cell-free preparations of Ac. calcoaceticus and Ps. putida grown on either d,l-carnitine or -butyrobetaine showed no detectable -butyrobetaine hydroxylase activity.     

Biodegradation of mixture containing monohydroxybenzoate isomers by <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

A bacterial strain capable of utilizing a mixture containing 2-hydroxybenzoic acid (2-HBA), 3-hydroxybenzoic acid (3-HBA) and 4-hydroxybenzoic (4-HBA) acid was isolated through enrichment from a soil sample. Based on 16SrDNA sequencing, the microorganism was identified as Acinetobacter calcoaceticus. The sequence of biodegradation of the three isomers when provided as a mixture (0.025%, w/v each) was elucidated. The dihydroxylated metabolites formed from the degradation of 2-HBA, 3-HBA and 4-HBA were identified as catechol, gentisate and protocatechuate, respectively, using the cell-free supernatant and cell-free crude extracts. Monooxygenases and dioxygenases that were induced in the cells of Acinetobacter calcoaceticus in response to growth on mixture containing 2-HBA, 3-HBA and 4-HBA could be detected in cell-free extracts. These data revealed the pathways operating in Acinetobacter calcoaceticus for the sequential metabolism of monohydroxybenzoate isomers when presented as a mixture.     

Growth on Phenol at Chemostress Levels Amplifies the Expression of the Phenol Degradation Pathway in Acinetobacter calcoaceticus

Bacteria often use pollutants as sole carbon and energy sources. However, if they are toxic and the concentrations are high these compounds inhibit growth, and eventually poison the biocatalysts. In order to identify mechanisms contributing to stability, the protein patterns of Acinetobacter calcoaceticus during growth on potentially toxic phenol were analysed using two‐dimensional gel electrophoresis. Enzymes involved in the catabolism or assimilation of phenol, such as phenol monooxygenase and catechol 1,2‐dioxygenase, were induced at a more than twofold level in response to long‐term exposure to high concentrations of the compound serving as the sole carbon and energy source. This would have clear adaptive benefits, since increased rates of consumption of phenol would reduce the susceptibility of Acinetobacter calcoaceticus to phenol poisoning. In contrast, transient induction of only one heat shock protein and one oxidative stress protein was detected during long‐term exposure to high concentrations of phenol.     

Extrazelluläre Lipase aus Acinetobacter calcoaceticus

Cell-free growth liquor of Acinetobacter calcoaceticus 69-V contains an extracellular lipase. Its activity depends on growth phase and carbon source. During growth on acetate or succinate the activity ist low or zero, respectively. Growth on alkanes causes an increase in the extracellular lipase activity. Activity reaches maximum values during the exponential phase of growth which significantly decrease in the stationary phase During the growth on alkanes some surfactants (Tauroglycocholate, Triton X-405) stimulate the excretion of the enzyme and some other (Tween, Brij, Triton X-100) inhibit the lipase and growth of cells, respectively Air-water and alkane-water interfaces inhibit the lipase activity. During starvation of the bacteria grown on alkanes lipase is excreted in the starvation medium.     

Glucose as an energy donor in acetate growing Acinetobacter calcoaceticus

Since glucose can be oxidized but not assimilated by Acinetobacter calcoaceticus 69-V the question arose whether energy generated by glucose oxidation can help incorporate carbon from heterotrophic substrates and, if so, what the efficiency of ATP production is like. For this reason this species was grown in the chemostat on acetate. After having reached steady state conditions an increasing concentration of glucose was added. This led to an increase in the biomass level from about 0.4 g/g for growth on acetate alone to 0.6–0.65 g/g in the presence of glucose, independently of either the growth rate or the steepness of the glucose gradient used. This upper value approximates about the limit of the carbon conversion efficiency calculated for non-glycolytic substrates. Glucose was almost exclusively oxidized to gluconic acid, 2- and 5-ketogluconates, and pentose 5-phosphates were found only in traces. These results demonstrate that glucose functions as an additional energy source in Acinetobacter calcoaceticus 69-V. From the transient behaviour of biomass increase and the mixing proportion at which the maximum growth yield on acetate in the presence of glucose was obtained it followed that two mol of ATP must have been generated per mol of glucose oxidized. This property is discussed in terms of coupling glucose dehydrogenase with the respiratory chain.Abbreviations G _ox glucose oxidized to gluconic acid - G _t amount of glucose necessary for complete substitution of S _d - S _o inlet concentration of the limiting carbon substrate - S _a and S _d assimilated and dissimilated part respectively of the carbon substrate - PQQ pyrrolo-quinoline-quinone - V _ATP ^Ac ATP gain from complete oxidation to CO₂ of acetate (P/O=2) - V _ATP ^Glc ATP gain from oxidation of glucose to gluconic acid     

In vivo phosphorylation of isocitrate lyase inEscherichia coli andAcinetobacter calcoaceticus

Isocitrate lyase inEscherichia coli and inAcinetobacter calcoaceticus is phosphorylated when the cells are grown with acetate as the sole carbon source in low-phosphate mineral salts medium containing³²P inorganic phosphate. The level of³²P incorporation into the enzyme in both microorganisms appears to be constant throughout the entire growth cycle. Further, theresults of immunoblots and rocket immunoelectrophoresis suggest that the amount of isocitrate lyase protein, although at different levels in each microorganism, also remains constant throughout the growth cycle.     

Fatty acid patterns of Acinetobacter calcoaceticus 69-V indicate sensitivity against xenobiotics

The growth of Acinetobacter calcoaceticus 69-V on the alcohols ethanol, n-propanol, n-pentanol, n-hexanol and on phenol caused an alteration in its fatty acid composition leading to a gradual increase in the degree of saturation of the C16 acids from 55.4% to 83.5%, which (apart from phenol-grown cells) correlated to an increase in the resistance of the electron-transport phosphorylation against the effect of 2,4-dinitrophenol. These changes are in principle paralleled by changes observed when the growth temperature was increased in the sequence 20, 30 and 40°C with acetate as the carbon and energy source. However, in cells grown at 40°C, as in the case of phenol-grown cells, resistance decreased. This effect could be caused by an increase in the fluidity of the target membrane since, by contrast, the increase in sensitivity induced by growth at 40°C can be partially annulled by provoking a decrease in fluidity by performing the inhibition measurements at a lower temperature (20°C). Both the degree of saturation of the fatty acids and the fluidity of the cytoplasmic membrane are features that should enable the resistance of Acinetobacter calcoaceticus 69-V to xenobiotics to be predicted.     

Metabolism of monofluorobenzoates by Acinetobacter calcoaceticus N.C.I.B. 8250 formation of monofluorocatechols

None of the monofluorobenzoates serves as sole source of carbon and energy for growth of Acinetobacter calcoaceticus but all can contribute to growth on other substrates. The monofluorobenzoates are oxidised by bacteria pre-induced for benzoate oxidation and can themselves induce the appropriate enzymes. The initial products of oxidation have been separated and identified by gas-liquid chromatography. 2-Fluorobenzoate is oxidised to catechol, fluoride and 3-fluorocatechol; 3-fluorobenzoate gives 3- and 4-fluorocatechol; 4-fluorobenzoate gives 4-fluorocatechol. The fluorocatechols appear to be partially oxidised beyond the stage of 3-oxoadipate by suitably pre-induced bacteria.     

Degradation of the Herbicide Mecoprop [2-(2-Methyl-4-Chlorophenoxy)Propionic Acid] by a Synergistic Microbial Community

A microbial community isolated from wheat root systems was capable of growth on mecoprop as the sole carbon and energy source. When exposed to fresh herbicide additions, the community was able to shorten the lag phase from 30 days to less than 24 h. The community comprised two Pseudomonas species, an Alcaligenes species, a Flavobacterium species, and Acinetobacter calcoaceticus. None of the pure cultures was capable of growing on mecoprop. Certain combinations of two or more community constituents were required before growth commenced. The mecoprop-degrading community could also degrade 2,4-dichlorophenoxyacetic acid and 2-methyl-4-chlorophenoxyacetic acid but not 2,4,5-trichlorophenoxyacetic acid.     

Glucose stimulates a decrease of the fatty acid saturation degree in Acinetobacter calcoaceticus

The fatty acid composition of Acinetobacter calcoaceticus 69-V was determined under various growth conditions. Saturated, unsaturated, and hydroxy fatty acids with chain lengths of 12–18 carbon atoms predominated in the fatty acid profile. With acetate or propanol as growth substrates, the ratio of saturated to unsaturated fatty acids varied with changes in the temperature. This was the only adaptive mechanism detected that compensated for the physical effects of temperature alterations on the cell membranes. The fatty acid composition of A. calcoaceticus grown at 40 °C had a saturation degree of approximately 50%; after growth at 20 °C it was approximately 35%. In the presence of a carbon and energy source, A. calcoaceticus was able to respond to temperature reductions under oxic conditions regardless of whether fatty acid biosynthesis was inhibited or not. This suggests an aerobic mechanism of fatty acid biosynthesis and the involvement of a fatty acid desaturase system. Addition of the non-growth substrate, glucose, helped the organism to adapt to lower temperature. The molecular mechanism of the aid is not really understood. The oxidation of glucose could provide the desaturase either with electrons directly via a pyrrolo-quinoline-quinone-linked glucose dehydrogenase or with NADH after fatty acid degradation has been initiated by ATP generated by the oxidation of glucose. Received: 19 June 1998 / Accepted: 28 December 1998     

Physiological regulation of competence induction for natural transformation in Acinetobacter calcoaceticus

Acinetobacter calcoaceticus induced competence for natural transformation maximally after dilution of a stationary culture into fresh medium. Competence was gradually lost during prolonged exponential growth and after entrance into the stationary state. Growth cessation and nutrient upshift were involved in the induction of competence. The level of competence of a chemostat culture of A. calcoaceticus was dependent on the nature of the growth limitation. Under potassium limitation a transformation frequency of ±1x10^-4 was obtained. This frequency was independent of the specific growth rate. In phosphate-, nitrogen-, and carbon-limited chemostat cultures, in contrast, the transformation frequency depended on the specific growth rate; the transformation frequency equalled±10^-4 at dilution rates close to µ_max of 0.6h^-1 and decreased to ±10^-7 at a dilution rate of 0.1 h^-1. We conclude that (1) DNA uptake for natural transformation in A. calcoaceticus does not serve a nutrient function and (2) competence induction is regulated via a promoter(s) that resembles the fis promoter from Escherichia coli.     

Intracellular Accumulation of the Monomeric Precursors of Polyphosphates and Polyhydroxyalkanoates in Acinetobacter calcoaceticusand Escherichia coliCells

The formation of polyhydroxyalkanoates granules in anaerobically grown Escherichia coliM-17 cells was found to be preceded by the intracellular accumulation of carbonic acids (predominantly, acetic acid), amounting to 9% of the cytosol. The intracellular concentration of acidic metabolites increased after the lyophilization of the bacterial biomass and decreased after its long-term storage (3.5–13.5 years). The decrease in the concentration of acidic metabolites is likely due to the dehydration of dimeric carbonic acids in the viscoelastic cytosol of resting bacterial cells. The hydrophobic obligately aerobic cells of Acinetobacter calcoaceticusIEGM 549 are able to utilize a wide range of growth substrates (from acetate and citrate to hydrophobic hydrocarbons), which is considerably wider than the range of the growth substrates of E. coli(predominantly, carbohydrates). The minimal essential and optimal concentrations of orthophosphates in the growth medium of A. calcoaceticuswere found to be tens of times lower than in the case of E. coli.The intracellular content of orthophosphates in A. calcoaceticuscells reached 35–77% of the total phosphorus content (P_total), providing for the intense synthesis of polyphosphates. The P_totalof the A. calcoaceticuscells grown in media with different proportions between the concentrations of acetate and phosphorus varied from 0.7 to 3.3%, averaging 2%. This value of P_totalis about two times higher than that observed for fermenting E. colicells. Lowering the cultivation temperature of A. calcoaceticusfrom 37–32 to 4°C augmented the accumulation of orthophosphates in the cytoplasm, presumably owing to a decreased requirement of growth processes for orthophosphate. In this case, if the concentration of phosphates in the cultivation medium was low, they were completely depleted.     

Direct production of L-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis alpha-amylase using cspB promoter and signal sequence

Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes α-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40°C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37°C, respectively. The α-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence.     

Rapid and Dense Culture of Acinetobacter calcoaceticus on Palm Oil

A soil microorganism, identified as Acinetobacter calcoaceticus KB-2, was cultivated on palm oil as a carbon source for cell production. This organism grew with a specific growth rate of l.lOh^?1. The pH optimum for growth was between 6.5 and 7.0, and the temperature optimum was 39°C. Compared with other strains on water-insoluble substrates such as hydrocarbons and natural oils and fats so far reported, the cultivation time for this strain was short and the cell mass productivity was relatively high. More than 90% of the palm oil was assimilated by this strain, and the overall cell yield was 1.02 (g of cells/g of palm oil) after 8 hr cultivation with the concentration of 3% palm oil.     

Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates ammonia and other forms of reduced nitrogen either through the GS/GOGAT pathway or by the concerted action of l-alanine dehydrogenase and aminotransferases. These routes are light-independent and very responsive to the carbon and nitrogen sources used for cell growth. GS was most active in cells grown on nitrate or l-glutamate as nitrogen sources, whereas it was heavily adenylylated and siginificantly repressed by ammonium, glycine, l-alanine, l-aspartate, l-asparagine and l-glutamine, under which conditions specific aminotransferases were induced. GOGAT activity was kept at constitutive levels in cells grown on l-amino acids as nitrogen sources except on l-glutamine where it was significantly induced during the early phase of growth. In vitro, GOGAT activity was strongly inhibited by l-tyrosine and NADPH. In cells using l-asparagine or l-aspartate as nitrogen source, a concerted induction of l-aspartate aminotransferase and l-asparaginase was observed. Enzyme level enhancements in response to nitrogen source variation involved de novo protein synthesis and strongly correlated with the cell growth phase.Abbreviations ADH l-alanine dehydrogenase - AOAT l-alanine:2-oxoglutarate aminotransferase - Asnase l-asparaginase - GOAT Glycine: oxaloacetate aminotransferase - GOGAT Glutamate synthase - GOT l-aspartate: 2-oxoglutarate aminotransferase - GS Glutamine synthetase - HPLC High-Pressure Liquid Chromatography - MOPS 2-(N-morpholino)propanesulfonic acid - MSX l-methionine-d,l-sulfoximine     

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on l-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on l-glutamate, l-proline, or l-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru^- phenotype of the mutants.