Citrate-grown Cells Research Articles

Characterization of citrate utilization inCorynebacterium glutamicumby transcriptome and proteome analysis

Corynebacterium glutamicum grows aerobically on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. To characterize the citrate utilization in C. glutamicum on a genomewide scale, a comparative analysis was carried out by combining transcriptome and proteome analysis. In cells grown on citrate, transcriptome analysis revealed highest expression changes for two different citrate-uptake systems encoded by citM and tctCBA, whereas genes encoding uptake systems for the glucose- (ptsG), sucrose- (ptsS) and fructose- (ptsF) specific PTS components and permeases for gluconate (gntP) and glutamate (gluC) displayed decreased mRNA levels in citrate-grown cells. This pattern was also observed when cells grown in Luria-Bertani (LB) medium plus citrate were compared with cells grown in LB medium, indicating some kind of catabolite repression. Genes encoding enzymes of the tricarboxylic acid cycle (aconitase, succinyl-CoA synthetase, succinate dehydrogenase and fumarase), malic enzyme, PEP carboxykinase, gluconeogenic glyceraldehyde-3-phosphate dehydrogenase and ATP synthase displayed increased expression in cells grown on citrate. Accordingly, proteome analysis revealed elevated protein levels of these enzymes and showed a good correlation with the mRNA levels. In conclusion, this study revealed the citrate stimulon in C. glutamicum and the regulated central metabolic genes when grown on citrate.

Metabolism of acrylate to beta-hydroxypropionate and its role in dimethylsulfoniopropionate lyase induction by a salt marsh sediment bacterium, Alcaligenes faecalis M3A.

Dimethylsulfoniopropionate (DMSP) is degraded to dimethylsulfide (DMS) and acrylate by the enzyme DMSP lyase. DMS or acrylate can serve as a carbon source for both free-living and endophytic bacteria in the marine environment. In this study, we report on the mechanism of DMSP-acrylate metabolism by Alcaligenes faecalis M3A. Suspensions of citrate-grown cells expressed a low level of DMSP lyase activity that could be induced to much higher levels in the presence of DMSP, acrylate, and its metabolic product, beta-hydroxypropionate. DMSP was degraded outside the cell, resulting in an extracellular accumulation of acrylate, which in suspensions of citrate-grown cells was then metabolized at a low endogenous rate. The inducible nature of acrylate metabolism was evidenced by both an increase in the rate of its degradation over time and the ability of acrylate-grown cells to metabolize this molecule at about an eight times higher rate than citrate-grown cells. Therefore, acrylate induces both its production (from DMSP) and its degradation by an acrylase enzyme. (1)H and (13)C nuclear magnetic resonance analyses were used to identify the products resulting from [1-(13)C]acrylate metabolism. The results indicated that A. faecalis first metabolized acrylate to beta-hydroxypropionate outside the cell, which was followed by its intracellular accumulation and subsequent induction of DMSP lyase activity. In summary, the mechanism of DMSP degradation to acrylate and the subsequent degradation of acrylate to beta-hydroxypropionate in the aerobic beta-Proteobacterium A. faecalis has been described.

Effects of octane on the fatty acid composition and transition temperature of Pseudomonas oleovorans membrane lipids during growth in two-liquid-phase continuous cultures

Growth of Pseudomonas oleovorans GPol in continuous culture containing a bulk n-octane phase resulted in changes of the fatty acid composition of the membrane lipids. Compared to citrate-grown cells, the ratio of C 18 to C 16 fatty acids and the ratio of unsaturated to saturated fatty acids increased as a result of growth on octane. Trans-unsaturated fatty acids, which are rarely found in bacteria, were formed during continuous growth of P. oleovorans on octane. Moreover, the mean acyl chain length and unsaturated fatty acids also increased as the growth rates increased both in octane-grown and citrate-grown cells. Differential scanning calorimetry measurements of extracted lipids showed the transition temperature of membrane lipids from octane-grown cells increased from about 24°C to 32°C as the growth rate increased, whereas cells grown on citrate showed a constant transition temperature of about 6°C at all growth rates tested, indicating a decrease of membrane lipid fluidity in octane-grown cells. Because alkanes are known to increase bilayer fluidity by intercalating between lipid fatty acyl chains, the increased transition temperature of the lipids of cells grown on octane may be a physiological response of P. oleovorans to compensate for the direct effects of octane on its cellular membranes.

Acquisition of iron from citrate by Pseudomonas aeruginosa

Transport of [14C]citrate, ferric [14C]citrate and [55Fe]ferric citrate into Pseudomonas aeruginosa grown in synthetic media containing citrate, succinate, or succinate and citrate as carbon and energy sources was measured. Cells grown in citrate-containing medium transported radiolabelled citrate and iron, whereas the succinate-grown cells transported iron but not citrate. Binding studies revealed that isolated outer and inner membranes of citrate-grown cells contain a citrate receptor, absent from membranes of succinate-grown cells. [55Fe]Ferric citrate bound to the isolated outer membranes of each cell type. The failure of citrate to compete with this binding suggests the presence of a ferric citrate receptor on the outer membranes of each cell type. Citrate induced the synthesis of two outer-membrane proteins of 41 and 19 kDa. A third protein of 17 kDa was more dominant in citrate-grown cells than in succinate-grown cells.

Aromatic metabolism by a 2,4-D degrading Arthrobacter sp.

A hypothesis that low molecular weight aromatic compounds, which occur naturally in the soil, may act as alternative substrates or inducers for the 2,4-dichlorophenoxyacetate (2,4-D) degrading enzyme system of an Arthrobacter sp. and maintain this enzyme system in the absence of 2,4-D, was examined experimentally. In a nonmanometric resting cell experiment, the 2,4-D adapted Arthrobacter sp. rapidly degraded 6 of the 12 aromatic compounds tested, and these 6 compounds were tested further with 2 additional aromatic compounds in manometric experiments with 2,4-D grown and citrate-grown cells. Homogentisic acid and catechol were oxidized more rapidly by 2,4-D grown cells than by cells grown on citrate medium. Thus, these two compounds could serve as alternative substrates for the 2,4-D degrading enzyme system of the Arthrobacter sp. In further manometric experiments, the 2,4-D degrading enzyme system in cells adapted to 2,4-dichlorophenol, catechol, or sodium benzoate was not induced.

Changes in the enzyme activities involved in nitrogen assimilation in Mycobacterium smegmatis under various growth conditions

Glutamate dehydrogenase (aminating) and glutamine synthetase activities were assayed in Mycobacterium smegmatis following growth on various carbon and nitrogen sources. The activities (expressed as nmoles product formed/min/mg crude extract protein) of these two enzymes were higher in crude extracts from glucose-grown cells than in glycerol- or fructose-grown cells. In the presence of succinate, pyruvate, fumarate or acetate in the growth medium, both these enzyme activities were lower than those in citrate-grown cells. The glutamate dehydrogenase (GDH) activity was the same in asparagine and glutamine-grown cells. Ammonium chloride, alanine or glutamic acid, when used as nitrogen source, resulted in low GDH activity as compared to asparagine-grown cells. Glutamine synthetase activity was considerably lower (2-4 fold) when the cells were grown on alanine, glutamine, glutamic acid or ammonium chloride as the nitrogen source than those in asparagine-grown cells. Glutamate and ammonium chloride, when present in the growth medium, repressed both glutamate dehydrogenase and glutamine synthetase, though the degree of repression was small. The results suggest that only a weak transcriptional control operates for these enzyme activities in M. smegmatis.

Citrate utilization by Escherichia coli: plasmid- and chromosome-encoded systems

Citrate utilization plasmids have previously been identified in atypical Escherichia coli isolates. A different citrate-utilizing (Cit+) variant of E. coli K-12 arose as a consequence of two chromosomal mutations (B. G. Hall, J. Bacteriol. 151:269-273, 1982). The processes controlling the transport of citrate in both a Cit+ chromosomal mutant and a Cit+ plasmid system were studied. Both systems were found to be inducible in growth experiments. In transport assays with whole cells, citrate-grown cells accumulated [1,5-14C]citrate at two to three times the rate of uninduced cells. Only the Vmax was affected by induction, and the Km for whole cells remained at 67 microM citrate for the chromosomal strain and 120 microM citrate for the plasmid-conferred system. There was no detectable accumulation of radioactivity with [6-14C]citrate, because of rapid metabolism and the release of 14CO2. Energy-dependent citrate transport was found with membrane vesicles obtained from both the chromosome-conferred and the plasmid Cit+ systems. The vesicle systems were inhibited by valinomycin and carbonyl cyanide m-chloro-phenylhydrazone but not by nigericin and monensin. In contrast to whole cells, the vesicle systems were resistant to Hg2+ and showed identical kinetics with [1,5-14C]citrate and [6-14C]citrate. H+ appeared to be important for citrate transport in whole cells and membranes. Monovalent cations such as Na+ and K+, divalent cations such as Mg2+ and Mn2+, and anions such as PO4(3-), SO4(2-), and NO3- were not required. The two systems differed in inhibition by citrate analogs.

The properties of citrate transport in membrane vesicles from Bacillus subtilis.

The uptake system for citrate is induced in Bacillus subtilis W23 by growth in the presence of citrate and only membrane vesicles isolated from these cells show energy-dependent citrate uptake. Citrate transport in membrane vesicles is strictly dependent on the presence of divalent cations such as Mg2+, Mn2+, Zn2+, Ba2+, Be2+, Ca2+, Cu2+, Co2+ or Ni2+. The initial rate of citrate transport increases with the divalent cation concentration up to a maximum. The maximum initial rate of citrate uptake is reached with 2 mM Mg2+. The cations form stable chelates with citrate. The metal citrate complex is the transported solute. This is demonstrated for citrate uptake in the presence of Ca2+. Membrane vesicles from citrate-grown cells accumulate Ca2+ and citrate only if both solutes are present. Citrate and Ca2+ are accumulated in equimolar quantities. The uptake of Ca2+ but not of citrate is inhibited by Mg2+. Uptake of the metal-citrate complex is inhibited by the uncoupler carbonylcyanide p-trifluoromethoxyphenyl-hydrazone and in the presence of K+ ions by valinomycin and nigericin. The inhibitory effects correlate with the effects observed on the components of the proton-motive force, indicating that the proton-motive force is a driving force for metal-citrate transport. The number of protons (n) symported with the metal-citrate complex has been determined under different experimental conditions from the steady state levels of citrate accumulation, the electrical potential and pH gradient. This number varies from 1 at pH 4.7 to 2 at pH 8.0.

Acetoin and Diacetyl Production by Lactobacillus plantarum Able to Use Citrate

Whole-cell suspensions of Lactobacillus plantarum grown on lactose in the presence of citrate did not produce acetoin and diacetyl (AD) (D) from citrate in succinate buffer, pH 4.4 unless both a source of energy and nitrogen was present, but did from pyruvate. The total AD and the amount of D, produced by citrate-grown cells, from citrate were about two times the amounts formed from pyruvate, calculated on a molar basis. It appears, that AD are formed not only from pyruvate resulting from cleavage of citrate but also from acetyl-coenzyme A arising during a probable breakdown of citrate in a reversible reaction of citrate synthetase. Neither acetate nor acetaldehyde had any effect on the total AD or the amount of D produced from pyruvate by pyruvate-grown cells. The rates of AD production from pyruvate by whole-cell suspensions of pyruvate-grown L. plantarum and Streptococcus subsp. diacetylactis represented only 69.7 and 6.6%, respectively, of that produced by Lactobacillus casei. These were 0.075 μmoles/mg dry wt−1 ml−1 min−1 for L. casei, 0.053 for L. plantarum and 0.005 for S. lactis subsp. diacetylactis.

Transport of citric acid by Aerobacter aerogenes

The kinetics of citrate transport by induced cells of Aerobacter aerogenes was measured and an apparent K m of 2.5 × 10 −6 was determined. Citrate was transported optimally at pH 7 and 30–37 °. Osmotic shock decreased total citrate uptake by 75% but the initial rate of transport was unaffected. Shocked cells incubated with citrate recovered their ability to take up citrate, but chloramphenicol prevented this recovery. Aconitase activity was 20-fold higher in extracts from citrate-grown cells of A. aerogenes than from glucose-grown cells. Aconitase activity was diminished by osmotic shock. Aconitase activity was recovered by incubation of shocked cells with citrate concomitant with the recovery of citrate uptake. There was no accumulation of label from uptake of [6- 14C]citrate and 14CO 2 was evolved immediately. Intracellular citrate could not be detected after a 10-sec pulse with [1,5- 14C]citrate or [2,4- 14C]citrate; instead, the major intracellular product was glutamate. Citrate lyase was not detected in cell-free extracts of citrate-grown cells. These results indicated that A. aerogenes metabolized citrate as rapidly as it entered the cells and that aerobic citrate catabolism proceeded via the tricarboxylic acid cycle.

Enzymatic analysis of the requirement for sodium in aerobic growth of Salmonella typhimurium on citrate.

Na(+) was required for the aerobic growth of Salmonella typhimurium on citrate, but not on l-malate, glucose, or glycerol. The maximal growth rate and the maximal total growth occurred with 6 to 7 mm Na(+). Na(+) could not be replaced by K(+), NH(4) (+), Li(+), Rb(+), or Cs(+). Sonically treated extracts of citrate-grown cells contained the enzymes of the citrate fermentation pathway (citritase and oxalacetate decarboxylase) and all of the enzymes of the citric acid cycle. Thus, two separate routes of citrate catabolism appeared to be operational in the cells. Two discrete oxalacetate (OAA) decarboxylases were also demonstrated. One was of the "classic" type, being activated by Mn(++) and inhibited by ethylenediaminetetracetate (EDTA). It was present in the cell sap. The second decarboxylase closely resembled the Na(+)-activated OAA decarboxylase of citrate-grown Aerobacter aerogenes, whose growth also requires, or is increased, by Na(+). This decarboxylase was EDTA-insensitive, specifically activated by Na(+) and inhibited by avidin, and it had a high affinity for OAA. It was induced by growth on citrate, but not l-malate or glycerol. It is suggested that the Na(+) requirement for growth reflects the need to activate this OAA decarboxylase as a component of the citrate fermentation pathway and that citrate catabolism via the citric acid cycle, which should be independent of Na(+), is somehow dependent upon the activity of the Na(+)-activated enzyme.

Phenols as intermediates in the decomposition of phenoxyacetates by an Arthrobacter species.

Decomposition of 2,4-dichlorophenoxyacetate (2,4-D) by a soil arthrobacter was studied using the technique of sequential induction. Compounds oxidized rapidly and without a lag by 2,4-D-grown cells, but slowly or not at all by citrate-grown cells, included 2,4-D, 2- and 4-chlorophenoxyacetate, 3,5-dichloro- and 4-chloro-catechol, catechol, 2,4-dichlorophenol, and 2- and 4-chlorophenol. The manometric data suggested that phenols and catechols were intermediates in the degradation of phenoxyacetates. Resting cells failed to accumulate chlorine-substituted phenols during the metabolism of halogenated phenoxyacetates, apparently because the phenols were oxidized as readily as they were formed. However, 2,4-D-induced cells contained enzymes which acted upon phenoxyacetate and 4-hydroxyphenoxyacetate, but these cells did not metabolize phenol and hydroquinone. Suspensions of such bacteria, but not citrate-grown cells, converted phenoxyacetate and 4-hydroxyphenoxyacetate almost completely to phenol and hydroquinone, respectively. The results indicate that the Arthrobacter sp. degrades 2,4-D via 2,4-dichlorophenol, and 2- and 4-chlorophenoxyacetate via 2- and 4-chlorophenol, respectively. The results also demonstrate a new technique for obtaining high yields of an intermediate in a metabolic sequence.

PREPARATION AND PROPERTIES OF PROTOPLASTS FROM LIPOMYCES LIPOFER.

Lipomyces lipofer was grown aerobically in chemically defined liquid media containing glucose or citrate as a carbon source. Protoplasts were prepared by digestion of young cells with the intestinal secretions from Helix pomatia in the presence of 10% (w/v) mannitol. Differences in substrate utilization between citrate- and glucose-grown cells were noted and compared with the substrate utilization of the corresponding protoplasts. Examination of protoplasts derived from glucose- and citrate-grown cells showed that the citrate-grown organisms yielded morphologically and metabolically more stable protoplasts. The effects of hypotonic conditions on the metabolic activity of protoplasts as well as the consequences of supplementation of these protoplasts with cofactors have been described.

Studies on the mechanism of acetate oxidation by bacteria. VI. Comparative patterns of acetate oxidation by citrate-grown and acetate-grown Aerobacter aerogenes.

The data presented in this paper indicate operation of different mechanisms for acetate oxidation by A. aerogenes, depending on the carbon source used for growth. The mechanism for citrate-grown cells appears to involve a conventional citric acid cycle, whereas acetate-grown cells appear to incorporate acetate carbon more readily via a dicarboxylic acid cycle.