A novel metabolic network leads to enhanced citrate biogenesis in <Emphasis Type="Italic">Pseudomonas </Emphasis><Emphasis Type="Italic">fluorescens</Emphasis> exposed to aluminum toxicity

Aluminum (Al), an environmental toxin, is known to have a negative impact on various biological systems. However, some microbes have devised intricate mechanisms to combat the toxic influence of this trivalent metal. In this study, Pseudomonas fluorescens grown in malate invoked a unique metabolic shift to promote the synthesis of citrate, a metabolite involved in the sequestration of Al. Electrophoretic and spectrophotometric assays revealed several malate-metabolizing enzymes including malate dehydrogenase (MDH) and malic enzyme (ME) displayed increases in activity and expression in the Al-treated cells. Whereas pyruvate dehydrogenase (PDH) also showed increased activity and expression in the Al-stressed cultures, phosphoenolpyruvate carboxykinase (PEPCK) displayed a marked diminution in the Al-treated cells. The upregulation of citrate synthase (CS) coupled with the diminished activities of aconitase (ACN) and NAD-isocitrate dehydrogenase (NAD-ICDH) appeared to be instrumental in the accumulation of citrate. HPLC experiments revealed high levels of citrate in the Al-stressed cultures. Thus, an Al-enriched environment provoked a metabolic shift in P. fluorescens dedicated to the conversion of malate to citrate.     

Ethanol catabolism in Corynebacterium glutamicum

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h(-1) and biomass yields up to 0.47 g dry weight (g ethanol)(-1). Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.     

Effect of aeration and sodium on the metabolism of citrate by Klebsiella aerogenes.

Anaerobic growth of Klebsiella aerogenes NCDO 711 (NCTC 418) on citrate was dependent on the presence of Na+ in the medium, and fermentation of citrate was mediated via the fermentation pathway enzymes, citrate lyase and a Na+-dependent oxalacetate decarboxylase. This confirms the previous findings on strain NCTC 418. Growth under aerobic conditions was independent of Na+. The mean generation time for cells grown aerobically on either Na+ or K+ citrate medium was about 60 min, with a molar growth yield of about 40 g (dry weight) of cells per mol of citrate utilized. Citrate was apparently metabolized aerobically in both the Na+ and K+ citrate cells via the citric acid cycle, since cell extracts contained alpha-ketoglutarate dehydrogenase but not the citrate fermentation enzymes. The presence of theother enzymes of the citric acid cycle in K. aerogenes was shown in earlier studies. Under aerated conditions (no detectable oxygen tension in the culture), growth was faster on the Na+ citrate medium (mean generation time, 85 min) than on the K+ citrate medium (mean generation time, 120 min). Both cultures grew slower than under aerobic conditions, presumably because of oxygen limitation. Despite the faster growth rate, the molar growth yield of the aerated Na+ citrate culture was one-half that observed for the aerated K+ citrate culture. Citrate was metabolized via the citric acid cycle in cells grown in the K+ citrate medium under aerated conditions since alpha-ketoglutarate dehydrogenase, but not the fermentation enzymes, was detected in extracts prepared from these cells. Metabolism of citrate in the Na+ citrate medium under aerated conditions occurred via both the fermentation pathway (approximately 75 percent) and the citric acid cycle (about 25 percent), as evidenced by (i) the presence of the fermentation enzymes and alpha-ketoglutarate dehydrogenase in extracts of cells grown under these conditions, (ii) a molar growth yield which was intermediate between that obtained for anaerobic and aerated K+ citrate cultures, and (iii) the excretion of acetate, which also occurred in anaerobic cultures but not in aerated K+ citrate or aerobic cultures.     

Comparative proteomes of Corynebacterium glutamicum grown on aromatic compounds revealed novel proteins involved in aromatic degradation and a clear link between aromatic catabolism and gluconeogenesis via fructose-1,6-bisphosphatase

The current study examined the aromatic degradation and central metabolism in Corynebacterium glutamicum by proteomic and molecular methods. Comparative analysis of proteomes from cells grown on gentisate and on glucose revealed that 30% of the proteins of which their abundance changed were involved in aromatic degradation and central carbon metabolism. Similar results were obtained from cells grown on benzoate, 4-cresol, phenol, and resorcinol. Results from these experiments revealed that (i) enzymes involved in degradation of benzoate, 4-cresol, gentisate, phenol, and resorcinol were specifically synthesized and (ii) that the abundance of enzymes involved in central carbon metabolism of glycolysis/gluconeogenesis, pentose phosphate pathway, and TCA cycles were significantly changed on various aromatic compounds. Significantly, three novel proteins, NCgl0524, NCgl0525, and NCgl0527, were identified on 4-cresol. The genes encoding NCgl0525 and NCgl0527 were confirmed to be necessary for assimilation of 4-cresol with C. glutamicum. The abundance of fructose-1,6-bisphosphatase (Fbp) was universally increased on all the tested aromatic compounds. This Fbp gene was disrupted and the mutant WT(Deltafbp) lost the ability to grow on aromatic compounds. Genetic complementation by the Fbp gene restored this ability. We concluded that gluconeogenesis is a necessary process for C. glutamicum growing on various aromatic compounds.     

A comparative proteomic approach to understand the adaptations of an H+ -ATPase-defective mutant of Corynebacterium glutamicum ATCC14067 to energy deficiencies

F172-8, an H(+)-ATPase-defective mutant of the glutamic acid-producing bacterium Corynebacterium glutamicum ATCC 14067, exhibits enhanced rates of glucose consumption and respiration compared to the parental strain when cultured in a biotin-rich medium with glucose as the carbon source. We conducted a comparative proteomic analysis to clarify the mechanism by which the enhanced glucose metabolism in this mutant is established using a proteome reference map for strain ATCC 14067. A comparison of the proteomes of the two strains revealed the up-regulated expression of the several important enzymes such as pyruvate kinase (Pyk), malate:quinone oxidoreductase (Mqo), and malate dehydrogenase (Mdh) in the mutant. Because Pyk activates glycolysis in response to cellular energy shortages in this bacterium, its increased expression may contribute to the enhanced glucose metabolism of the mutant. A unique reoxidation system has been suggested for NADH in C. glutamicum consisting of coupled reactions between Mqo and Mdh, together with the respiratory chain; therefore, the enhanced expression of both enzymes might contribute to the reoxidation of NADH during increased respiration. The proteomic analysis allowed the identification of unique physiological changes associated with the H(+)-ATPase defect in F172-8 and contributed to the understanding of the adaptations of C. glutamicum to energy deficiencies.