Intermediary carbon metabolism of Azospirillum brasilense.

Azospirillum brasilense Sp 7 grew rapidly in AZO medium containing reduced nitrogen and succinate as an energy source, with a doubling time of 43 min. No growth was measured with glucose as the sole carbon source. In contrast, Azospirillum lipoferum Sp 59b could grow in media containing either succinate or glucose with a doubling time of 69 min and 223 min, respectively. Warburg-Barcroft respirometry showed that the rate of oxygen consumption by A. brasilense Sp 7 on glucose medium (0.034 mumol of O2 min-1 mg-1 of cell protein) was only one-quarter of that on succinate medium (0.14 mumol of O2 min-1 mg-1). Radioisotopic labeling showed that very little glucose was assimilated by A. brasilense Sp 7 as compared to succinate. High respiration rates were measured on A. lipoferum Sp 59b with either succinate (0.15 mumol of O2 min-1 mg-1) or glucose (0.13 mumol of O2 min-1 mg-1) as the sole carbon source. The pattern of CO2 evolution from differentially labeled succinate indicated that A. brasilense Sp 7 had a complete tricarboxylic acid cycle. Assimilation of most of the radioactivity from labeled succinate, pyruvate, and acetate into lipids suggested a strong anabolic metabolism and the presence of an active malic enzyme of phosphoenolpyruvate carboxykinase. The distribution of radioactivity from differentially labeled pyruvate showed that gluconeogenesis competed with pyruvate dehydrogenase. Uptake and incorporation of labeled acetate also indicated the presence of a glyoxylate cycle in A. brasilense Sp 7.     

Phosphate and carbon source regulation of alkaline phosphatase and phospholipase in Vibrio vulnificus

In this study, the effects of phosphate concentration and carbon source on the patterns of alkaline phosphatase (APase) and phospholipase (PLase) expression in Vibrio vulnificus ATCC 29307 were assessed under various conditions. The activities of these enzymes were repressed by excess phosphate (4 mM) in the culture medium, but this repression was reversed upon the onset of phosphate starvation in low phosphate defined medium (LPDM) containing 0.2 mM of phosphate at approximately the end of the exponential growth phase. The expressions of the two enzymes were also influenced by different carbon sources, including glucose, fructose, maltose, glycerol, and sodium acetate at different levels. The APase activity was derepressed most profoundly in LPDM containing fructose as a sole carbon source. However, the repression/derepression of the enzyme by phosphate was not observed in media containing glycerol or sodium acetate. In LPDM-glycerol or sodium acetate, the growth rate was quite low. The highest levels of PLase activity were detected in LPDMsodium acetate, followed by LPDM-fructose. PLase was not fully repressed by high phosphate concentrations when sodium acetate was utilized as the sole carbon source. These results showed that multiple regulatory systems, including the phosphate regulon, may perform a function in the expression of both or either APase and PLC, in the broader context of the survival of V. vulnificus.     

Sucrose transport and hydrolysis inRhizobium tropici

TheRhizobium tropici strain CFN 299 was maintained on PY medium and was grown in minimal medium (MM) with sucrose, glucose, fructose and glutamate (or their combination) as carbon sources. Bacteria were able to simultaneously use different carbon sources and, with a combination sucrose and glutamate, the growth rate was faster than with either carbon source alone. Sucrose transport was induced by sucrose and partially repressed by glucose and glutamate if they were included in MM as additional carbon sources. The transport of sucrose was active because both an uncoupler (dinitrophenol, DNP) and inhibitors of terminal oxidation (KCN, NaN₃) severely reduced sucrose uptake. Sucrose transport was also sensitive to a functional sulfhydryl reagent but was much less sensitive to EDTA and arsenate. We obtained nonlinear Lineweaver-Burk plots for the uptake of sucrose (by sucrose-grown bacteria), and this implied the existence of at least two uptake mechanisms. Invertase (EC 3.2.1.26) is the main enzyme for sucrose hydrolysis in this organism. This enzyme was induced by sucrose and had high activity in mid-log phase cells when sucrose was the sole carbon source (0.2%). Invertase activity was not detected in growth medium. In general, the results obtained support the idea, thatR. tropici is adapted to sucrose utilization and to multicarbon nutrition during its interaction with plants.     

Organic acid utilization,succinate excretion,encystation and oscillating nitrogenase activity inAzospirillum brasilense under microaerobic conditions

Oscillating nitrogenase activity in long lasting batch cultures ofAzospirillum brasilense ATCC 29145 is independent of the carbon source malate. With fumarate, succinate or pyruvate as sole carbon source nitrogenase activity is also oscillating. Cultivation in a medium with 20-fold the buffer concentration also results in oscillating nitrogenase activity. Nitrogen-fixing cultures ofAzospirillum brasilense ATCC 29145 excrete ammonia into the culture medium varying between 0.02 and 0.04 mM concentrations. This is not sufficient to cause a drop of nitrogenase activity inAzospirillum brasilense after the first maximum. During growth under nitrogen-fixing conditions with malate as carbon source, the cells excrete significant quantities of succinate into the culture medium. Cultures with only 0.05% malate reutilized the excreted succinate as soon as malate disappeared from the medium. Azospirillum brasilense ATCC 29145 is shown to have the capability of encystation. Encysted cells are different from vegetative cells in their resistance to desiccation, by the spherical shape and by immotility. The results indicate that oscillating nitrogenase activity in long lasting cultures reflects the development from vegetative cells to cysts and again to vegetative cells under microaerobic conditions.     

Kinetics of cell growth and heterologous glucoamylase production in recombinant Aspergillus nidulans

In the work, a study of cell growth and the regulation of heterologous glucoamylase synthesis under the control of the positively regulated alcA promoter in a recombinant Aspergillus nidulans is presented. We found that similar growth rates were obtained for both the host and recombinant cells when either glucose or fructose was employed as sole carbon and energy source. Use of the potent inducer cyclopentanone in concentrations greater than 3 mM resulted n maximum glucoamylase concentration and maximum overall specific glucoamylase concentration over 80 h of batch cultivation. However, cyclopentanone concentrations in excess of 3 mM also showed an inhibitory effect on spore germination as well as fungal growth. In contrast, another inducer, threonine, had no negative effect on spore germination even when concentrations of up to 100 mM were used with either glucose or fructose as carbon source. Glucoamylase synthesis in the presence of glucose plus either inducer did not begin until glucose was totally depleted, suggesting strong catabolite repression. Similar results were obtained when fructose was employed, although low levels of glucoamylase were detected before fructose depletion, suggesting partial catabolite repression. The highest enzyme concentration (570 mg/L) and overall specific enzyme concentration (81 mg/g cell) were observed in batch culture when cyclopentanone was the inducer and fructose the primary carbon source. A maximum glucoamylase concentration of 1.1 g/L and an overall specific glucoamylase concentration of 167 mg/g cell were obtained in a bioreactor using cyclopentanone as the inducer and limited-fructose feeding strategy, which nearly doubles the glucoamylase productivity from batch cultures. (c) 1993 John Wiley & Sons, Inc.     

Control of inducer accumulation plays a key role in succinate-mediated catabolite repression in Sinorhizobium meliloti

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the alpha-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other alpha-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of beta-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of alpha-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce beta-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced beta-galactoside transporters and cause the induction of alpha-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a beta-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.     

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the -glutamyltransferase method were similar for NH₄-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH₄)₂SO₄ and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.Abbreviations GS glutamine synthetase - SMM succinate minimal medium - ASMM ammonium/succinate minimal medium - GT -glutamyl transferase - SVP snake venom phosphodiesterase     

Nutrient utilization during biomass and anthocyanin accumulation in suspension cultures of wild carrot cells

The medium used for the growth of anthocyanin-accumulating wild carrot (D. carota) suspension cultures contained ammonia as a sole nitrogen source and was buffered with succinate. Ammonia was the first nutrient to be completely utilized.The uptake of carbohydrate, phosphate and succinate continued after ammonia depletion. Biomass accumulation was faster and greater when sucrose was initially present in the medium than when glucose was present. When sucrose was provided in the medium it was rapidly hydrolysed to glucose and fructose and the fructose was used preferentially to glucose. Anthocyanin accumulation was rapid after ammonia fell below 3 mM and until the pH of the medium rose from 4.5 to 5.1 or 5.2.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

NH(4)-Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH(4). Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH(4). The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O(2)) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH(4)Cl. Both mutants failed to incorporate [C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense, as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10 cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a N(2)-enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of N inside root and shoot material than the wild type did. The results of our nitrogen balance and N enrichment studies indicated that NH(4)-excreting A. brasilense strains potentially support the nitrogen supply of the host plants.     

Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled

Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38 degrees C in a standard minimal medium (MMAB) containing 2.5 gl(-1)l-malate and 50 microgml(-1) tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene (ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense.     

Diauxic growth of Agrobacterium tumefaciens 15955 on succinate and mannopine.

Diauxic growth was observed upon incubation of Agrobacterium tumefaciens 15955 on a mixture of succinate and mannopine as the carbon source. Diauxic growth was also observed when either fumarate or L-malate was mixed with mannopine. No diauxie was detectable when A. tumefaciens 15955 was grown on a mixture of mannopine and glucose, fructose, sucrose, or L-arabinose. Preferential utilization of succinate was observed in the initial growth phase of diauxie, whereas the final growth phase occurred at the expense of mannopine. Cells harvested during the initial growth phase exhibited a capacity for uptake of [14C]succinate but not of [14C] mannopine. A capacity for [14C]mannopine uptake was expressed during the final growth phase. Extracts from cells grown on a mixture of succinate and mannopine exhibited a low level of mannopine cyclase activity in the initial phase of diauxie. This activity increased substantially in the final phase of growth. Added succinate had no effect on the rate of [14C]mannopine uptake or mannopine cyclase activities of cells previously grown on mannopine. Diauxie was also observed during growth of strain 15955 on a mixture of succinate and octopine.     

A Crh-specific function in carbon catabolite repression in Bacillus subtilis

Carbon catabolite repression in Bacillus subtilis is mediated by phosphorylation of the phosphoenolpyruvate:carbohydrate phosphotransferase system intermediate HPr at a serine residue catalyzed by HPr kinase. The orthologous protein Crh functions in a similar way, but, unlike HPr, it is not functional in carbohydrate uptake. A specific function for Crh is not known. The role of HPr and Crh in repressing the citM gene encoding the Mg(2+)-citrate transporter was investigated during growth of B. subtilis on different carbon sources. In glucose minimal medium, full repression was supported by both HPr and Crh. Strains deficient in Crh or the regulatory function of HPr revealed the same repression as the wild-type strain. In contrast, in a medium containing succinate and glutamate, repression was specifically mediated via Crh. Repression was relieved in the Crh-deficient strain, but still present in the HPr mutant strain. The data are the first demonstration of a Crh-specific function in B. subtilis and suggest a role for Crh in regulation of expression during growth on substrates other than carbohydrates.     

Effects of anaerobiosis and nitrate on the expression of succinate dehydrogenase and enzymes associated with nitrogen metabolism in Klebsiella pneumoniae

We have shown that the low histidase activity found in anaerobic, nitrogen-limited cultures of Klebsiella pneumoniae is due to repression of the right-hand hut operon. In addition, we have examined the effects of NO3- on the aerobic and anaerobic expression of catabolite- and NH4+-repressible enzymes in this organism. NO3- permitted anaerobic growth of K. pneumoniae in minimal medium containing histidine as the sole carbon source, and histidase and succinate dehydrogenase were derepressed during anaerobic growth in histidine/NO3- medium. Use of sucrose rather than histidine as the carbon source reversed the effects of NO3- and repressed histidase and succinate dehydrogenase activities. Anaerobic growth in sucrose/NO3- medium also uncoupled the expression of urease and glutamine synthetase.     

Identification of a phosphoenolpyruvate:fructose 1-phosphotransferase system in Azospirillum brasilense

An inducible phosphoenolpyruvate:fructose phosphotransferase system has been detected in Azospirillum brasilense, which requires a minimum of two components of the crude extracts for activity: (i) a soluble fraction (enzyme I) and (ii) a membrane fraction (enzyme II). The uninduced cells neither show any uptake of fructose nor express activity of either of these two enzyme fractions. C-1 of fructose is the site of phosphorylation. This phosphotransferase system does not accept glucose as a substrate for phosphorylation.     

Biochemical characterization of a fructokinase mutant of Rhizobium meliloti.

A double mutant strain (UR3) of Rhizobium meliloti L5-30 was isolated from a phosphoglucose isomerase mutant (UR1) on the basis of its resistance to fructose inhibition when grown on fructose-rich medium. UR3 lacked both phosphoglucose isomerase and fructokinase activity. A mutant strain (UR4) lacking only the fructokinase activity was derived from UR3; it grew on the same carbon sources as the parent strain, but not on fructose, mannitol, or sorbitol. A spontaneous revertant (UR5) of normal growth phenotype contained fructokinase activity. A fructose transport system was found in L5-30, UR4, and UR5 grown in arabinose-fructose minimal medium. No fructose uptake activity was detected when L5-30 and UR5 were grown on arabinose minimal medium, but this activity was present in strain UR4. Free fructose was concentrated intracellularly by UR4 > 200-fold above the external level. A partial transformation of fructose into mannitol and sorbitol was detected by enzymatic analysis of the uptake products. Polyol dehydrogenase activity was detected in UR4 grown in arabinose-fructose minimal medium. The induction pattern of polyol dehydrogenase activities in this strain might be due to slight intracellular fructose accumulation.     

Nutrient effects on anthracycline production by Streptomyces peucetius in a defined medium

A defined medium was developed for Streptomyces peucetius that optimally contained 0.5 mM magnesium, 1 mM phosphate, 75-125 mM glucose, 10 mM nitrate, and microelements. Poorer results were obtained with nitrite, aspartate, or ammonia as sole nitrogen sources. Other carbon sources which supported best growth and highest anthracycline titers were fructose, maltose, and soluble starch. In each case, substantial residual carbon remained at the end of 6 days, suggesting a lack of catabolite repression by the carbohydrate carbon sources on anthracycline biosynthesis. Studies involving limiting and nonlimiting concentrations of glucose supplemented with arabinose, a poorly utilizable carbon source, indicated that high carbon concentrations were not necessary for osmotic stabilization. Inorganic phosphate was found to have an inhibitory effect on anthracycline production. Furthermore, when cultures at early stages of anthracycline production were spiked with inorganic phosphate, a delay in further anthracycline production resulted until the added phosphate was depleted. A 10% inoculum of stationary phase cells yielded the best growth and most consistent anthracycline production. Spectrophotometric analyses at 495 nm of chloroform--methanol-extracted material were also found to be useful for the determination of total anthracyclines in culture extracts.     

Two types of glucose effects on beta-galactosidase synthesis in a membrane fraction of Escherichia coli: correlation with repression observed in intact cells.

A membrane fraction obtained from an osmotic lysate of Escherichia coli spheroplasts retains capability to synthesize beta-galactosidase. The system also retains cellular regulatory functions, one of which is known as catabolite repression. Two types of repression of beta-galactosidase synthesis were observed in this membrane system: one was caused by the addition of 2-deoxyglucose or glucose at a low concentration (3 times 10- minus 4 M), and the other was caused by glucose-6-phosphate or glucose at a high concentration (3 times 10- minus 2 M). In the presence of cyclic adenosine 3',5'-monophosphate (10 mM), repression caused by the former was completely reversed, whereas repression by the latter was only partially reversed. Conditions in intact cells causing transient and permanent repression were also investigated. Upon addition of 2-deoxyglucose or glucose at a low concentration to intact cells, only transient repression of beta-galactosidase synthesis was observed. Glucose at a high concentration caused both transient and subsequent permanent repression, and intensity of permanent repression depended upon glucose concentration, whereas duration and intensity of transient repression were independent of glucose concentration. Mutants deficient in phosphoenolpyruvate-phosphotransferase system (Hpr minus and enzyme I minus) showed transient repression but failed to show permanent repression. In mutants deficient in glucose catabolism beyond glucose-6-phosphate, both transient and permanent repression were observed. Correlation between the observations in the membrane system and in intact cells is discussed. The results obtained here strongly suggest that transient repression is caused by glucose itself, and that permanent repression is caused by glucose-6-phosphate of high intracellular levels of glucose.