Diauxic Growth in Rice Suspension Cells Grown on Mixed Carbon Sources of Acetate and Glucose

Diauxic growth was observed in rice (Oryza sativa L.) suspension cells growing on acetate (10 mM) and glucose (10 mM). Cells used acetate during the first growth phase and the acetate level in the medium was rapidly decreased, whereas the level of glucose remained essentially unchanged. After acetate was depleted from the medium, cells started to use glucose, forming the second growth phase. It appears that uptake of [14C]glucose was repressed during the first growth phase and became active during the second growth phase. In contrast, uptake of [14C]acetate occurred actively throughout the diauxic growth. By further demonstrating the specific induction of isocitrate lyase (EC 4.1.3.1), a glyoxylate cycle enzyme, and hexokinase (EC 2.7.1.1), a glycolysis enzyme, during the first and second growth phases, respectively, it was clearly shown that rice cells use acetate first and do not use both carbon sources simultaneously. This kind of diauxic growth pattern has been observed in bacteria. To our knowledege, this study is the first report demonstrating the presence of diauxic growth in plant cells.     

Mannopine and mannopinic acid as substrates for Arthrobacter sp. strain MBA209 and Pseudomonas putida NA513.

The characteristics of mannopine and mannopinic acid utilization by Agrobacterium tumefaciens B6S3, Arthrobacter sp. strain MBA209, and Pseudomonas putida NA513 were studied. Strain B6S3 utilized the four mannityl opines, mannopine, mannopinic acid, agropine, and agropinic acid. It also utilized several mannityl opine analogs, which were modified in either the sugar or the amino acid moiety. It utilized mannopine more rapidly after preincubation on mannopine, mannopinic acid, or glutamine than after pregrowth on glucose, mannose, or mannitol. Strains MBA209 and NA513 utilized mannopine and mannopinic acid, but not the other two mannityl opines. They utilized few mannityl opine analogs, sometimes because of failure to utilize the products of initial cleavage of the analog. Utilization of mannopine and mannopinic acid by strain NA513 was strictly dependent on prior growth on these substrates. A spontaneous regulatory variant of strain NA513 remained unable to utilize most of the mannityl opine analogs. Glutamine, mannose, and several analogs had no inhibitory effect on [14C]mannopine utilization by strain NA513.     

Spontaneous mutation conferring the ability to catabolize mannopine in Agrobacterium tumefaciens.

Two nopaline-type strains of Agrobacterium tumefaciens, C58 and T37, as well as strain A136, which is a Ti plasmid-cured derivative of strain C58, gave rise to spontaneous mutants that were able to grow on mannopine. The observation of mutagenesis with strain A136 demonstrated that the ability to acquire this new catabolic potential was independent of the presence of a Ti plasmid. The mutants were isolated after 4 weeks of incubation on minimal medium containing mannopine as the sole carbon source. They also utilized mannopinic acid, but not agropine or agropinic acid. In addition, the spontaneous mutant LM136, but not its parent strain A136, degraded many mannityl opine analogs. [14C]mannopine disappeared in the presence of LM136 cells which had been pregrown on opine or nonopine substrates. These results suggested that the catabolic system of this mutant was not subject to a stringent regulation. A clone conferring the ability to utilize mannopine on a recipient pseudomonad was selected from a genomic library from both the mutant LM136 and its parent strain. Only the LM136 clone was expressed in the parent Agrobacterium strain A136. Southern analysis showed that the genes for mannopine catabolism in the spontaneous mutants differed from the corresponding Ti plasmid-encoded genes of octopine-type or agropine-type Agrobacterium strains. Cells of LM136 utilized [14C]mannopine without generating detectable amounts of intracellular agropine. In contrast, a major fraction of the radioactivity recovered from cells of the octopine-type strain Ach5, after incubation on [14C]mannopine, was in the form of agropine.     

Regulation of fructose uptake and catabolism by succinate in Azospirillum brasilense.

Fructose uptake and catabolism in Azospirillum brasilense is dependent on three fructose-inducible enzymes (fru-enzymes): (i) enzyme I and (ii) enzyme II of the phosphoenolpyruvate:fructose phosphotransferase system and (iii) 1-phosphofructokinase. In minimal medium containing 3.7 mM succinate and 22 mM fructose as sources of carbon, growth of A. brasilense was diauxic, succinate being utilized in the first phase of growth and fructose in the second phase with a lag period between the two growth phases. None of the fru-enzymes could be detected in cells grown with succinate as the sole source of carbon, but they were detectable toward the end of the first phase of diauxie. All the fru-enzymes were coinduced by fructose and coordinately repressed by succinate. Studies on the effect of succinate on differential rates of syntheses of the fru-enzymes revealed that their induced syntheses in fructose minimal medium were subject to transient as well as permanent (catabolite) repression by succinate. Succinate also caused a similar pattern of transient and permanent repression of the fructose transport system in A. brasilense. However, no inducer (fructose) exclusionlike effect was observed as there was no inhibition of fructose uptake in the presence of succinate with fructose-grown cells even when they were fully induced for succinate uptake activity.     

Pyruvate-dependent diauxic growth of Rhodospirillum rubrum in light.

When Rhodospirillum rubrum mutant C was first exposed to radiant energy after long-term anaerobic dark growth, the cells often exhibited a diauxic growth response. This happened with pyruvate in the medium and when cultures were exposed to a less-than-growth-saturating white light intensity of about 6,460 lx. Under the growth-saturating light condition, mutant C photometabolized and growth was not affected by Na hypophosphite, an inhibitor of pyruvate fermentation. In lower intensity light, in which diauxie occurred, initial (phase I) growth occurred by fermentation of Na pyruvate and was sensitive to Na hypophosphite inhibition. Once pyruvate was depleted, phase I growth stopped, the bacteriochlorophyll content of the cells began to increase from about 3 nmol/mg of protein, and growth finally resumed phototrophically (phase II). The lag period and phase II growth were influenced by radiant energy. By changing the white light intensity from 2,150 to 753 lx between experiments, the duration of both the lag period and the generation time of cells in phase II growth increased. Diauxic growth was pyruvate dependent. It occurred with pyruvate even if malate, a photometabolizable substrate, was added to the growth medium. Moreover, the biphasic growth response was reversible. It was observed not only with R. rubrum mutant C grown cells photosynthetically, but also when other strains of R. rubrum were placed in pyruvate medium under lowered light conditions. Only R. rubrum S1 did not exhibit the typical pyruvate-dependent diauxic growth response.     

Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes.

The agropine/mannopine synthesis region of the TR region of the Ri plasmid of Agrobacterium rhizogenes strain A4 was localized on the basis of sequence similarity with probes from Ti plasmids of Agrobacterium tumefaciens and analysis of transposon insertions. The nucleotide sequence of the right part of the TR-DNA of pRiA4, encompassing the three genes involved in mannityl-opine synthesis, was determined and compared to the sequence of the corresponding region of the octopine-type Ti plasmid pTi15955. The organization of this region is strongly conserved between Ri and Ti plasmids, but the similarity is restricted to the coding sequences: no homology was detected in the 5' and 3' flanking sequences. The mas1' and ags proteins are the most conserved, showing more than 68% amino acid conservation, whereas the mas2' proteins are only 59% identical. Significant G/C content and codon usage differences are observed between pTi15955 and pRiA4. An open reading frame strongly similar to that of bacterial repressors is situated immediately to the right of the TR region.     

Hyperbolic growth of Thermoanaerobacter thermohydrosulfuricus (Clostridium thermohydrosulfuricum) increases ethanol production in pH-controlled batch culture

Thermoanaerobacter thermohydrosulfuricus Rt8.B1 exhibited hyperbolic growth (i.e. a continuous rate of growth, without diauxie, during growth and utilization of two carbon sources) on mixed carbohydrate substrates when grown in pH-controlled batch culture. Hyperbolic growth was observed with xylose in combination with either glucose or cellobiose. Diauxic growth ways observed when T. thermohydrosulfuricus Rt8.B1 was grown on a glucose plus cellobiose substrate mix. The major fermentation end-products under all substrate conditions were ethanol and acetate. Ethanol production varied depending on the substrate supplied and was always greatest on mixtures that included xylose (i.e. hyperbolic growth). High ethanol-to-acetate ratios could not be explained on the basis of a greater substrate uptake and thus more ethanol production under these conditions, or by variations in the levels of acetate kinase and NADP-linked alcohol dehydrogenase synthesis. The high ethanol-to-acetate ratio could not be increased by growing T.thermohydrosulfuricus Rt8.B1 under a partial pressure of hydrogen (1 atm) or by growth at different pH. Growth under these conditions decreased the ethanol-to-acetate ratio.Correspondence to: G. M. Cook     

Diauxic growth in Azotobacter vinelandii.

Azotobacter vinelandii exhibited diauxie when grown in a medium containing both acetate and glucose as carbon sources. Acetate was used as the primary carbon source during the acetate-glucose diauxie. Uptake of acetate was constitutively expressed during both diauxic phases of growth. Induction of the glucose uptake system was inhibited in the presence of acetate. Acetate was also the preferred growth substrate for A. vinelandii grown in a medium containing either fructose, maltose, xylitol, or mannitol. The tricarboxylic acid cycle intermediates citrate, isocitrate, and 2-oxoglutarate inhibited glucose utilization in cells grown in glucose medium containing these substrates, and diauxic growth was observed under these growth conditions. Temporal expression of isocitrate-lyase, ATPase, and nitrogenase was exhibited during acetate-glucose diauxie.     

Radiorespirometry evidence for the discrimination between 13C-enriched glucose and unlabelled glucose molecules by Paracoccus denitrificans

Paracoccus denitrificans was grown on either unlabelled glucose, [1-13C]glucose or [6-13C]glucose as the sole carbon source for growth. The cells were then incubated with a range of 14C-glucose substrates to compare the 14CO2-evolution rates between cells grown on the glucose and the 13C-labelled glucose. Cells grown on 13C-glucose had significantly faster rates of 14CO2-evolution than those grown on unlabelled glucose. The % yields of 14CO2, per [1-14C]-, [6-14C]- and [U-14C]glucose supplied were also substantially greater than those measured for cells grown on unlabelled glucose. The data indicated that growth of Paracoccus on 13C-enriched glucose substrates resulted in cells with notably different 14C-glucose oxidation metabolism compared to that observed in cells grown on unlabelled glucose.     

Relationship between succinate transport and production of extracellular poly(3-hydroxybutyrate) depolymerase in Pseudomonas lemoignei

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6. 8. The apparent KS0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [14C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pKa2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate2- accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.     

Intermediary metabolism in Legionella pneumophila: utilization of amino acids and other compounds as energy sources.

The utilization of amino acids and other compounds as carbon and energy sources by Legionella pneumophila was examined. Based on the stimulation of oxygen consumption in washed-cell suspensions, glutamate, serine, threonine, and tyrosine were the only amino acids which were utilized as energy sources. Other stimulators of oxygen uptake were lactate, pyruvate, acetate, fumarate, and succinate. Citrate was a good stimulator only when the bacteria were grown in the presence of the substrate. Radiolabeling studies showed that [14C]glutamate was rapidly metabolized, with the label distributed evenly in all cell fractions. [14C]pyruvate and [14C]acetate were incorporated into the lipid-containing cell fraction, whereas glucose and glycerol were found in both the lipid- and polysaccharide-containing cell fractions. Radiorespirometry of differentially labeled [14C]glucose indicated that this compound was metabolized primarily by the pentose phosphate and Entner-Doudoroff pathways rather than by the glycolytic pathway.     

Eight DNA insertion events of Agrobacterium tumefaciens Ti-plasmids in isogenic sunflower genomes are all distinct

We investigated whether the same or different T-DNA insertions occur every time Agrobacterium tumefaciens, the octopine type strain pTi 15955 strr, infects genetically identical sunflower plants. Eight newly established crown gall tissue culture lines were analyzed for their T-DNA content. Our data showed that all isogenic crown gall callus DNA produced distinct hybridization patterns. These eight patterns were also different from three standard lines included for comparison. In addition, all the tumor lines analyzed produced octopine, albeit in different quantities, and five produced agropine and mannopine. We concluded, that each A. tumefaciens crown gall tissue line derived from isogenic sunflower plants contained a distinct insertion pattern of T-DNA. Possible causes and reasons for this diversity will be discussed.     

Transport of nonmetabolizable opines by Agrobacterium tumefaciens.

We have examined the uptake of [14C]octopine and [14C]nopaline by Agrobacterium tumefaciens strains containing the C58 chromosomal background in medium suitable for the induction of vir genes. All strains tested could transport both of these opines, regardless of the presence or type of Ti plasmid (octopine or nopaline) present in the bacterium. The transport of these opines required active cellular metabolism. Nonradioactive octopine, nopaline, and arginine competed effectively with [14C]octopine and [14C]nopaline for transport into A. tumefaciens A136, suggesting that the transport of these opines occurs via an arginine transport pathway not encoded by the Ti plasmid.     

Growth of Agrobacterium tumefaciens under octopine limitation in chemostats.

Agrobacterium tumefaciens B6 and ATCC 15955 were grown under octopine or glutamate limitation in chemostats. Examination of the maximum specific growth rate (mu max) and substrate affinity (KS) for each strain indicated that strain B6 was highly inefficient in its use of octopine as either a nitrogen or carbon source compared with strain ATCC 15955. Examination of the yield coefficients showed that in both strains octopine was used more efficiently as a nitrogen source than as a carbon source. The data permitted predictions to be made concerning the outcome of competition for a single limiting substrate. Under octopine limitation, strain ATCC 15955 should dominate; under glutamate limitation, strain B6 should dominate. The results of an observed competition with glutamate as the limiting substrate confirmed the latter prediction, although B6 did dominate at a rate faster than was predicted from simple competition theory. B6 displayed higher growth rates and substrate affinities than ATCC 15955 on all concentrations of glutamate. The yield of B6 on octopine was also considerably higher. This latter attribute could provide an ecological advantage to B6 because of the importance of yield in the fate of competitions under multisubstrate regimens. These will be the most prevalent regimens in the area around the tumor (tumorosphere) or the rhizosphere. The increased performance on glutamate could provide an advantage in an opine-free environment when B6 is growing as a saprophyte.     

Alteration of glucose transport and diauxic growth in 5-thio-D-glucose-resistant mutants of Azotobacter vinelandii.

Spontaneous mutants of Azotobacter vinelandii defective for glucose utilization were selected as resistant to 5-thio-D-glucose. Mutant strains AM2, AM38, and AM39 exhibited longer generation times than the wild type when grown on glucose. Mutant strain AM2 also exhibited an altered Km and Vmax for glucose uptake. During acetate-glucose diauxie, glucose utilization in the 5-thio-D-glucose-resistant mutants was subject to severe inhibition by acetate. These mutants did not exhibit the normal glucose phase of diauxie. Transport studies during diauxie indicated that glucose uptake was not induced in mutant strain AM2. However, increasing the glucose concentration from 25 to 200 mM relieved the severe acetate inhibition, and under these conditions the mutant strain AM2 exhibited normal diauxie. Revertants of mutant strain AM2 exhibited normal glucose and diauxie growth. The results are discussed in terms of a model for acetate regulation of glucose utilization in A. vinelandii.     

Functional and molecular identification of sodium-coupled dicarboxylate transporters in rat primary cultured cerebrocortical astrocytes and neurons

Na+-coupled carboxylate transporters (NaCs) mediate the uptake of tricarboxylic acid cycle intermediates in mammalian tissues. Of these transporters, NaC3 (formerly known as Na+-coupled dicarboxylate transporter 3, NaDC3/SDCT2) and NaC2 (formerly known as Na+-coupled citrate transporter, NaCT) have been shown to be expressed in brain. There is, however, little information available on the precise distribution and function of both transporters in the CNS. In the present study, we investigated the functional characteristics of Na+-dependent succinate and citrate transport in primary cultures of astrocytes and neurons from rat cerebral cortex. Uptake of succinate was Na+ dependent, Li+ sensitive and saturable with a Michaelis constant (Kt) value of 28.4 microM in rat astrocytes. Na+ activation kinetics revealed that the Na+ to succinate stoichiometry was 3:1 and the concentration of Na+ necessary for half-maximal transport was 53 mM. Although uptake of citrate in astrocytes was also Na+ dependent and saturable, its Kt value was significantly higher (approximately 1.2 mM) than that of succinate. Unlabeled succinate (2 mM) inhibited Na+-dependent [14C]succinate (18 microM) and [14C]citrate (4.5 microM) transport completely, whereas unlabeled citrate inhibited Na+-dependent [14C]succinate uptake more weakly. Interestingly, N-acetyl-L-aspartate, which is the second most abundant amino acid in the nervous system, also completely inhibited Na+-dependent succinate transport in rat astrocytes. The inhibition constant (Ki) for the inhibition of [14C]succinate uptake by unlabeled succinate, N-acetyl-L-aspartate and citrate was 15.9, 155 and 764 microM respectively. In primary cultures of neurons, uptake of citrate was also Na+ dependent and saturable with a Kt value of 16.2 microM, which was different from that observed in astrocytes, suggesting that different Na+-dependent citrate transport systems are expressed in neurons and astrocytes. RT-PCR and immunocytochemistry revealed that NaC3 and NaC2 are expressed in cerebrocortical astrocytes and neurons respectively. These results are in good agreement with our previous reports on the brain distribution pattern of NaC2 and NaC3 mRNA using in situ hybridization. This is the first report of the differential expression of different NaCs in astrocytes and neurons. These transporters might play important roles in the trafficking of tricarboxylic acid cycle intermediates and related metabolites between glia and neurons.     

Growth of Agrobacterium tumefaciens under octopine limitation in chemostats

Agrobacterium tumefaciens B6 and ATCC 15955 were grown under octopine or glutamate limitation in chemostats. Examination of the maximum specific growth rate (mu max) and substrate affinity (KS) for each strain indicated that strain B6 was highly inefficient in its use of octopine as either a nitrogen or carbon source compared with strain ATCC 15955. Examination of the yield coefficients showed that in both strains octopine was used more efficiently as a nitrogen source than as a carbon source. The data permitted predictions to be made concerning the outcome of competition for a single limiting substrate. Under octopine limitation, strain ATCC 15955 should dominate; under glutamate limitation, strain B6 should dominate. The results of an observed competition with glutamate as the limiting substrate confirmed the latter prediction, although B6 did dominate at a rate faster than was predicted from simple competition theory. B6 displayed higher growth rates and substrate affinities than ATCC 15955 on all concentrations of glutamate. The yield of B6 on octopine was also considerably higher. This latter attribute could provide an ecological advantage to B6 because of the importance of yield in the fate of competitions under multisubstrate regimens. These will be the most prevalent regimens in the area around the tumor (tumorosphere) or the rhizosphere. The increased performance on glutamate could provide an advantage in an opine-free environment when B6 is growing as a saprophyte.     

The pathway of carbon dioxide assimilation in rhodospirillum rubrum grown in turbidostat continuous-flow culture

Summary A comparison of light and dark short-term incorporation of [¹⁴C]-carbon dioxide by Rhodospirillum rubrum grown in turbidostat continuous-flow culture at two different steady states on medium containing malate has shown that the labelling of phosphate esters was the main light-dependent process. Thus, the reductive pentose phosphate cycle appears to be the major pathway of carbon dioxide assimilation in the light under these growth conditions.The labelling of glutamate was also light-dependent and was most marked in the most rapidly growing steady state culture.The assimilated [¹⁴C]carbon was transferred to metabolites of the tricarboxylic acid cycle, particularly C₄-dicarboxylic acids, and the transfer involved additional carboxylations which were not light-dependent. The activity of these reactions accounted for initial high rates of carbon dioxide assimilation in the dark.In the dark assimilated [¹⁴C]carbon accumulated in succinate.     

Activity of the quorum-sensing regulator TraR of Agrobacterium tumefaciens is inhibited by a truncated, dominant defective TraR-like protein

Horizontal transfer of Agrobacterium tumefaciens tumour-inducing plasmids requires opines, which are released from plant tumours as nutrients for the bacteria. The opine octopine causes synthesis of the quorum-sensing TraR protein, which activates several tra promoters in the presence of a pheromone called Agrobacterium autoinducer (AAI). A gene, traS , was previously found on the same Ti plasmid in an operon that directs the uptake of mannopine, another opine. TraS strongly resembles TraR but lacks a DNA-binding module. TraS did not activate a TraR-dependent promoter and blocked TraR function, probably by forming inactive heteromultimers. Expression of traS was induced by mannopine, although this induction was strongly inhibited by the favoured catabolites succinate, glutamine and tryptone. Mannopine inhibited conjugation in a TraS-dependent fashion, and artificial overexpression of TraS also inhibited conjugation. Favoured catabolites restored tra gene expression in wild-type strains but not in strains that overexpress TraS. Downstream of traS is a gene encoding a truncated, defective chemoreceptor whose expression abolished chemotaxis.