Transient response of Enterobacter aerogenes under a dual nutrient limitation in a chemostat.

Utilizing a chemostat with a dual nutrient limitation of nitrogen and phosphate, we examined the transient response of the culture following a pulse of one of the limiting nutrients (ammonia). This method provided quantitative evidence that cells can be grown under dual nutrient limitation. Furthermore, the pattern of response was consistent with the hypothesis that phosphate limitation restricts nucleic acid synthesis in the cell and that nitrogen limitation restricts protein synthesis. The net result is that under a phosphate limitation there is a restricted biosynthetic capacity which we feel is closely associated with the RNA content of the cell.     

Control of teichoic acid synthesis in Bacillus licheniformis ATCC 9945

Analysis of cell walls of Bacillus licheniformis ATCC 9945 grown under phosphate limitation showed that teichoic acid could be replaced by teichuronic acid under these conditions. Teichuronic acid, however, was always present in the walls to some extent irrespective of the growth conditions. The enzymes involved in teichoic acid synthesis were investigated and the synthesis of these was shown to be repressed when the intracellular Pi level fell. CDP-glycerol pyrophosphorylase was studied in some detail and evidence is presented to show that the enzyme is inactivated under phosphate-limited conditions. The mechanism of inactivation is unknown but it has been shown that it does not require protein synthesis de novo.     

Glycogen metabolism in resting and growing cells of Saccharomyces carlsbergensis

Saccharomyces carlsbergensis cells, growing under carbohydrate or nitrogen limitation, initially deplete their glycogen, which is resynthesized only during the late exponential phase. Cells, harvested in the carly exponential phase, are even unable to synthesize glycogen in glucose-containing phosphate buffer. This is in contrast to cells from the stationary phase which rapidly synthesize glycogen under the same conditions. Lack of O₂ slows down glycogen synthesis.Contrary to cells suspended in complete medium, addition of ammonia alone to nitrogen free-media induced neither breakdown of glycogen, nor complete cessation of glycogen synthesis. Ammonia slowed down glycogen synthesis (both aerobic and anaerobic), only, in cells grown either under carbohydrate or under nitrogen limitation.Glycogen synthesis was observed 1 min after addition of glucose to a starved cell suspension in phosphate buffer. Removal of the sugar from the buffer resulted in an instantanous decrease of the glycogen level in the cells. The results indicate that glycogen-metabolism is regulated by a variety of endogenous and environmental factors.     

Control of teichoicand teichuronic acid biosynthesis in Bacillus subitlis 168trp−: Evidence for repression of enzyme synthesis and inhibition of enzyme activity

Phosphate starvatiion induced teichuronic acid synthesis in cells of Bacillus subtilis 168trp^? which had previously been grown with excess phophate. This induction was prevented when protein synthesis was inhibited immediately prior to phosphate starvation and under these conditions cells continued to form teichoic acid. The converse was true when phosphate was added to cells previously grown in phosphate-limited chemostat. The increase in teichoic acid synthesis normally following phosphate addition was prevented by chlorampehnicol or amino acid starvation and cells continued to make teichuronic acid. The suggestion that repression of enzyme synthesis is involved in controlling the type of wall polymer made was supported by the low levels of UDP-glucose dehydrogenase found in cells grown with excess phosphate and of CDP-glycerol pyrophosphorylase in phophate-limited cells. The greater amounts of teichoic acid made under phosphate limitation and of teichuronic acid with excess phosphate when protein synthesis was also inhibited indicated that modulation of enzyme activity occurs. Glycerol starvation of a glycerol-requiring mutant did not derepress teichuronic acid synthesis, indicating that glycerol-containing intermediates do not act as repressors.     

Functional analysis of relA and rshA, two relA/spoT homologues of Streptomyces coelicolor A3(2)

Deletion of the (p)ppGpp synthetase gene, relA, of Streptomyces coelicolor A3(2) results in loss of production of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) and delayed morphological differentiation when the mutant is grown under conditions of nitrogen limitation. To analyze the role of (p)ppGpp as an intracellular signaling molecule for the initiation of antibiotic production, several C-terminally deleted derivatives of S. coelicolor relA that could potentially function in the absence of ribosome activation were placed under the control of the thiostrepton-inducible tipA promoter. While 0.82- and 1.28-kb N-terminal segments failed to restore (p)ppGpp and antibiotic production upon induction in a relA null mutant, 1.46- and 2.07-kb segments did. Under conditions of phosphate limitation, deletion of relA had little or no effect on Act or Red synthesis, potentially reflecting an alternative mechanism for ppGpp synthesis. A second S. coelicolor RelA homologue (RshA, with 42% identity to S. coelicolor RelA) was identified in the genome sequence. However, deletion of rshA had no effect on the ability of the relA mutant to make Act and Red when grown under conditions of phosphate limitation. While high-level induction of tipAp::rshA in the relA mutant resulted in growth inhibition, low-level induction restored antibiotic production and sporulation. In neither case, nor in the relA mutant that was grown under phosphate limitation and producing Act and Red, could (p)ppGpp synthesis be detected. Thus, a ppGpp-independent mechanism exists to activate antibiotic production under conditions of phosphate limitation that can be mimicked by overexpression of rshA.     

Effects of phosphorous, nitrogen, and carbon limitation on biomass composition in batch and continuous flow cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii

We have used phosphate, nitrogen, or carbon limited batch and continuous flow cultures to study how growth and biochemical composition of the dinoflagellate Crypthecodinium cohnii CCMP 316 is affected by nutrient limitation. Specific contents of phosphorous, proteins, and starch were differently affected by nutrient limitation. The specific phosphorous content in C. cohnii varied 10-20 times depending on phosphate availability in the medium. When phosphate was available it was taken up in excess and stored to be re-utilized during phosphate limitation. The specific protein content varied twofold. At most conditions, proteins made up 12-15% of the biomass dry weight but when cells were nitrogen limited, the specific protein content was only half this value. Floridean starch was the major cell constituent of C. cohnii accounting for 40-50% of the biomass dry weight. Only during carbon limitation did the specific starch content decrease. In contrast was the specific lipid content almost unaffected by nutrient availability and lipids accounted for 12-15% of the biomass dry weight irrespectively of which nutrient that was limiting. Lipid production does therefore not depend on nutrient limitation in C. cohnii and lipids are produced even by carbon limited cells. Cultures grown under phosphate limitation resulted in formation of cells with maximal specific contents of all the three major cell constituents; starch, lipid, and protein.     

EFFECTS OF NITRATE AVAILABILITY AND IRRADIANCE ON INTERNAL NITROGEN CONSTITUENTS IN CORALLINA ELONGATA (RHODOPHYTA)1

Short-term (5-h) phycobiliprotein photoacclimation was a NO₃^?, dependent process in the red alga Corallina elongata Ellis et Soland. At low irradianre levels, phycobiliprotein synthesis (both r-phycocyacin and r-phycocyanin) took place when N supply was sufficient but was restricted by N limitation. Exposure to saturating irradiance resulted in pigment degradation under N limitation; however, under N-sufficient conditions a partial r-phycoerythrin synthesis was observed, despite the repressing role of high photon flux densities on phycobiliprotein synthesis. Soluble protein was less affected than phycobiliprotein by N limitation at low photon flux densities indicating that N limitation stimulates the flow of internal N metabolites toward the synthesis of nonpigmented proteins rather than pigmented proteins. The addition of protein synthesis inhibitors revealed that new phycobiliprotein synthesis occurs in response to sufficient N conditions. When protein synthesis was blocked in the chloroplast and cytoplasm simultaneously (addition of chloramphemcol and cycloheximide), both pigmented and nonpigmented protein synthesis was inhibited. Howeever, when protein systhesis was blocked in the chloroplast, only phycobiliprotein synthesis was clearly inhibited, whereas nonpigmented protein was less affected, indicating that phycobiliprotein is the main fraction of protein synthesized in the chloroplast at low photon flux densities when external N is available. This inhibition of phycobiliprotein synthesis was consistent with a maximal increase in metabolites of protein synthesis (internal NH₄⁺ and amino acids). Our results suggest that phycobiliproteins may be an important N reservoir to meet internal N demands during N limitations in C. elongata. Moreover, r-phycoerythrin, synthesized even at saturating irradiance levels, and the major constituent of the phycobiliprotein pigments, may be more sensitive to changes in N supply than r-phycocyanin. The influence of limited irradiance levels on N assimilation and the effects of repressing protein synthesis on internal N accumulation are also discussed.     

Polymyxin biosynthesis by Bacillus polymyxa during growth limitation by the nutritional sources

The synthesis of the antibiotic polymyxin M was studied under the conditions of batch and continuous cultivation of Bacillus polymyxa var. Ross whose growth was limited with glucose, phosphate and ammonium nitrogen. Polymyxin M was synthesized when the culture growth decelerated as a result of its limitation with the above compounds. Different amounts of the antibiotic were synthesized depending on the type of a limiting factor. The highest productiveness was found in the case of glucose limitation. The optimal conditions for polymyxin M synthesis were established under the conditions of one-step continuous cultivation.     

Influence of different substrate limitations on the yield, composition and molecular mass of exopolysaccharides produced by Lactococcus lactis subsp. cremoris in continuous cultures

The type of substrate limitation had a remarkable influence on the molecular mass of exopolysaccharides (EPS) produced by Lactococcus lactis subsp. cremoris NIZO B40 and NIZO B891. Under glucose/energy limitation, the molecular mass was much smaller than under leucine or phosphate limitation, resulting in a marked decrease of the intrinsic viscosity of this EPS. The sugar composition of EPS produced by both strains, and the phosphate content of EPS produced by NIZO B40, were not affected by the type of nutrient limitation. Both strains produced comparable amounts of EPS under leucine and glucose limitation, but the efficiency of EPS production was highest under glucose limitation. The EPS yields of the phosphorylated B40 EPS as well as the unphosphorylated B891 EPS were reduced, with about 40% under conditions of phosphate limitation.     

Light-, nitrogen-, and phosphorus-limited growth of Phaeodactylum tricornutum Bohlin strain TFX-1: Chemical composition,carbon partitioning,and the diel periodicity of physiological processes

Phaeodactylum tricomutum Bohlin (strain TFX-1) was grown under light-, nitrogen-, and phosphorus-limited conditions in continuous or semicontinuous cultures under a 12L-12D light regime. The C, N, and P contents were determined at each steady state, as was the partitioning of cellular organic carbon into protein, lipids, polysaccharides, and metabolic intermediates. All determinations were made at the beginning and again at the end of the light period. The rates of nutrient assimilation and of synthesis of biochemical constituents during the light and dark periods were calculated from the above data, and the periodicities of these processes characterized. The elemental composition of the cells was different under each limitation. In particular, phosphorus limitation severely restricted the ability of the cell to store nitrogen in non-protein forms. Biochemical composition and the diel periodicity of cellular processes also differed between limitations. Nutrient uptake was most strongly periodic under light limitation. Protein synthesis showed increased periodicity under nitrogen limitation, relative to the other limitations, while the periodicity of lipid synthesis was reduced under phosphate limitation. Polysaccharide was synthesized at high rates during the light period and consumed in the dark under all limitations.     

Sequential processes of phosphate limitation and of phosphate release in streptomycin fermentations

The significance of the sequential processes of phosphate limitation and of phosphate release from medium constituents is demonstrated in technical streptomycin fermentations. The phosphate limitation initiated the streptomycin synthesis as well as the formation of phosphatases and protease. In later periods of the process the phosphate release influences especially the enzyme formation.     

Protein secretion in phosphate-limited cultures of Bacillus subtilis 168

The secretion of proteins from Bacillus subtilis was studied under physiologically well-defined conditions in continuous cultures at a range of specific growth rates. The kinetics of secretion was analysed by using pulse-chase and immunoprecipitation techniques that allowed both processing and release to be monitored. Growth conditions were selected that were known to lead to significant changes in the anionic polymer composition of the cell wall. Under magnesium limitation only low levels of native proteins were released into the growth medium. In contrast, much higher amounts of released protein were observed under phosphate limitation. Although synthesis of native secretory proteins appeared to be highly regulated, only minor changes in the secretion of heterologous proteins were detected. Comparable kinetics of protein release of cells grown under different conditions indicated similar cell wall permeabilities. The large changes in the amounts of released proteins were not reflected in the production of chaperones and components required for protein secretion. The data suggest that the capacity of the secretion machinery is not a major limiting step in the export of native secretory proteins. Received: 23 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997     

The plastid outer membrane localized LPTD1 is important for glycerolipid remodelling under phosphate starvation

Glycerolipid synthesis in plants is coordinated between plastids and the endoplasmic reticulum (ER). A central step within the glycerolipid synthesis is the transport of phosphatidic acid from ER to chloroplasts. The chloroplast outer envelope protein TGD4 belongs to the LptD family conserved in bacteria and plants and selectively binds and may transport phosphatidic acid. We describe a second LptD‐family protein in A. thaliana (atLPTD1; At2g44640) characterized by a barrel domain with an amino‐acid signature typical for cyanobacterial LptDs. It forms a cation selective channel in vitro with a diameter of about 9 Å. atLPTD1 levels are induced under phosphate starvation. Plants expressing an RNAi construct against atLPTD1 show a growth phenotype under normal conditions. Expressing the RNAi against atLPTD1 in the tgd4–1 background renders the plants more sensitive to light stress or phosphate limitation than the individual mutants. Moreover, lipid analysis revealed that digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol levels remain constant in the RNAi mutants under phosphate starvation, while these two lipids are enhanced in wild‐type. Based on our results, we propose a function of atLPTD1 in the transport of lipids from ER to chloroplast under phosphate starvation, which is combinatory with the function of TGD4.     

A mutant of Arabidopsis lacking the triose-phosphate/phosphate translocator reveals metabolic regulation of starch breakdown in the light

The chloroplast envelope triose-phosphate/phosphate translocator (TPT) is responsible for carbohydrate export during photosynthesis. Using measurements of carbohydrates, partitioning of assimilated 14CO2, photosynthetic gas exchange, and chlorophyll fluorescence, we show that a mutant of Arabidopsis lacking the TPT increases synthesis of starch compared to the wild type, thereby compensating for a deficiency in its ability to export triose-phosphate from the chloroplast. However, during growth under high light, the capacity for starch synthesis becomes limiting so that the chloroplastic phosphate pool is depleted, resulting in a restriction on electron transport, a reduction in the rate of photosynthesis, and slowed plant growth. Under the same conditions but not under low light, we observe release of 14C label from starch, indicating that its synthesis and degradation occur simultaneously in the light. The induction of starch turnover in the mutant specifically under high light conditions leads us to conclude that it is regulated by one or more metabolic signals, which arise as a result of phosphate limitation of photosynthesis.     

Cellular levels, excretion, and synthesis rates of cyclic AMP in Escherichia coli grown in continuous culture.

Changes in dilution rate did not elicit large and systematic changes in cellular cyclic AMP levels in Escherichia coli grown in a chemostat under carbon or phosphate limitation. However, the technical difficulties of measuring low levels of cellular cyclic AMP in the presence of a large background of extracellular cyclic AMP precluded firm conclusions in this point. The net rate of cyclic AMP synthesis increased exponentially with increasing dilution rate through either the entire range of dilution rates examined (phosphate limitation) or a substantial part of the range (lactose and glucose limitations). Thus, it is probable that growth rate regulates the synthesis of adenylate cyclase. The maximum rate of net cyclic AMP synthesis was greater under lactose than under glucose limitation, which is consistent with the notion that the uptake of phosphotransferase sugars is more inhibitory to adenylate cyclase than the uptake of other carbon substrates. Phosphate-limited cultures exhibited the lowest rate of net cyclic AMP synthesis, which could be due to the role of phosphorylated metabolites in the regulation of adenylate cyclase activity. Under all growth conditions examined, greater than 99.9% of the cyclic AMP synthesized was found in the culture medium. The function of this excretion, which consumed up to 9% of the total energy available to the cell and which evidently resulted from elaborate regulatory mechanisms, remains entirely unknown.     

Evidence for nitrogen catabolite repression during citric acid production by Aspergillus niger under phosphate-limited growth conditions

Experiments performed in batch fermentation under phosphate-limited growth conditions showed that the citric acid yield was inversely related to the excess nitrogen concentration in the medium. Results from chemostat culture confirmed a negative relationship between the citric acid yield and both the specific growth rate and the nitrogen consumption rate. This is evidence for nitrogen catabolite repression. A fed-batch fermentation performed under dual phosphate/nitrogen limitation produced results very similar to those from a culture limited by nitrogen alone. There is no advantage in maintaining an excess of phosphate during citric acid production and the process will therefore be more economic when operated under dual limitation conditions.