Osmoregulation in Rhizobium meliloti: Production of Glutamic Acid in Response to Osmotic Stress

Rhizobium meliloti, like many other bacteria, accumulates high levels of glutamic acid when osmotically stressed. The effect was found to be proportional to the osmolarity of the growth medium. NaCl, KCI, sucrose, and polyethylene glycol elicited this response. The intracellular levels of glutamate and K⁺ began to increase immediately when cells were shifted to high-osmolarity medium. Antibiotics that inhibit protein synthesis did not affect this increase in glutamate production. Cells growing in conventional media at any stage in the growth cycle could be suspended in medium causing osmotic stress and excess glutamate accumulated. The excess glutamate did not appear to be excreted, and the intracellular level eventually returned to normal when osmotically stressed cells were suspended in low-osmolarity medium. A glt mutant lacking glutamate synthase and auxotrophic for glutamate accumulated excess glutamate in response to osmotic stress. Addition of isoleucine, glutamine, proline, or arginine stimulated glutamate accumulation to wild-type levels when the mutant cells were suspended in minimal medium with NaCl to cause osmotic stress. In both wild-type and mutant cells, inhibitors of transaminase activity, including azaserine and aminooxyacetate, reduced glutamate levels. The results suggest that the excess glutamate made in response to osmotic stress is derived from degradation of amino acids and transamination of 2-ketoglutarate.     

Glutamate synthesis in Streptomyces coelicolor.

Both glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) are involved in glutamate synthesis in Streptomyces coelicolor. The highest levels of GDH were seen in extracts of cells grown with high levels of ammonium as the nitrogen source. GOGAT activity was reduced two- to threefold in extracts of cells grown with good sources of glutamate. S. coelicolor mutants deficient in GOGAT (Glt-) required glutamate for growth with L-alanine, asparagine, arginine, or histidine as the nitrogen source but grew like wild-type cells when ammonium, glutamine, or aspartate was the nitrogen source. The glt mutations were tightly linked to hisA1. Mutants deficient in both GOGAT and GDH (Gdh-) required glutamate for growth in all media. The gdh-5 mutation was mapped to the left region of the S. coelicolor chromosomal map, between proA1 and uraA1.     

Factors reducing and promoting the effectiveness of proline as an osmoprotectant in Escherichia coli K12

Proline accumulation in Escherichia coli is mediated by three proline porters. Proline catabolism is effected by proline porter I (PPI) and proline/delta 1-pyrroline carboxylate dehydrogenase. Proline did not accumulate cytoplasmically when E. coli was subjected to osmotic stress in minimal salts medium. Although PPI is induced when proline is provided as carbon or nitrogen source, its activity decreased following growth of the bacteria in minimal salts medium of high osmotic strength. Proline dehydrogenase was induced by proline in low or high osmotic strength media. Proline porter II (PPII) was both activated and induced in osmotically stressed bacteria, though the dependencies of the two responses on medium osmolarity differed. Osmotic downshift during the transport measurement decreased the uptake of proline, serine and glutamine by bacteria cultured in media of high osmotic strength. Thus, while osmotic upshift caused specific activation of PPII, osmotic downshift caused a non-specific reduction in amino acid uptake. Glycine betaine inhibited the uptake of [14C]proline via PPII and PPIII but not via PPI. The dependence of that inhibition on glycine betaine concentration was similar when PPII was uninduced, induced or activated by osmotic stress, or induced by amino acid limited growth. Thus PPII and PPIII, not PPI, contribute to the mechanism of osmoprotection by proline and glycine betaine. The tendency for exogenous proline to accumulate in the cytoplasm of bacteria exposed to osmotic stress would, however, be countered by increased proline catabolism.     

¹³C-metabolic enrichment of glutamate in glutamate dehydrogenase mutants of Saccharomyces cerevisiae

Glutamate dehydrogenases (GDH) interconvert α-ketoglutarate and glutamate. In yeast, NADP-dependent enzymes, encoded by GDH1 and GDH3, are reported to synthesize glutamate from α-ketoglutarate, while an NAD-dependent enzyme, encoded by GDH2, catalyzes the reverse. Cells were grown in acetate/raffinose (YNAceRaf) to examine the role(s) of these enzymes during aerobic metabolism. In YNAceRaf the doubling time of wild type, gdh2Δ, and gdh3Δ cells was comparable at ～4 h. NADP-dependent GDH activity (Gdh1p+Gdh3p) in wild type, gdh2Δ, and gdh3Δ was decreased ～80% and NAD-dependent activity (Gdh2p) in wild type and gdh3Δ was increased ～20-fold in YNAceRaf as compared to glucose. Cells carrying the gdh1Δ allele did not divide in YNAceRaf, yet both the NADP-dependent (Gdh3p) and NAD-dependent (Gdh2p) GDH activity was ～3-fold higher than in glucose. Metabolism of [1,2-(13)C]-acetate and analysis of carbon NMR spectra were used to examine glutamate metabolism. Incorporation of (13)C into glutamate was nearly undetectable in gdh1Δ cells, reflecting a GDH activity at <15% of wild type. Analysis of (13)C-enrichment of glutamate carbons indicates a decreased rate of glutamate biosynthesis from acetate in gdh2Δ and gdh3Δ strains as compared to wild type. Further, the relative complexity of (13)C-isotopomers at early time points was noticeably greater in gdh3Δ as compared to wild type and gdh2Δ cells. These in vivo data show that Gdh1p is the primary GDH enzyme and Gdh2p and Gdh3p play evident roles during aerobic glutamate metabolism.     

Myceloid cell formation in Arthrobacter globiformis during osmotic stress

C.E. DEUTCH AND G.S. PERERA. 1992. Arthrobacter globiformis was grown in a semi-defined liquid medium containing added solutes to determine the effects of osmotic stress on its reproduction and cell morphology. There was a progressive reduction in the specific growth rate during exponential phase as the concentration of NaCl was increased, although the final yields of the cultures during stationary phase were not affected. Clusters of branching myceloid cells rather than the typical bacillary forms predominated during exponential phase. These myceloids did not undergo complete septation and persisted into stationary phase. Similar responses were observed with potassium sulphate as the exogenous solute but less dramatic morphological effects were found with added polyethylene glycol or sucrose. The myceloids formed in response to osmotic stress could not be disrupted mechanically but were more sensitive than normal cells to lysozyme, particularly during stationary phase. Addition of osmoprotective compounds such as proline, glutamate, glycine betaine, or trehalose to the growth medium did not significantly relieve the effects of osmotic stress on growth rate or morphology. A. simplex also formed myceloid cells during osmotic stress but A. crystallopoietes did not. These results indicate that arthrobacters exhibit characteristic responses to osmotic stress and suggest these bacteria may contain novel osmoprotective compounds.     

Myceloid cell formation in Arthrobacter globiformis during osmotic stress.

Arthrobacter globiformis was grown in a semi-defined liquid medium containing added solutes to determine the effects of osmotic stress on its reproduction and cell morphology. There was a progressive reduction in the specific growth rate during exponential phase as the concentration of NaCl was increased, although the final yields of the cultures during stationary phase were not affected. Clusters of branching myceloid cells rather than the typical bacillary forms predominated during exponential phase. These myceloids did not undergo complete septation and persisted into stationary phase. Similar responses were observed with potassium sulphate as the exogenous solute but less dramatic morphological effects were found with added polyethylene glycol or sucrose. The myceloids formed in response to osmotic stress could not be disrupted mechanically but were more sensitive than normal cells to lysozyme, particularly during stationary phase. Addition of osmoprotective compounds such as proline, glutamate, glycine betaine, or trehalose to the growth medium did not significantly relieve the effects of osmotic stress on growth rate or morphology. A. simplex also formed myceloid cells during osmotic stress but A. crystallopoietes did not. These results indicate that arthrobacters exhibit characteristic responses to osmotic stress and suggest these bacteria may contain novel osmoprotective compounds.     

Exopolysaccharide production by the epipelic diatom Cylindrotheca closterium: effects of nutrient conditions

During the stationary phase of a batch culture of the epipelic diatom Cylindrotheca closterium, accumulation of exopolysaccharides and intracellular carbohydrates was observed. When nitrogen was added to the culture in the stationary phase, growth was resumed and the accumulation of exopolysaccharides was delayed. This indicated that nitrogen depletion caused cessation of growth, and stimulated exopolysaccharide accumulation. Exopolysaccharide accumulation was also stimulated when cells were either resuspended in medium lacking N or P, or when they were inoculated in medium with low concentrations of N or P. Growth was not immediately affected by low N or P concentrations. S depletion only resulted in exopolysaccharide accumulation when growth was affected. Si or Fe depletion did not stimulate exopolysaccharide accumulation, even when growth rates were lowered. Apparently, stimulation of exopolysaccharide accumulation is dependent on the type of nutrient depletion. Intracellular storage carbohydrates did not accumulate when cells were incubated at low N or P concentrations. Cells grown with ammonium as nitrogen source produced more carbohydrates (both extracellular and intracellular) than cells grown with nitrate as nitrogen source, indicating that both exopolysaccharides and intracellular carbohydrates accumulated as a result of overflow metabolism.     

13C-NMR study of lactate metabolism in Propionibacterium freudenreichii subsp. shermanii

The adaptation to osmotic stress in Propionibacterium freudenreichii subsp. shermanii was investigated by using natural-abundance ¹³C nuclear magnetic resonance spectroscopy. Cells incubated either in a standard laboratory medium or in a medium designed to simulate the physicochemical conditions of Swiss-type cheese were found to accumulate different levels of osmotic-stress-protectant molecules. Proline, betaine, trehalose and glutamate were found simultaneously. Moreover, two types of polysaccharides were in evidence in this strain. Lactate catabolism was not mainly directed towards cell growth requirements and organic acid production but also towards biosynthesis of osmolytes requested for adaptation in a cheese environment. The possible involvement of such type of metabolite accumulation in the main cheese-ripening bacteria in Swiss-type cheeses is discussed.     

Role of Glycine Betaine and Related Osmolytes in Osmotic Stress Adaptation in Yersinia enterocolitica ATCC 9610

Yersinia enterocolitica is a gram-negative, food-borne pathogen that can grow in 5% NaCl and at refrigerator temperatures. In this report, the compatible solutes (osmolytes) which accumulate intracellularly and confer the observed osmotic tolerance to this pathogen were identified. In minimal medium, glutamate was the only detectable osmolyte that accumulated in osmotically stressed cells. However, when the growth medium was supplemented with glycine betaine, dimethylglycine, or carnitine, the respective osmolyte accumulated intracellularly to high levels and the growth rates of the osmotically stressed cultures improved from 2.4- to 3.5-fold. Chill stress also stimulated the intracellular accumulation of glycine betaine, but the growth rate was only slightly improved by this osmolyte. Both osmotic upshock and temperature downshock stimulated the rate of uptake of [(sup14)C]glycine betaine by more than 30-fold, consistent with other data indicating that the osmolytes are accumulated from the growth medium via transport.     

Osmoprotection by pipecolic acid in Sinorhizobium meliloti: specific effects of D and L isomers

DL-Pipecolic acid (DL-PIP) promotes growth restoration of Sinorhizobium meliloti cells facing inhibitory hyperosmolarity. Surprisingly, D and L isomers of this imino acid supplied separately were not effective. The uptake of L-PIP was significantly favored in the presence of the D isomer and by a hyperosmotic stress. Chromatographic analysis of the intracellular solutes showed that stressed cells did not accumulate radiolabeled L-PIP. Rather, it participates in the synthesis of the main endogenous osmolytes (glutamate and the dipeptide N-acetylglutaminylglutamine amide) during the lag phase, thus providing a means for the stressed cells to recover the osmotic balance. (13)C nuclear magnetic resonance analysis was used to determine the fate of D-PIP taken into the cells. In the absence of L-PIP, the imported D isomer was readily degraded. Supplied together with its L isomer, D-PIP was accumulated temporarily and thus might contribute together with the endogenous osmolytes to enhance the internal osmotic strength. Furthermore, it started to disappear from the cytosol when the L isomer was no longer available in the culture medium (during the late exponential phase of growth). Together, these results show an uncommon mechanism of protection of osmotically stressed cells of S. meliloti. It was proved, for the first time, that the presence of the two isomers of the same molecule is necessary for it to manifest an osmoprotective activity. Indeed, D-PIP seems to play a major role in cellular osmoadaptation through both its own accumulation and improvement of the utilization of the L isomer as an immediate precursor of endogenous osmolytes.     

Preferential Osmolyte Accumulation: a Mechanism of Osmotic Stress Adaptation in Diazotrophic Bacteria

A common cellular mechanism of osmotic-stress adaptation is the intracellular accumulation of organic solutes (osmolytes). We investigated the mechanism of osmotic adaptation in the diazotrophic bacteria Azotobacter chroococcum, Azospirillum brasilense, and Klebsiella pneumoniae, which are adversely affected by high osmotic strength (i.e., soil salinity and/or drought). We used natural-abundance ¹³C nuclear magnetic resonance spectroscopy to identify all the osmolytes accumulating in these strains during osmotic stress generated by 0.5 M NaCl. Evidence is presented for the accumulation of trehalose and glutamate in Azotobacter chroococcum ZSM4, proline and glutamate in Azospirillum brasilense SHS6, and trehalose and proline in K. pneumoniae. Glycine betaine was accumulated in all strains grown in culture media containing yeast extract as the sole nitrogen source. Alternative nitrogen sources (e.g., NH₄Cl or casamino acids) in the culture medium did not result in measurable glycine betaine accumulation. We suggest that the mechanism of osmotic adaptation in these organisms entails the accumulation of osmolytes in hyperosmotically stressed cells resulting from either enhanced uptake from the medium (of glycine betaine, proline, and glutamate) or increased net biosynthesis (of trehalose, proline, and glutamate) or both. The preferred osmolyte in Azotobacter chroococcum ZSM4 shifted from glutamate to trehalose as a consequence of a prolonged osmotic stress. Also, the dominant osmolyte in Azospirillum brasilense SHS6 shifted from glutamate to proline accumulation as the osmotic strength of the medium increased.     

Influence of nitrogen source and growth status on glutamine synthetase and glutamate synthase activity in Mycobacterium avium

An investigation was made of the activity of glutamine synthetase and glutamate synthase from batch-cultured cells of Mycobacterium avium. The bacteria were grown in medium with ammonium chloride concentrations of 0, 0.1, 0.25, 1, 5, or 25 mumol/mL or with glutamine at 0.1 or 1 mumol/mL. The specific activity of the two enzymes was determined at 0, 22, 45, and 70 h of incubation. Regardless of the ammonia concentration in the medium, glutamate synthase specific activity was two to five times higher in extracts from elongating cells, incubated 22 h, than in those from shortened cells, incubated 45 or 70 h. In contrast, there was no apparent difference in glutamine synthetase specific activity with regard to culture age; however, glutamine synthetase specific activity varied inversely with the concentration of ammonium chloride in the medium. Cells grown in glutamine had high activity of glutamine synthetase.     

Uptake of oxygen,release and degradation of hydrogen peroxide by Streptococcus mutans NCTC 10449

Streptococcus mutans NCTC 10499 was cultured under glucose limitation in a chemostat at varying oxygen supply. The rates of oxygen uptake and hydrogen peroxide degradation by cells from the cultures were measured polarographically using a Clark electrode. Oxygenation of the chemostat culture led to adaptation of the organism to oxygen, in that the maximum oxygen uptake rate of the cells was higher when the cells were grown at higher rate of oxygen supply. It is noted that anaerobically grown cells still exhibited significant oxygen uptake. The rate of oxygen uptake followed saturation-type kinetics and Ks values of cells for oxygen were in the micromole range. Hydrogen peroxide accumulation was not observed in aerated chemostat cultures. However, anaerobically grown cells accumulated H2O2 when exposed to oxygen. Cells from aerated cultures did not accumulate hydrogen peroxide. This may be explained by the fact that the rate of hydrogen peroxide degradation was consistently higher than the rate of oxygen uptake.     

渗透胁迫下不同抗旱性小麦幼苗氨同化差异

在渗透胁迫下,测定了不同抗旱性小麦（抗旱性强的品种洛旱6号和抗旱性弱的品种周麦18）幼苗氨同化酶及相关参数的变化.结果表明:小麦生物量在渗透胁迫下明显降低,且抗旱性弱的周麦18降幅较大.铵态氮含量随胁迫程度的增加而增加,且周麦18增加较明显;谷氨酰胺合成酶(GS)活性在不同抗旱性品种间表现不同,抗旱性强的洛旱6号在低渗透胁迫下显著增加,在高渗透胁迫下明显降低,而周麦18随胁迫程度的增加逐渐降低;依赖还原型辅酶Ⅰ的谷氨酸脱氢酶(NADH-GDH)活性随胁迫程度的增加逐渐加大,低渗透胁迫下周麦18增加较明显,高渗透胁迫下洛旱6号增幅较大;依赖氧化型辅酶Ⅰ的谷氨酸脱氢酶(NAD^+-GDH)和依赖氧化型辅酶Ⅱ的异柠檬酸脱氢酶(NADP-ICDH)活性均随胁迫程度的增加而增加,周麦18的NAD^+-GDH活性、洛旱6号的NADP-ICDH活性增幅较大.表明小麦抗旱性的提高与铵态氮同化的增强有关,低渗透和高渗透胁迫下分别依赖GS和NADH-GDH活性的增加.     

Selection for citrate synthase deficiency in icd mutants of Escherichia coli.

Cultures of isocitrate dehydrogenase-deficient (icd) mutants were overgrown by double mutants (icd glt) lacking citrate synthase activity also. The icd mutants grew more slowly than wild-type cells or the double mutants because they accumulated an inhibitory metabolite (possibly citrate). Intracellular citrate levels were several hundred-fold higher in icd cells than in wild-type or icd glt cells. Final growth yields of the wild type and the icd mutant on limiting glucose were equivalent and greater than the growth yield of icd glt double mutants. The icd gene mapped between 60 and 74 min. icd mutants were resistant to nalidixic acid, but glt and icd glt mutants and wild-type cells were sensitive, indicating that resistance results from accumulation of isocitrate, citrate, or a derivative of these compounds.     

Ammonium Assimilation and the Role of [gamma]-Aminobutyric Acid in pH Homeostasis in Carrot Cell Suspensions

In vivo 15N NMR spectroscopy was used to monitor the assimilation of ammonium by cell-suspension cultures of carrot (Daucus carota L. cv Chantenay). The cell suspensions were supplied with oxygen in the form of either pure oxygen ("oxygenated cells") or air ("aerated cells"). In contrast to oxygenated cells, in which ammonium assimilation had no effect on cytoplasmic pH, ammonium assimilation by aerated cells caused a decrease in cytoplasmic pH of almost 0.2 pH unit. This led to a change in nitrogen metabolism resulting in the accumulation of [gamma]-aminobutyric acid. The metabolic effect of the reduced oxygen supply under aerated conditions could be mimicked by artificially decreasing the cytoplasmic pH of oxygenated cells and was abolished by increasing the cytoplasmic pH of aerated cells. The activity of glutamate decarboxylase increased as the cytoplasmic pH declined and decreased as the pH recovered. These findings are consistent with a role for the decarboxylation of glutamate, a proton-consuming reaction, in the short-term regulation of cytoplasmic pH, and they demonstrate that cytoplasmic pH influences the pathways of intermediary nitrogen metabolism.     

Rhizobium meliloti glutamate synthase: cloning and initial characterization of the glt locus.

The genetic locus glt, encoding glutamate synthase from Rhizobium meliloti 1021, was selected from a pLAFR1 clone bank by complementation of the R. meliloti 41 Glt- mutant AK330. A fragment of cloned DNA complementing this mutant also served to complement the Escherichia coli glt null mutant PA340. Complementation studies using these mutants suggested that glutamate synthase expression requires two complementation groups present at this locus. Genomic Southern analysis using a probe of the R. meliloti 1021 glt region showed a close resemblance between R. meliloti 1021, 41, and 102f34 at glt, whereas R. meliloti 104A14 showed many differences in restriction fragment length polymorphism patterns at this locus. R. meliloti 102f34, but not the other strains, showed an additional region with sequence similarity to glt. Insertion alleles containing transposable kanamycin resistance elements were constructed and used to derive Glt- mutants of R. meliloti 1021 and 102f34. These mutants were unable to assimilate ammonia and were Nod+ Fix+ on alfalfa seedlings. The mutants also showed poor or no growth on nitrogen sources such as glutamate, aspartate, arginine, and histidine, which are utilized by the wild-type parental strains. Strains that remained auxotrophic but grew nearly as well as the wild type on these nitrogen sources were readily isolated from populations of glt insertion mutants, indicating that degradation of these amino acids is negatively regulated in R. meliloti as a result of disruptions of glt.     

Microtubule dynamics in relation to osmotic stress-induced ABA accumulation in Zea mays roots

Microtubules play important roles in many physiological processes such as plant responses to drought stress. Abscisic acid (ABA) accumulates significantly in plants in response to drought conditions, which has been considered as a major response for plants to enhance drought tolerance. In this work, the focus was on the possible roles of microtubules in the induction of ABA biosynthesis in the roots of Zea mays when subjected to osmotic stress. The dynamic changes of microtubules in response to the stress were investigated by immunofluorescence staining, enzyme-linked immunosorbent assay, and a pharmacological approach. Disruption and stabilization of microtubules both significantly stimulated ABA accumulation in maize root cells, although this stimulation was markedly lower than that caused by osmotic stress. Cells in which the microtubule stability had been changed did not respond further to osmotic stress in terms of ABA biosynthesis. However, treatment with both a microtubule de-stabilizer and a stabilizer enhanced the sensitivity of cells to osmotic stress in terms of ABA accumulation. It is suggested that both osmotic stress and changes in microtubule dynamics would trigger maize root cells to biosynthesize ABA, and interactions between osmotic stress and microtubule dynamics would have an effect on ABA accumulation in root cells, although the exact mechanism is not clear at present.