Nitrogen fixaton in Klebsiella pneumoniae during osmotic stress effect of exogenous proline or a proline overproducing plasmid

Osmotic stress, imposed by 0.5 M NaCl or other electrolytes and non-electrolytes, caused over a 100-fold reduction in the whole-cell nitrogen fixation activity in Klebsiella pneumoniae, wild-type strain M₅A₁. This reduction of nitrogen fixation activity could be reversed by the addition of proline to the culture medium at 0.5 mM concentration. With 0.5 M NaCl, in the presence of proline, nitrogenase activity was 47-fold greater than in the absence of proline. A mutation, originally isolated in Salmonella typhimurium, which resulted in proline over-production and enhanced osmotolerance, was transferred into K. pneumoniae by F′ conjugation. Intracellular proline, synthesized at high levels because of the mutation, had similar stimulatory effects on nitrogen fixation under osmotic stress as proline provided exogenously. In the overproducing strain, the cellular level of proline is elevated as much as 125-fold during stress over that seen in the control strain. To determine the mechanism of stimulation of nitrogen fixaton by proline during stress, the biosynthesis of nitrogenase polypeptides was studied. Net nitrogenase biosynthesis and the biosynthesis of other unidentified peptides, is strongly inhibited during osmotic stress; proline reverses the inhibition. The role of proline in enhancing nitrogen fixation during osmotic stress is discussed.     

Glycine betaine, an osmotic effector in Klebsiella pneumoniae and other members of the Enterobacteriaceae

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance.     

Glycine betaine, an osmotic effector in Klebsiella pneumoniae and other members of the Enterobacteriaceae.

Osmoregulation was examined in members of the Enterobacteriaceae. Exogenous glycine betaine at a concentration as low as 1 mM was found to stimulate the growth rate of Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae in media of inhibitory osmotic strength. The stimulation was shown to be independent of any specific solutes, electrolytes, or nonelectrolytes. Therefore, the stimulatory effect of glycine betaine was a consequence of high osmotic potential. This effect was found to be far greater than the proline effect previously observed in S. typhimurium. Whereas nitrogen fixation by K. pneumoniae is completely inhibited under conditions of osmotic stress, nitrogenase activity could be partially restored by the addition of exogenous glycine betaine to the culture medium. Furthermore, glycine betaine in combination with proline, especially proline produced internally at a high level because of regulatory mutations affecting proline biosynthesis, strongly stimulated nitrogen fixation activity during osmotic stress. Glycine betaine was accumulated by the cells, and the amount taken up was correlated with the osmolarity of the medium. These findings are discussed in relation to the possible mechanisms by which glycine betaine might cause enhanced osmotolerance.     

Mutational Engineering of the cyanobacterium Nostoc muscorum for resistance to growth-inhibitory action of LiCl and NaCl

The effect of NaCl on two vital processes of cyanobacterial metabolism, viz. N(2) fixation and oxygenic photosynthesis, was studied in the cyanobacterium Nostoc muscorum grown diazotrophically. An increase in NaCl concentration suppressed the formation of heterocyst and adversely affected the nitrogenase activity in the parent, whereas in Li(+)-R and Na(+)-R mutants NaCl stress did not cause any adverse effect. The rate of photosynthetic O(2)-evolution was also adversely affected by the NaCl stress, but the magnitude was less than that of nitrogenase activity. L-Proline, the well-known osmoprotectant, provided protection to the cyanobacterium against NaCl stress. The parent strain utilized L-proline as a nitrogen source and suppressed heterocyst formation and nitrogenase activity, while mutants showed normal heterocyst frequency and nitrogenase activity. Therefore, it may be that the proline metabolism is altered as a result of mutation. The intracellular levels of proline in the parent were enhanced about threefold in the medium containing 1 mol x m(-3) proline, while in mutants there was no significant increase in the intracellular level of proline. In the medium containing both NaCl and proline, the intracellular level of proline was enhanced in the parent as well as in both mutant strains. This suggests that the parent strain possessed both normal proline uptake and salt-induced proline uptake systems, whereas the mutant strains were defective in normal proline uptake and had only salt-induced proline uptake. The over-accumulation of proline in the presence of NaCl stress is due either to the loss of proline oxidase activity or to the accumulation of exogenous proline.     

Regulation of cytoplasmic proline levels in Salmonella typhimurium: effect of osmotic stress on synthesis, degradation, and cellular retention of proline.

I investigated the effects of osmotic stress on the synthesis and catabolism of proline in Salmonella typhimurium by measuring the intracellular and extracellular proline levels in various strains. In the wild-type strain, exposure to 0.8 M NaCl did not cause a significant change in the intracellular proline level; however, it brought about a 6.5-fold increase in the intracellular glutamate pool size. These results indicate that gamma-glutamyl kinase is inhibited by proline in wild-type cells in media of normal or elevated osmolarity. I also tested whether proline is subject to turnover in cells wild type with respect to the enzymes of the proline degradation pathway. In strains that were wild type for proline biosynthesis, the loss of the proline catabolic enzymes, due to putA mutations, did not result in a statistically significant increase in the intracellular proline levels. Therefore, in the wild-type strain, proline turnover does not seem to be important for control of the intracellular proline levels. However, in a proline-overproducing mutant, a putA lesion caused a threefold increase in the intracellular proline level and a 6.5-fold increase in the extracellular proline level, indicating that proline is subject to turnover in the overproducing mutant. The proline-overproducing mutants excreted large quantities of the proline into the culture medium; osmotic stress altered the partitioning of proline such that the ratio of intracellular to extracellular levels of proline increased with increased osmotic stress. The increased cellular retention of proline in media of high osmolarity is probably due to the functioning of the ProP and ProU proline transport systems, which are stimulated under conditions of osmotic stress.     

Mutational Engineering of the Cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis> for Resistance to Growth-Inhibitory Action of LiCl and NaCl

The effect of NaCl on two vital processes of cyanobacterial metabolism, viz. N₂ fixation and oxygenic photosynthesis, was studied in the cyanobacterium Nostoc muscorum grown diazotrophically. An increase in NaCl concentration suppressed the formation of heterocyst and adversely affected the nitrogenase activity in the parent, whereas in Li⁺-R and Na⁺-R mutants NaCl stress did not cause any adverse effect. The rate of photosynthetic O₂-evolution was also adversely affected by the NaCl stress, but the magnitude was less than that of nitrogenase activity. L-Proline, the well-known osmoprotectant, provided protection to the cyanobacterium against NaCl stress. The parent strain utilized L-proline as a nitrogen source and suppressed heterocyst formation and nitrogenase activity, while mutants showed normal heterocyst frequency and nitrogenase activity. Therefore, it may be that the proline metabolism is altered as a result of mutation. The intracellular levels of proline in the parent were enhanced about threefold in the medium containing 1 mol m⁻³ proline, while in mutants there was no significant increase in the intracellular level of proline. In the medium containing both NaCl and proline, the intracellular level of proline was enhanced in the parent as well as in both mutant strains. This suggests that the parent strain possessed both normal proline uptake and salt-induced proline uptake systems, whereas the mutant strains were defective in normal proline uptake and had only salt-induced proline uptake. The over-accumulation of proline in the presence of NaCl stress is due either to the loss of proline oxidase activity or to the accumulation of exogenous proline. Received: 10 July 2002 / Accepted: 13 August 2002     

Genetically modified cyanobacteriumNostoc muscorum overproducing proline in response to salinity and osmotic stresses

In the parentNostoc muscorum an active proline oxidase enzyme is required to assimilate exogenous proline as a fixed nitrogen source. Cyanobacterial mutants, resistant to growth inhibitory action of proline analogue L-azetidine-2-carboxylate (Ac-R), were deficient in proline oxidase activity, and were over-accumulators of proline. Proline over-accumulation, resulting either from mutational acquisition of the Ac-R phenotype, or from salinity-induced uptake of exogenous proline, confirmed enhanced salinity/osmotic tolerance in the mutant strain. The nitrogenase activity and photosynthetic O₂ evolution of the parent were sensitive to both salinity as well as osmotic stresses than of Ac-R mutant strain. In addition, the mutation to Ac-resistant phenotype showed no alteration in salinity inducible potassium transport system in the cyanobacterium.     

Osmoregulation in Klebsiella pneumoniae: enhancement of anaerobic growth and nitrogen fixation under stress by proline betaine, gamma-butyrobetaine, and other related compounds

Exogenous proline betaine ( stachydrine or N- dimethylproline ) or gamma-butyrobetaine (gamma-trimethylaminobutyrate), at a concentration as low as 1 mM, were found to stimulate the growth rate of Klebsiella pneumoniae, wild type M5A1 , in media of inhibitory osmotic strength (0.8 M NaC1). Simultaneously, nitrogen fixation by whole cells, a process particularly sensitive to osmotic stress, was strongly enhanced by these compounds. However, in the absence of sodium chloride, both the growth and nitrogen fixation were not affected by the addition of the methylammonium derivatives in the medium. The sensitivity of the nitrogen fixation to osmotic stress was used as a bioassay to evaluate the potentiality of osmoprotective compound in relation to the number of methyl groups on the nitrogen atom of glycine, proline, and gamma-aminobutyrate. Experiments with sarcosine ( monomethylglycine ), dimethylglycine, and glycine betaine ( trimethylglycine ), or experiments with mono- and di- methylproline or gamma-mono-, gamma-di, gamma-tri- methylaminobutyrate , indicated that the greatest stress tolerance was always obtained with the more N-methylated compounds.     

Nonspecific inhibition of proline dehydrogenase synthesis in Escherichia coli during osmotic stress

L-Proline, which is accumulated by Escherichia coli during growth in media of high osmolality, also induces the synthesis of the enzyme degrading it to glutamate. To determine if proline catabolism is inhibited during osmotic stress, proline utilization and the formation of proline dehydrogenase were examined in varying concentrations of NaCl and sucrose. Although the specific growth rate of E. coli with proline as the sole nitrogen source diminished as the solute osmolality increased, a comparable reduction in growth rate occurred with ammonium as the primary nitrogen source. Proline catabolism, as measured in whole cells by the conversion of [14C]proline to [14C]glutamate, was only slightly inhibited by solute osmolalities up to 1.0 osmol/kg; more than 50% of the initial activity was still found at 2.0 osmol/kg. By contrast, the specific activity of proline dehydrogenase in bacteria grown in the presence of added solutes decreased to less than 20% of the control level. This reduction was related to a lower rate of synthesis, but was independent of genes currently known to be involved in osmoregulation or proline metabolism. The specific activities of tryptophanase, beta-galactosidase, and histidinol dehydrogenase were also reduced under similar growth conditions. These results indicate that while proline catabolism is not directly inhibited by high solute concentrations, prolonged exposure to osmotic stress leads to its reduction as part of a more general metabolic response.     

Osmotically regulated transport of proline by Lactobacillus acidophilus IFO 3532.

We reported previously that, when exposed to high osmotic pressure, Lactobacillus acidophilus IFO 3532 cells accumulated N,N,N-trimethylglycine (glycine betaine), which serves as a compatible intracellular solute. When grown in medium with high osmotic pressure, these cells also accumulated one amino acid, proline. The uptake of [3H]proline by resting, glucose-energized cells was stimulated by increasing the osmotic pressure of the assay medium with 0.5 to 1.0 M KCl, 1.0 M NaCl, or 0.5 M sucrose. The accumulated [3H]proline was not metabolized further. In contrast, there was no osmotic stimulation of [3H]leucine uptake. The uptake of proline was activated rather than induced by exposure of the cells to high osmotic pressure. Only one proline transport system could be discerned from kinetics plots. The affinity of the carrier for proline remained constant over a range of osmotic pressures from 650 to 1,910 mosM (Kt, 7.8 to 15.5 mM). The Vmax, however, increased from 15 nmol/min/mg of dry weight in 0.5 M sucrose to 27 and 40 nmol/min/mg of dry weight in 0.5 M KCl and in 1.0 M KCl or NaCl, respectively. The efflux of proline from preloaded cells occurred rapidly when the osmotic pressure of the suspending buffer was lowered.     

Osmotically regulated transport of proline by Lactobacillus acidophilus IFO 3532.

We reported previously that, when exposed to high osmotic pressure, Lactobacillus acidophilus IFO 3532 cells accumulated N,N,N-trimethylglycine (glycine betaine), which serves as a compatible intracellular solute. When grown in medium with high osmotic pressure, these cells also accumulated one amino acid, proline. The uptake of [3H]proline by resting, glucose-energized cells was stimulated by increasing the osmotic pressure of the assay medium with 0.5 to 1.0 M KCl, 1.0 M NaCl, or 0.5 M sucrose. The accumulated [3H]proline was not metabolized further. In contrast, there was no osmotic stimulation of [3H]leucine uptake. The uptake of proline was activated rather than induced by exposure of the cells to high osmotic pressure. Only one proline transport system could be discerned from kinetics plots. The affinity of the carrier for proline remained constant over a range of osmotic pressures from 650 to 1,910 mosM (Kt, 7.8 to 15.5 mM). The Vmax, however, increased from 15 nmol/min/mg of dry weight in 0.5 M sucrose to 27 and 40 nmol/min/mg of dry weight in 0.5 M KCl and in 1.0 M KCl or NaCl, respectively. The efflux of proline from preloaded cells occurred rapidly when the osmotic pressure of the suspending buffer was lowered.     

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.     

Expression analysis of proline metabolism-related genes from halophyte Arabis stelleri under osmotic stress conditions

Arabis stelleri var.japonica evidenced stronger osmotic stress tolerance than Arabidopsis thaliana.Using an A.thaliana microarray chip,we determined changes in the expression of approximately 2 800genes between A.stelleri plants treated with 0.2 M mannitol versus mock-treated plants.The most significant changes in the gene expression patterns were in genes defining cellular components or in genes associated with the endomembrane system,stimulus response,stress response,chemical stimulus response,and defense response.The expression patterns of three de novo proline biosynthesis enzymes were evaluated in A.stelleri var.japonica seedlings treated with 0.2 M mannitol,0.2 M sorbitol,and 0.2 M NaCl.The expression of Δ1-pyrroline-5-carboxylate synthetase was not affected by NaCl stress but was similarly induced by mannitol and sorbitol.The proline dehydrogenase gene,which is known to be repressed by dehydration stress and induced by free L-proline,was induced at an early stage by mannitol treatment,but the level of proline dehydrogenase was increased later by treatment with both mannitol and NaCl.The level of free L-proline accumulation increased progressively in response to treatments with mannitol,sorbitol,and NaCl.Mannitol induced L-proline accumulation more rapidly than NaCl or sorbitol.These findings demonstrate that the osmotic tolerance of the novel halophyte,Arabis stelleri,is associated with the accumulation of L-proline.     

Structure of the Escherichia coli 0104 polysaccharide and its identity with the capsular K9 polysaccharide

Spontaneous mutants of Rhizobium leguminosarum biovar viciae strain C1204b were selected for their ability to tolerate 0.2 M NaCl, a growth-inhibiting level of salt for the parental strain. Transposon-mediated salt-sensitive mutants of strain C1204b were screened for their inability to grow in 0.08 M NaCl. Quantitation of the free-amino acid pools in the mutants grown in NaCl revealed a dramatic increase in glutamine, serine, glutamate and proline, and to a lesser extent alanine and glycine in the salt-tolerant mutants in comparison with the parental strain exposed to NaCl; but only glutamate and proline increased in the salt-sensitive mutants under NaCl stress. Extracellular polysaccharide levels were quantitated for the salt-tolerant mutants and determined to be approximately two-fold higher than for the parental strain. Although the mutations that occurred in the NaCl-tolerant and NaCl-sensitive strains did not interfere with nodule formation, no nitrogenase activity could be observed in the NaCl tolerant mutants as evaluated by acetylene reduction.     

Water deficit enhancement of proline and α-amino nitrogen accumulation in potato plants and its association with susceptibility to drought

Potato plants ( Solanum tuberosum L. cvs 'Up-to-Date', 'Desiree', 'Alpha', 'Spunta', 'Elvira' and 'Troubadour') were exposed to cycles of water stress and relief during growth. Severe water deficit induced increased proline content 6- to 7-fold in nonturgid leaves which just started to wilt, and 8- to 27-fold in fully wilted leaves of potatoes. However, proline content was not affected during the early stages of stress development over a range of osmotic potentials in the leaves. The rising proline content was related to turgor loss of leaves independent of changes in the osmotic potentials, which indicates that proline involvement in osmoregulation of potato leaves is unlikely.
Repeated cycles of water stress and relief resulted in increased proline and α-amino nitrogen content in the tuber tissue of some of the cultivars. The smallest increase in proline content was obtained in 'Alpha' tubers and the content of α-amino nitrogen remained unaffected by the water stress. Concomitantly, 'Alpha' was the most drought-tolerant cultivar, as determined by its capacity to accumulate dry matter in tubers under stress conditions. On the other hand, in tubers of cultivars which were more susceptible to drought, a marked increase in proline and α-amino nitrogen was observed in response to water stress. The possible association of these findings with tolerance of potatoes to repeated short periods of drought is discussed.     

Effects of salt stress on growth, photosynthesis and nitrogen fixation in chick-pea (Cicer arietinum L.)

Plants of chick-pea (Cicer arietinum L. cv. ILC1919) inoculated with Mesorhizobium ciceri strain ch-191 were grown in a controlled environmental chamber, and were administered salt (0, 50, 75, and 100 mM NaCl) during the vegetative period. Four harvests (4, 7, 11, and 14d after treatment) were analysed. The aim was to ascertain whether the negative effect of saline stress on nitrogen fixation is due to a limitation on the photosynthate supply to the nodule or a limitation on the nodular metabolism which sustains nitrogenase activity.Plant growth was affected only by the highest NaCl concentration, whereas nitrogenase activity was affected from 50 mM. At the first harvest, Rubisco, PEPC and MDH activities in leaves rose with salt, but fell during the following harvests. The increase of PEPC and MDH in nodules at the two first samplings was clearly related to salt concentration. While 50 mM NaCl increased GS and GOGAT in nodules at some harvests, 100 mM strongly inhibited these activities at all the harvests. The accumulation of proline, amino acids and carbohydrates was clearly related to salt especially in the leaves, whereas in the nodules the protein content was boosted by salt. Although photosynthesis declined with NaCl, the response of nitrogen fixation to salt was more pronounced. This situation, together with carbohydrate accumulation, suggests that the lack of photosynthate does not cause the inhibition of nitrogenase activity under this type of stress. The similar trend observed for the PEPC-MDH pathway and the ARA support the hypothesis concerning the limitation in the supply of energy substrate, mainly malate, to the bacteroids. The accumulation of compatible solutes is more a consequence of damage produced by salt stress than of a protective strategy.     

Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.