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     

Evidence for a sodium-dependent proline and glycine-betaine uptake in the cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis>

The cyanobacterium Nostoc muscorum is able to utilized proline and glycine-betaine as a nitrogen source under unstressed growth conditions. This cyanobacterium when grow in modified Chu No. 10 medium (without Na⁺) unable to utilized proline and glycine-betaine as a nitrogen source. Spontaneously occurring mutant clones defective in Na⁺ transport (Na⁺-R) was isolated and analyzed for proline and glycine-betaine utilization. The mutant phenotype showed normal heterocyst frequency and nitrogenase activity even in the medium containing 1 mM proline or 1 mM glycine-betaine, indicates the role of Na⁺ for proline/glycine-betaine uptake. The Na⁺-R mutant showed 100% survival at pH 11 and was simultaneously able to uptake and utilize proline/glycine-betaine at higher alkaline pH. This indicates that proline and glycinebetaine uptake systems are more efficient at higher alkaline pH. Since, the hypersaline environments are rich in Na⁺ contents and have alkaline pH, therefore it is suggested that the origin and evolution of specific compatible solutes may not depend only on the osmoregulatory role they play, but also on the other ecological factors operating simultaneously in the organism’s niche.     

An azide-resistant mutant of the blue-green algaNostoc muscorum producing heterocyst and nitrogenase in the presence of fixed inorganic nitrogen source

The N₂, NO ₃ ^– , NO ₂ ^– , NH ₄ ⁺ and glutamine growing cultures of parentNostoc muscorum are found more or less equally sensitive to azide inhibition of growth. A mutant strain resistant to sodium azide was isolated from the parent strain in NO ₃ ^– medium and the two strains were compared with regard to their heterocyst formation and nitrogenase activity in NO ₃ ^– , NO ₂ ^– , NH ₄ ⁺ and glutamine media. While the parent strain stops production of both heterocyst and nitrogenase in all the fixed nitrogen media, the azide resistant strain forms both in the fixed inorganic nitrogen media but only heterocyst and no nitrogenase in the glutamine medium. Clearly a single genetic determinant of regulatory nature appears to mediate azide-resistance as well as relief of heterocyst and nitrogenase formation from inhibition by the fixed inorganic nitrogen source. The results of glutamine effect on the heterocyst and nitrogenase formation of the two strains indicate the operation of two levels of glutamine-sensitive regulation, one which operates through the common genetic determinant of heterocyst and nitrogenase regulation and the other exclusive to nitrogenase regulation. The in vivo functional nitrogenase does not appear to be the reason for azide-resistance and neither ammonia nor glutamine or its close metabolic product seems to function in the control of heterocyst spacing.     

Sodium requirement and metabolism in nitrogen-fixing cyanobacteria

Sodium affects the metabolism of eukaryotes and prokaryotes in several ways. This review collates information on the effects of Na⁺ on the metabolism of cyanobacteria with emphasis on the N₂,fixing filamentous species. Na⁺ is required for nitrogenase activity inAnabaena torulosa, Anabaena L-31 andPlectonema boryanum. The features of this requirement have been mainly studied inAnabaena torulosa. The need for Na⁺ is specific and cannot be replaced by K⁺, Li⁺, Ca 2 + or Mg²⁺. Processes crucial for expression of nitrogenase such as molybdenum uptake, protection of the enzyme from oxygen inactivation and conformational activation of the enzyme are not affected by Na⁺. Mo-Fe protein and Fe protein, the two components of nitrogenase are synthesized in the absence of Na⁺ but the enzyme complex is catalytically inactive. Photoevolution of O₂ and CO₂ fixation, which are severely inhibited in the absence of Na⁺, are quickly restored by glutamine or glutamate indicating that Na⁺ deprivation affects photosynthesis indirectly due to deficiency in the products of N₂ fixation. Na⁺ deprivation decreases phosphate uptake, nucleoside phosphate pool and nitrogenase activity. These effects are reversed by the addition of Na⁺ suggesting that a limitation of available ATP caused by reduced phosphate uptake results in loss of nitrogenase activity during Na⁺ starvation. Na⁺ influx inAnabaena torulosa andAnabaena L-31 is unaffected by low K⁺ concentration, is carrier mediated, follows Michaelis-Menten kinetics and is modulated mainly by membrane potential. Treatments which cause membrane depolarisation and hyperpolarisation inhibit and enhance Na⁺ influx respectively. These cyanobacteria exhibit rapid active efflux of Na⁺, in a manner different from the Na⁺/H⁺ antiporter mechanism found inAnacystis nidulans. Na⁺ requirement in nitrogen metabolism including nitrate assimilation, synthesis of amino acids and proteins, in respiration and oxidative phosphorylation, in transport of sugars and amino acids, cellular distribution of absorbed sodium, physiological basis of salt tolerance and prospects of reclamation of saline soils by cyanobacteria are the other aspects discussed in this review.     

Contributions of arbuscular mycorrhizal fungi to growth,photosynthesis, root morphology and ionic balance of citrus seedlings under salt stress

A pot study was conducted to determine the effects of arbuscular mycorrhizal (AM) fungi (Glomus mosseae and Paraglomus occultum) and salt (NaCl) stress on growth, photosynthesis, root morphology and ionic balance of citrus (Citrus tangerine Hort. ex Tanaka) seedlings. Eighty-five-day-old seedlings were exposed to 100 mM NaCl for 60 days to induce salt stress. Mycorrhizal colonization of citrus seedlings was not affected by salinity when associated with P. occultum, but significantly decreased when with G. mosseae. Compared with the non-mycorrhizal controls, mycorrhizal seedlings generally had greater plant height, stem diameter, shoot, root and total plant biomass, photosynthetic rate, transpiration rate and stomatal conductance under the 0 and 100 mM NaCl stresses. Root length, root projected area and root surface area were also higher in the mycorrhizal than in the non-mycorrhizal seedlings, but higher root volume in seedlings with G. mosseae. Leaf Na⁺ concentrations were significantly decreased, but leaf K⁺ and Mg²⁺ concentrations and the K⁺/Na⁺ ratio were increased when seedlings with both G. mosseae and P. occultum. Under the salt stress, Na⁺ concentrations were increased but K⁺ concentrations decreased in the mycorrhizal seedlings. Under the salt stress, Ca²⁺ concentrations were increased in the seedlings with P. occultum or without AM fungi (AMF), but decreased with G. mosseae. Ratios of both Ca²⁺/Na⁺ and Mg²⁺/Na⁺ were also increased in seedlings with G. mosseae under the non-salinity stress, while only the Mg²⁺/Na⁺ ratio was increased in seedlings with P. occultum under the salt stress. Our results suggested that salt tolerance of citrus seedlings could be enhanced by associated AMF with better plant growth, root morphology, photosynthesis and ionic balance.     

Caesium uptake and toxicity in the cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis>

A NH₄⁺ transport-defective mutant and a K⁺ transport-defective mutant of the cyanobacterium Nostoc muscorum were analysed with regard to percentage survival as a function of CsCl toxicity and Cs⁺ uptake activity. Neither survival nor Cs⁺ uptake was affected in either of the two mutants when compared with the wild type. The results indicate that the toxicity of Cs⁺ is determined at more than one cellular site in this organism.     

The Na+/H+ antiporter of alkaliphilic Bacillus sp.

The Na⁺/H⁺ antiporter, which appears to predominantly contribute to the alkaliphily of Bacillus halodurans C-125, was studied in an alkali-sensitive mutant of this strain and a transformant with restored alkaliphily. The alkali-sensitive mutant, strain 38154, which has lost the ability to grow above pH 9.5, was found to lack electro-genic Na⁺/H⁺ antiport activity driven by ΔΨ (membrane potential, interior negative), and it showed defective regulation of intracellular pH under alkaline conditions. On the other hand, a transformant carrying a 2.0-kb DNA fragment from the parental genome that complemented this defect was able to maintain an intracellular pH lower than that of the external milieu, and it was found to have recovered the Na⁺/H⁺ antiport activity driven by ΔΨ. Sequence analyses found that a 5.1-kb DNA region contained four open reading frames (ORF-1 to ORF-4). Direct sequencing of the corresponding region in mutant 38154 revealed a G-to-A substitution, which resulted in an amino acid substitution from Gly-393 to Arg in the putative ORF-1 product. It has been recently found that a region homologous to the DNA fragment responsible for the alkaliphily of strain C-125 exists in the genomes of Bacillus subtilis, Sinorhizobium (Rhizobium) meliloti, and Staphylococcus aureus. These homologues are present as a cluster of seven ORFs in each case. The shaA gene product of B. subtilis shows significant similarity to the ORF-1 product of strain C-125. Disruption of the shaA gene resulted in a decrease in Na⁺/H⁺ antiport activity, and growth of the shaA-disrupted strain was impaired when the external Na⁺ concentration was increased. We conclude that the shaA gene encodes a Na⁺/H⁺ antiporter, which plays an important role in extrusion of cytotoxic Na⁺. Received: May 29, 2000 / Accepted: July 18, 2000     

Growth stimulation and inhibition by salt in relation to Na<Superscript>+</Superscript> manipulating genes in xero-halophyte <Emphasis Type="Italic">Atriplex halimus</Emphasis> L.

In the present study, Na⁺ manipulating genes could contribute not only to ion homeostasis but also to growth stimulation with exposing the halophyte Atriplex halimus L. to moderate NaCl concentration. The stimulation of growth was attributed to Na⁺ accumulation inside the vacuole leading to increase leaf cell size as well as accelerate leaf cell division. Increasing the assimilatory surface could result in enhancing the photosynthetic rate. The reduction of A. halimus growth compared to optimum growth at 50 and 200 mM NaCl could be attributed to osmotic effect rather than the ionic one of salt stress. The inhibition of photosynthesis seemed to be resulted from limitation of CO₂ due to the osmotic effect on stomatal conductance rather than the activity loss of photosynthetic machinery. The depletion of starch content along with the increase in sucrose content could imply that photosynthesis may be a limiting for A. halimus growth. The fast coordinate induction of Na⁺ manipulating genes could reveal that the tolerance of A. halimus to high concentrations evolved from its ability to regulate and control Na⁺ influx and efflux. V-H ⁺-PPase may play a vital role in A. halimus tolerance to osmotic and/or ionic stress due to its kinetics of induction. It seemed that H⁺-ATPase plays a pivotal role in A. halimus tolerance to stress due to the increase in its protein level was detected with all NaCl concentrations as well as with PEG treatments. Both of these genes might be useful in improving stress tolerance in transgenic crops.     

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.     

A two-gene ABC-type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore

A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na at elevated pH, was deficient in energy-dependent Na extrusion. The capacity of the mutant JC901 for Na -dependent pH homeostasis was unaffected relative to the wild-type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3 -terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane-spanning regions at its C-terminus, and exhibits modest sequence similarity to regions of eukaryotic Na⁺/H⁺ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP-binding proteins that are components of transport systems of the ATP-binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natAB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p-chlorophenylhydrazone (CCCP), and, more modestly, by Na⁺, and K⁺, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na⁺-sensitive phe-notype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol-resistance does not result from NatAB-dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na⁺     

Malaria parasites tolerate a broad range of ionic environments and do not require host cation remodelling

Malaria parasites grow within erythrocytes, but are also free in host plasma between cycles of asexual replication. As a result, the parasite is exposed to fluctuating levels of Na⁺ and K⁺, ions assumed to serve important roles for the human pathogen, Plasmodium falciparum. We examined these assumptions and the parasite's ionic requirements by establishing continuous culture in novel sucrose‐based media. With sucrose as the primary osmoticant and K⁺ and Cl^? as the main extracellular ions, we obtained parasite growth and propagation at rates indistinguishable from those in physiological media. These conditions abolish long‐known increases in intracellular Na⁺ via parasite‐induced channels, excluding a requirement for erythrocyte cation remodelling. We also dissected Na⁺, K⁺ and Cl^? requirements and found that unexpectedly low concentrations of each ion meet the parasite's demands. Surprisingly, growth was not adversely affected by up to 148 mM K⁺, suggesting that low extracellular K⁺ is not an essential trigger for erythrocyte invasion. At the same time, merozoite egress and invasion required a threshold ionic strength, suggesting critical electrostatic interactions between macromolecules at these stages. These findings provide insights into transmembrane signalling in malaria and reveal fundamental differences between host and parasite ionic requirements.     

Temporal separation of nitrogen fixation and photosynthesis in the filamentous,non-heterocystous cyanobacterium Oscillatoria sp.

When growing in laternating light-dark cycles, nitrogenase activity (acetylene reduction) in the filamentous, non-heterocystous cyanobacterium Oscillatoria sp. strain 23 (Oldenburg) is predominantly present during the dark period. Dark respiration followed the same pattern as nitrogenase. Maximum activities of nitrogenase and respiration appeared at the same time and were 3.6 mol C₂H₄ and 1.4 mg O₂ mg Chl a ^-1·h^-1, respectively. Cultures, adapted to light-dark cycles, but transferred to continuous light, retained their reciprocal rhythm of oxygenic photosynthesis and nitrogen fixation. Moreover, even in the light, oxygen uptake was observed at the same rate as in the dark. Oxygen uptake and nitrogenase activity coincided. However, nitrogenase activity in the light was 6 times as high (22 mol C₂H₄ mg Chl a ^-1·h^-1) as compared to the dark activity. Although some overlap was observed in which both oxygen evolution and nitrogenase activity occurred simultaneously, it was concluded that in Oscillatoria nitrogen fixation and photosynthesis are separated temporary. If present, light covered the energy demand of nitrogenase and respiration very probably fulfilled a protective function.     

Comparative effects of salt and alkali stresses on growth, osmotic adjustment and ionic balance of an alkali-resistant halophyte Suaeda glauca (Bge.)

Effects of salt and alkali stresses on growth, osmotic adjustment and ionic balance of Suaeda glauca (Bge.), an alkali-resistant succulent halophyte, were compared. The results showed that alkali stress clearly inhibited the growth of S. glauca. Moreover, the concentrations of Na⁺ and K⁺ both increased with increasing salinity under both stresses, suggesting no competitive inhibition between absorptions of Na⁺ and K⁺. The mechanism underlying osmotic adjustment during salt stress was similar to alkali stress in shoots. The shared essential features were that organic acids, betaine and inorganic ions (dominated by Na⁺) mostly accumulated. On the other hand, the mechanisms governing ionic balance under both stresses were different. Under salt stress, S. glauca accumulated organic acids and inorganic anions to maintain the intracellular ionic equilibrium, but the anion contribution of inorganic ions was greater than that of organic acids. However, the concentrations of inorganic anions under alkali stress were significantly lower than those under salt stress of the same intensity, suggesting that alkali stress might inhibit uptake of anions, such as NO₃ ^– and H₂PO₄ ^–. Under alkali stress, organic acids were the dominant factor in maintaining ionic equilibrium. The contribution of organic acids to anions was 74.1%, while that of inorganic anions was only 25.9%. S. glauca enhanced the synthesis of organic acids, dominated by oxalic acid, to compensate for the shortage of inorganic anions.     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Sodium Requirement for Photosynthesis and Its Relationship with Dinitrogen Fixation and the External CO(2) Concentration in Cyanobacteria

Cells of Anabaena PCC 7119 and of a mutant strain of Nostoc muscorum unable to fix dinitrogen, grown at pH 8 and under low CO₂ tension (air), showed a reduced capacity for photosynthesis when cultured in the absence of sodium, this inhibition being followed by symptoms of photooxidation, such as chlorosis, oxygen consumption in the light, and decrease of superoxide dismutase activity. The impairment of photosynthesis preceded that of nitrogenase activity, indicating that the requirement for sodium in photosynthesis was independent of its effects on nitrogen metabolism. However, when cyanobacteria were grown at pH 6.3 or under high CO₂ tensions, sodium was not required for photosynthesis and no symptoms of photooxidation were observed.     

Functional differentiation in the anterior gills of the aquatic air-breathing fish, <Emphasis Type="Italic">Trichogaster leeri</Emphasis>

The fish gill is a multifunctional organ responsible for gas exchange and ionic regulation. It is hypothesized that both morphological and functional differentiation can be found in the gills of the aquatic air-breathing fish, Trichogaster leeri. To test this, we used the air-breathing fish, Trichogaster leeri, to investigate various morphological/functional parameters. First, we evaluated the importance of performing the aquatic surface respiration behavior in T. leeri. A reduced survival rate was observed when fish were kept in the restrained cages in hypoxic conditions. On the gross anatomy of gills, we found evidence of both morphological and functional modification in the first and the second gills and are responsible for ionic regulation. There were large-bore arterioarterial shunts in the fourth gill arch. It is specialized for the transport of oxygenated blood and is less responsive to environmental stress. In addition, the anterior and the posterior gills differed in the Na⁺, K⁺-ATPase activity upon ionic stresses. That is, only the Na⁺, K⁺-ATPase activity of the anterior two gills was up-regulated significantly in the deionized water. Lastly, we found that the number of mitochondria-rich cells in the first and the second gills increased following ionic stress and no difference was found in the third and the fourth gills following such an exposure. These results supported the hypothesis that there are morphological and functional differences between anterior and posterior gill arches within the air-breathing Trichogaster leeri. In contrast, no significant difference was found among gills in gross anatomy, filament density and Na⁺, K⁺-ATPase activity in the non-air-breather, Barbodes schwanenfeldi.     

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.     

[14C]Carbon-dioxide fixation by isolated leaf epidermes with stomata closed or open

Following small hypo-osmotic shocks, ion concentrations (Na⁺, K⁺, Cl^-) in Platymonas subcordiformis decreased; this was due mainly to an increase of cell volume. With larger hypo-osmotic stresses, the decrease of ion concentration continued and, additionally, extrusion of mannitol was observed. The ion and mannitol concentrations were not regained after 240 min. In contrast, following hyperosmotic shocks, the ion concentrations increased transitorily during the first 20–40 min. The same was true for K⁺ following small hyperosmotic stresses and for Na⁺ and — partially — Cl^- with larger shocks. Large hyperosmotic stresses caused permanent accumulation of mannitol, which levelled off after 60–80 min. Thus the transient increase of ions bridged the concentration gap until mannitol was accumulated to a high enough concentration to account for the osmotic adaptation of Platymonas, together with a basal level of the ions K⁺, Na⁺, Cl^-.Abbreviations PS photosynthesis - Resp respiration     

Fermenting Escherichia coli Is Able to Grow in Media of High Osmolarity, But Is Sensitive to the Presence of Sodium Ion

Escherichia coli is able to grow at increased NaCl concentrations that provides an increase in medium osmolarity and cellular Na⁺ content. The addition of 0.5 M NaCl to the growth medium led to a substantial decrease in growth rate during anaerobic fermentation on glucose at pH of 7.3 or 9.0. This inhibitory effect of 0.5 M NaCl was at least threefold stronger than that seen under aerobic conditions, and stronger than equivalent concentrations of sucrose, KCl, or potassium glutamate under anaerobic conditions. Further, proline was found to stimulate the growth rate at high NaCl concentration under anaerobic and to a lesser extent, under aerobic conditions. Wild-type cells and mutants having a functional NhaA or ChaA alone grown under anaerobic conditions at pH 9.0 and subsequently loaded with Na⁺ were shown to extrude Na⁺ at a rate that were lower than the extrusion rate reported for appropriate aerobically grown bacteria (Sakuma et al. [1998] Biochim Biophys Acta 1363:231–237). The growth rate and Na⁺ extrusion activity of a mutant having a functional NhaA were similar to that of the wild type and higher than that of a mutant with an active ChaA. A mutant defective for both NhaA and ChaA was unable to grow under anaerobic conditions at pH 9.0 in the presence of 0.15 M Na⁺. It is suggested that the observed strong inhibition in the growth of E. coli during fermentation under anaerobic conditions in the presence of increased NaCl concentration could be due to a decrease in Na⁺ extrusion activity. Received: 18 September 1998 / Accepted: 2 April 1999