Isolation and cloning of a Azospirillum lipoferum locus that complements Escherichia coli proU mutant

Glycine betaine relieved sodium chloride-mediated inhibition of growth in Azospirillum lipoferum ATCC 29708. ³⁵S-methionine labelling of proteins after salinity up-shock revealed strong induction of a 30 kDa protein which cross-reacted with the anti-glycine betaine binding protein antibody from Escherichia coli. This suggested that A. lipoferum had a salinity-induced ProU-like high-affinity glycine betaine transport system. A genomic library of A. lipoferum ATCC 29708 was screened for the proU-like gene by complementation of a proU mutant of E. coli. Four recombinant cosmids, capable of restoring growth of the proU mutant on plates containing 600 mM NaCl and 1 mM glycine betaine were selected. Selected recombinant cosmids hybridized with a proU gene probe from E. coli. Complementation of E. coli proU mutant with the A. lipoferum genomic DNA was evident by the ability of proU mutant (containing selected recombinant cosmids) to grow on minimal medium supplemented with 600 mM NaCl and 1 mM glycine betaine.     

Functional Expression of Sinorhizobium meliloti BetS, a High-Affinity Betaine Transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Østerås, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na⁺/H⁺ antiporters in B. japonicum could explain its very high Na⁺ sensitivity.     

In vitro evaluation of phenolic and osmolite compounds,ionic content,and antioxidant activity in safflower (<Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.) under salinity stress

Carthamus tinctorius L., rich in antioxidant compounds, is a herbal medicine. Biochemical mechanisms of adaptation to salinity stress in safflower are still poorly understood at the cellular level. For this purpose, callus cultures of four different genotypes of safflower were used in this study to evaluate changes in their biochemical (ionic content, proline, and glycine betaine), total phenolics content (TPC), total flavonoids content (TFD), antioxidant responses (2,2-diphenyl-1-picrylhydrazyl: DPPH assay and carotenoid content), and lipid peroxidation (malon dialdehyde content: MDA) under salinity stress. The calluses derived from hypocotyls were exposed to in vitro salt stress at different concentrations of sodium chloride (0, 100, 200, and 300 mM). A reducing trend was observed in K⁺ and carotenoid reserves of the calluses with increasing NaCl concentration while an increasing trend was observed in Na⁺ content, proline, MDA, TPC, TFD, and DPPH activity under the same conditions. Callus glycine betaine content was found to decrease in the medium containing 100 mM NaCl but increased beyond this concentration up to 300 mM NaCl. Positive and significant correlations were recognized between DPPH and total phenolics as well as DPPH and total flavonoid contents, demonstrating that phenolics are the main contributors to the potential antioxidant activity of safflower at the cellular level. Overall, the salt-tolerant genotypes of Mex.2-137 and Mex.2-138 were found capable of being processed for the production of secondary metabolites via NaCl elicitation.     

Formation and role of glycine betaine in the moderate halophile Vibrio costicola

The moderate halophile Vibrio costicola, growing on a chemically-defined medium, transformed choline into glycine betaine (betaine) by the membrane-bound enzyme choline dehydrogenase and the cytoplasmic enzyme betainal (betaine aldehyde) dehydrogenase. Choline dehydrogenase was strongly induced and betainal dehydrogenase less strongly induced by choline. The formation of these enzymes was also regulated by the NaCl concentration of the growth medium, increasing with increasing NaCl concentrations. Intracellular betaine concentrations also increased with increasing choline and NaCl concentrations in the medium. This increase was almost completely blocked by chloramphenicol, which does not block the increase in salt-tolerant active transport on transfer from a low to a high salt concentration.Choline dehydrogenase was inhibited by chloride salts of Na⁺, K⁺, and NH _inf4 ^su+ , the inhibition being due to the Cl^- ions. Betainal dehydrogenase was stimulated by 0.5 M salts and could function in up to 2.0 M salts.Cells grew as well in the presence as in the absence of choline in 0.5 M and 1.0 M NaCl, but formed no intracellular betaine. Choline stimulated growth in 2.0 M NaCl and was essential for growth in 3.0 M NaCl. Thus, while betaine is important for some of the adaptations to high salt concentration by V. costicola, it by no means accounts for all of them.Abbreviations CDMM chemically-defined minimal medium - PPT proteose-peptone tryptone medium - SDS sodium dodecyl sulfate Deceased, 1987     

Betaine Transport Imparts Osmotolerance on a Strain of Lactobacillus acidophilus

Unlike most Lactobacillus acidophilus strains, a specific strain, L. acidophilus IFO 3532, was found to grow in rich medium containing 1 M sodium acetate, KCl, or NaCl. This strain could also grow with up to 1.8 M NaCl or 3 M nonelectrolytes (fructose, xylose, or sorbitol) added. Thus, this strain was tolerant to osmotic pressures up to 2.8 osM. A search for an intracellular solute which conferred osmoprotection led to the identification of glycine betaine (betaine). Betaine was accumulated to high concentrations in cells growing in MRS medium supplemented with 1 M KCl or NaCl. Uptake of [¹⁴C]betaine by L. acidophilus 3532 cells suspended in buffer was stimulated by increasing the medium osmotic pressure with 1 M KCl or NaCl. The accumulated betaine was not metabolized further; transport was relatively specific for betaine and was dependent on an energy source. Other lactobacilli, more osmosensitive than strain 3532, including L. acidophilus strain E4356, L. bulgaricus 8144, and L. delbrueckii 9649, showed lower betaine transport rates in response to an osmotic challenge than L. acidophilus 3532. Experiments with chloramphenicol-treated L. acidophilus 3532 cells indicated that the transport system was not induced but appeared to be activated by an increase in osmotic pressure.     

Choline Derivatives Involved in Osmotolerance of Penicillium fellutanum

Penicillium fellutanum is osmotolerant and xerotolerant when cultured in a low-phosphate medium containing 3 M NaCl. Glycerol and erythritol accumulated in cultures with NaCl concentrations up to 2 M; glycerol was the only detectable polyol in cultures containing 3 M NaCl. In cultures with 3 M NaCl, the intracellular levels of glycine betaine and choline-O-sulfate were 22- and 2.6-fold greater (70 and 46 mM), respectively, than those of cultures without added NaCl. The levels of glycine betaine and glycerol decreased in mycelia transferred from a medium containing 3 M NaCl into a fresh medium without added NaCl. NaCl at 3 M inhibited mycelial mass accumulation; this inhibition was partially corrected by supplementation of cultures with glycine betaine (2 mM) or choline-O-sulfate (10 mM). The presence of exogenous choline chloride (2 mM) in plate cultures protected the cells from stress from 3 M NaCl. The data suggest that glycine betaine and choline-O-sulfate are secondary osmoprotectants which are effective at the point that the cell is incapable of synthesizing more glycerol.     

Glycine Betaine, Carnitine, and Choline Enhance Salinity Tolerance and Prevent the Accumulation of Sodium to a Level Inhibiting Growth of Tetragenococcus halophila

Natural-abundance ¹³C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium Tetragenococcus halophila. When grown in complex growth media supplemented or not with NaCl, T. halophila accumulates glycine betaine and carnitine. Unlike other moderate halophiles, T. halophila was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint. Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl. These compounds were taken up when available in the surrounding medium. The transport activity occurred at low and high salinities and seems to be constitutive. Glycine betaine and carnitine were accumulated by T. halophila in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine. This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium. An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity. Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg [dry weight]), independent of the NaCl concentration of the medium. In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M. Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na⁺ content and confer a much higher salt tolerance to T. halophila.     

Engineering whole-cell biosensors to evaluate the effect of osmotic conditions on bacteria

Enhanced green fluorescent protein (eGFP) is a variant of wild-type GFP humanized for optimal expression in mammalian cell lines. A computational approach comparing wtGFP and eGFP showed the occurrence of rare proline codons within the eGFP gene that could interfere with and hamper protein production in prokaryotic expression systems. The eGFP gene excised from mammalian plasmid pEGFP N3 was used for construction of two inducible promoter-reporter fusions, T7-eGFP and P_proU-eGFP, through directional cloning. The T7-eGFP fusion confirmed expression of eGFP protein within the bacterial strain, showing a fluorescent green cell pellet and overexpression of the ~29 kDa eGFP protein upon induction with IPTG. The proU operon aids in osmoadaptation by encoding a transport system for uptake of various compatible solutes, including glycine-betaine and proline. Expression of the proU operon is induced upon growth of bacteria in media of elevated osmolarity. When coupled to an eGFP reporter, a time course study using fluorometry demonstrated that induction of P_proU in Escherichia coli occurred rapidly. The P_proU induction and recombinant eGFP production depends on time and concentration of solute (NaCl) in the medium. Cells containing the P_proU-eGFP fusion showed maximum promoter activity at 500 mM concentration of NaCl with a sensitivity of the P_proU promoter being 50 mM. The relative fluorescence reflected the amount of protein synthesized proportional to the activity of induced promoter and effect of NaCl on growth was also taken into consideration. Thus, such environmentally regulated highly sensitive promoters with enhanced reporters could possibly be used as whole-cell biosensors.     

Identification of an ATP-Driven, Osmoregulated Glycine Betaine Transport System in Listeria monocytogenes

The ability of the gram-positive, food-borne pathogen Listeria monocytogenes to tolerate environments of elevated osmolarity and reduced temperature is due in part to the transport and accumulation of the osmolyte glycine betaine. Previously we showed that glycine betaine transport was the result of Na⁺-glycine betaine symport. In this report, we identify a second glycine betaine transporter from L. monocytogenes which is osmotically activated but does not require a high concentration of Na⁺ for activity. By using a pool of Tn917-LTV3 mutants, a salt- and chill-sensitive mutant which was also found to be impaired in its ability to transport glycine betaine was isolated. DNA sequence analysis of the region flanking the site of transposon insertion revealed three open reading frames homologous to opuA from Bacillus subtilis and proU from Escherichia coli, both of which encode glycine betaine transport systems that belong to the superfamily of ATP-dependent transporters. The three open reading frames are closely spaced, suggesting that they are arranged in an operon. Moreover, a region upstream from the first reading frame was found to be homologous to the promoter regions of both opuA and proU. One unusual feature not shared with these other two systems is that the start codons for two of the open reading frames in L. monocytogenes appear to be TTG. That glycine betaine uptake is nearly eliminated in the mutant strain when it is assayed in the absence of Na⁺ is an indication that only the ATP-dependent transporter and the Na⁺-glycine betaine symporter occur in L. monocytogenes.     

The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12

The ProP and ProU transport systems of Escherichia coli mediate the uptake of several osmoprotectants including glycine betaine. Here we report that both ProP and ProU are involved in the transport of the potent osmoprotectant proline betaine. A set of isogenic E. coli strains carrying deletions in either the proP or proU loci was constructed. The growth properties of these mutants in high osmolarity minimal media containing 1 mM proline betaine demonstrated that the osmoprotective effect of this compound was dependent on either an intact ProP or ProU uptake system. Proline betaine competes with glycine betaine for binding to the proU-encoded periplasmic substrate binding protein (ProX) and we estimate a K_D of 5.2 μM for proline betaine binding. This value is similar to the binding constant of the ProX protein determined previously for the binding of glycine betaine (K_D of 1.4 μM). Our results thus demonstrate that the binding-protein-dependent ProU transport system of E. coli mediates the efficient uptake of the osmoprotectants glycine betaine and proline betaine.     

betaine modulates intracellular thiol and potassium levels in Escherichia coli in medium with high osmolarity and alkaline pH

Glycine betaine stimulates the growth rate of various bacteria in high osmolarity medium. In our studies, glycine betaine stimulated the growth rate of Escherichia coli K 12 in minimal medium with normal osmolarity at alkaline pH (pH 8.2). Betaine also caused a reduction in the intracellular pools of K⁺ and low molecular weight thiols in E. coli growing both in medium with high osmolarity and at alkaline pH. These effects of betaine were absent at pH 7.0. In cells growing in high osmolarity medium, 10 mM sodium acetate or 10 M N-ethylmaleimide reduced expression of the osmosensitive gene proU to the same extent as treatment with betaine; however, under these conditions, sodium acetate and N-ethylmaleimide did not stimulate the growth of E. coli. It is proposed that low molecular weight thiols and intracellular pH may participate in the response of E. coli to betaine.     

Growth of <Emphasis Type="Italic">Paenarthrobacter aurescens</Emphasis> strain TC1 on atrazine and isopropylamine during osmotic stress

Paenarthrobacter aurescens strain TC1 can use the herbicide atrazine and its degradation product isopropylamine as nutrients. Because osmotic stress can change the morphology of arthrobacters and decrease their metabolism of some carbon compounds, the effects of increasing NaCl concentrations on strain TC1 and its ability to utilize atrazine and isopropylamine were determined. Strain TC1 was cultured in minimal media with different NaCl concentrations and varying combinations of d-glucose, ammonium sulfate, atrazine, or isopropylamine. Growth was measured quantitatively as an increase in turbidity. Physiological effects were assessed using Biolog? GP test plates and BD BBL Crystal GP or bioMérieux API 20E test systems. The effects of osmoprotective compounds were determined in liquid media and on agar plates. Strain TC1 formed multicellular myceloids and its growth rate slowed as the salt concentration increased, but the culture yields were similar up to 0.6 mol l^?1 NaCl. The bacteria metabolized about half the carbon sources in Biolog? GP test plates, but their use of some compounds and several hydrolytic activities decreased with high salt concentrations. However, strain TC1 grew well with atrazine and isopropylamine as the nitrogen source in media containing up to 0.6 mol l^?1 NaCl. Growth in 0.8 mol l^?1 NaCl was more limited but could be enhanced by glycine betaine, L-proline, and L-glutamate. P. aurescens strain TC1 can continue to use atrazine and isopropylamine as nutrients during osmotic stress and so may be particularly useful for remediation of contaminated soils with low water activity.     

Anabaena sp. PCC7120 transformed with glycine methylation genes from Aphanothece halophytica synthesized glycine betaine showing increased tolerance to salt

Photosynthetic, nitrogen-fixing Anabaena strains play an important role in the carbon and nitrogen cycles in tropical paddy fields although they are salt sensitive. Improvement in salt tolerance of Anabaena cells by expressing glycine betaine–synthesizing genes is an interesting subject. Due to the absence of choline in cyanobacteria, choline-oxidizing enzyme could not be used for the synthesis of glycine betaine. Here, the genes encoding glycine-sarcosine and dimethylglycine methyltransferases (ApGSMT-DMT) from a halotolerant cyanobacterium Aphanothece halophytica were expressed in Anabaena sp. strain PCC7120. The ApGSMT-DMT-expressing Anabaena cells were capable of synthesizing glycine betaine without the addition of any substance. The accumulation level of glycine betaine in Anabaena increased with rise of salt concentration. The transformed cells exhibited an improved growth and more tolerance to salinity than the control cells. The present work provides a prospect to engineer a nitrogen-fixing cyanobacterium having enhanced tolerance to stress by manipulating de novo synthesis of glycine betaine.     

Role for Glycine Betaine Transport in Vibrio cholerae Osmoadaptation and Biofilm Formation within Microbial Communities

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.     

Studies on halotolerance in a moderately halophilic bacterium. Effect of betaine on salt resistance of the respiratory system

The role of betaine as a factor influencing the salt resistance of the respiratory system in resting cells of the moderately halophilic halotolerant bacterium Ba(1) was studied. Betaine accelerated succinate oxidation in cells obtained from low-salt medium, and stimulation of the respiratory rate was stronger the higher the sodium chloride concentration in the assay medium. The stimulatory effect also depended on the ratio of betaine concentration to the amount of bacteria present. Accumulation of labelled betaine by the bacterial cells was demonstrated; like the respiratory stimulation, it was favourably influenced by an increase in the sodium chloride concentration of the medium. In cells harvested from a high-salt medium and washed with 2.0m-sodium chloride, betaine caused no increase in the respiratory rate, nor was the already high salt resistance of the respiratory system further improved by the addition of betaine. When, however, these cells lost their salt resistance as a result of washing in the absence of sodium chloride, betaine was able to restore it to its original level. In contrast with respiration in low-salt-grown bacteria, that in high-salt-grown cells was not affected by betaine, even after they were washed in the absence of sodium chloride, when the sodium chloride concentration was optimum.     

Choline and Glycine Betaine Uptake in Various Strains of Rhizobia Isolated from Nodules of Vicia faba var. major and Cicer arietinum L.: Modulation by Salt,Choline, and Glycine Betaine

Two strains of Rhizobia isolated from nodules of Vicia faba var. major and one strain isolated from nodules of Cicer arietinum L. were characterized for salt resistance. The presence of 1 mM glycine betaine or choline in a minimal medium with added NaCl had a beneficial role on the growth of the three strains. Both molecules were found to be taken up by cells obtained at low osmolarity, and whereas glycine betaine uptake activity was stimulated significantly in cells grown in the presence of 0.15 M NaCl, choline uptake activity was strongly inhibited by salt in all tested strains. However, in cells grown with exogenous choline, the uptake inhibition exerted by salt was relieved, mainly in the strain isolated from nodules of C. arietinum L. On the basis of kinetics determinations, in control cells as well as in salt-stressed cells, only high-affinity activities were observed for glycine betaine and choline (apparent K _m s between 3 and 18 μM). Periplasmic proteins that bound glycine betaine or choline were identified. In nondenaturing conditions, these proteins extracted from the various strains showed different electrophoretic mobility with always a less negative entire charge than the analogous proteins from Rhizobium meliloti. Received: 29 July 1996 / Accepted: 10 September 1996     

Functional expression of Sinorhizobium meliloti BetS, a high-affinity betaine transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Osteras, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na(+)/H(+) antiporters in B. japonicum could explain its very high Na(+) sensitivity.     

Glycine Betaine Enhances Growth of Nitrogen-Fixing Bacteria <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> PAL5 Under Saline Stress Conditions

In this study, the effect of glycine betaine as osmoprotectant compound for Gluconacetobacter diazotrophicus PAL5 was evaluated by kinetic growth parameters. Batch fermentation assays were performed employing media supplemented with different sodium chloride concentrations to simulate saline stress conditions. Salt concentrations of 50–300 mM led to decreased cell concentrations, while the maximum specific growth rates and cell productivities were reduced at concentrations above 100-mM NaCl. Salt inhibition was mainly observed in media with 200- and 300-mM NaCl, in which drastic changes in cell morphology were also noted. The addition of glycine betaine to the media showed to be efficient to counteract the salt inhibitory effect by increasing some fermentation parameters. However, the osmoprotectant was not able to revert the polymorphism promoted by higher salt concentrations.     

Atrazine degradation in saline wastewater by Pseudomonas sp strain ADP

Wastewater from atrazine manufacturing plants contains large amounts of residual atrazine and atrazine synthesis products, which must be removed before disposal. One of the obstacles to biological treatment of these wastewaters is their high salt content, eg, up to 4% NaCl (w/v). To enable biological treatment, bacteria capable of atrazine mineralization must be adapted to high-salinity conditions. A recently isolated atrazine-degrading bacterium, Pseudomonas sp strain ADP, originally isolated from contaminated soils was adapted to biodegradation of atrazine at salt concentrations relevant to atrazine manufacturing wastewater. The adaptation mechanism was based on the ability of the bacterium to produce trehalose as its main osmolyte. Trehalose accumulation was confirmed by natural-abundance ¹H NMR spectral analysis. The bacterium synthesized trehalose de novo in the cells, but could not utilize trehalose added to the growth medium. Interestingly, the bacterium could not produce glycine betaine (a common compatible solute), but addition of 1 mM of glycine betaine to the medium induced salt tolerance. Osmoregulated Pseudomonas sp strain ADP, feeding on citrate decreased the concentration of atrazine in non-sterile authentic wastewater from 25 ppm to below 1 ppm in less than 2 days. The results of our study suggest that salt-adapted Pseudomonas sp strain ADP can be used for atrazine degradation in salt-containing wastewater. Received 26 August 1997/ Accepted in revised form 06 December 1997     

Bacterial chitin utilization at halophilic conditions

Chitin is a dominant structural polymer produced in large amounts by brine shrimp Artemia in hypersaline lakes. Microbiological analysis of chitin utilization as a growth substrate in hypersaline chloride–sulfate lakes in the south Kulunda Steppe (Altai, Russia) revealed two groups of bacteria able to grow on chitin at moderate salinity. Under aerobic conditions, an enrichment culture was obtained at 2 M NaCl. Further purification resulted in the isolation of strains HCh1 and strain HCh2, identified as representatives of the genera Saccharospirillum and Arhodomonas (both in the Gammaproteobacteria). The chitin-utilizing potential has not been previously recognized in these genera. The Saccharospirillum sp. strain HCh1 grew on chitin within the salinity range from 0.5 to 3.25 M NaCl (optimum at 1 M), while Arhodomonas sp. strain HCh2 grew up to 2.5 M NaCl but had a higher salt optimum at 1.5 M. Anaerobic enrichments grew with chitin at 2 and 4 M NaCl, but growth in the latter was extremely slow and the culture eventually lost viability. The enrichment at 2 M NaCl resulted in the isolation of strain HCh-An1, identified as a distant new species of the genus Orenia in the clostridial order Halanaerobiales. It was able to grow on chitin within a salinity range from 1.0 to 2.5 M NaCl (optimum at 1.5 M). The strain is proposed as a new species of the genus Orenia—O. chitinitropha.