Osmotic regulation of cyclic 1,2-ß-glucan synthesis

Nora Iñón de Iannino1, Gabriel Briones1, Florencia Iannino1 and Rodolfo A. Ugalde1

Instituto de Investigaciones Biotecnólogicas, Universidad Nacional de General San Martín, Av. General Paz entre Constituyentes y Albarellos, PO Box 30, (1650) Buenos Aires, Argentina1

Author for correspondence: Nora Iñón de Iannino. Tel: +54 11 4 580 7285. Fax: +54 11 4 752 9639. e-mail: norai{at}inti.gov.ar


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In contrast to what happens in Agrobacterium tumefaciens and Rhizobium meliloti, synthesis of periplasmic cyclic 1,2-ß-glucan in Brucella spp. was not inhibited when bacteria were grown in media of high osmolarity. Studies performed with crude membrane preparations showed that cyclic 1,2-ß-glucan synthetase of Brucella spp. was not inhibited by 0·5 M KCl or potassium glutamate; concentrations that completely inhibit the osmosensitive enzymes of A. tumefaciens A348 or R. meliloti 102F34, respectively encoded by the chvB or ndvB genes. The Brucella abortus cyclic 1,2-ß-glucan synthetase gene (cgs) was introduced into A. tumefaciens A1011 chvB and R. meliloti GRT21s ndvB mutants. Synthesis of cyclic 1,2-ß-glucan by the recombinant strains was not inhibited when grown in media of high osmolarity (0·25 M NaCl or 0·5 M mannitol). On the other hand, when the A. tumefaciens cyclic 1,2-ß-glucan synthetase gene was introduced into the R. meliloti GRT21s ndvB mutant, the recombinant strain displayed marked inhibition of cyclic 1,2-ß-glucan synthesis when grown in high-osmolarity media. However, the same gene introduced into a B. abortus cgs mutant background resulted in no inhibition of glucan synthesis at high osmolarity. In vitro studies with crude membranes isolated from recombinant strains revealed that Brucella cyclic 1,2-ß-glucan synthetase was not inhibited by high concentrations of KCl or potassium glutamate even when expressed in Agrobacterium or Rhizobium backgrounds. It was concluded that the lack of effect of high osmolarity on 1,2-ß-glucan synthesis in Brucella is due to two convergent mechanisms: a) the presence of a cyclic 1,2-ß-glucan synthetase that is not affected by concentrations of solutes such as KCl or potassium glutamate and b) either the possible accumulation of compatible solutes that might protect the enzyme from the inhibition by potassium glutamate or the accumulation of other osmolytes that do not affect the 1,2-ß-glucan synthetase.

Keywords: cyclic 1,2-ß-glucan, osmoregulation, Brucella, Agrobacterium


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Agrobacterium tumefaciens chromosomal virulence genes chvB and chvA are required for synthesis and secretion of cyclic 1,2-ß-glucans (Douglas et al., 1985 ; Puvanesarajah et al., 1985 ). chvB encodes the inner-membrane-bound cyclic 1,2-ß-glucan synthetase and chvA encodes a 75 kDa inner-membrane protein required for secretion of cyclic 1,2-ß-glucan (Zorreguieta & Ugalde, 1986 ; Iñón de Iannino & Ugalde, 1989 ). In Rhizobium spp. the ndvB and ndvA genes, homologous to A. tumefaciens chvB and chvA, are required for 1,2-ß-glucan synthesis and secretion respectively (Dylan et al., 1986 ; Geremía et al., 1987 ). ndvB and chvB mutants are defective in nodule invasion and virulence, and show impaired growth in low osmolarity medium (Zorreguieta et al., 1990 ; Dylan et al., 1990 ; Cangelosi et al., 1990 ). The cyclic 1,2-ß-glucan synthetase enzyme contains all the enzymic activities required for synthesis of cyclic 1,2-ß-glucan, i.e. initiation, elongation and cyclization (Altabe et al., 1990 ; Castro et al., 1996 ); the glucan-linked synthetase is, therefore, an intermediate in glucan synthesis (Zorreguieta & Ugalde, 1986 ).

Osmotic regulation of Rhizobium meliloti and A. tumefaciens cyclic 1,2-ß-glucan synthesis is similar to that of membrane-derived oligosaccharides (MDOs) in Escherichia coli (Breedveld & Miller, 1994 ). High osmolarity was found to inhibit the in vivo accumulation of cellular 1,2-ß-glucans in Agrobacterium and Rhizobium or MDO in E. coli (Breedveld & Miller, 1994 ). Exceptions have been reported with respect to osmoregulation. In several strains of R. leguminosarum and in R. meliloti strain GR4, the levels of cell-associated cyclic 1,2-ß-glucans were independent of osmolarity of the growth medium (Breedveld et al., 1991 ; Soto et al., 1993 ).

It was demonstrated that high osmolarity has no effect on gene expression of cyclic 1,2-ß-glucan synthetase, since membranes prepared from cells grown at high osmolarity displayed normal glucan synthesis activity in vitro (Zorreguieta et al., 1990 ). Osmotic regulation of periplasmic glucan biosynthesis by Agrobacterium and Rhizobium appeared to occur at a post-translational level, as with the osmotic regulation of biosynthesis of MDOs in E. coli (Rumley et al., 1992 ). A. tumefaciens and R. meliloti 1,2-ß-glucan synthesis is inhibited in vitro by KCl and potassium glutamate.

Bacteria respond to changes of osmolarity of the environment by varying the intracellular concentration of specific solutes. The accumulation of K+, followed by glutamate, is a primary response to osmotic upshift in E. coli and other bacteria in minimal salt media. Increase of the intracellular concentration of potassium glutamate allows adaptation of the cell to environments of moderately high osmolarities. At high osmolarity, bacteria replace K+ ions with high intracellular concentrations of one or more compatible solutes. These include polyols such as trehalose, amino acids such as proline and methylamines such as glycine betaine (Lucht & Bremer, 1994 ). Rhizobium and Agrobacterium cytoplasmic concentrations of K+ and glutamate also increase in response to increases of osmolarity of the growth media (Miller & Wood 1996 ).

Brucella is a pathogenic bacterium that causes a chronic disease in mammals, including humans (Smith & Ficht, 1990 ). Brucella, like Agrobacterium and Rhizobium, forms cyclic 1,2-ß-glucans (Bundle et al., 1987 , 1988 ). As described in Agrobacterium and Rhizobium, the biosynthesis of cyclic 1,2-ß-glucans in Brucella spp. proceeds through a membrane-bound 1,2-ß-glucan synthetase, with the formation of protein-bound intermediates. A characteristic of Brucella cyclic 1,2-ß-glucan synthesis is that high osmolarity of the growth medium has no effect on cellular cyclic 1,2-ß-glucan accumulation (Briones et al., 1997 ). Recently, the B. abortus S19 cyclic 1,2-ß-glucan synthetase (cgs) gene was cloned, characterized and sequenced. The gene complemented A. tumefaciens chvB and R. meliloti ndvB mutants, restoring virulence, nodule development, motility and biosynthesis of cyclic 1,2-ß-glucan (Iñón de Iannino et al., 1998 ). It was also determined that a B. abortus cgs mutant displayed a reduced ability to survive in the spleen of mice, thus suggesting that, as in Agrobacterium, cyclic 1,2-ß-glucans may behave as virulence factors (Iñón de Iannino et al., 1998 ).

In this communication we characterized osmoregulation of the synthesis of cyclic 1,2-ß-glucan by Brucella spp., Agrobacterium, Rhizobium and various recombinant strains harbouring Brucella or Agrobacterium cyclic 1,2-ß-glucan synthetase genes.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and media.
Bacterial strains and plasmids used are listed in Table 1. A. tumefaciens A348 wild-type strain, A. tumefaciens chvB mutant A1011 and plasmid pCD523 (Douglas et al., 1985 ) were provided by Dr E. Nester (Dept Microbiology, Washington University). R. meliloti GR4 and GRT21s ndvB mutant (Geremía et al., 1987 ), and cosmid pBA19, containing a 19 kb B. abortus DNA fragment (Iñón de Iannino et al., 1998 ) have been described previously. A. tumefaciens strains were grown on tryptone–yeast extract (TY) medium (0·5% tryptone, 0·3% yeast extract). R. meliloti strains were grown on mannitol–yeast extract (AMA) medium (0·5 g K2HPO4 l-1, 0·2 g MgSO4 . 7H2O l-1, 0·2 g NaCl l-1, 1 g yeast extract l-1, 10 g mannitol l-1). Brucella strains were grown in Brucella broth medium (BB; Difco). Kanamycin or tetracycline were added when required to a final concentration of 100 µg ml-1 and 10 µg ml-1, respectively. When indicated, medium was supplemented with 0·25 M NaCl or 0·5 M mannitol.


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Table 1. Bacteria and plasmids used in this study

 
DNA techniques and gene manipulations.
B. abortus S19 cgs was cloned as described previously (Iñón de Iannino et al., 1998 ). R. meliloti ndvB mutant GRT21s and A. tumefaciens chvB mutant A1011 were complemented with cosmid pBA19 as described previously (Iñón de Iannino et al., 1998 ). R. meliloti GRT21s(pCD523) was obtained by using the triparental system described by Ditta et al. (1980) . Mating was carried out by patching similar numbers of donor cells [E. coli HB101(pCD523)], recipient cells (R. meliloti GRT21s) and helper cells [E. coli HB101(pRK2013)]. Transconjugants were selected on AB media containing the appropriate antibiotics. B. abortus BAI129(pCD523) was obtained by a biparental system. E. coli S17.1 carrying pCD523 was used as the donor. Transconjugants were selected on BB agar medium containing 5 µg nalidixic acid ml-1, 100 µg ampicillin ml-1, 100 µg kanamycin ml-1 and 10 µg tetracycline ml-1.

Isolation of cyclic 1,2-ß-glucan from cells and osmotic regulation studies.
Cells from 100 ml cultures were harvested by centrifugation at 10000 g for 10 min. Cell pellets were extracted with 10% TCA for 30 min at room temperature. The TCA extracts were neutralized with ammonium hydroxide, concentrated and subjected to gel filtration on Bio-Gel P4 columns (78x1·8 cm; Bio-Rad). The columns were eluted with 0·1 M pyridine–acetate buffer (pH 5·5). Fractions of 1·5 ml were collected and carbohydrates determined by the anthrone–sulfuric acid method (Dische, 1962 ). DEAE-Sephadex chromatography was carried out as described previously (Iñón de Iannino & Ugalde, 1989 ). Studies on the effect of osmolarity on cellular-glucan accumulation were carried out by growing the cells to stationary phase with 0·5 M mannitol or 0·25 M NaCl. Cells were harvested, cellular glucans extracted with TCA and subjected to Bio-Gel P4 chromatography as described above.

In vitro synthesis of cyclic 1,2-ß-glucans.
In vitro synthesis of 1,2-ß-glucan was studied with Brucella, Rhizobium or Agrobacterium total or inner membranes as described previously (Iñón de Iannino & Ugalde 1989 ; Briones et al., 1997 ). Membranes (0·1 mg protein) were incubated with 0·2 µCi UDP-[14C]glucose (specific activity 300 mCi mmol-1) at 28 °C (Agrobacterium or Rhizobium membranes) or at 37 °C (Brucella membranes). Formation of cyclic 1,2-ß-glucan and the presence of the inner-membrane-protein intermediate were estimated as described previously (Iñón de Iannino & Ugalde 1989 ). The enzymic activity was expressed as pmol [14C]glucose accumulated in the intermediate protein (10% TCA-insoluble products) per mg protein. When indicated, different concentrations of KCl or potassium glutamate were added. The basal activity was the level of activity in the standard assay mixture with no additional salts.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Effect of KCl on in vitro cyclic 1,2-ß-glucan synthesis
All the evidence accumulated so far supports the interpretation that the effect of high osmolarity on Agrobacterium and Rhizobium cyclic 1,2-ß-glucan accumulation is due to a direct inhibition of the cyclic 1,2-ß-glucan synthetase enzymic activity and not to an effect on cgs gene expression. Increasing the osmolarity of the growth medium led to an increase in the ionic strength of the cytosol, mainly due to an uptake of potassium ions (Miller & Wood, 1996 ). Inhibition of the cyclic 1,2-ß-glucan synthetase by high ionic strength might be the mechanism by which its activity is reduced when Agrobacterium and Rhizobium cells are grown in a medium of high osmolarity (Zorreguieta et al., 1990 ; Ingram-Smith & Miller, 1998 ).

To determine if the lack of effect of high osmolarity on Brucella cyclic 1,2-ß-glucan is due to an insensitivity of the Brucella cyclic 1,2-ß-glucan synthetase to high cytoplasmatic ionic strength, the effect of KCl on the in vitro cyclic 1,2-ß-glucan synthetase activity was studied. The effect of different concentrations of KCl on the incorporation of [14C]glucose into TCA-insoluble protein intermediates by membranes from different sources incubated with UDP-[14C]glucose is shown in Table 2. This assay quantified the formation of protein–glucosyl intermediates during initiation, the first of three reactions that take place during the synthesis of cyclic glucans. It can be seen that an inhibition of approximately 50% was obtained when 0·1 M KCl was added to membranes of A. tumefaciens A348 and R. meliloti 102F34, two strains in which the accumulation of cyclic 1,2-ß-glucan is osmoregulated. On the other hand, at that KCl concentration, membranes of R. meliloti GR4 and B. abortus S19, in which synthesis in vivo is not inhibited by the osmolarity of the growth media, showed an inhibition of less than 8%. The same behaviour was observed with B. abortus 2308 and Brucella ovis REO198 (data not shown). It can be observed that in A. tumefaciens A1011 chvB and R. meliloti GRT21s ndvB mutants complemented with B. abortus cgs, glucan synthesis was not inhibited by 0·25 M KCl, thus showing the same behaviour observed with B. abortus membranes.


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Table 2. Effect of KCl on the incorporation of radioactivity into 1,2-ß-glucan protein intermediates by membranes of different strains

 
Effect of potassium glutamate on in vitro synthesis
Immediately after an increase in external osmolarity, water is released from cells, reducing turgor and inducing shrinking. Under these conditions, K+ is accumulated in order to restore turgor. The uptake of K+ is the first response to osmotic stress, and glutamate is the most common counter-ion of K+. The effect of potassium glutamate on the incorporation of [14C]glucose into glucan–protein intermediates (initiation reaction) was studied and results very similar to those observed with KCl were obtained (data not shown).

To characterize the products accumulated in the TCA-insoluble fraction, SDS-PAGE was carried out. We demonstrated previously that the only protein that became labelled with [14C]glucose after incubation of membranes with UDP-[14C]glucose is the cyclic 1,2-ß-glucan synthetase (Iñón de Iannino et al., 1998 ). No protein was labelled when the incubation was carried out with membranes isolated from a cyclic 1,2-ß-glucan synthetase null mutant. As shown in Fig. 1, 0·5 M potassium glutamate inhibited the incorporation of [14C]glucose into the protein intermediate by A. tumefaciens A348 and by R. meliloti 102F34 (Fig. 1a, b). In contrast, the incorporation of [14C]glucose into protein intermediates was not inhibited by 0·5 M potassium glutamate when B. abortus S19 or R. meliloti GR4 membranes were used as the enzyme source (Fig. 1c, d). Membranes of A. tumefaciens A1011 chvB and R. meliloti GRT21s ndvB mutants complemented with the B. abortus S19 cgs gene were not inhibited by 0·5 M potassium glutamate (Fig. 1e, f). On the other hand, membranes of R. meliloti GRT21s ndvB or B. abortus BAI129 cgs mutants complemented with the A. tumefaciens cyclic 1,2-ß-glucan synthetase were inhibited by 0·5 M potassium glutamate (Fig. 1g, h).



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Fig. 1. Effect of potassium glutamate on the incorporation of [14C]glucose into glucan–protein intermediates of different strains. Total membranes (0·2 mg protein) of different strains were incubated with UDP-[14C]glucose as described in Methods, with or without the addition of 0·5 M potassium glutamate. Reactions were stopped by the addition of 10% TCA and the precipitates subjected to SDS-PAGE. Radioactivity was detected by autoradiography. (a), A. tumefaciens A348; (b) R. meliloti 102F34; (c), B. abortus S19; (d), R. meliloti GR4; (e), A. tumefaciens A1011(pBA19); (f), R. meliloti GRT21s(pBA19); (g), R. meliloti GRT21s(pCD523); (h), B. abortus BAI129(pCD523).

 
These results showed that Brucella cyclic 1,2-ß-glucan synthetase activity is not affected by concentrations as high as 0·5 M KCl or potassium glutamate. Both compounds strongly inhibited the in vitro activity of Rhizobium and Agrobacterium cyclic 1,2-ß-glucan synthetase at similar concentrations. These results suggest that the lack of inhibition of the Brucella enzyme is due to an intrinsic characteristic of the enzyme, the activity of which is not affected by concentrations of solutes that are known to be accumulated inside the cells upon growth in high-osmolarity media.

Effect of glycine betaine on A. tumefaciens glucosyl transferase activity
The effect of the osmoprotectant glycine betaine to reverse the inhibitory effect of potassium glutamate on the A. tumefaciens glucosyltransferase activity was studied as described in Methods. The addition of 0·1 M potassium glutamate to the reaction mixture inhibited the glucosyl transferase activity by 45%. However, the addition of 1 M glycine betaine prevented this inhibition, restoring the glucosyltransferase activity to 96%. We observed an inhibition of 60% when 0·5 M potassium glutamate was added; this inhibition was only partially reversed to 44% inhibition of enzyme activity when 1 M glycine betaine was added [100% enzymic activity corresponded to 210 pmol glucose h-1 (mg protein)-1]. These results demonstrated that the intracellular accumulation of glycine betaine may prevent inhibition of the glucosyltransferase activity by potassium glutamate accumulated intracellularly under osmotically stressful conditions.

Effect of osmolarity on cellular cyclic 1,2-ß-glucan accumulation
Various strains were grown in media of low or high osmolarity, as indicated under Methods. TCA extracts obtained from stationary-phase cultures were subjected to Bio-Gel P4 chromatography. Products recovered between fractions 5 and 18 (Fig. 2) were pooled and characterized as cyclic 1,2-ß-glucans by TLC as described by Iñón de Iannino et al. (1998) . Total accumulation of cellular cyclic 1,2-ß-glucan [mg glucose equivalents (g cellular wet weight)-1] was calculated from the total amount of glucan recovered from fractions 5 to 18 of Bio-Gel P4 columns. The addition of 0·25 M NaCl to the growth media abolished accumulation of cellular glucans by A. tumefaciens A348 wild-type strain (fractions 8 to 18 of Fig. 2a). Cosmid pBA19, containing the complete B. abortus cgs gene, restored synthesis of cyclic 1,2-ß-glucan in R. meliloti GRT21s ndvB and A. tumefaciens A1011 chvB mutants (Iñón de Iannino et al., 1998 ). It is shown in Fig. 2(b, g), that the accumulation of cellular cyclic 1,2-ß-glucan by A. tumefaciens A1011 chvB or R. meliloti GRT21s ndvB mutants complemented with B. abortus cgs, was not inhibited by the presence of 0·25 M NaCl in the growth medium. Similar results were obtained when these complemented strains were grown in medium containing 0·5 M mannitol (data not shown). Although increasing the osmolarity had no effect on the total amount of cellular glucan accumulated by R. meliloti GRT21s(pBA19) strain, when cells were grown in media containing 0·25 M NaCl, glucans were recovered from the Bio-Gel P4 column at an elution volume slightly higher than glucans recovered from the cells grown in medium without salt (Fig. 2g). The same results were obtained with the R. melitoti GR4 wild-type strain (Fig. 2e). As described below, differences in Bio-Gel P4 elution were caused by differences in glucan substitution. Glucans accumulated by the A. tumefaciens chvB mutant complemented with Brucella cgs grown in medium with 0·25 M NaCl eluted from the Bio-Gel P4 column at the same elution volume as those obtained from cells grown without NaCl. (Fig. 2b). The addition of 0·5 M mannitol to B. abortus S19 cultures had no effect on the accumulation of cellular glucans recovered from cells growing at high or low osmolarity (Fig. 2c). The same results were obtained with the Brucella cgs mutant harbouring the A. tumefaciens chvB gene (Fig. 2d). On the other hand, the R. meliloti GRT21s ndvB mutant complemented with the Agrobacterium cyclic 1,2-ß-glucan synthetase gene, displayed strong inhibition when grown in high-osmolarity medium (Fig. 2f).



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Fig. 2. Bio-Gel P4 chromatography of cyclic 1,2-ß-glucans accumulated in vivo by different strains in cells grown in medium of low and high osmolarity. Cells from 100 ml cultures were harvested and extracted with 10% TCA. Extracts were subjected to Bio-Gel P4 chromatography. Fractions were collected and carbohydrates measured as indicated in Methods. Carbohydrates were expressed as mg glucose equivalents (g wet weight)-1. (a), A. tumefaciens A348; (b), A. tumefaciens A1011(pBA19); (c), B. abortus S19; (d), B. abortus BAI129(pCD523); (e), R. meliloti GR4; (f), R. meliloti GRT21s(pCD523); (g), R. meliloti GRT21s(pBA19). (a), (b), (e), (f) and (g) contained 0·25 M NaCl; (c) and (d) contained 0·5 M mannitol. {bullet}, control; {circ}, with 0·25 M NaCl or 0·5 M mannitol.

 
Osmolarity affects the substitution of R. meliloti cellular glucans
Cellular glucans accumulated by R. meliloti GR4 or R. meliloti GRT21s complemented with the Brucella cyclic 1,2-ß-glucan synthetase grown in AMA medium supplemented or not with 0·25 M NaCl (fractions 5 to 18 of Fig. 2e, g) were recovered from the Bio-Gel P4 column and subjected to DEAE-Sephadex chromatography. As shown in Fig. 3, glucans recovered from cells grown without the addition of NaCl eluted from the DEAE-Sephadex column with 250 mM NaCl (fractions 4 to 6 of Fig. 3a, b), thus indicating that most of cellular glucans accumulated under this growth condition were modified with anionic substituents. However, glucans recovered from cells grown with 0·25 M NaCl eluted from the DEAE-Sephadex column with water (fractions 1 to 3 of Fig. 3c), thus indicating that most of them were non-substituted neutral glucans. An A. tumefaciens chvB mutant complemented with the B. abortus cgs gene produced, when grown in medium with 0·25 M NaCl, glucans with the same net charge as control cells (Fig. 3d–f), thus indicating that in the Agrobacterium background high osmolarity had no effect on glucan substitution.



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Fig. 3. DEAE–Sephadex chromatography of cellular glucans accumulated by different strains. Glucans were recovered from Bio-Gel P4 columns (fractions 5 to 18 of Fig. 2) and subjected to DEAE–Sephadex chromatography. Columns (0·25x3 cm) were eluted with 1·5 ml water (fractions 1–3), 1·5 ml 0·25 M NaCl (fractions 4–6) and 0·5 M NaCl (fractions 7–9). Fractions of 0·5 ml were collected and carbohydrates measured by the anthrone–sulfuric acid method (Dische et al., 1962 ). (a), R. meliloti GR4 grown in AMA medium; (b), R. meliloti GRT21s(pBA19) grown in AMA medium; (c), R. meliloti GRT21s(pBA19) grown in AMA medium with 0·25 M NaCl; (d), A. tumefaciens A348 grown in TY medium; (e), A. tumefaciens A1011(pBA19) grown in TY medium; (f), A. tumefaciens A1011(pBA19) grown in TY medium with 0·25 M NaCl.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We studied the effect of high osmolarity of the growth media on the accumulation of cyclic 1,2-ß-glucan in Agrobacterium, Rhizobium and Brucella. It is known that K+ and glutamate are solutes accumulated in the cytoplasm when bacteria are exposed to high osmolarity, in order to restore cell turgor (Csonka, 1991 ). The absence of inhibition by these solutes of cyclic 1,2-ß-glucan synthetase activity might explain in part the osmoresistant accumulation of glucan at high osmolarity by Brucella and R. meliloti strain GR4.

Synthesis of cyclic glucans by A. tumefaciens and R. meliloti complemented with Brucella cyclic 1,2-ß-glucan synthetase was not inhibited by high osmolarity of growth media. Since this is the behaviour observed with Brucella spp., we concluded that the enzyme is responsible for the lack of inhibition at high osmolarity. Brucella cyclic 1,2-ß-glucan synthetase in the Agrobacterium or Rhizobium background was not inhibited by 0·5 M KCl or potassium glutamate, thus indicating that the Brucella enzyme is insensitive to inhibition by those endocellular osmolytes accumulated under osmotic stress. Since the in vivo accumulation of cyclic glucan by the recombinant B. abortus BAI129(pCD523) strain (B. abortus BAI129 cgs mutant complemented with the A. tumefaciens cyclic 1,2-ß-glucan synthetase gene) was not inhibited by the high osmolarity of the medium, but the enzyme was inhibited in vitro by KCl or potassium glutamate, we may postulate the following hypothesis: I) cyclic 1,2-ß-glucan synthesis is not regulated by the osmotic inhibition of the activity of the cyclic 1,2-ß-glucan synthetase or II) Brucella may have additional mechanisms that prevent inhibition of cyclic 1,2-ß-glucan synthesis under osmotically stressful conditions, in addition to possessing an enzyme resistant to these osmolytes. These additional mechanisms may be the ability of Brucella to accumulate different osmolytes or osmoprotective solutes, such as glycine betaine. Interestingly we have found that B. abortus has three genes highly homologous to proV, proW and proX involved in the transport of glycine betaine (D. O. Sanchez, R. Zandomeni & S. Cravero, unpublished). Glycine betaine reversion of the inhibitory effect of potassium glutamate on A. tumefaciens 1,2-ß-glucan synthetase activity, which has been described by Ingram-Smith & Miller (1998) in R. meliloti, may be the mechanism operative in Brucella. Either one, or both, of these two possibilities (lack of glucan synthetase inhibition by salts or/and accumulation of protective osmolytes) may be responsible for the lack of inhibition of cyclic glucan accumulation under high osmolarity by a Brucella mutant expressing the A. tumefaciens enzyme.

We have shown the synthesis of anionic cyclic 1,2-ß-glucans by R. meliloti GR4. Although synthesis of cyclic 1,2-ß-glucans was not osmoregulated, the anionic substitution of cyclic 1,2-ß-glucans was inhibited by high osmolarity, indicating that they are independent processes. This behaviour is different from that observed in Agrobacterium, in which high osmolarity has no effect on anionic substitution of cyclic glucans synthesized by the osmoresistant Brucella enzyme. These results are consistent with earlier studies, which showed that synthesis of glycerophosphorylated cyclic glucans in R. meliloti 1021 was inhibited in cells grown at high osmolarity (Breedveld & Miller, 1995 ; Wang et al., 1999 ).


   ACKNOWLEDGEMENTS
 
We thank J. J. Cazzulo and Armando J. Parodi for critical reading of the manuscript, E. W. Nester for providing A. tumefaciens strains A348, A1011 and plasmid pCD523. This work was supported in part by grants from the Ministerio de Cultura y Educación, República Argentina, the Instituto de Investigaciones Biotecnológicas de la Universidad Nacional de General San Martín, the Agencia Nacional de Promoción Científica y Tecnológica PICT 97-00080-01768 and from Consejo Nacional de Investigaciones Científicas y Técnicas (PEI No 0183/98). We acknowledge the financial support of the Universidad Nacional de General San Martín. N.I. and R.A.U. are members of the Research Career of CONICET.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Briones, G., Iñón de Iannino, N., Steinberg, M. & Ugalde, R. A. (1997). Periplasmic cyclic 1,2-ß-glucan in Brucella spp. is not osmoregulated.Microbiology 143, 1115-1124.[Abstract]

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Received 2 November 1999; revised 7 February 2000; accepted 27 March 2000.