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Keywords:

  • Exopolysaccharide;
  • Capsular polysaccharide;
  • Cell aggregation;
  • Flocculation;
  • Azospirillum brasilense

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

The exopolysaccharide (EPS) and capsular polysaccharide (CPS) composition of four Azospirillum brasilense strains differing in their aggregation capacity was analyzed by high performance anion exchange chromatography. When growing the different strains in an aggregation inducing medium containing a high carbon:nitrogen (C:N) ratio, both EPS and CPS showed a positive correlation between aggregation and the relative amount of arabinose. Arabinose was not detected in polysaccharides from Sp72002, a pleiotrophic Tn5 mutant strain impaired in aggregation. Arabinose was also not detected in extracellular polysaccharides of bacteria grown in a low C:N ratio, non-inducing aggregation medium, with exception for a relatively small amount found in the CPS of FAJ0204, a super-aggregating mutant strain. The only monosaccharides able to significantly inhibit aggregation at low sugar concentration when tested in a bioassay were arabinose (at a higher extent) and galactose. The possibility that residues of arabinose present in the extracellular polysaccharides are involved in the aggregation of A. brasilense is discussed.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

The free-living N2-fixing rhizobacteria of the genus Azospirillum live in close association with plant roots, and may exert beneficial effects on plant growth and yield of many crops of agronomic importance [1,2]. Azospirillum brasilense cells possess the capacity to aggregate and flocculate, and this property may positively affect their dispersal and survival in soil [3,4]. Azospirilla are particularly responsive to variations in the carbon:nitrogen (C:N) ratio. Rich media with a low C:N ratio tend to promote a dispersive growth mode, whereas in the presence of a medium with high C:N ratio the cells tend to aggregate and flocculate [4–8].

Along with the fact that flocs can be produced on a large scale and separated easily from the growth medium, the phenomenon of bacterial aggregation is of great interest in the production of bacterial inoculants for agriculture. Neyra et al. [9] proposed the generation of inoculants containing flocs of Azospirillum and Rhizobium for common bean plants.

Data suggesting the involvement of extracellular polysaccharides (exopolysaccharide, EPS; capsular polysaccharide, CPS) in aggregation of Azospirillum have been published. The production of surface polysaccharide by Azospirillum has been demonstrated by growing colonies on media containing the fluorescent dye calcofluor, which binds predominantly to β-1,4- and β-1,3-linked glucans [10]. Non-fluorescent mutants of A. brasilense and Azospirillum lipoferum have lost the ability to form flocs and to anchor to wheat roots, indicating that the calcofluor-binding polysaccharide may be necessary for cell aggregation and root attachment [11,12]. Sadasivan and Neyra [4] related floc formation in A. brasilense Sp7 and A. lipoferum to β-linked EPSs. It has recently been found that a spontaneous mutant of A. brasilense strain Sp7 which forms colonies that stain weakly with Congo red does not flocculate and lacks cell-surface materials found in the parental strain [13]. Genetic complementation of this mutant led to the identification of flcA, a gene involved in the regulation of different processes in A. brasilense, including flocculation [14].

In a previous work, a medium for consistent induction of aggregation of A. brasilense cells was developed and the effects of chemical and physical factors on this phenomenon were studied [8]. Growth of A. brasilense strain Cd in a medium with a high C:N ratio using fructose and ammonium chloride as C and N sources, respectively, resulted in flocculation visible to the naked eye after 24 h, whereas no cell aggregates were formed after 72 h of growth in a low C:N medium. The concentration of EPS produced by different strains of A. brasilense varying in their capacity to aggregate was shown to strongly correlate to the extent of aggregation [8]. Since the extent of aggregation may be determined not only by quantitative but also by qualitative differences in surface polysaccharide production, the present study was carried out with the objective of comparing these strains for the composition of their extracellular polysaccharides.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

2.1Bacterial strains and growth conditions

The A. brasilense strains used in this study were: wild-type strains Cd [15] and Sp7 [16]; FAJ0204, a Tn5 mutant of Sp7 defective in the production of both lateral and polar flagella, kindly supplied by J. Vanderleyden (Catholic University of Leuven, Belgium); Sp72002 [14], a pleiotrophic Tn5 mutant of Sp7 impaired in flcA, and affected in its aggregation capacity, kindly supplied by C. Elmerich (Institute Pasteur, Paris, France). Bacteria were maintained on nutrient agar (Difco, Detroit, MI, USA) slants. Kanamycin was added at 50 μg ml−1 for the Tn5 mutant strains. For aggregation assays and extracellular material extraction, bacteria were grown in 250-ml Erlenmeyer flasks, with 100 ml liquid medium. High and low C:N media were as previously described [8]. Flasks were inoculated with exponential phase cultures at an initial OD540 of approximately 0.05 (about 107 colony forming units ml−1), and incubated on a rotary shaker (150 rpm) at 30°C.

2.2Extraction of extracellular polysaccharides

Polysaccharides were extracted as described by Del Gallo and Haegi [6] with some modifications. Following growth for 24 h, bacterial cultures were centrifuged at 4000×g for 20 min at 4°C. The supernatant was collected, filtered through a 0.22-μm membrane and left to stand in three volumes of cold ethanol for 24 h at 4°C, resulting in EPS precipitation. The precipitated polysaccharides were isolated by centrifugation (30 000×g, 20 min, 4°C), resuspended in and dialyzed against distilled water for 72 h at 4°C, using a dialysis membrane with a molecular mass cutoff of 2 kDa. The bacterial pellet from the first centrifugation was resuspended in 10 volumes of phosphate-buffered saline and left to stand for 7 days at 4°C. Thereafter, cells were removed by centrifugation (4000×g, 20 min, 4°C), and the CPS containing supernatant was filtrated through a 0.22-μm membrane and dialyzed against distilled water as described for the EPS. The amounts of sugar in the polysaccharidic fractions were evaluated by the anthrone method, using glucose as a standard [17], and were in accordance with previous results [8].

2.3High performance anion exchange (HPAE) chromatography

Following polysaccharide extraction, samples were lyophilized and then hydrolyzed in 0.5 M H2SO4 at 90°C for 18 h. After neutralization with Ba(OH)2 and centrifugation (7000×g, 15 min, 4°C), the supernatants were filtered through 0.45-μm membranes and samples of 25 μl were applied onto a Dionex HPAE chromatography coupled with pulsed amperometric detection using a Carbo Pac™-PA1 column (250×4 mm; Dionex Co., Sunnyvale, CA, USA) with 16 mM NaOH as eluant and a flow rate of 1.0 ml min−1. Glucose, galactose, fucose, rhamnose and arabinose, which are five from the main components of the extracellular polysaccharides of A. brasilense grown on fructose as carbon source [18,19], were well resolved using this procedure.

2.4Aggregation bioassay

The bioassay is based on findings from previous work in which sonication of A. brasilense flocs for a relatively short period (20 s) was shown to be efficient in temporarily disrupting the cell aggregates [8]. The assay is fully described in Burdman et al. [20]. Briefly, it consists of adding the supernatant of 20-s sonicates of high C:N-grown (aggregating) strain Cd cells to low C:N-grown (non-aggregating) cells. The latter are then able to aggregate and flocculate after an incubation period of 2–3 h in a conical tube [20]. In the present study, the effects of different monosaccharides on aggregation were tested by adding them to the bioassay at various concentrations. The percentage of aggregation was measured as described previously [8]. The aggregate containing suspension was allowed to stand and after 20 min, aggregates had settled to the bottom of the tube, and the suspension was mostly composed of free (dispersed) cells. Turbidity was measured from the suspension using a Genesis 5 spectrophotometer (Spectronic Ins.) at 540 nm (ODs). The culture was then dispersed by a tissue homogenizer (Heidolph RzR 50) for 1 min, and the total turbidity was measured (ODt). Percentage of aggregation was estimated as follows: % aggregation=(ODt−ODs)×100/ODt.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

The relative concentrations (in %) of the tested monosaccharides detected from the EPS and CPS of four A. brasilense strains, grown in aggregation and non-aggregation inducing conditions (high and low C:N, respectively), are presented in Figs. 1 and 2. These percentages do not take into account other sugar components such as residues of galacturonic acid, glucosamine and galactosamine that were also reported to be present in the extracellular polysaccharides of some A. brasilense strains [6,18,19], but at lower concentrations than the sugars tested in the present study. These sugars are not well resolved by the used method.

image

Figure 1. Relative concentration (%) among five of the main monosaccharides in the EPS of four A. brasilense strains grown for 24 h in high (H) and low (L) C:N medium. Columns represent averages (from three replicates) from one of two independent experiments with similar results. nd=not detectable. Different letters indicate significant differences (P=0.05) among the various sugars in each strain, at high or low C:N ratio, as determined by one-way analysis of variance (ANOVA). Asterisks in (H) indicate significant differences (P=0.05) in the relative amount of the referred sugar in each strain between high and low C:N conditions, as determined by t-test analysis. Differences in the relative amount of each sugar among the different strains as determined by one-way ANOVA are referred in the text.

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image

Figure 2. Relative concentration (%) among five of the main monosaccharides in the CPS of four A. brasilense strains grown for 24 h in high (H) and low (L) C:N medium. Columns represent averages (from three replicates) from one of two independent experiments with similar results. nd=not detectable. Statistical analysis was carried out and expressed as for the EPS (see Fig. 1 for legend).

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In general, glucose was statistically significant present at the highest concentrations in all strains, both in the EPS and the CPS fractions. This was true for most of the strains grown under high as well as low C:N. The highest relative amounts of glucose were observed in the EPS of low C:N-grown strains (Fig. 1). In contrast, increased relative amounts of glucose in the CPS of high C:N-grown bacteria as compared to low C:N-grown bacteria were observed (Fig. 2). Rhamnose was also found to be generally present in relatively high amounts, especially in the CPS fractions. In the tested strains, the relative amounts of rhamnose in the CPS fractions obtained from low C:N medium were significantly higher when compared to CPS fractions originating from high C:N medium (Fig. 2). This trend was not observed in the different EPS fractions (Fig. 1).

Similarly to previous reported data [8], the extent of aggregation of the different strains after 24 h of growth in high C:N medium was determined to be approximately 15, 40 and 60% for strains Cd, Sp7 and FAJ0204, respectively. As previously indicated, strain Sp72002 is a pleiotrophic Tn5 mutant impaired in aggregation. The relative amounts of arabinose present in the EPS of these four strains when grown in the high C:N medium (Fig. 1) were significantly different between each other (P=0.05), and strongly correlated with the extent of aggregation they exhibited (r2=0.85). Moreover, in the super-aggregative mutant strain FAJ0204, arabinose was significantly found to be the predominant sugar (Fig. 1). In the CPS from high C:N-grown bacteria (Fig. 2), the relative amount of arabinose also correlated with the aggregation values but to a smaller extent (r2=0.75). No arabinose could be detected in the extracellular polysaccharides of strain Sp72002. Moreover, no arabinose was found in the extracellular polysaccharides of bacteria grown in low C:N (Figs. 1 and 2), except for a small amount present in the CPS of strain FAJ0204, which, under these growth conditions, exhibited an extent of aggregation of about 20% after 24 h. No correlation was found between the relative amount of any of the other tested monosaccharides and extent of aggregation.

The EPS fractions of the four tested strains grown under high C:N conditions were also analyzed by gas liquid chromatography-mass spectrometry (GLC-MS). This analysis was carried out on the O-trimethylsilyl derivatives of the methyl glycosides [21]. In addition to the previously HPAE-detected monosaccharides, residues of mannose, xylose and allose were also found in the EPS of the different strains when analyzed by GLC-MS. In contrast, fucose could not be detected using this technique. In general, similar results to those obtained by HPAE chromatography were obtained by GLC-MS, confirming the relatively high amount of arabinose present in the EPS of high C:N-grown FAJ0204 cells. Indeed, this strain showed concentration ratios of 2.0, 1.0, 0.5, 0.5, 0.3, 0.2 and 0.1 for arabinose, glucose, galactose, rhamnose, xylose, allose and mannose, respectively. In contrast, arabinose could not be detected also by this technique when analyzing the EPS composition of the mutant Sp72002, which exhibited concentration ratios of 1.0, 0.6, 0.3, 0.2 and 0.1 for glucose, galactose, rhamnose, mannose and allose, respectively.

An aggregation bioassay was utilized to evaluate the influence of various monosaccharides (most of them present in the extracellular polysaccharides of A. brasilense) on aggregation. If a specific component in the extracellular polysaccharides plays a role in aggregation, it is expected that its addition will cause an inhibition of this process by obstructing the adhesion sites. Fig. 3 shows the results of one of three independent experiments, all exhibiting statistically similar results. At a low sugar concentration of 0.01 M, arabinose (at a higher extent) and galactose significantly inhibited cell aggregation while the other tested monosaccharides were ineffective, not being significantly different from the untreated controls. The extent of inhibition was shown to be concentration-dependent (Fig. 3). Only at higher concentrations, relatively low inhibitory effects (in comparison to arabinose and galactose) were observed with the other sugars with the exception of fructose, which among the tested sugars is the only monosaccharide not present in the extracellular polysaccharides of A. brasilense.

image

Figure 3. Effects of different monosaccharides at various concentrations on aggregation of low C:N-grown A. brasilense strain Cd after 4 h of incubation with 20-s sonicates of high C:N-grown cells, according to Burdman et al. [20]. Bars represent averages from one representative experiment (three replicates per treatment). Different letters indicate significant differences (P=0.05) among the various monosaccharides for each sugar concentration, as determined by one-way ANOVA. Ar, arabinose; Ga, galactose; Rh, rhamnose; Fu, fucose; Gl, glucose; Fr, fructose; Co, control (without sugar addition).

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

Published data support the hypothesis that extracellular polysaccharides are involved in aggregation of Azospirillum (see Section 1). We previously reported on evidences indicating that outer membrane proteins (OMPs) are involved in this phenomenon [20]. The strains used in the present study differ in their aggregation capacity but exhibit similar OMPs profiles. Moreover, no differences are observed in OMPs profiles between cells grown under high and low C:N conditions [8]. However, these strains did differ in the amounts of EPS produced, which were shown to correlate to their extents of aggregation [8]. As was shown in the present study, the composition of the EPS and CPS also differed between the tested strains. Therefore, it is possible that the OMP(s) involved in aggregation is (are) constitutively present in A. brasilense, with differences in the extent of aggregation among the various strains being related to the amount, composition and structure of the extracellular polysaccharide. Interactions between proteins and polysaccharides leading to cell aggregation are well established in the microbial world, with good examples being the well characterized systems of Myxobacteria[22] and dental plaques [23].

Analysis of the relative concentration of five from the main monosaccharides present in the extracellular polysaccharides of the different strains suggests that arabinose present in the EPS and in the CPS may play an important role in determining the aggregation capability of A. brasilense. This hypothesis was strengthened by the fact that L-arabinose is able to strongly inhibit aggregation when added to the bacterial medium in an aggregation bioassay. Recently, in a screening for cell-surface lectins of A. brasilense, it was found that arabinose was the only sugar capable of completely inhibiting agglutination of A. brasilense Sp245 cells using latex beads covered with five different neoglycoproteins [24], thus providing other evidence on the possible involvement of this sugar in aggregation. These findings do not exclude that residual sugars in the extracellular polysaccharides of the bacterium other than arabinose may also be involved in cell aggregation although evidences for this possibility are lacking from this as well as from previous works.

We are currently investigating other features of the OMPs and of the extracellular polysaccharides of A. brasilense in order to elucidate the biological factors involved in cell to cell adhesion of this bacterium. Since it seems that surface components that are associated with the aggregation process are also important in plant root colonization [11,12,19], advances in the understanding of the aggregation process may also broaden our knowledge on the interactions that lead to the adhesion and colonization of plant roots by the bacteria.

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References

We thank J. Vanderleyden (Catholic University of Leuven, Belgium) and C. Elmerich (Institute Pasteur, Paris, France) for supplying strains for this research, and M. Megias (University of Sevilla, Spain) for helpful discussions. This work was supported by a grant from ‘The Israel Science Foundation’ founded by ‘The Academy of Sciences and Humanities’.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgments
  8. References
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