Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.     

Identification and isolation of genes involved in poly(beta-hydroxybutyrate) biosynthesis in Azospirillum brasilense and characterization of a phbC mutant

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(beta-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway, phbA (beta-ketothiolase), phbB (acetoacetyl coenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned, and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome. An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbC gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the phbC mutant strain than in the wild type.     

Poly β-hydroxybutyrate depolymerase (PhaZ) in<Emphasis Type="Italic"> Azospirillum brasilense</Emphasis> and characterization of a<Emphasis Type="Italic"> phaZ</Emphasis> mutant

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly--hydroxybutyrate (PHB) under sub-optimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments. PHB depolymerase (PhaZ) is an essential enzyme in PHB degradation. The phaZ gene was identified in Azospirillum brasilense, cloned, sequenced, and shown to be located on the chromosome. Insertion of a kanamycin-resistant cassette within phaZ of A. brasilense resulted in a phaZ mutant that was unable to degrade PHB; however, carbon source utilization was similar in both the wild-type and the mutant strain. The ability of the wild-type to endure starvation conditions, ultraviolet irradiation, heat, and osmotic shock, and to grow in the presence of hydrogen peroxide was higher than that of the mutant strain. By contrast, the ability of the phaZ mutant strain to endure desiccation was higher than that of the wild-type strain. No differences between the strains were seen in their ability to endure sonication, or to survive in carrier materials used for soil inoculants. In addition, motility was the same between the two strains, whereas cell aggregation and exopolysaccharide production were higher in the wild-type than in the phaZ mutant strain.     

Identification and Isolation of Genes Involved in Poly(β-Hydroxybutyrate) Biosynthesis in Azospirillum brasilense and Characterization of a phbC Mutant

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(β-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway, phbA (β-ketothiolase), phbB (acetoacetyl coenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned, and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome. An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbC gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the phbC mutant strain than in the wild type.     

Poly-β-hydroxybutyrate metabolism in Azospirillum brasilense and the ecological role of PHB in the rhizosphere

Abstract Azospirillum brasilense is a rhizosphere microorganism which has potential use for promoting plant growth in economically important crops. Its ability to survive the adverse conditions imposed by nutrient starvation and competition in the rhizosphere is of great importance. A. brasilense accumulates up to 70% of its cell dry weight with poly-β-hydroxybutyrate (PHB). In the presence of stress factors such as ultraviolet radiation, desiccation and osmotic stress, PHB-rich cells survived better than PHB-poor cells. Polymer-rich cells of Azospirillum fixed N₂ in the absence of exogenous carbon and combined nitrogen. The enzymes of the PHB cycle in both the synthesis and degradation processes as well as during starvation were more active in PHB-rich cells. After 24 h of starvation there was a peak of activity of d (−)β-hydroxybutyrate dehydrogenase, β-ketothiolase and thiophorase due to PHB degradation. Additionally, acetoacetyl-CoA reductase dropped to a minimum level because PHB could not be synthesized. The possible utilization of PHB as a sole carbon and energy source by A. brasilense and other bacteria during establishment, proliferation and survival in the rhizosphere will be discussed.     

Glycogen phosphorylase is involved in stress endurance and biofilm formation in Azospirillum brasilense Sp7

Here we report the identification of a glycogen phosphorylase ( glgP ) gene in the plant growth-promoting rhizobacterium Azospirillum brasilense , Sp7, and the characterization of a glgP marker exchange mutant of this strain. The glgP mutant showed a twofold reduction of glycogen phosphorylase activity and an increased glycogen accumulation as compared with wild-type Sp7, indicating that the identified gene indeed encodes a protein with glycogen phosphorylase activity. Interestingly, the glgP mutant had higher survival rates than the wild type after exposure to starvation, desiccation and osmotic pressure. The mutant was shown to be compromised in its biofilm formation ability. Analysis of the exopolysaccharide sugar composition of the glgP mutant revealed a decrease in the amount of glucose, accompanied by increases in rhamnose, fucose and ribose, as compared with the Sp7 exopolysaccharide. To the best of our knowledge, this is the first study that demonstrates GlgP activity in A. brasilense , and shows that glycogen accumulation may play an important role in the stress endurance of this bacterium.     

Survival of poly-3-hydroxybutyrate-producing bacteria in soil microcosms

Bacillus megaterium is a potential bioremediation and biocontrol agent. The accumulation of reserve polymers, such as poly-3-hydroxybutyrate (PHB), increases survival of B. megaterium in water. We used wild-type strains of this species and mutant strains deficient in PHB synthesis in soil microcosms for testing the hypothesis that differences in survival capabilities and spore quality between strains is maintained in heterogeneous environments enriched with organic matter. No differences in survival between strains, nor a decrease in bacterial cell numbers were observed in sterile soil microcosms. In non-sterile soil, the total cell number (vegetative cells plus spores) of the PHB wild-type strain was 3.5 times higher than that of the PHB-negative mutant. We suggest that for predictive purposes, validation of survival in a variety of conditions is necessary.     

Arabinose content of extracellular polysaccharide plays a role in cell aggregation of Azospirillum brasilense

Extracellular polysaccharides play an important role in aggregation and surface colonization of plant-associated bacteria. In this work, we report the time course production and monomer composition of the exopolysaccharide (EPS) produced by wild type strain and several mutants of the plant growth promoting rhizobacterium (PGPR) Azospirillum brasilense. In a fructose synthetic medium, wild type strain Sp7 produced a glucose-rich EPS during exponential phase growth and an arabinose-rich EPS during stationary and death phase growth. D-glucose or L-arabinose did not support cell growth as sole carbon sources. However, glucose and arabinose-rich EPSs, when used as carbon source, supported bacterial growth. Cell aggregation of Sp7 correlated with the synthesis of arabinose-rich EPS. exoB (UDP-glucose 4'-epimerase), exoC (phosphomannomutase) and phbC (poly-beta-hydroxyburyrate synthase) mutant strains, under tested conditions, produced arabinose-rich EPS and exhibited highly cell aggregation capability. A mutant defective in LPS production (dTDP 4-rhamnose reductase; rmlD) produced glucose-rich EPS and did not aggregate. These results support that arabinose content of EPS plays an important role in cell aggregation. Cell aggregation appears to be a time course phenomenon that takes place during reduced metabolic cell activity. Thus, aggregation could constitute a protected model of growth that allows survival in a hostile environment. The occurrence of exoC and rmlD was detected in several species of Azospirillum.     

The ntrB and ntrC genes are involved in the regulation of poly-3-hydroxybutyrate biosynthesis by ammonia in Azospirillum brasilense Sp7

Azospirillum brasilense Sp7 and its ntrA (rpoN), ntrBC, and ntrC mutants have been evaluated for their capabilities of poly-3-hydroxybutyrate (PHB) accumulation in media with high and low ammonia concentrations. It was observed that the ntrBC and ntrC mutants can produce PHB in both low- and high-C/N-ratio media, while no significant PHB production was observed for the wild type or the ntrA mutant in low-C/N-ratio media. Further investigation by fermentation analysis indicated that the ntrBC and ntrC mutants were able to grow and accumulate PHB simultaneously in the presence of a high concentration of ammonia in the medium, while little PHB was produced in the wild type and ntrA (rpoN) mutant during active growth phase. These results provide the first genetic evidence that the ntrB and ntrC genes are involved in the regulation of PHB synthesis by ammonia in A. brasilense Sp7.     

The role of chemotaxis genes in establishing the associative relations between Azospirillum brasilense and wheat

The chemotactic properties of a number of Azospirillum brasilense natural strains have been studied. Azospirillum demonstrate the positive chemotactic reaction towards the organic acids salts but a poor reaction towards the presence of the attractants like hydrocarbons and aminoacids except for arabinose and glutamic acid. The series of Che- mutants deficient in general chemotaxis has been selected by introducing the transposon Tn5 into the cells of rifampicinresistant mutant strain Azospirillum brasilense 5T-2. The ability of the mutant cells to fast and solid adsorption on the roots of the sterile wheat sprouts is shown to be decreased 2-3 folds as compared with the one of the wild type strain. Chemotaxis is suggested to affect the adsorbtion ability of azospirillum and their associative properties.     

Effects of heavy metals on plant-associated rhizobacteria: comparison of endophytic and non-endophytic strains of Azospirillum brasilense.

The plant-associated nitrogen-fixing rhizobacterium Azospirillum brasilense attracts world-wide attention owing to its plant growth-promoting activities. Among hundreds of its strains known up to date, wild-type strain Sp245 has been proved to be capable of colonising both the plant-root interior and exterior (i.e. a facultative endophyte), whereas others are non-endophytes colonising the root surface only. Thus, the different ecological niches occupied by these strains in the rhizosphere suggest that their responses to environmental conditions might differ as well. In this study, responses of A. brasilense strains Sp245 and Sp7 to several heavy metals (Co2+, Cu2+, Zn2+), present in the medium at tolerable concentrations (up to 0.2 mmol/l) and taken up by the bacteria, were compared. Fourier transform infrared (FTIR) spectroscopy was used for controlling the compositional features of whole cells. The results obtained show that in strain Sp7 (non-endophyte) the heavy metals induced an enhanced accumulation of polyester compounds (poly-3-hydroxybutyrate; PHB). In contrast, the response of the endophytic strain Sp245 to heavy metal uptake was found to be much less pronounced. These dissimilarities in their behaviour may be caused by different adaptation abilities of these strains to stress conditions owing to their different ecological status. It was also found that adding 0.2 mmol/l Cu2+ or Cd2+ in the culture medium resulted in noticeably reducing the levels of indole-3-acetic acid (IAA, auxin) produced by both the strains of the bacterium. This can directly affect the efficiency of associative plant-bacterial symbioses involving A. brasilense in heavy-metal-contaminated soil.     

Effect of poly(3-hydroxybutyrate) (PHB) content on the starvation-survival of bacteria in natural waters

Abstract The effect of poly(3-hydroxybutyrate) (PHB) content on the survival of wild-type strains and PHB negative mutants of Bacillus megaterium and Alcaligenes eutrophus in natural waters was studied. The survival strategy of B. megaterium was dominated by the development of resistant forms, but the number of the wild-type vegetative cells was higher than that of PHB mutant strain. In some environmental conditions the mutant spores needed a heat shock for germination, a fact that suggests, for the first time, that PHB plays a role in this phenomenon. Survival of A. eutrophus wild-type strain in all experiments was higher compared to the PHB mutant, and differences were significant. In raw river water, survival of both species was lower than in sterile river water.     

Transfer of transformation and conjugation plasmids from Escherichia coli to Bacillus subtilis and Azospirillum brasilense]

The conjugative transfer of RP4 plasmid from Escherichia coli to Azospirillum brasilense was detected after introduction and subsequent incubation of these microorganisms in soil. The plasmid transfer via transformation from Escherichia coli to Bacillus subtilis was observed in case both bacteria were growing together in sand containing sucrose solution. The possible reason for low frequency interspecies plasmid transformation under conditions close to natural habitats is poor survival of "domesticated" rather than wild type Bacillus subtilis strains and lack of competence state in this case.     

Control of Nitrogenase Reactivation by the GlnZ Protein in Azospirillum brasilense

The glnZ mutant of Azospirillum brasilense (strain 7611) showed only partial recovery (20 to 40%) after 80 min of ammonia-induced nitrogenase switch-off, whereas the wild type recovered totally within 10 min. In contrast, the two strains showed identical anoxic-induced switch-on/switch-off, indicating no cross talk between the two reactivation mechanisms.     

The role of cell surface bacterial lectins in the aggregation of Azospirilla

The mutant strain Azospirillum brasilense Sp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilense Sp7(S) both in the exponential and stationary growth phases. The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla. The specific binding of the A. brasilense Sp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters. The interaction of the lectins of A. brasilense 75, A. brasilense Sp7, and A. lipoferum 59b with the polysaccharide-containing complexes that were isolated from these strains was not specific. No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found. A coflocculation of A. brasilense Sp7 cells with Bacillus polymyxa 1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.     

Comparison of the symbiotic and competition phenotypes of Sinorhizobium meliloti PHB synthesis and degradation pathway mutants

The competitive abilities of Sinorhizobium meliloti mutant strains containing lesions in the PHB synthesis (phbC) and degradation (bdhA) pathways were compared. While the bdhA mutant showed no noticeable symbiotic defects on alfalfa host plants when inoculated alone, in mixed inoculation experiments it was found to be less competitive than the wild type for nodule occupancy. Long-term survival of the bdhA mutant on a carbon-limiting medium was not affected. However, when subjected to competition with the wild-type strain in periodic subculturing through alternating carbon-limiting and carbon-excess conditions, the bdhA mutant performed poorly. A more severe defect in competition for growth and nodule occupancy was observed with a mutant unable to synthesize PHB (phbC). These results indicate that the ability to efficiently deposit cellular PHB stores is a key factor influencing competitive survival under conditions of fluctuating nutrient carbon availability, whereas the ability to use these stores is less important.     

Involvement of glnB, glnZ, and glnD genes in the regulation of poly-3-hydroxybutyrate biosynthesis by ammonia in Azospirillum brasilense Sp7

The role of three key nitrogen regulatory genes, glnB (encoding the P(II) protein), glnZ (encoding the P(z) protein), and glnD (encoding the GlnD protein), in regulation of poly-3-hydroxybutyrate (PHB) biosynthesis by ammonia in Azospirillum brasilense Sp7 was investigated. It was observed that glnB glnZ and glnD mutants produce substantially higher amounts of PHB than the wild type produces during the active growth phase. glnB and glnZ mutants have PHB production phenotypes similar to that of the wild type. Our results indicate that the P(II)-P(z) system is apparently involved in nitrogen-dependent regulation of PHB biosynthesis in A. brasilense Sp7.     

Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense.

The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [32P]phosphate or [3H]uracil labeling or by cross-reaction with an anti-A. brasilense PII-antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII. Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.     

Characterization of cell surface and extracellular matrix remodeling of Azospirillum brasilense chemotaxis-like 1 signal transduction pathway mutants by atomic force microscopy

To compete in complex microbial communities, bacteria must sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the regulation of multiple behaviors in response to changes in the environment, including motility patterns, exopolysaccharide production, and cell-to-cell interactions. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting flocculation. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Whereas the wild-type strain produces a smooth mucosal extracellular matrix after 24 h, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition, lectin-binding assays, and comparison of lipopolysaccharides profiles suggest that the extracellular matrix differs between the cheA1 and the cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition.