The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis.

Azospirillum brasilense is a plant root-colonizing bacterium that exerts beneficial effects on the growth of many agricultural crops. Extracellular polysaccharides of the bacterium play an important role in its interactions with plant roots. The pRhico plasmid of A. brasilense Sp7, also named p90, carries several genes involved in synthesis and export of cell surface polysaccharides. We generated two Sp7 mutants impaired in two pRhico-located genes, noeJ and noeL, encoding mannose-6-phosphate isomerase and GDP-mannose 4,6-dehydratase, respectively. Our results demonstrate that in A. brasilense Sp7, noeJ and noeL are involved in lipopolysaccharide and exopolysaccharide synthesis. noeJ and noeL mutant strains were significantly altered in their outer membrane and cytoplasmic/periplasmic protein profiles relative to the wild-type strain. Moreover, both noeJ and noeL mutations significantly affected the bacterial responses to several stresses and antimicrobial compounds. Disruption of noeL, but not noeJ, affected the ability of the A. brasilense Sp7 to form biofilms. The pleiotropic alterations observed in the mutants could be due, at least partially, to their altered lipopolysaccharides and exopolysaccharides relative to the wild-type.

A thioredoxin of Sinorhizobium meliloti CE52G is required for melanin production and symbiotic nitrogen fixation.

A miniTn5-induced mutant of a melanin-producing strain of Sinorhizobium meliloti (CE52G) that does not produce melanin was mapped to a gene identified as a probable thioredoxin gene. It was proved that the thiol-reducing activity of the mutant was affected. Addition to the growth medium of substrates that induce the production of melanin (L-tyrosine, guaiacol, orcinol) increased the thioredoxin-like (trxL) mRNA level in the wild-type strain. The mutant strain was affected in the response to paraquat-induced oxidative stress, symbiotic nitrogen fixation, and both laccase and tyrosinase activities. The importance of thioredoxin in melanin production in bacteria, through the regulation of laccase or tyrosinase activities, or both, by the redox state of structural or catalytic SH groups, is discussed.

Effects of inoculation with plant growth-promoting rhizobacteria on resident rhizosphere microorganisms.

Plant growth-promoting rhizobacteria (PGPR) are exogenous bacteria introduced into agricultural ecosystems that act positively upon plant development. However, amendment reproducibility as well as the potential effects of inoculation upon plant root-associated microbial communities can be sources of concern. To address these questions, an understanding of mutual interactions between inoculants and resident rhizosphere microorganisms is required. Mechanisms used by PGPR can be direct or indirect; the former entails the secretion of growth regulators and the latter occurs through the production of antimicrobial compounds that reduce the deleterious effects of phytopathogens. The different modes of action may lead to different relationships between an inoculant and root microbial communities. Rhizobacterial communities are also affected by the plant, engineered genes, environmental stresses and agricultural practices. These factors appear to determine community structure more than an exogenous, active PGPR introduced at high levels.

Carotenoid production and phenotypic variation in Azospirillum brasilense.

We assessed the occurrence of phenotypic variation in Azospirillum brasilense strains Sp7, Cd, Sp245, Az39 and phv2 during growth in rich media, screening for variants altered in colony pigmentation or extracellular polysaccharide (EPS) production. Previous studies showed that EPS-overproducing variants of Sp7 appear frequently following starvation or growth in minimal medium. In contrast, no such variants were detected during growth in rich media in the tested strains except for few variants of phv2. Regarding alteration in colony pigmentation (from pink to white in strain Cd and from white to pink in the others), strain Sp7 showed a relatively high frequency of variation (0.009-0.026%). Strain Cd showed a lower frequency of alteration in pigmentation (0-0.008%), and this type of variation was not detected in the other strains. In A. brasilense, carotenoid synthesis is controlled by two RpoE sigma factors and their cognate ChrR anti-sigma factors, the latter acting as negative regulators of carotenoid synthesis. Here, all tested (n = 28) pink variants of Sp7 carried mutations in one of the anti-sigma factor genes, chrR1. Our findings indicate that, in A. brasilense, phenotypic variation is strain- and environment-dependent and support the central role of ChrR1 in regulation of carotenoid production.

cDNA-AFLP reveals differentially expressed genes related to cell aggregation of Azospirillum brasilense.

The response regulatory gene flcA controls the differentiation process of Azospirillum brasilense from vegetative state to cyst-like forms, both in culture and in association with plants. In contrast to the wild type parental strain Sp7, strain Sp72002, a Tn5 induced flcA(-) mutant, does not aggregate, does not differentiate from motile, vibroid cells into nonmotile, cyst-like forms and lacks some of the exopolysaccharide material on its cell surface. Here we aimed to identify differentially expressed genes whose expression could be modulated by flcA. Using cDNA-amplified fragment length polymorphism, we detected 81 transcript-derived fragments showing differential expression during exponential growth phase in an aggregation-inducing medium containing high C : N ratio. The fragments were sequenced and analyzed, and expression of ten genes encoding known proteins was compared between the two strains by reverse transcriptase polymerase chain reaction-(RT-PCR). RT-PCR analyses confirmed the differential patterns observed by cDNA-AFLP in nine of those genes. Among them are genes homologous to nodQ, involved in sulfation; narK, involved in nitrite/nitrate transport, and flp, involved in autoaggregation; as well as genes encoding a biopolymer transport protein, and the signal recognition particle. This work demonstrates the usefulness of the cDNA-AFLP approach to reveal genes that are differentially expressed during aggregation in Azospirillum brasilense and provides insights into the aggregation process of this bacterium.

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.

Laccase activity in melanin-producing strains of Sinorhizobium meliloti.

Laccase-like activity was detected in melanin-producing strains of Sinorhizobium meliloti mainly in cells at the stationary growth phase when copper was added to the medium. The laccase showed both syringaldazine and ABTS (2,2'-azino-bis-ethylbenzthiazoline-6-sulfonic acid) oxidase activities and was activated by the addition of 1.7 mM sodium dodecyl sulfate. Activity was totally inhibited by the addition of 1.0 mM EDTA, suggesting that the enzyme is a metal-dependent one. The enzyme was found to be cytosolic having an optimum pH of 5.0, an estimated molecular mass of 95 kDa and a K(m) of 4 microM for syringaldazine. Both laccase and tyrosinase activities were detected in melanin-producing S. meliloti strains. Plant growth-promoting (PGP) effect in rice by a laccase-producing S. meliloti strain when co-inoculated with Azospirillum brasilense Cd was observed. PGP effect by co-inoculation significantly increased plant yield compared to A. brasilense by itself. To the best of our knowledge this is the first report on laccase production in rhizobia and cooperation between Azospirillum and Sinorhizobium in rice.

Key physiological properties contributing to rhizosphere adaptation and plant growth promotion abilities of Azospirillum brasilense.

Azospirillum brasilense is a plant growth promoting rhizobacterium (PGPR) that is being increasingly used in agriculture in a commercial scale. Recent research has elucidated key properties of A. brasilense that contribute to its ability to adapt to the rhizosphere habitat and to promote plant growth. They include synthesis of the auxin indole-3-acetic acid, nitric oxide, carotenoids, and a range of cell surface components as well as the ability to undergo phenotypic variation. Storage and utilization of polybetahydroxyalkanoate polymers are important for the shelf life of the bacteria in production of inoculants, products containing bacterial cells in a suitable carrier for agricultural use. Azospirillum brasilense is able to fix nitrogen, but despite some controversy, as judging from most systems evaluated so far, contribution of fixed nitrogen by this bacterium does not seem to play a major role in plant growth promotion. In this review, we focus on recent advances in the understanding of physiological properties of A. brasilense that are important for rhizosphere performance and successful interactions with plant roots.

Phenotypic variation in Azospirillum brasilense exposed to starvation.

Bacteria have developed mechanisms that allow them maintaining cell viability during starvation and resuming growth when nutrients become available. Among these mechanisms are adaptive mutations and phase variation, which are often associated with DNA rearrangements. Azospirillum brasilense is a Gram-negative, nitrogen-fixing, plant growth-promoting rhizobacterium. Here we report phenotypic variants of A. brasilense that were collected after exposure to prolonged starvation or after re-isolation from maize roots. The variants differed in several features from the parental strains, including pigmentation, aggregation ability, EPS amount and composition and LPS structure. One of the phenotypic variants, overproducing EPS and showing an altered LPS structure, was further characterized and showed differential response to several stresses and antibiotics relative to its parental strain. Characterization of the variants by repetitive-PCR revealed that phenotypic variation was often associated with DNA rearrangements.

Effects of Azospirillum brasilense with genetically modified auxin biosynthesis gene ipdC upon the diversity of the indigenous microbiota of the wheat rhizosphere.

The phytostimulatory properties of Azospirillum inoculants, which entail production of the phytohormone indole-3-acetic acid (IAA), can be enhanced by genetic means. However, it is not known whether this could affect their interactions with indigenous soil microbes. Here, wheat seeds were inoculated with the wild-type strain Azospirillum brasilense Sp245 or one of three genetically modified (GM) derivatives and grown for one month. The GM derivatives contained a plasmid vector harboring the indole-3-pyruvate/phenylpyruvate decarboxylase gene ipdC (IAA production) controlled either by the constitutive promoter PnptII or the root exudate-responsive promoter PsbpA, or by an empty vector (GM control). All inoculants displayed equal rhizosphere population densities. Only inoculation with either ipdC construct increased shoot biomass compared with the non-inoculated control. At one month after inoculation, automated ribosomal intergenic spacer analysis (ARISA) revealed that the effect of the PsbpA construct on bacterial community structure differed from that of the GM control, which was confirmed by 16S rDNA-based denaturing gradient gel electrophoresis (DGGE). The fungal community was sensitive to inoculation with the PsbpA construct and especially the GM control, based on ARISA data. Overall, fungal and bacterial communities displayed distinct responses to inoculation of GM A. brasilense phytostimulators, whose effects could differ from those of the wild-type.

The wzm gene located on the pRhico plasmid of Azospirillum brasilense Sp7 is involved in lipopolysaccharide synthesis.

Several genes involved in the interaction between Azospirillum brasilense Sp7 and plants are located on the pRhico plasmid. Here we report the characterization of an Sp7 mutant strain with impairment of the pRhico-located gene wzm. This gene encodes an inner-membrane component of an ATP-binding cassette (ABC) transporter with similarity to transporters involved in surface polysaccharide export. Indeed, SDS-PAGE revealed that LPS synthesis is affected in the wzm mutant. No significant differences were observed between wild-type and mutant strains in exopolysaccharide (EPS) amount; however, several differences were observed between them in EPS monosaccharide composition, and only wild-type colonies stained positively with Congo red. Microscopy revealed that wzm mutant cells are longer and thinner, and exhibit several differences in their cell surface relative to the wild-type. The wzm mutant was more resistant to oxidative stress, starvation, desiccation, heat and osmotic shock than the wild-type. In contrast, the mutant was more susceptible than the wild-type to UV radiation and saline stress. The strains also differed in their susceptibility to different antibiotics. Differences between the strains were also observed in their outer-membrane protein composition. No differences were observed between strains in their ability to attach to sweet corn roots and seeds, and to promote growth under the tested conditions. As LPS plays an important role in cell envelope structural integrity, we propose that the pleiotropic phenotypic changes observed in the wzm mutant are due to its altered LPS relative to the wild-type.

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.

Ecological and agricultural significance of bacterial polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are a group of carbon andenergy storage compounds that are accumulated during suboptimal growth by many bacteria, and intracellularly deposited in the form of inclusion bodies. Accumulation of PHAs is thought to be used by bacteria to increase survival and stress tolerance in changing environments, and in competitive settings where carbon and energy sources may be limited, such as those encountered in the soil and the rhizosphere. Understanding the role that PHAs play as internal storage polymers is of fundamental importance in microbial ecology, and holds great potential for the improvement of bacterial inoculants for plants and soils. This review summarizes the current knowledge on the ecological function of PHAs, and their strategic role as survival factors in microorganisms under varying environmental stress is emphasized. It also explores the phylogeny of the PHA cycle enzymes, PHA synthase, and PHA depolymerase, suggesting that PHA accumulation was earlier acquired and maintained during evolution, thus contributing to microbial survival in the environment.

Azospirillum brasilense does not affect population structure of specific rhizobacterial communities of inoculated maize (Zea mays).

Positive response of plant species to plant growth-promoting rhizobacteria have led to an increased interest in their use as bacterial inoculants. However, the introduction of exogenous bacteria into natural ecosystems may perturb bacterial populations within the microbial community and lead to the disruption of indigenous populations performing key functional roles. In this study the effect of Azospirillum brasilense inoculation on maize (Zea mays) rhizosphere Actinobacteria, Bacteroidetes, alpha-Proteobacteria, Pseudomonas and Bdellovibrio spp. was assessed using a polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) approach in conjunction with group-specific primers. The DGGE fingerprints analysis revealed that the introduction of A. brasilense did not alter or disrupt the microbial system at the group-specific level. However, some communities such as the alpha-Proteobacteria and Bdellovibrio were influenced by plant age while the other bacterial groups remained unaffected. Based on these as well as previous data, it can be inferred that inoculation with A. brasilense does not perturb the natural bacterial populations investigated.

Inoculation with the plant-growth-promoting rhizobacterium Azospirillum brasilense causes little disturbance in the rhizosphere and rhizoplane of maize (Zea mays).

Inoculation with Azospirillum brasilense exerts beneficial effects on plant growth and crop yields. In this study, a comparative analysis of maize (Zea mays) root inoculated or not inoculated with A. brasilense strains was performed in two soils. Colonization dynamics of the rhizobacteria were tracked in various root compartments using 16S rRNA-targeted probes and 4',6'diamidino-2-phenylindole staining, and the structure of bacterial populations in the same samples was analyzed by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction products of the 16S rRNA gene. Based on whole cell hybridization, a large fraction of the bacterial community was found to be active in both the rhizoplane-endorhizosphere and rhizosphere soil compartments, in both soil types. A DGGE fingerprint analysis revealed that plant inoculation with A. brasilense had no effect on the structural composition of the bacterial communities, which were also found to be very similar at the root tip and at zones of root branching. However, rhizobacterial populations were strongly influenced by plant age, and their complexity decreased in the rhizoplane-endorhizosphere in comparison to rhizosphere soil. A clone library generated from rhizosphere DNA revealed a highly diverse community of soil and rhizosphere bacteria, including an indigenous Azospirillum-like organism. A large proportion of these clones was only distantly related to known species.

Involvement of the reserve material poly-beta-hydroxybutyrate in Azospirillum brasilense stress endurance and root colonization.

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-beta-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.

Poly beta-hydroxybutyrate depolymerase (PhaZ) in Azospirillum brasilense and characterization of a phaZ mutant.

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly-beta-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.

Involvement of outer-membrane proteins in the aggregation of Azospirillum brasilense.

A bioassay was developed to investigate biological factors involved in the aggregation of Azospirillum brasilense strain Cd. Cells were grown for 24 h under aggregation-inducing and non-aggregation-inducing conditions (high and low C:N, respectively) and sonicated for 20 s. The cells were washed by centrifugation and resuspended in potassium phosphate buffer containing the two types of sonication extract. A greater extent of aggregation and higher flocculation were observed after 2-3 h incubation in the presence of sonicates from cells grown at high C:N (H-cells) compared to cells grown at low C:N. Flocculation did not occur after incubation of these cells in phosphate buffer. Boiled or proteinase K-treated sonicates originating from H-cells had lower aggregation-inducing capacity. After fractionation of the crude sonicate, both the outer-membrane protein (OMP) and the total membrane (mostly OMP) fractions possessed relatively high aggregation specific activities. The aggregation-inducing capacity of the OMP fraction strongly correlated with its protein concentration in the bioassay. Treatment of this fraction with proteinase K also decreased its aggregation-inducing activity. These findings suggest that OMPs are involved in the aggregation process of cells of A. brasilense.

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.

Aggregation in Azospirillum brasilense: effects of chemical and physical factors and involvement of extracellular components.

A medium for consistent induction of aggregation of Azospirillum brasilense cells was developed and used to study the effects of chemical and physical factors as well as extracellular components involved in this phenomenon. Growth of A. brasilense strain Cd in a high C:N medium using fructose and ammonium chloride as C and N sources, respectively, resulted in flocculation visible to the naked eye after 24 h. No cell aggregates were formed after 72 h growth in low C:N medium. Aggregating cells, but not cells grown under low C:N, accumulated high amounts of poly-beta-hydroxybutyrate and the cell envelope contained a well-defined electron-dense layer outside the outer membrane. Suspending the aggregates in 0.2 or 0.5 M urea was the only treatment effective for disrupting aggregates. The concentration of exopolysaccharide produced by four different strains of A. brasilense, differing in their capacity to aggregate, strongly correlated with the extent of aggregation. Electrophoretic protein profiles from different fractions of aggregating and non-aggregating cells were compared. Differences were observed in the pattern of low-molecular-mass proteins and in the polar flagellin that has previously been proposed to be involved in adhesion processes. However, a mutant lacking both lateral and polar flagella showed the strongest aggregation. The involvement of polysaccharides and/or proteins in aggregation of A. brasilense is discussed.