Mutants with enhanced nitrogenase activity in hydroponic Azospirillum brasilense-wheat associations

The effect of a mutation affecting flocculation, differentiation into cyst-like forms, and root colonization on nitrogenase expression by Azospirillum brasilense is described. The gene flcA of strain Sp7 restored these phenotypes in spontaneous mutants of both strains Sp7 and Sp245. Employing both constitutive pLA-lacZ and nifH-lacZ reporter fusions expressed in situ, the colony morphology, colonization pattern, and potential for nitrogenase activity of spontaneous mutants and flcA Tn5-induced mutants were established. The results of this study show that the ability of Sp7 and Sp245 mutant strains to remain in a vegetative form improved their ability to express nitrogenase activity in association with wheat in a hydroponic system. Restoring the cyst formation and colonization pattern to the spontaneous mutant Sp7-S reduced nitrogenase activity rates in association with plants to that of the wild-type Sp7. Although Tn5-induced flcA mutants showed higher potentials for nitrogenase expression than Sp7, their potentials were lower than that of Sp7-S, indicating that other factors in this strain contribute to its exceptional nitrogenase activity rates on plants. The lack of lateral flagella is not one of these factors, as Sp7-PM23, a spontaneous mutant impaired in swarming and lateral-flagellum production but not in flocculation, showed wild-type nitrogenase activity and expression. The results also suggest factors of importance in evolving an effective symbiosis between Azospirillum and wheat, such as increasing the availability of microaerobic niches along the root, increased supply of carbon sources by the plant, and the retention of the bacterial cells in vegetative form for faster metabolism.     

Identification and characterization of a periplasmic nitrate reductase in Azospirillum brasilense Sp245

The Azospirillum brasilense Sp245 napABC genes, encoding nitrate reductase activity, were isolated and sequenced. The derived protein sequences are very similar throughout the whole Nap segment to the NapABC protein sequences of Escherichia coli, Pseudomonas sp. G-179, Ralstonia eutropha, Rhodobacter sphaeroides, and Paracoccus denitrificans. Based on whole-cell nitrate reductase assays with the artificial electron donors benzyl viologen and methyl viologen, and assays with periplasmic cell-free extracts, it was concluded that the napABC-encoded enzyme activity in Azospirillum brasilense Sp245 corresponds to a periplasmic dissimilatory nitrate reductase, which was expressed under anoxic conditions and oxic conditions. A kanamycin-resistant Azospirillum brasilense Sp245 napA insertion mutant was constructed. The mutant still expressed assimilatory nitrate reductase activity, but was devoid of its periplasmic dissimilatory nitrate reductase activity.     

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.     

The use of fragments of the 85- and 120-MDa plasmids of Azospirillum brasilense Sp245 to study the plasmid rearrangement in this bacterium and to search for homologous sequences in plasmids of Azospirillum brasilense Sp7

A spontaneous loss of the 85- (p85) and 120-MDa (p120) replicons and simultaneous generation of a plasmid of more than 300 MDa were associated with defects in synthesis of O-specific and Calcofluor-binding polysaccharides and had no effect on flagellation and motility of the Azospirillum brasilense Sp245.5 mutant. The plasmid rearrangement was studied by hybridization of DNAs from the wild-type Sp245 strain and the Sp245.5 mutant with p85 and p120 fragments that contained loci involved in formation of the polar (fla) and lateral (laf) flagella, synthesis of O-specific and Calcofluor-binding polysaccharides (lps/cal), swimming (mot), and swarming (swa) of bacteria. Hybridization with the p120 fragments revealed incorporation of the intact fla/swa loci and the altered lps/cal loci into a new megaplasmid. Two EcoRI fragments homologous to the fla/laf/mot/swa loci of p85 were found in A. brasilense Sp245 DNA, whereas only one copy was preserved in the Sp245.5 mutant. Hybridization of the p120 and p85 fragments of Sp245 to the A. brasilense Sp7 DNA for the first time revealed regions of substantial homology to these fragments in the 90- and 115-MDa Sp7 plasmids, respectively.     

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.     

Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation

M ichiels , K., V erreth , C. & V anderleyden , J. 1990. Azospirillum lipoferum and Azospirillum brasilense surface polysaccharide mutants that are affected in flocculation. Journal of Applied Bacteriology 69 , 705–711.
Surface polysaccharide production by Azospirillum is demonstrated by fluorescence of colonies grown on media containing the fluorescent dye Calcofluor, which binds to β-linked polysaccharides. Mutants showing decreased and increased levels of fluorescence are obtained from Azospirillum lipoferum strain Sp59b by chemical mutagenesis, and from A. brasilense strain 7030 by Tn5 mutagenesis.
The A. brasilense 7030 fluorescence mutants produce wild-type levels of exo-polysaccharide in their culture supernatant fluids, but are affected in flocculation in liquid culture. On the basis of these observations, we postulate that an A. brasilense surface polysaccharide, different from the exopolysaccharide, is involved in both Calcofluor staining and flocculation.
It is shown by DNA hybridization that the genetic loci affected in the A. brasilense 7030 fluorescence mutants are different from the A. brasilense exoB and exoC loci, which are involved in exopolysaccharide production.     

Identification and mapping of loci involved in motility, adsorption to wheat roots, colony morphology, and growth in minimal medium on the Azospirillum brasilense Sp7 90-MDa plasmid.

We have constructed a cosmid library of the Azospirillum brasilense Sp7 90-MDa plasmid (p90) and established the EcoRI restriction map of this plasmid. The central regions of cloned p90 DNA fragments from several recombinant cosmids were deleted by restriction endonuclease digestion and replaced by a DNA cassette encoding kanamycin resistance. Using these in vitro constructed deletions for marker exchange in Sp7, we made six different p90 deletion derivatives spanning all together 50% of the total length of p90. Comparison of the deletion derivatives with Sp7 for several properties revealed p90 loci involved in colony morphology, growth on minimal medium, motility, and adsorption to wheat roots. In analogy with the rhizobial symbiotic plasmids (pSym), we propose to denote the p90 plasmid as a rhizocoenotic plasmid (pRhico), carrying several genes involved in the A. brasilense-plant root interaction.     

The Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol is a signal inducing rhizoplane expression of Azospirillum genes involved in plant-growth promotion

During evolution, plants have become associated with guilds of plant-growth-promoting rhizobacteria (PGPR), which raises the possibility that individual PGPR populations may have developed mechanisms to cointeract with one another on plant roots. We hypothesize that this has resulted in signaling phenomena between different types of PGPR colonizing the same roots. Here, the objective was to determine whether the Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol (DAPG) can act as a signal on Azospirillum PGPR and enhance the phytostimulation effects of the latter. On roots, the DAPG-producing Pseudomonas fluorescens F113 strain but not its phl-negative mutant enhanced the phytostimulatory effect of Azospirillum brasilense Sp245-Rif on wheat. Accordingly, DAPG enhanced Sp245-Rif traits involved in root colonization (cell motility, biofilm formation, and poly-β-hydroxybutyrate production) and phytostimulation (auxin production). A differential fluorescence induction promoter-trapping approach based on flow cytometry was then used to identify Sp245-Rif genes upregulated by DAPG. DAPG enhanced expression of a wide range of Sp245-Rif genes, including genes involved in phytostimulation. Four of them (i.e., ppdC, flgE, nirK, and nifX-nifB) tended to be upregulated on roots in the presence of P. fluorescens F113 compared with its phl-negative mutant. Our results indicate that DAPG can act as a signal by which some beneficial pseudomonads may stimulate plant-beneficial activities of Azospirillum PGPR.     

Analysis of DNA, lipopolysaccharide structure, and some cultural and morphological properties in closely related strains of Azospirillum brasilense

We studied closely related Azospirillum brasilense strains Sp7 and Cd. For probing of their genomes, the fragments of 85-MDa (p85) and 120-MDa (p120) from A. brasilense Sp245 plasmids were hybridized with 115-MDa (p115) and 90-Mda (p90) plasmids of strain Sp7, respectively. Strain Cd was found to lose the 115-Mda plasmid and one of the two EcoRI restriction fragments of the total DNA (localized within p115 and the chromosome) that was homologous to an EcoRI-generated p85 fragment of 2.4 kb. On the contrary, in the total DNA of strain Sp7-S, in spite of the previously established disappearance of the 115-Mda replicon, two fragments homologous to p85 were revealed, as with strain Sp7. It is suggested that the Sp7-S genome contains the total p115 DNA or at least a certain part of it. Strains Sp7 and Cd were found to differ in size and morphology of colonies on solid and semisolid media, in the levels of resistance to a cation surfactant cetavlon, and in the antigen structure of lipopolysaccharides.     

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.     

巴西固氮螺菌Yu62nifA基因克隆、测序及功能分析

用TD-PCR法克隆了巴西固氮螺菌(Azospirillun brasilense)Yu62的nifA基因.序列分析表明它与巴西固氮螺菌Sp7的nifA序列高度同源(96.5%),其编码的产物NifA蛋白与Sp7菌株NifA的氨基酸序列同源性为97.6%.该基因可以完全互补巴西固氮螺菌Sp7 nifA-突变株的Nif-表型.研究了NH4+和O2对Yu62 nifA基因的表达及NifA活性的影响.结果表明mfA基因在Yu62菌株中是部分组成型表达的,氨和氧不能完全阻遏其表达,在5mmol/LNH4Cl与微氧(0.5%O2)条件下表达最高;NifA蛋白在0.4%～0.5%O2时活性最高,氧分压降低和提高都使NifA活性下降,1mmol/L NH4Cl足以抑制NifA的活性.     

Comparative in situ analysis of ipdC-gfpmut3 promoter fusions of Azospirillum brasilense strains Sp7 and Sp245

Inoculation of wheat roots with Azospirillum brasilense results in an increase of plant growth and yield, which is proposed to be mainly due to the bacterial production of indole-3-acetic acid in the rhizosphere. Field inoculation experiments had revealed more consistent plant growth stimulation using A. brasilense strain Sp245 as compared with the strain Sp7. Therefore, the in situ expression of the key gene ipdC (indole-3-pyruvate decarboxylase) was examined in these two strains. Within the ipdC promoter of strain Sp245 a region of 150 bases was identified, which was missing in strain Sp7. Thus, three different translational ipdC promoter fusions with gfpmut3 were constructed on plasmid level: the first contained the part of the Sp245 promoter region homologous to strain Sp7, the second was bearing the complete promoter region of Sp245 including the specific insertion and the third comprised the Sp7 promoter region. By comparing the fluorescence levels of these constructs after growth on mineral medium with and without inducing amino acids, it could be demonstrated that ipdC expression in A. brasilense Sp245 was subject to a stricter control compared with strain Sp7. Microscopic detection of these reporter strains colonizing the rhizoplane documented for the first time an in situ expression of ipdC.     

Annotation of the pRhico plasmid of Azospirillum brasilense reveals its role in determining the outer surface composition

The plant growth-promoting soil bacterium Azospirillum brasilense enhances growth of economically important crops, such as wheat, corn and rice. In order to improve plant growth, a close bacterial association with the plant roots is needed. Genes encoded on a 90-MDa plasmid, denoted pRhico plasmid, present in A. brasilense Sp7, play an important role in plant root interaction. Sequencing, annotation and in silico analysis of this 90-MDa plasmid revealed the presence of a large collection of genes encoding enzymes involved in surface polysaccharide biosynthesis. Analysis of the 90-MDa plasmid genome provided evidence for its essential role in the viability of the bacterial cell.