巴西固氮螺菌Yu62在玉米根的定植

将GFPmut2质粒中的gfp基因 (编码绿色荧光蛋白)克隆到载体pVK100中,构建成重组质粒pVK1001.将pVK1001通过电转化方法导入到联合固氮菌巴西固氮螺菌Yu62中,获得GFP标记的巴西固氮螺菌Yu62菌株.用标记菌株接种限菌培养条件下生长的玉米(农大3318)幼苗,在接种后8 d、12 d,用激光共聚焦扫描显微镜进行观测,结果表明巴西固氮螺菌Yu62菌株能定植于玉米根部皮层的薄壁细胞间隙.用扫描电镜和超薄切片电镜观察表明,大多数细菌主要定植于根表,少数菌可进入玉米根组织内.     

The serotyping of Azospirillum spp. by cell-gold immunoblotting

Ten Azospirillum strains were serotyped using the method of cell-gold immunoblotting (dot-blot immune-overlay assay). Colloidal gold-protein A conjugate was used. Antibodies raised against the whole cells showed strain specificity and interacted mainly with carbohydrate antigens on the cell surface. Immunological identity for A. brasilense Sp 245 and Sp 107 strains was found. Cell-gold immunoblotting can be recommended for serotyping of a wide variety of bacterial strains.     

A CheR/CheB fusion protein is involved in cyst cell development and chemotaxis in Azospirillum brasilense Sp7

We here report the sequence and functional analysis of cstB of Azospirillum brasilense Sp7. The predicted cstB contains C-terminal two PAS domains and N-terminal part which has similarity with CheB-CheR fusion protein. cstB mutants had reduced swarming ability compared to that of A. brasilense wild-type strain, implying that cstB was involved in chemotaxis in A. brasilense. A microscopic analysis revealed that cstB mutants developed mature cyst cells more quickly than wild type, indicating that cstB is involved in cyst formation. cstB mutants were affected in colony morphology and the production of exopolysaccharides (EPS) which are essential for A. brasilense cells to differentiate into cyst-like forms. These observations suggested that cstB was a multi-effector involved in cyst development and chemotaxis in A. brasilense.     

巴西固氮螺菌中P_Ⅱ和P_Z在固氮调节中的不同作用

在巴西固氮螺菌 (Azospirillumbrasilense)中 ,glnB和glnZ是两个高度同源基因 ,分别位于 3 7kb EcoRI+PstI和 3 7kb SalI的两个不同的染色体片段上。用卡那霉素盒 (Kmr cas sette)插入法 ,对glnB和glnZ分别进行定位诱变 ,并获得相应的突变株 ,即glnB- 和glnZ- 。研究表明 ,glnB- 突变株丧失固氮酶活性 ,表现为Nif- ,而glnZ- 象野生型菌株一样具有固氮酶活性。为了进一步研究这两个基因的功能 ,将glnB和glnZ分别构建在pVK1 0 0载体上形成重组质粒pVK -Ⅱ和pVK -Z ,对glnB- 和glnZ- 突变株进行互补实验 ,进一步证明了glnB与固氮酶活有直接相关性 ,而glnZ无此作用。同时 ,通过三亲接合法将pVK -Ⅱ和pVK -Z分别转移到巴西固氮螺菌野生型Yu62和具有一定抗铵能力的draT- 突变株中 ,使glnB和glnZ的拷贝数增加 ,进一步比较它们的固氮酶活性。结果表明多拷贝的glnB基因 ,能显著提高固氮酶活性 ,而多拷贝的glnZ对固氮酶活性无影响。同时 ,将pVK Ⅱ和pVK -…     

Emergence of a laccase-positive variant of Azospirillum lipoferum occurs via a two-step phenotypic switching process

Two variants have been isolated from the wild-type Azospirillum lipoferum strain 4B. The first variant, 4V(I), spontaneously emerged from the wild-type at frequencies in the order of 10(-4) to 10(-3) per cell generation. Compared to the wild-type, the 4V(I) variant gained (production of a carotenoid-like pigment, assimilation of certain carbohydrates) and lost (swimming motility, reduction of triphenyl tetrazolium chloride, acid production from certain sugars) apparently unrelated phenotypic characteristics. Only from the 4V(I) variant, a second atypical stable form, variant 4V(II), which acquired laccase activity and ability to produce melanin, appeared under very specific conditions, namely growth at extremely low oxygen concentrations. Neither of the variants was able to revert to the parental phenotype. The results suggest that atypical non-motile laccase-positive isolates of A. lipoferum that are found in the rice rhizosphere originate from wild-type (motile, laccase-negative) cells via a two-step phenotypic switching event, a non-motile laccase-negative variant being an intermediate phase.     

Detection of a sheath on <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> polar flagellum

The presence of a polysaccharide sheath on the surface of the polar flagellum of Azospirillum brasilense was revelted by immunoelectron microscopy and immunodiffusion analysis with strain-specific antibodies to lipopolysaccharides (LPS). The antigenic identity of A. brasilense Sp245 sheath material and one of the two O-specific polysaccharides of its somatic LPS was demonstrated. The screening effect of the sheath in respect to flagellin was determined by agglutination tests and by the inhibition of azospirilla motility in liquid and semisolid agarized media caused by strain-specific antibodies to LPS; no pronounced effect of genus-specific antibodies to flagellin was observed.     

INVOLVEMENT OF INDOLE‐3‐ACETIC ACID PRODUCED BY THE GROWTH‐PROMOTING BACTERIUM AZOSPIRILLUM SPP. IN PROMOTING GROWTH OF CHLORELLA VULGARIS1

Involvement of indole‐3‐acetic acid (IAA), produced by the microalgae‐growth‐promoting bacteria Azospirillum brasilens and A. lipoferum, in promoting growth of the microalga Chlorella vulgaris Beij. was studied. Four wildtype strains of Azospirillum and their IAA‐deficient mutants were co‐immobilized with C. vulgaris in alginate beads. Cultures were grown in synthetic growth medium supplemented with tryptophan. Growth promotion of microalgae and production of exogenous IAA by Azospirillum spp. were monitored. All wildtype Azospirillum spp. produced significant but varying amounts of IAA, while their mutant forms produced significantly less. The results demonstrated a significant growth promotion in Chlorella cultures when immobilized with the four wildtype strains of Azospirillum, while very low or no enhanced growth was induced by the four IAA‐deficient mutants, compared to when C. vulgaris is immobilized alone. A complementation experiment, where an IAA‐attenuated mutant (A. brasilense SpM7918) was supplemented with IAA produced by its parental wildtype strain (A. brasilense Sp6), restored growth promotion in the microalgae‐mutant culture.     

ROLE OF GLUTAMATE DEHYDROGENASE AND GLUTAMINE SYNTHETASE IN CHLORELLA VULGARIS DURING ASSIMILATION OF AMMONIUM WHEN JOINTLY IMMOBILIZED WITH THE MICROALGAE‐GROWTH‐PROMOTING BACTERIUM AZOSPIRILLUM BRASILENSE1

Enzymatic activities of glutamate dehydrogenase (GDH) and glutamine synthetase (GS) participating in the nitrogen metabolism and related ammonium absorption were assayed after the microalga Chlorella vulgaris Beij. was jointly immobilized with the microalgae‐growth‐promoting bacterium Azospirillum brasilense. At initial concentrations of 3, 6, and 10 mg · L^?1 NH₄⁺, joint immobilization enhances growth of C. vulgaris but does not affect ammonium absorption capacity of the microalga. However, at 8 mg · L^?1 NH₄⁺, joint immobilization enhanced ammonium absorption by the microalga without affecting the growth of the microalgal population. Correlations between absorption of ammonium per cell and per culture showed direct (negative and positive) linear correlations between these parameters and microalga populations at 3, 6, and 10 mg · L^?1 NH₄⁺, but not at 8 mg · L^?1 NH₄⁺, where the highest absorption of ammonium occurred. In all cultures, immobilized and jointly immobilized, having the four initial ammonium concentrations, enzymatic activities of Chlorella are affected by A. brasilense. Regardless of the initial concentration of ammonium, GS activity in C. vulgaris was always higher when jointly immobilized and determined on a per‐cell basis. When jointly immobilized, only at an initial concentration of 8 mg · L^?1 NH₄⁺ was GDH activity per cell higher.     

Azospirillum brasilense Sp245 triggers cytokinin signaling in root tips and improves biomass accumulation in Arabidopsis through canonical cytokinin receptors

The plant growth promoting rhizobacterium Azospirillum brasilense Sp245 enhances biomass production in cereals and horticultural species and is an interesting model to study the physiology of the phytostimulation program. Although auxin production by Azospirillum appears to be critical for root architectural readjustments, the role of cytokinins in the growth promoting effects of Azospirillum remains unclear. Here, Arabidopsis thaliana seedlings were co-cultivated in vitro with A. brasilense Sp245 to assess whether direct contact of roots with bacterial colonies or exposure to the bacterial volatiles using divided Petri plates would affect biomass production and root organogenesis. Both interaction types increased root and shoot fresh weight but had contrasting effects on primary root length, lateral root formation and root hair development. Cell proliferation in root meristems analyzed with the CYCB1;1::GUS reporter decreased over time with direct contact, but was augmented by plant exposure to volatiles. Noteworthy, the expression of the cytokinin-inducible reporters TCS::GFP and ARR5::GUS increased in root tips in response to bacterial contact, without being affected by the volatiles. In A. thaliana having single (cre1-12, ahk2-2, ahk3-3), double (cre1-12/ahk2-2, cre1-12/ahk3-3, ahk2-2/ahk3-3) or triple (cre1-12/ahk2-2/ahk3-3) mutations in canonical cytokinin receptors, only the triple mutant had a marked effect on plant growth in response to A. brasilense. These results show that different mechanisms are elicited by A. brasilense, which influence the cytokinin-signaling pathway.     

Molecular cloning and sequence analysis of an Azospirilium brasilense indole-3-pyruvate decarboxylase gene

Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome library in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. brasilense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA production in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that this fragment contains one complete open reading grame. The predicted protein sequence shows extensive homology with the indole-3-pyruvate decarboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense mutant Sp245a, constructed by homogenotization of a Tn5 insertion derivative of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 1021 resulted in increased IAA production. Cell-free extracts prepared from R. meliloti and A. brasilense transconjugants harboring this gene could convert indole-3-pyruvic acid to indole-3-acetaldehyde and tryptophol. These results clearly demonstrate that IAA production in A. brasilense is mediated by indole-3-pyruvate decarboxylase.