Intracellular colonization of roots of Arabidopsis and crop plants by Gluconacetobacter diazotrophicus

Summary We have investigated the interaction of Gluconacetobacter diazotrophicus, a non-nodulating endophytic nitrogen-fixing bacterium isolated from the intercellular spaces of sugarcane, with Arabidopsis thaliana and the crop plants maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), oilseed rape (Brassica napus), tomato (Lycopersicon esculentum), and white clover (Trifolium repens). Using seedlings grown aseptically in sucrose-containing culture media, we have shown that inoculation with very low numbers of G. diazotrophicus results in extensive intracellular colonization of root meristems and progressive systemic intracellular root colonization. Light microscopic examination of thin sections of resin-embedded root tips of Arabidopsis and these crop plants inoculated with β-glucuronidase (GUS)-labeled and with NifH promoter-GUS-labeled G. diazotrophicus showed blue-stained G. diazotrophicus within the cytoplasm of root cells, indicating that intracellular conditions were suitable for nitrogenase gene expression. Electron microscopy confirmed that these bluestained intracellular G. diazotrophicus were within membrane-bounded vesicles. We discuss whether these novel inoculations with G. diazotrophicus are likely to enable non-nodular endosymbiotic nitrogen fixation and whether these inoculations can also provide a plant system to investigate the endosymbiotic theory of the origin of eukaryotic organelles.     

Energy Generation by Extracellular Aldose Oxidation in N2-Fixing Gluconacetobacter diazotrophicus

Gluconacetobacter diazotrophicus PAL3 was grown in a chemostat with N₂ and mixtures of xylose and gluconate. Xylose was oxidized to xylonate, which was accumulated in the culture supernatants. Biomass yields and carbon from gluconate incorporated into biomass increased with the rate of xylose oxidation. By using metabolic balances it is demonstrated that extracellular xylose oxidation led N₂-fixing G. diazotrophicus cultures to increase the efficiency of energy generation.     

Contribution of Gluconacetobacter diazotrophicus to phosphorus nutrition in strawberry plants

Plant and Soil - Gluconacetobacter diazotrophicus (family Acetobacteraceae), is a N2-fixing bacterium with capability of mineral solubilization through organic acid production. The aim of this work...     

Glucose Metabolism in Batch and Continuous Cultures of Gluconacetobacter diazotrophicus PAL 3

Periplasmic glucose oxidation (by way of a pyrrolo-quinoline-quinone [PQQ]–linked glucose dehydrogenase [GDH]) was observed in continuous cultures of Gluconacetobacter diazotrophicus regardless of the carbon source (glucose or gluconate) and the nitrogen source (N₂ or NH₃). Its synthesis was stimulated by conditions of high energetic demand (i.e., N₂-fixation) and/or C-limitation. Under C-excess conditions, PQQ-GDH synthesis increased with the glucose concentration in the culture medium. In batch cultures, PQQ-GDH was actively expressed in very early stages with higher activities under conditions of N₂-fixation. Hexokinase activity was almost absent under any culture condition. Cytoplasmic nicotinamide adenine dinucleotide (NAD)–linked glucose dehydrogenase (GDH) was expressed in continuous cultures under all tested conditions, and its synthesis increased with the glucose concentration. In contrast, low activities of this enzyme were detected in batch cultures. Periplasmic oxidation, by way of PQQ-GDH, seems to be the principal pathway for metabolism of glucose in G. Diazotrophicus, and NAD-GDH is an alternative route under certain environmental conditions.     

Natural association of Gluconacetobacter diazotrophicus and diazotrophic Acetobacter peroxydans with wetland rice

The family Acetobacteraceae currently includes three known nitrogen-fixing species, Gluconacetobacter diazotrophicus, G. johannae and G. azotocaptans. In the present study, acetic acid-producing nitrogen-fixing bacteria were isolated from four different wetland rice varieties cultivated in the state of Tamilnadu, India. Most of these isolates were identified as G. diazotrophicus on the basis of their phenotypic characteristics and PCR assays using specific primers for that species. Based on 16S rDNA partial sequence analysis and DNA: DNA reassociation experiments the remaining isolates were identified as Acetobacter peroxydans, another species of the Acetobacteraceae family, thus far never reported as diazotrophic. The presence of nifH genes in A. peroxydans was confirmed by PCR amplification with nifH specific primers. Scope for the findings: This is the first report of the occurrence and association of N2-fixing Gluconacetobacter diazotrophicus and Acetobacter peroxydans with wetland rice varieties. This is the first report of diazotrophic nature of A. peroxydans.     

Fructo-oligosaccharides production by the Gluconacetobacter diazotrophicus levansucrase expressed in the methylotrophic yeast Pichia pastoris

Levansucrase (LsdA) (EC 2.4.1.10) from Gluconacetobacter diazotrophicus (formerly Acetobacter diazotrophicus) yields high levels of fructo-oligosaccharides (FOS) from sucrose. A DNA fragment encoding the precursor LsdA lacking the first 57 amino acids was fused to the pho1 signal sequence under the control of the Pichia pastoris-alcohol oxidase 1 (AOX1) promoter. Methanol induction of a P. pastoris strain harboring a single copy of the lsdA expression cassette integrated in the genome resulted in the production of active levansucrase. After fermentation of the recombinant yeast, LsdA activity was detected in the periplasmic fraction (81%) and in the culture supernatant (18%) with an overall yield of 1% of total protein. The recombinant LsdA was glycosylated and displayed optimal pH and temperature for enzyme activity similar to those of the native enzyme, but thermal stability was increased. Neither fructosylpolymerase activity nor FOS production was affected. Incubation of recombinant LsdA in sucrose (500 g l(-1)) yielded 43% (w/w) of total sugar as 1-kestose, with a conversion efficiency about 70%. Intact recombinant yeast cells also converted sucrose to FOS although for a 30% efficiency.     

Energy generation by extracellular aldose oxidation in N(2)-fixing Gluconacetobacter diazotrophicus

Gluconacetobacter diazotrophicus PAL3 was grown in a chemostat with N(2) and mixtures of xylose and gluconate. Xylose was oxidized to xylonate, which was accumulated in the culture supernatants. Biomass yields and carbon from gluconate incorporated into biomass increased with the rate of xylose oxidation. By using metabolic balances it is demonstrated that extracellular xylose oxidation led N(2)-fixing G. diazotrophicus cultures to increase the efficiency of energy generation.     

N-fertilizer saving by the inoculation of Gluconacetobacter diazotrophicus and Herbaspirillum sp. in micropropagated sugarcane plants

Colonization of micropropagated sugarcane plants by Gluconacetobacter diazotrophicus and Herbaspirillum sp. was confirmed by a dot-immunoblot assay. In all, a 45-day short-term and 180-day long-term experiments conducted on micropropagated sugarcane plants of Co 86032, a sugar rich popular variety in South India, indicated the usefulness of these diazotrophs as plant growth promoting bacteria. Co-inoculation of these two bacteria enhanced the biomass considerably under N-limited condition in the short duration experiment. In the long-term experiment, the establishment of inoculated Herbaspirillum sp. remained stable with the age of the crop up to 180 days, while there was a reduction in population of G. diazotrophicus for the same period. The total bio-mass and leaf N were higher in plants inoculated with G. diazotrophicus and Herbaspirillum sp. without N fertilization and also in plants with 50% of the recommended N (140 kg ha(-1)) than the plants fertilized with recommended dose of inorganic N (280 kg ha(-1)). This experiment showed that inoculation with these bacteria in sugarcane variety Co 86032 could mitigate fertilizer N application considerably in sugarcane cultivation.     

Exopolysaccharide production is required for biofilm formation and plant colonization by the nitrogen-fixing endophyte Gluconacetobacter diazotrophicus

The genome of the endophytic diazotrophic bacterial species Gluconacetobacter diazotrophicus PAL5 (PAL5) revealed the presence of a gum gene cluster. In this study, the gumD gene homologue, which is predicted to be responsible for the first step in exopolysaccharide (EPS) production, was insertionally inactivated and the resultant mutant (MGD) was functionally studied. The mutant MGD presented normal growth and nitrogen (N(2)) fixation levels but did not produce EPS when grown on different carbon sources. MGD presented altered colony morphology on soft agar plates (0.3% agar) and was defective in biofilm formation on glass wool. Most interestingly, MGD was defective in rice root surface attachment and in root surface and endophytic colonization. Genetic complementation reverted all mutant phenotypes. Also, the addition of EPS purified from culture supernatants of the wild-type strain PAL5 to the mutant MGD was effective in partially restoring wild-type biofilm formation and plant colonization. These data provide strong evidence that the PAL5 gumD gene is involved in EPS biosynthesis and that EPS biosynthesis is required for biofilm formation and plant colonization. To our knowledge, this is the first report of a role of EPS in the endophytic colonization of graminaceous plants by a nitrogen-fixing bacterium.     

Antagonism among Gluconacetobacter diazotrophicus strains in culture media and in endophytic association

In this study the antagonistic activity among 55 Gluconacetobacter diazotrophicus strains, belonging to 13 electrophoretic types (ETs), in culture media was analyzed. Antagonistic effects were seen only in strains belonging to two ETs named ET-1 and ET-3. Two out of 29 ET-1 strains, and 3 out of 7 ET-3 strains of G. diazotrophicus showed antagonistic effects against many other strains belonging to all the ETs of this species analyzed, and against closely related strains of Gluconacetobacter species, including Gluconacetobacter johannae, Gluconacetobacter azotocaptans and Gluconacetobacter liquefaciens but not against other phylogenetically distant bacterial species. Results showed that the substance responsible of such antagonistic activity is a low molecular mass molecule (approximately 3400 Da), stable from pH 3.5 to 8.5, and very stable at 4 degrees C for 10 months. This substance was sensitive to proteases, and the antagonistic activity was lost after 2 h at 95 degrees C. All of these features show that the substance is related to bacteriocin-like molecules. The antagonistic substance should be chromosomally encoded because ET-3 strains of G. diazotrophicus do not harbor any plasmids. The antagonistic ability of ET-3 strains of G. diazotrophicus could be an advantage for the natural colonization of the sugarcane environment, as was observed in experiments with micropropagated sterile sugarcane plantlets co-inoculated with a bacteriocin-producer strain and a bacteriocin-sensitive strain of G. diazotrophicus. In these experiments, both in the rhizosphere as well as inside the roots, the bacteriocin-sensitive population decreased drastically. In addition, this study shows that inside the plants there may exist antagonistic interactions among endophytic bacteria like to those described among the rhizospheric community.     

Effect of some abiotic factors on the biological activity of Gluconacetobacter diazotrophicus

AIMS: The effect of some abiotic factors, dryness, heat and salinity on the growth and biological activity of Gluconacetobacter diazotrophicus, and the influence of a salt stress on some enzymes involved in carbon metabolism of these bacteria is studied under laboratory conditions. METHODS AND RESULTS: Strain PAL-5 of G. diazotrophicus was incubated under different conditions of drying, heat and salinity. Cells showed tolerance to heat treatments and salt concentrations, and sensitivity to drying conditions. Higher NaCl dosage of 150 and 200 mmol l -1 limited its growth and drastically affected the nitrogenase activity and the enzymes glucose dehydrogenase, alcohol dehydrogenase, fumarase, isocitrate dehydrogenase and malate dehydrogenase. CONCLUSIONS: Gluconacetobacter diazotrophicus, despite its endophytic nature, tolerated heat treatments and salinity stress, but its nitrogenase activity and carbon metabolism enzymes were affected by high NaCl dosage. SIGNIFICANCE AND IMPACT OF THE STUDY: The investigation of the biological activity of G. diazotrophicus in response to different abiotic factors led to more knowledge of this endophyte and may help to clarify pathways involved in its transmission into the host plant.     

Effect of inorganic N on the population,in vitro colonization and morphology of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus)

We investigated whether Acetobacter diazotrophicus (syn.Gluconacetobacter diazotrophicus) could be recovered only from sugarcane plants either with low or no application of fertiliser N. We report here the enrichment and enumeration of A. diazotrophicus from high N-fertilised samples where high heterotrophic populations reduce the numbers of A. diazotrophicus ultimately diminshing its isolation frequency as reported earlier. The growth medium of micropropagated sugarcane seedlings of the varieties Co 8021, Co 86249, Co 86010, Co 86032, and Co 87025 was amended with potassium nitrate, ammonium nitrate, ammonium chloride and urea. The colonisation and AR activity of A. diazotrophicus were affected in the presence of high levels (25 mM) of ammonium chloride and ammonium nitrate but remained unaffected in low levels of N (i.e 1/10th of MS liquid medium) and with high levels of potassium nitrate (25 mM) and urea (500 ppm). A. diazotrophicus was detected in the inoculated plants both at low and high levels of N based on the amplification of a specific 16S rRNA gene fragment using PCR based method targeting a stretch of 445 bp with primers AC and DI. High levels of N in the growth medium induced morphological changes on A. diazotrophicus cells resulting in long pleomorphic cells. The percentage of pleomorphic cells was in the decending order from NH₄NO₃, NH₄Cl, KNO₃, and urea. These changes were more prominent in ammonium chloride and ammonium nitrate than potassium nitrate, urea and N free medium. The morphological changes and the increased heterotrophic populations may play a role on the survival ofA. diazotrophicus in high N-fertilised samples/environments.     

Evidence for conformational protection of nitrogenase against oxygen in Gluconacetobacter diazotrophicus by a putative FeSII protein

The mechanisms protecting nitrogenase in Gluconacetobacter diazotrophicus from damage by oxygen were studied. Evidence is provided suggesting that in G. diazotrophicus these mechanisms include respiratory protection as well as conformational protection in which a putative FeSII Shethna protein is involved.     

Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media

The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.     

Identification and characterization of an iron ABC transporter operon in Gluconacetobacter diazotrophicus Pal 5

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane. We have cloned and sequenced the genes coding for the components of the iron ABC-type acquisition system of G. diazotrophicus. Sequence analysis revealed three ORFs, (feuA, feuB, and feuC) organized as an operon and encoding polypeptides of 346 (38 kDa), 342 (34.2 kDa), and 240 (26 kDa) amino acids, respectively. The deduced translation products of the feu operon showed similarity with a periplasmic solute-binding protein (FeuA), permease (FeuB), and ATPase (FeuC) involved in Fe transport. The role of FeuB in the survival of G. diazotrophicus under iron depletion was evaluated by comparing the ability of wild-type and FeuB-Km^R -mutant strains in a medium without iron supplementation and in a medium containing 2, 2′-dipyridyl (DP). Growth of the mutant was affected in the medium containing DP. The operon was expressed at higher levels in cells depleted for iron than in those that contained the metal. A decrease in nitrogenase activity was observed with the FeuB-Km^R -mutant strain that with the wild-type under iron deficiency conditions, suggesting that the Feu operon play role in Fe nutrition of G. diazotrophicus.     

Occurrence of Gluconacetobacter diazotrophicus in tropical and subtropical plants of Western Ghats, India

Endophytic bacteria were isolated from the tissues of surface sterilized roots, stems, and leaves of fifty different crop plants. Phenotypic, biochemical tests and species-specific PCR assay permitted identification of four isolates of Gluconacetobacter diazotrophicus from root tissues of carrot (Daucus carota L.), raddish (Raphanus sativus L.), beetroot (Beta vulgaris L.) and coffee (Coffea arabica L.). Further the plant growth promoting traits such as nitrogenase activity, production of phytohormone indole acetic acid (IAA), phosphorus and zinc solubilization were assessed. Significant nitrogenase activity was recorded among the isolates and all the isolates produced IAA in the presence of tryptophan. Though all the four isolates efficiently solubilized phosphorus, the zinc solubilizing ability differed among the isolates.