Lipopolysaccharides from six strains of Acetobacter diazotrophicus

Abstract Lipopolysaccharides from six nitrogen-fixing strains of Acetobacter diazotrophicus (PR2, PAL3, PAL5, PR4, PR14, PR20), isolated from sugarcane, were purified by phenol-water extraction and ultracentrifugation. The relatively large molecular mass observed by SDS-PAGE indicated that the lipopolysaccharides of each strain possessed an O-side chain. Analysis of each lipopolysaccharide by colorimetric assays and by gas liquid chromatography/mass spectrometry combination showed that the core and lipid A composition was similar for all strains, containing 3-deoxy-d-manno-2-octulosonic acid, glucosamine and fatty acid (16-0, 3-OH-14, 2-OH-16:0, 3-OH-16:0). The neutral sugar composition showed the predominance of 6-deoxy-hexose (rhamnose and fucose) and ribose, in comparison with hexose (glucose, galactose, mannose). The presence of 6-deoxy-hexose and ribose containing O-side chains is discussed as a way of discriminating A. diazotrophicus from other Acetobacter species.     

Molecular analysis of the chromosomal region encoding the nifA and nifB genes of Acetobacter diazotrophicus

The Acetobacter diazotrophicus nifA gene was isolated by its ability to restore a Nif⁺ phenotype to a nifA mutant of Azotobacter vinelandii. Sequencing revealed that the nifA gene was upstream and adjacent to the nifB gene and both are transcribed in the same direction but independently from different promoters. The 3′ end of the nifB gene was located approximately 2.5 kb upstream of the nitrogenase structural gene cluster, nifHDK. The deduced amino acid sequences of the A. diazotrophicus nifA and nifB gene products were most similar to the NifA and NifB proteins of Azorhizobium caulinodans and Rhodobacter capsulatus, respectively. A. diazotrophicus nifA expression was repressed in cultures exposed to high levels of ammonium while oxygen apparently had no influence. Both oxygen and ammonium prevented expression of a nifB-reporter strain, consistent with the observation that ammonium repressed nifA expression, and indicating that A. diazotrophicus NifA activity is inhibited by oxygen as in other Proteobacterial α group diazotrophs.     

Effect of high sugar concentration on nitrogenase activity of Acetobacter diazotrophicus

Acetobacter diazotrophicus is a nitrogen-fixing bacterium that grows inside sugar cane plant tissue where the sucrose concentration is approximately 10%. The influence of high sugar content on nitrogenase was measured in the presence of oxygen and of nitrogen added in the form of ammonium and amino acids. In all parameters analyzed, 10% sucrose protected nitrogenase against inhibition by oxygen, ammonium, some amino acids, and also to some extent by salt stress. The oxygen concentration at which inhibition occurred increased from 2 kPa in 1% glucose or gluconic acid, to 4 kPa (0.4 atm) in 10% sucrose. Nitrogenase activity was partially inhibited by increased ammonium levels (2.0, 5.0, and 10.0 mM) in the presence of 1% sucrose, but the cells maintained their nitrogenase activity at 10% sucrose. This could be explained by the slow ammonium assimilation by the cells in the presence of high sucrose concentrations, i.e., independent of its concentration between 2 and 10 mM, the assimilation of ammonium was reduced to one-third in cells grown with 10% sucrose. Some amino acids were also tested in the presence of 1 and 10% sucrose. Cells grown in 1% sucrose had their nitrogenase activity reduced by 50–98% in the presence of glutamic acid, glutamine, alanine, asparagine, or threonine, whereas with 10% sucrose, nitrogenase activity was increased by glutamic acid and was reduced by only 61–73% by the other amino acids. The effect of NaCl concentrations (0.0, 0.25, 0.5, 0.75, or 1.0%) was also studied at the two concentrations of sucrose. Nitrogenase activity and growth of A. diazotrophicus, which was visualized by the pellicle formation in semi-solid medium, showed sensitivity even to low NaCl concentrations, which was somewhat relieved at the higher sucrose level. These observations indicate different osmotolerance mechanisms for sucrose and salt. Received: 23 June 1998 / Accepted: 6 October 1998     

Production of indole-3-acetic acid and gibberellins A1 and A3 by Acetobacter diazotrophicus and Herbaspirillum seropedicae in chemically-defined culture media

The characterization by capillary gas chromatography-mass spectrometry of the plant hormones indole-3-acetic acid and the gibberellins GA₁ and GA₃ from chemically-defined cultures of Acetobacter diazotrophicus and Herbaspirillum seropedicae is reported. Both bacteria are endophytic in gramineae species where they promote growth and yield. Quantification was also done by selected ion monitoring with [17,17-²H₂]-Gibberellin A₁, [17,17-²H₂]-Gibberellin A₃ and [¹³C₆]-indole-3-acetic acid as internal standards. The results presented show the importance of studying phytohormonal production when the interrelationships between plants and microorganisms are analyzed and may help explain the beneficial effects of endophytic bacteria to the host plant, as has been demonstrated previously for Azospirillum spp.     

Azide resistant mutants of Acetobacter diazotrophicus and Azospirillum brasilense increase yield and nitrogen content of cotton

Abstract

Evolution of symbiotic plant-microbe interactions has provided mankind a powerful and environment-friendly means to increase yield of agricultural crops. Here, we report that some azide resistant mutants of two microbial strains can significantly enhance the productivity of cotton varieties, as an attractive and cheap biological substitute of chemical fertilizers, for improved yield of an important cash crop, without any untoward impacts. Sodium azide resistant mutants were isolated from each strain of Azospirillum brasilense and Acetobacter diazotrophicus on different concentrations of sodium azide ranging from 5–60µg/ml. These azide resistant mutants were assessed for their performance on cotton (varieties H-117, HD-123) for various parameters. Inoculation of cottonseeds with mutants obtained better results than inoculation with their respective parental strains. Azide resistant mutants, when used as biofertilizers, showed increased plant height, early flowering, more yield, and high biomass and total nitrogen content. They also increased, in cotton genotypes, the indole acetic acid production and ammonia excretion due to high nitrogenase activity.     

Differentiation of Capsular Polysaccharides from Acetobacter diazotrophicus Strains Isolated from Sugarcane

Capsular polysaccharides (CPSs) from six representative strains of Acetobacter diazotrophicus were isolated and fractionated by gel filtration and anion-exchange chromatography. Purified CPSs obtained in the non-adsorbed fraction of a DEAE-Sephadex A-25 column were qualitatively and quantitatively analyzed for sugar composition. Uronic acid and amino sugars were not detected in all purified CPSs. Basically the CPSs of A. diazotrophicus are composed of rhamnose, mannose, galactose and glucose. The presence of fucose was only observed in the CPS of strains PR2 and PAL3. Based on these results, the six strains of A. diazotrophicus could be divided into four groups according to the sugar content of their capsules: (i) fucose-containing capsules (PR2 and PAL3, localized in roots), (ii) mannose-rich capsule (PAL5, localized in root), (iii) capsules with a high ratio of hexose to rhamnose (PR4 and PR20, localized in stems) and (iv) capsules with a low ratio of hexose to rhamnose (PR14, localized in rhizosphere). For all CPSs, sodium dodecy sulfate-polyacrylamide gel electrophoresis showed diffuse bands of slow mobility in silver-stained gels. The different CPS migration patterns could not be correlated with sugar composition. The purified CPS of strain PAL3 was found to be immunogenic and immunochemically similar to the CPS of strain PR2. The serological specificity to CPS of strains PAL3 and PR2 correlated well with the presence of fucose, indicating that this deoxyhexose is immunodominant. These findings demonstrated the feasibility of preparing specific antibodies to fucose-containing CPS of A. diazotrophicus, indicating the possibility of utilization of this antiserum for future taxonomic studies or to select strains with chemically related capsular polysaccharides from their natural habitat.     

Levansucrase from Acetobacter diazotrophicus SRT4 Is Secreted via Periplasm by a Signal-Peptide-Dependent Pathway

Acetobacter diazotrophicus SRT4 secretes a constitutive levansucrase (LsdA) (EC 2.4.1.10) that is responsible for sucrose utilization. Immunogold electron microscopical studies revealed that LsdA accumulates in the periplasm before secretion. The periplasmic and extracellular forms of the enzyme were purified to homogeneity. Both proteins exhibited similar physical and biochemical characteristics indicating that LsdA adopts its final conformation in the periplasm. The N-terminal sequence of mature LsdA was pGlu-Gly-Asn-Phe-Ser-Arg as determined by PSD-MALDI-TOFMS (post-source decay—matrix-assisted laser desorption/ionization—time-of-flight mass spectrometry). Comparison of this sequence with the predicted precursor protein revealed the cleavage of a 30-residue typical signal peptide followed by the formation of the pyroglutamic acid (pGlu) residue. Thus, in contrast with other Gram-negative bacteria, A. diazotrophicus secretes levansucrase by a signal-peptide-dependent mechanism. Received: 24 March 1999 / Accepted: 30 April 1999     

Cloning and sequencing of the levansucrase gene from Acetobacter xylinum NCI 1005.

The levansucrase gene (lsxA) was cloned from the genomic DNA of Acetobacter xylinum NCI 1005, and the nucleotide sequence of the lsxA gene (1,293 bp) was determined. The deduced amino acid sequence of the lsxA gene showed 57.4% and 46.2% identity with the levansucrases from Zymomonas mobilis and Erwinia amylovora, respectively, while only 35.2% identity with that from Acetobacter diazotrophicus. The gene product of lsxA (LsxA) that was overproduced in E. coli coded for a polypeptide of molecular mass 47 kDa. The LsxA released glucose and produced polysaccharide from sucrose, the structure of which was analyzed by nuclear magnetic resonance spectroscopy and determined to be a beta-(2,6)-linked polyfructan.     

Improved methodology for isolation of Acetobacter diazotrophicus and confirmation of its endophytic habitat

Nitrogen-free, semi-solid defined medium with crystallized cane sugar (100 g/l) supplemented with cane juice (5 ml/l) was the most selective for isolating Acetobacter diazotrophicus. Surveys of A. diazotrophicus using this medium showed that >10³ cells/g fresh wt were present at all sites in all parts of the sugar cane plant and in all trash samples examined, reaching up to 10⁷/g. Additional samples, from forage grasses and cereals and from weed species collected within the sugar cane fields, were all negative. Heat treatment (50°C for 30 min) of the sugar cane setts did not affect A. diazotrophicus numbers within the plant. Nitrogenase activity of intact soil-plant systems in pots planted with heat-treated setts did not respond to inoculation with A. diazotrophicus. The endophytic habitat of this diazotroph and its propagation within the stem cuttings was confirmed.The authors are with EMBRAPA-CNPAB, Cx Postal 74.505, Seropédica, Rio de Janeiro, 23851-970, Brazil     

Molecular analysis of plasmid pAP4 from Acetobacter pasteurianus

The complete nucleotide sequence of plasmid pAP4 isolated from Acetobacter pasteurianus 2374^T has been determined. Plasmid pAP4 was analysed and found to be 3,870 bp in size with a G+C content of 50.1%. Computer assisted analysis of sequence data revealed 2 possible ORFs with typical promoter regions. ORF1 codes for a protein responsible for kanamycin resistance similar with Tn5 transposone, ORF2 encodes a resistance to ampicillin identical with Tn3 transposone. Plasmid has in A. pasteurianus five copies and in E. coli DH1 about 30 copies per chromosome and it segregation stability in both strains is very high. Based on the data on replication region, plasmid does not code for a replication protein and origin region is similar with ColE1-like plasmid.     

Comparison of characteristics of levan produced by different preparations of levansucrase from Zymomonas mobilis

The characteristics of levan formation by different preparations of levansucrase (free and immobilized enzyme and toluene-permeabilized whole cells), derived from recombinant levansucrase from Zymomonas mobilis expressed in Escherichia coli, were investigated. The maximal yield of levan by the three preparations were similar and were about 70–80% on a fructose-released basis with sucrose as nutrient at 100 g l^–1. Immobilized enzyme and toluene-permeabilized whole cells produced low molecular weight levan (2–3 × 10⁶), as determined by HPLC while high molecular weight levan (>6 × 10⁶) was the major product with the free levansucrase. The size of levan can thus be controlled by immobilized levansucrase and toluene-permeabilized whole cells in high yield.     

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.     

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.     

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.     

Influence of 1‐methylcyclopropene (1‐MCP) on the production of bacterial cellulose biosynthesized by Acetobacter xylinum under the agitated culture

Aims: Bacterial cellulose is an extracellular polysaccharide secreted by Acetobacter xylinum, which has become a novel material increasingly used in food and medical industries. However, its broad application is limited by its low yield and high cost. 1‐Methylcyclopropene (1‐MCP) is a potent inhibitor to either exogenous or endogenous ethylene during the biological senescence of plants, which has been broadly applied in commercial preservation of fruits and vegetables. The purpose of this study was to investigate the effects of 1‐MCP on both the growth of Acet.  xylinum and its cellulose production to demonstrate the potential enhancement of bacterial cellulose yield. Methods and Results: Three groups of samples were fermented under agitated culture with 125 rev min^?1 rotational speed. To the culture media, 0·14 mg of 1‐MCP contained in 100 mg dextrose powder was added on assigned days or on the first culture day only. Results from the measurement of bacterial cell concentration and bacterial cellulose yield at the end of a 12‐day culture demonstrated that cultures excluding 1‐MCP displayed a higher cell concentration and a lower cellulose production, while cultures containing 1‐MCP produced 15·6% more cellulose (1‐MCP added on day 1) and 25·4% (1‐MCP added on each assigned day) with less biomass. Conclusions: 1‐MCP was able to affect the growth of Acet. xylinum cells and resulted in increasing bacterial cellulose yield up to 25·4% over controls, which did not contain 1‐MCP. Significance and Impact of the Study: This was the first study to use the growth inhibitor of plants to investigate its effects on bacterial growth and production. It also demonstrated a significant enhancement of bacterial cellulose yield by the addition of 1‐MCP during the common agitated culture of Acet. xylinum.     

山西醋醅中醋酸菌的分离及初步鉴定

目的从山西省某醋厂能正常发酵的醋醅中分离出优势醋酸菌株并加以鉴定。方法经过菌种的增殖培养,采用稀释涂布法分离菌株,得到127株醋酸菌,再经过初筛和复筛,筛选出9株产醋酸优势菌株,对9株优势菌株进行传代培养。结果筛选出在传代培养过程中,产醋酸酸度高且产量稳定的菌株为L4,其产酸量为66.92 g/L,酒精转化率为72.42%。结论根据菌株L4形态观察及生理生化特征初步判定为醋酸菌属醋化醋杆菌奥尔兰亚种。