Carotenoids' influence on radiotolerance of Pantoea agglomerans, a plant pathogen

Aim:  The aim of this study was to evaluate the effect of γ radiation on the carotenoid content of two strains of the Enterobacteriaceae : Pantoea agglomerans .
Methods and Results:  Pantoea agglomerans strains ATCC 49174 and RL1 were used for this study. Successive radiation treatments were performed to study the radiotolerance. Total carotenoids were obtained by multiple extraction using chloroform/methanol (2 : 1), quantified by measuring the optical density at 453 nm and their antioxidant activity measured by a colorimetric method. The D ₁₀ studies were conducted using a UC-15A irradiator loaded with ⁶⁰Co. Bacterial counts from various dilutions were carried out after irradiation. Strain ATCC 49174 irradiated at 1 kGy produced 4·3 times more carotenoids than the control, whereas carotenoid synthesis increased by 2·9-fold in the strain RL1. However, there was no significant difference in the D ₁₀ values.
Conclusion:  Carotenoid increased production is influenced by γ radiation but does not modify the tolerance to radiations.
Significance and Impact of the Study:  To our knowledge, this is the first study to demonstrate the effects of γ radiation on carotenoid production levels.     

Rice endophyte Pantoea agglomerans YS19 forms multicellular symplasmata via cell aggregation

Pantoea agglomerans is characterized by the formation of multicellular symplasmata. One unanswered question regarding this bacterium is how these structures are formed. In this study, the rice diazotrophic endophyte P. agglomerans YS19 was selected for exploration of this theme. YS19 was labeled with green fluorescent protein and the resulting recombinant YS19::gfp was observed to grow only slightly more slowly (a decrease of 5.5%) than the wild-type strain, and to show high GFP label stability (label loss rate 8.9218 x 10(-6) per generation, nearly reaching the generally accepted spontaneous mutation rate for most bacteria). YS19::gfp resembled the wild-type YS19 in symplasmata formation and growth profiles. Based on associated cultivation of both strains by mixing their individually cultivated single cells, symplasmata were formed and composed of both YS19::gfp and YS19, suggesting that YS19 formed symplasmata via aggregation, not proliferation, of the original single cells.     

Optimization of medium for indole-3-acetic acid production using Pantoea agglomerans strain PVM

Aims: To optimize the medium components for the production of indole‐3‐acetic acid (IAA) by isolated bacterium Pantoea agglomerans strain PVM. Methods and Results: Present study deals with the production of an essential plant hormone IAA by a bacterial isolate P. agglomerans strain PVM identified by 16S rRNA gene sequence analysis. The medium containing 8 g l^?1 of meat extract and 1 g l^?1 of l ‐tryptophan (precursor) at optimum pH 7, 30°C and 48‐h incubation gave the maximum production of IAA (2·191 g l^?1). Effect of IAA synthesized on in vitro root induction in Nicotiana tobacum (leaf) explants was compared with that of control. IAA was characterized by high‐performance thin‐layer chromatography, high‐performance liquid chromatography and gas chromatography–mass spectroscopy. Conclusions: Pantoea agglomerans strain PVM was a good candidate for the inexpensive and utmost production of IAA in short period, as it requires simple medium (meat extract and l ‐tryptophan). Significance and Impact of the Study: The present report first time showed the rapid, cost‐effective and maximum production of IAA. No reports are available on the optimization of particular medium components for the production of IAA. This study demonstrates a novel approach for in vitro root induction in N. tobacum (leaf) explants.     

Rice endophyte Pantoea agglomerans YS19 promotes host plant growth and affects allocations of host photosynthates

AIMS: The aims of the study were to identify the effects of rice endophyte Pantoea agglomerans YS19 on host plant growth and allocations of photosynthates. METHODS AND RESULTS: Endophytic diazotrophic YS19 showed nitrogen-fixing activity in N-free medium, and produced four categories of phytohormones which were indole-3-acetic acid, abscisic acid, gibberellic acid and cytokinin in Luria-Bertani medium. Inoculation of YS19 improved the biomass of the 12-day-cultivated host rice seedlings by 63.4% on N-free medium or by 18.7% on N-supplemented medium. Spraying of YS19 cell culture onto the rice plants at the premilk stage enhanced the transportation of the photosynthetic assimilation product from the source (flag leaves) to the sink (stachys) significantly. The formation of the plant sink was obviously inhibited when YS19 cell culture was applied at the late milk stage. CONCLUSIONS: This research suggests that endophyte YS19 promotes host rice plant growth and affects allocations of host photosynthates. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggested that YS19 possesses the potential for increasing rice production in field application. Meanwhile, a suitable plant growth stage must be selected for the foliar spraying of YS19 cell culture.     

水稻内生优势成团泛菌GFP标记菌株的性质与标记丢失动力学

为研究内生细菌对宿主植物侵染定殖的机理和其共生生物学作用 ,对水稻内生优势成团泛菌 (Pantoeaagglomerans)YS19与绿色荧光蛋白 (GFP)标记的YS19B ::gfp菌株的生长动力学进行了比较研究 ,探讨了成团泛菌YS19B ::gfp的标记稳定性和荧光性质 .标记菌株与野生型菌株相比 ,最大比生长速率和最大生物量仅减小 12 4 %和 6 % ,代时延长 14 0 % .成团泛菌YS19B ::gfp在指数期连续传代培养 10 0代后 ,GFP标记的保持率为 89 1% ,建立了标记菌株在有标记丢失存在时的生长动力学模型 :dX+ dt =μ+ (1-p)X+ ,解析出细胞分裂时标记丢失的概率p =9 75 6× 10 -7,确定了方程的模型参数 .标记菌株的荧光光谱在激发波长为 4 0 0nm时 ,最大发射波长为 5 0 8nm ,与供体菌株完全相同 .在LB培养基上生长时 ,成团泛菌YS19B ::gfp的GFP产生时间在指数期末期到稳定期较快 ,并于培养至 2 0h时达到最高 ,同时单位菌体生物量的荧光强度也达到最大 .结果说明 ,在GFP标记后成团泛菌YS19B ::gfp的生长仅受到较小影响 ,不致对成团泛菌的生理活动造成大的改变 ,同时由于该菌对宿主的侵染能力比其它内生细菌要强得多 ,因而该菌对植物的侵染活性影响也较小 ,该菌仍然可以保持其内生优势地位 .该标记的稳定性比较高 ,荧光产生正常 ,很适     

Isolation of endophytic diazotroph Pantoea agglomerans and nondiazotroph Enterobacter asburiae from sweetpotato stem in Japan

AIMS: To isolate and identify diazotrophic endophytes in the stem of Japanese sweetpotato cv. Koganesengan. METHODS AND RESULTS: Surface-sterilized and thinly sliced (1-2 mm) sweetpotato stem samples were incubated in test tubes with semi-solid modified Rennie (MR) medium. The test tubes were assayed for acetylene reduction activity (ARA) 5 days after incubation at 30 degrees C. Twelve isolates were obtained from MR plates inoculated with a loop of semi-solid MR medium from ARA+ tubes. However, ARA test showed that only nine isolates were diazotrophic and three were nondiazotrophic strains. Using the API 20E diagnostic kit, four diazotrophic isolates were identified as strains of Pantoea spp. and five isolates as Klebsiella spp. The nondiazotrophic bacteria were strains of Enterobacter spp. A diazotrophic isolate Pantoea sp. MY1 and nondiazotrophic isolate Enterobacter sp. MY2 were identified to the species level by full sequence analysis of 16S rRNA gene. The results showed that MY1 had 99.2% similarity to Pantoea agglomerans ATCC 27155 and MY2 had 99.5% similarity to Enterobacter asburiae ATCC 35953. CONCLUSION: The stem of sweetpotato cv. Koganesengan was colonized by diazotrophic endophyte P. agglomerans and nondiazotrophic endophyte E. asburiae. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is an essential step toward understanding the ecology and interaction between endophytic bacteria and sweetpotato.     

Root colonization by symplasmata-forming Enterobacter agglomerans

Abstract Enterobacter agglomerans strains are able to form cell aggregates called symplasmata when grown in a liquid medium. The nitrogen-fixing E. agglomerans strain NO30, isolated from the rhizosphere soil of rice, was inoculated onto roots of axenically grown wheat and rice seedlings and could colonize the roots of both plants. The ability of NO30 cells to colonize the plant roots seemed comparable in the host and non-host plants, as far as colony forming units (cfu) measurements were concerned. Nevertheless, electron microscopy (SEM, TEM) revealed that, in the case of rice, the normal host plant for NO30, the colonization was characterized by the formation of symplasmata, whereas only individual cells were found on wheat roots. Symplasmata formation seems to be specific for colonization of the host plant, rice. This finding also means that colonization of the host plant may be largely underestimated when measured by conventional techniques. Symplasmata formed in liquid medium or on the roots of rice were stained using Thiery's and Swift's technique, and the presence of polysaccharides and proteins was revealed in the extracellular matrix as well as in fibrils anchoring symplasmata to other symplasmata or to plant cells.     

Effect of γ-irradiation on membrane fatty acids and peptidoglycan's muropeptides of Pantoea agglomerans, a plant pathogen

Aims:  The aim of this study was to evaluate the effect of γ-irradiation on the fatty acids (FA) and muropeptides content of two strains of an Enterobacteriacea : Pantoea agglomerans .
Methods and Results:  Pantoea agglomerans strains ATCC 49174 and RL1 isolated from irradiated carrots were used for this study. Radiation treatments (1 and 3·5 kGy) were performed to study the radiotolerance. Total lipids were obtained by multiple extractions using methanol/chloroform (2 : 1) and were quantified by GC. Muropeptides were purified by successive enzymatic digestions and analysed using a reverse phase C₁₈ column in high performance liquid chromatography. A significant ( P  ≤ 0·05) modification of the bacterial wall was noticed for the membrane FA composition and the muropeptides.
Conclusion:  Effects of irradiation on the bacterial membrane are noticeable and could play an important role on the cellular response and ability to survive this harsh environment.
Significance and Impact of the Study:  To our knowledge, it is the first study to demonstrate the effects of ionizing irradiation on the modification of the FA and one of the few to confirm its effects on the muropeptides of the peptidoglycan.     

Production of 2,3-butanediol by Klebsiella pneumoniae BLh-1 and Pantoea agglomerans BL1 cultivated in acid and enzymatic hydrolysates of soybean hull

We investigated the production of 2,3-butanediol by two enterobacteria isolated from an environmental consortium, Klebsiella pneumoniae BLh-1 and Pantoea agglomerans BL1, in a bioprocess using acid and enzymatic hydrolysates of soybean hull as substrates. Cultivations were carried out in orbital shaker under microaerophilic conditions, at 30°C and 37°C, for both bacteria. Both hydrolysates presented high osmotic pressures, around 2,000 mOsm/kg, with varying concentrations of glucose, xylose, and arabinose. Both bacteria were able to grow in the hydrolysates, at both temperatures, and they efficiently converted sugars into 2,3-butanediol, showing yields varying from 0.25 to 0.51 g/g of sugars and maximum 2,3-butanediol concentrations varying from 6.4 to 21.9 g/L. Other metabolic products were also obtained in lower amounts, notably ethanol, which peaked at 3.6 g/L in cultures using the enzymatic hydrolysate at 30°C. These results suggest the potential use of these recently isolated bacteria to convert lignocellulosic biomass hydrolysates into value-added products.     

Pseudomonas putida NBRIC19 dihydrolipoamide succinyltransferase (SucB) gene controls degradation of toxic allelochemicals produced by Parthenium hysterophorus

Aims: The aim of this study was to identify the gene responsible for degradation of toxic allelochemicals of Parthenium by generating Tn5‐induced mutant of Pseudomonas putida NBRIC19. Furthermore, the study characterizes the mutant at physiological, biochemical and molecular level that helped in understanding the mechanisms of reducing the allelopathic inhibition of Parthenium by Ps. putida NBRIC19. Methods and Results: Tn5 mutant S‐74.3 showing inability to degrade toxic allelochemicals was selected after screening 22 000 transconjugants. Tn5 flanking SucB gene (dihydrolipoamide succinyltransferase) of Ps. putida NBRIC19 was found to be responsible for the degradation of toxic allelochemicals that also affected biofilm formation, chemotaxis and alginate production under toxic environment of allelochemicals. Phenotypic microarray data revealed that the respiratory activity of Ps. putida NBRIC19 and S‐74.3 differed on 47 substrates including amino acids, carboxylic acids, peptides and some chemical inhibitors. Conclusions: Study revealed that SucB gene regulates processes either directly or indirectly in Ps. putida NBRIC19, which on inactivation made the mutant less compatible for tolerating stress. Significance and Impact of the Study: This work provides the first evidence for a functional role of Ps. putida SucB gene in degradation of toxic allelochemicals of Parthenium that lead to reversal of plant growth inhibition by these toxic allelochemicals. The investigation also revealed interesting features about the involvement of microbes in plant–plant allelopathic interactions.     

Effect of temperature and soil moisture content on the colonization of the wheat rhizosphere by antiphytopathogenic bacilli

Vegetative experiments showed that the population density of antiphytopathogenic bacillar species introduced into the rhizosphere of spring wheat seedlings essentially depended on the soil temperature and not on the soil moisture content. As a rule, the population of introduced bacilli increased with the temperature. Under both low and optimal soil moisture contents, introduced bacilli were efficiently acclimated in the wheat rhizosphere.     

Relative importance of amino acids, glycine-betaine and ectoine synthesis in the biocontrol agent Pantoea agglomerans CPA-2 in response to osmotic, acidic and heat stress

AIMS: The objective of this work was to determine the role of different compatible solutes in adaptation of Pantoea agglomerans CPA-2 at different stages of growth to solute (0.98, 0.97, 0.96 aw), heat (35 and 40 degrees C) and acidic (pH 4.0, 5.0, 6.0) stress. METHODS AND RESULTS: Solute stress was imposed by using NaCl, glucose or glycerol, and pH was imposed with malic and citric acids. The accumulation of glycine-betaine, ectoine and amino acids in bacterial cells was quantified using high performance liquid chromathography (HPLC). There was a significant (P<0.05) accumulation of glycine-betaine (NaCl modified, 100-150 micromol g(-1) dry weight of cells) and ectoine (glucose modified media, >340 micromol g(-1) dry weight of cells) in the cells over a 48 h incubation period when compared with controls (<10 micromol g(-1) dry weight of cells). Chromatographic profile of amino acids was different with respect to control when NaCl or glucose was used as osmolyte. CONCLUSIONS: Pantoea agglomerans CPA-2 cells synthesised significant amounts of glycine-betaine and ectoine in response to imposed solute stress. However, these compounds and tested amino acids were not involved in cellular adaptation to either heat or pH stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This type of information can be effectively applied to improve ecophysiological quality of cells of bacterial biocontrol agents for better survival and biocontrol efficacy in the phyllosphere of plants.     

Production of indole-3-acetic acid, aromatic amino acid aminotransferase activities and plant growth promotion by Pantoea agglomerans rhizosphere isolates

The production of auxins, such as indole-3-acetic acid (IAA), by rhizobacteria has been associated with plant growth promotion, especially root initiation and elongation. Six indole-producing bacteria isolated from the rhizosphere of legumes grown in Saskatchewan soils and identified as Pantoea agglomerans spp. were examined for their ability to promote the growth of canola, lentil and pea under gnotobiotic conditions and for tryptophan (Trp)-dependent IAA production. Five of the isolates enhanced root length, root weight or shoot weight by 15–37% in at least one of the plant species, but isolates 3–117 and 5–51 were most consistent in enhancing plant growth across the three species. Indole concentrations in the rhizosphere of plants grown under gnotobiotic conditions increased in the presence of the rhizosphere isolates and when Trp was added 3 days prior to plant harvest. Isolates 3–117, 5–51 and 5–105 were most effective in increasing rhizosphere indole concentrations. Colony hybridization confirmed that all of the isolates possessed the ipdC gene which codes for a key enzyme in the Trp-dependent IAA synthetic pathway. The activity of amino acid aminotransferase (AAT), catalyzing the first step in the Trp-dependent synthetic pathway, was examined in the presence of Trp and other aromatic amino acids. All of the isolates accumulated Trp internally and released different amounts of IAA. The production of IAA from the isolates was greatest in the presence of Trp, ranging from 2.78 to 16.34 μg mg protein⁻¹ in the presence of 250 μg of Trp ml⁻¹. The specific activity of AAT was correlated with the concentration of IAA produced in the presence of Trp but not when tyrosine (Tyr), phenylalanine (Phe) or aspartate (Asp) was used as a sole nitrogen source. Isolate 3–117, which produced significant concentrations of IAA in the presence and absence of Trp, was able to use aromatic amino acids as sole sources of nitrogen and was most consistent in enhancing the growth of canola, lentil and pea may have potential for development as a plant growth-promoting inoculant. Responsible Editor: Peter A. H. Bakker.     

接种量对费氏中华根瘤菌HN01DL根圈定殖与结瘤的影响

在灭菌土和未灭菌土盆栽系统中 ,研究了大豆种子的表面初始接种量对费氏中华根瘤菌HN0 1DL在大豆根圈中的定殖动态与结瘤的影响。结果表明 ,与 3个接种量对应的早期根圈定殖动态和水平有明显差异 ,但随着宿主植物根系的生长其差异逐渐减小 ,整个定殖动态曲线的变化和定殖密度趋向一致 ,并且发现 3个不同的初始接种量对HN0 1DL在大豆黑农 33根系上的结瘤数量和占瘤率没有显著影响。     

Contribution of calystegine catabolic plasmid to competitive colonization of the rhizosphere of calystegine-producing plants by Sinorhizobium meliloti Rm41

Calystegines are plant secondary metabolites produced by the roots of a few plant species, and the ability to catabolize calystegines is infrequent in rhizosphere bacteria. In Sinorhizobium meliloti Rm41, the endosymbiont of the legume Medicago sativa, this ability results from the presence of the genes cac (for calystegine catabolism) located on the nonsymbiotic plasmid pRme41a. The effect of the cac catabolic plasmid pRme41a on the ability of Rm41 to colonize the rhizosphere of calystegine-positive plants was studied using derivatives of Rm41 with or without cac catabolic plasmid. When strains were inoculated alone, the presence of a cac catabolic plasmid had no effect on their colonization of the rhizosphere, regardless of whether plants produced calystegines or not. However, a spontaneous rifampicin-resistant mutant of Rm41 containing a cac catabolic plasmid reached population levels in the rhizosphere of calystegine-positive plants that were several orders of magnitude higher than those of the same strain without the plasmid, when each was co-inoculated with a derivative of Rm41 cured of pRme41a. In contrast, the cac catabolic plasmid provided little or no selective advantage in the rhizosphere of calystegine-negative plants. In conclusion, the cac catabolic plasmid pRme41a can contribute to the ability of S. meliloti Rm41 to colonize the rhizosphere of alternative, nonlegume plant hosts producing calystegines.     

The structure of the O-specific polysaccharide of the lipopolysaccharide from Pantoea agglomerans strain FL1

A neutral O-specific polysaccharide consisting of d-rhamnose was obtained by mild acid hydrolysis of the lipopolysaccharide of the plant pathogenic bacterium Pantoea agglomerans strain FL1, a common epiphyte of many plant species, and associated with Pseudomonas savastanoi pv. savastanoi in young and apparently intact olive knots. By means of compositional and methylation analyses, and NMR spectroscopy, the chemical repeating unit of the polymer was identified as a linear tetrasaccharide of the structure:     

Sulfur Disproportionation by the Facultative Anaerobe Pantoea agglomerans SP1 as a Mechanism for Chromium(VI) Reduction

The facultative anaerobe Pantoea agglomerans SP1 was previously shown to couple anaerobic growth to the dissimilatory reduction of a variety of electron acceptors, including Fe(III), Mn(IV), and Cr(VI), but not sulfate. In this study, we describe the additional capacity of this organism to grow via the disproportionation of elemental sulfur to sulfate and sulfide, a process that has previously only been reported in strictly anaerobic members of the i - Proteobacteria . The sulfide scavengers, Fe(III) Mn(IV), and for the first time, Cr(VI), were found to enhance growth coupled to S 0 -disproportionation. To our knowledge, this is the first facultative anaerobe reported to couple growth to sulfur disproportionation. This organism may play a role in the attenuation of Cr(VI) pollution.