Genome sequence of Pantoea agglomerans strain IG1

Pantoea agglomerans is a gram-negative bacterium that grows symbiotically with various plants. Here we report the 4.8-Mb genome sequence of P. agglomerans strain IG1. The lipopolysaccharides derived from P. agglomerans IG1 have been shown to be effective in the prevention of various diseases, such as bacterial or viral infection, lifestyle-related diseases. This genome sequence represents a substantial step toward the elucidation of pathways for production of lipopolysaccharides.     

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.     

Virulence mechanisms and host specificity of gall-forming Pantoea agglomerans

Pantoea agglomerans has been transformed from a commensal bacterium associated with many plants into a host-specific gall-forming pathogen by acquiring a plasmid-borne pathogenicity island. This pathogenicity island harbors the hrp/hrc gene cluster, in addition to genes encoding type III effector proteins, biosynthesis of the phytohormones indole-3-acetic acid and cytokinin, multiple diverse insertion sequences and pseudogenes. This review describes a unique model for understanding the emergence of new pathogens or new pathogenic variants, offering an insight into the function of type III effectors in host specificity and the evolution of a pathogen into pathovars. It also addresses the primary role of type III effectors in gall initiation as compared with a secondary role of phytohormones secreted by the pathogen.     

Colonization dynamics of Pantoea agglomerans in the wheat root habitat

Plants are colonized by microbial communities that have diverse implications for plant development and health. The establishment of a stable plant–bacteria interaction depends on a continuous coexistence over generations. Transmission via the seed is considered as the main route for vertical inheritance of plant-associated bacteria. Nonetheless, the ecological principles that govern the plant colonization by seed endophytes remain understudied. Here we quantify the contribution of arrival time and colonization history to bacterial colonization of the wheat root. Establishing a common seed endophyte, Pantoea agglomerans, and wheat as a model system enabled us to document bacterial colonization of the plant roots during the early stages of germination. Using our system, we estimate the carrying capacity of the wheat roots as 10⁸ cells g⁻¹, which is robust among individual plants and over time. Competitions in planta reveal a significant advantage of early incoming colonizers over late-incoming colonizers. Priming for the wheat environment had little effect on the colonizer success. Our experiments thus provide empirical data on the root colonization dynamics of a seed endophyte. The persistence of seed endophyte bacteria with the plant population over generations may contribute to the stable transmission that is one route for the evolution of a stable host-associated lifestyle.     

Sugarcane Growth Promotion by the Endophytic Bacterium Pantoea agglomerans 33.1

The promotion of sugarcane growth by the endophytic Pantoea agglomerans strain 33.1 was studied under gnotobiotic and greenhouse conditions. The green fluorescent protein (GFP)-tagged strain P. agglomerans 33.1::pNKGFP was monitored in vitro in sugarcane plants by microscopy, reisolation, and quantitative PCR (qPCR). Using qPCR and reisolation 4 and 15 days after inoculation, we observed that GFP-tagged strains reached similar density levels both in the rhizosphere and inside the roots and aerial plant tissues. Microscopic analysis was performed at 5, 10, and 18 days after inoculation. Under greenhouse conditions, P. agglomerans 33.1-inoculated sugarcane plants presented more dry mass 30 days after inoculation. Cross-colonization was confirmed by reisolation of the GFP-tagged strain. These data demonstrate that 33.1::pNKGFP is a superior colonizer of sugarcane due to its ability to colonize a number of different plant parts. The growth promotion observed in colonized plants may be related to the ability of P. agglomerans 33.1 to synthesize indoleacetic acid and solubilize phosphate. Additionally, this strain may trigger chitinase and cellulase production by plant roots, suggesting the induction of a plant defense system. However, levels of indigenous bacterial colonization did not vary between inoculated and noninoculated sugarcane plants under greenhouse conditions, suggesting that the presence of P. agglomerans 33.1 has no effect on these communities. In this study, different techniques were used to monitor 33.1::pNKGFP during sugarcane cross-colonization, and our results suggested that this plant growth promoter could be used with other crops. The interaction between sugarcane and P. agglomerans 33.1 has important benefits that promote the plant''s growth and fitness.     

Dissimilatory metal reduction by the facultative anaerobe Pantoea agglomerans SP1

Anaerobic enrichments with acetate as the electron donor and Fe(III) as the terminal electron acceptor were obtained from sediments of Salt Pond, a coastal marine basin near Woods Hole, Mass. A pure culture of a facultatively anaerobic Fe(III) reducer was isolated, and 16S rRNA analysis demonstrated that this organism was most closely related to Pantoea (formerly Enterobacter) agglomerans, a member of the family Enterobacteriaceae within the gamma subdivision of the Proteobacteria. This organism, designated strain SP1, can grow by coupling the oxidation of acetate or H(2) to the reduction of a variety of electron acceptors, including Fe(III), Mn(IV), Cr(VI), and the humic substance analog 2,6-anthraquinone disulfonate, but not sulfate. To our knowledge, this is the first mesophilic facultative anaerobe reported to couple acetate oxidation to dissimilatory metal reduction.     

Dissimilatory Metal Reduction by the Facultative Anaerobe Pantoea agglomerans SP1

Anaerobic enrichments with acetate as the electron donor and Fe(III) as the terminal electron acceptor were obtained from sediments of Salt Pond, a coastal marine basin near Woods Hole, Mass. A pure culture of a facultatively anaerobic Fe(III) reducer was isolated, and 16S rRNA analysis demonstrated that this organism was most closely related to Pantoea (formerly Enterobacter) agglomerans, a member of the family Enterobacteriaceae within the gamma subdivision of the Proteobacteria. This organism, designated strain SP1, can grow by coupling the oxidation of acetate or H₂ to the reduction of a variety of electron acceptors, including Fe(III), Mn(IV), Cr(VI), and the humic substance analog 2,6-anthraquinone disulfonate, but not sulfate. To our knowledge, this is the first mesophilic facultative anaerobe reported to couple acetate oxidation to dissimilatory metal reduction.     

5'-Deoxyisopentenyladenosine and other cytokinins in culture filtrates of the bacterium Pantoea agglomerans

A Pantoea agglomerans isolate from barley seeds, selected for inducing growth promotion in tomato test plants, was found to excrete several hormones of the cytokinin class into its culture medium. In addition to isopentenyladenine, isopentenyladenosine and the 2-methylthiol derivates of these, an unknown compound with affinity to anticytokinin antibodies was also isolated. Mass spectroscopy indicated the structure of this to be a deoxyisopentenyladenosine. The structure of 9-(5'-deoxy- β - d -ribofuranosyl)-6-(3-methyl-2-butenylamino)purine was verified after synthesis of standards and analysis with GC–MS. The synthesized 5'-deoxyisopentenyl-adenosine showed activity in the Amaranthus bioassay, specific for cytokinins. To our knowledge this is the first report of a naturally occurring cytokinin containing 5'-deoxyribose.     

Biological Control of Bacterial Wilt of Bean Using a Bacterial Endophyte, Pantoea agglomerans

Indoor studies were conducted to determine the potential use of Pantoea agglomerans isolate LRC 8311 as a biocontrol agent for control of bacterial wilt of bean caused by Curtobacterium flaccumfaciens pv. flaccumfaciens. Soaking seeds of great northern bean cv. US1140 in a suspension of 3 × 10⁸ cfu/ml P. agglomerans resulted in thorough endophytic colonization of the entire bean seedling from root to apical stem after 7 days, regardless of whether the inoculated seeds were hilum injured or not. Colonization of seedlings by P. agglomerans increased seedling height after 10 days, and had no negative effect on seedling emergence. Treatment of hilum‐injured bean seeds of great northern bean cv. US1140 or navy bean cv. Morden003 with a mixture of P. agglomerans + C. flaccumfaciens pv. flaccumfaciens resulted in a high rate of colonization of seedlings by P. agglomerans, reduced frequency of infection by C. flaccumfaciens pv. flaccumfaciens, improved seedling emergence and height, and reduced disease severity, compared with seeds treated with the wilt pathogen alone. Application of P. agglomerans as a soil drench 24 h after planting was also effective in suppressing bacterial wilt in some instances, but was generally not as effective as seed treatment. The study suggests that seed treatment with P. agglomerans may be an effective and practical method for control of bacterial wilt of bean.     

Water activity,temperature, and pH effects on growth of the biocontrol agent Pantoea agglomerans CPA-2

The growth response of the biocontrol agent Pantoea agglomerans to changes in water activity (a(w)), temperature, and pH was determined in vitro in nutrient yeast extract-sucrose medium. The minimum temperature at which P. agglomerans was able to grow was 267-272 kelvins (-6 to -1 degrees C), and growth of P. agglomerans did not change at varying pH levels (4.5-8.6). The minimum a(w) for growth was 0.96 in media modified with glycerol and 0.95 in media modified with NaCl or glucose. Solute used to reduce water activity had a great influence on bacterial growth, especially at unfavourable conditions (e.g., low pH or temperature). NaCl stimulated bacterial growth under optimum temperatures but inhibited it under unfavourable pH conditions (4.5 or 8.6). In contrast, the presence of glucose in the medium allowed P. agglomerans to grow over a broad range of temperature (3-42 degrees C) or pH (5-8.6) regimes. This study has defined the range of environmental conditions (a(w), pH, and temperature) over which the bacteria may be developed for biological control of postharvest diseases.     

Purification and Characterization of Gallic Acid Decarboxylase from Pantoea agglomerans T71

Oxygen-sensitive gallic acid decarboxylase from Pantoea (formerly Enterobacter) agglomerans T71 was purified from a cell extract after stabilization by reducing agents. This enzyme has a molecular mass of approximately 320 kDa and consists of six identical subunits. It is highly specific for gallic acid. Gallic acid decarboxylase is unique among similar decarboxylases in that it requires iron as a cofactor, as shown by plasma emission spectroscopy (which revealed an iron content of 0.8 mol per mol of enzyme subunit), spectrophotometric analysis (absorption shoulders at 398 and 472 nm), and inhibition of the enzyme activity by 2,2′-bipyridyl, o-phenanthroline, and EDTA. Another interesting feature of this strain is the fact that it contains a tannase, which is used together with the gallic acid decarboxylase in a two-enzyme resting cell bioconversion to synthesize valuable pyrogallol from readily available tannic acid.     

Effects of a lipopolysaccharide from Pantoea agglomerans on the cocaine-induced place preference

A lipopolysaccharide from Pantoea agglomerans (LPSp) was purified, and its effect on the cocaine-induced place preference was examined in rats. Cocaine (4 mg/kg, i.p.) produced a significant place preference. Administration of LPSp (5 – 1000 μg/kg, i.p.) alone resulted in neither preference nor aversion for either the drug- or saline-associated place. However, pretreatment with LPSp (500 and 1000 μg/kg, i.p.) abolished the place preference that had been induced by cocaine. Furthermore, treatment with LPSp (500 μg/kg, i.p.) abolished cocaine (20 mg/kg, i.p.)-induced locomotor enhancement in mice. These results suggest that while LPSp itself may possess neither reinforcing nor locomotor enhancing effects, it blocks both the reinforcing and the locomotor enhancing effects of cocaine. Therefore, LPSp might be useful in pharmacotherapy for prevention of recurrent cocaine abuse.     

Absence of lysogeny in wild populations of Erwinia amylovora and Pantoea agglomerans

Lytic bacteriophages are in development as biological control agents for the prevention of fire blight disease caused by Erwinia amylovora. Temperate phages should be excluded as biologicals since lysogeny produces the dual risks of host resistance to phage attack and the transduction of virulence determinants between bacteria. The extent of lysogeny was estimated in wild populations of E. amylovora and Pantoea agglomerans with real–time polymerase chain reaction primers developed to detect E. amylovora phages belonging to the Myoviridae and Podoviridae families. Pantoea agglomerans, an orchard epiphyte, is easily infected by Erwinia spp. phages, and it serves as a carrier in the development of the phage‐mediated biological control agent. Screening of 161 E. amylovora isolates from 16 distinct geographical areas in North America, Europe, North Africa and New Zealand and 82 P. agglomerans isolates from southern Ontario, Canada showed that none possessed prophage. Unstable phage resistant clones or lysogens were produced under laboratory conditions. Additionally, a stable lysogen was recovered from infection of bacterial isolate Ea110R with Podoviridae phage ΦEa35‐20. These laboratory observations suggested that while lysogeny is possible in E. amylovora, it is rare or absent in natural populations, and there is a minimal risk associated with lysogenic conversion and transduction by Erwinia spp. phages.     

Inhibition of morphine dependence by a lipopolysaccharide from Pantoea agglomerans.

A lipopolysaccharide from Pantoea agglomerans (LPSp) was purified and examined for relief of morphine dependence by observing its inhibition of the jumping of mice on naloxone-precipitate withdrawal. Administration of LPSp either intravenously or intradermally showed marked inhibition of the jumping. Beta-endorphin in mouse serum and brain tissue were recognized to be in synchrony with the time course of the relief. Administration of TNF-alpha gave similar effect, suggesting that LPSp induces a cytokine cascade to produce endogenous TNF followed by ACTH/beta-LPH gene products and beta-endorphin. The effect of LPSp was better than that of LPS from E. coli or Bordetella pertussis, and thus is considered to be applicable for clinical use.     

Transport and utilization of ferrioxamine-E-bound iron inErwinia herbicola (Pantoea agglomerans)

Summary We have analyzed ferrioxamine-E-mediated iron uptake and metabolization inErwinia herbicola K4 (Pantoea agglomerans) by means of in vivo Mössbauer spectroscopy and radioactive labeling techniques. A comparison of cell spectra with the spectrum of ferrioxamine clearly demonstrates that ferrioxamine E is not accumulated in the cell, indicating a fast metal transfer. Only two major components of iron metabolism can be detected, a ferric and a ferrous species. At 30 min after uptake, 86% of the internalized metal corresponded to a ferrous ion compound and 14% to a ferric iron species. Metal transfer apparently involves a reductive process. With progressing growth, the oxidized species of the two major proteins becomes dominant. The two iron metabolites closely resemble species previously isolated fromEscherichia coli. These components of iron metabolism differ from bacterio-ferritin, cytochromes and most iron-sulfur proteins. All other iron-containing cellular components are at least one order of magnitude lower in concentration. We suggest that the ferrous and ferric iron species correspond to two different oxidation states of a low-molecular mass protein.     

Effects of inoculation of EPS-producing Pantoea agglomerans on wheat rhizosphere aggregation

We investigated plant and soil nitrogen pools and soil processes in monospecific stands of the C₃ sedge Scirpus olneyi and the C₄ grass Spartina patens grown in the field in open top chambers in a brackish marsh on the Chesapeake Bay. Stands of S. olneyi responded to eight years of elevated CO₂, by increased rates of net ecosystem gas exchange and a large stimulation of net ecosystem production. We conducted our study in the summer of 1994 and 1995 when soil cores were collected and aboveground biomass was estimated. Nitrogen concentration in elevated CO₂ treatments was reduced 15% in stems of S. olneyi and 8% in the upper 10 cm of the soil profile. While total plant nitrogen per unit of land area remained the same between treatments, total soil nitrogen showed a non-significant tendency to decrease in the upper 10 cm of the soil profile in elevated CO₂ both years of study. A significant decrease in soil bulk density largely contributed to the observed decrease in soil nitrogen. Exchangeable nitrogen and potential denitrification rates were also reduced in elevated CO₂, but net nitrogen mineralization was unchanged by elevated CO₂ treatment in S. olneyi both years. Plants and soils in a pure stand of the C₄ grass, S. patens, showed none of these effects of elevated CO₂ treatment. Our data provides evidence of changes in nitrogen dynamics of an ecosystem exposed to elevated CO₂ for eight years; however due to the variability in these data, we cannot say if or how these changes are likely to impact the effect of rising CO₂ on primary production or carbon accumulation in this ecosystem in the future.     

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     

In vitro symplasmata formation in the rice diazotrophic endophyte Pantoea agglomerans YS19

Pantoea (formerly Enterobacter) agglomerans YS19 is an endophytic diazotrophic bacterium isolated from rice (Oryza sativa cv. Yuefu) grown in temperate climatic regions in west Beijing (China). The bacterium forms aggregate structures called `symplasmata'. A symplasmatum is a multicellular aggregate structure in which several (at least two) to hundreds of individual cells tightly bind together. The studies on the symplasmata formation of YS19 showed that there were two growth stages for YS19, including the single cell stage existing before exponential growth phase and the symplasmata forming stage starting at the early stationary growth phase in liquid GY (glucose yeast extract) medium or at the end of the exponential growth phase in liquid LB (Luria-Bertani) medium. There was a correlation between symplasmata formation and bacterial growth phase. When the medium was acidified, the cell growth rate was affected by the low pH of the medium, but the time required for symplasmata formation was not influenced by it. YS19 also formed symplasmata on agar medium, where more symplasmata were formed than in liquid medium. The volume of individual constitutional cells of symplasmata was sharply decreased by more than a half in comparison with that of the single cells existing before symplasmata formation. On all the media tested, YS19 formed symplasmata in most of the cell growth phases. The genome DNA/DNA homology between P. agglomerans YS19 and type strain P. agglomerans JCM1236^T (ATCC27155^T) was determined as 90.1%, confirming its membership of P. agglomerans. In order to investigate the phylogenetic relationships of YS19 at the intraspecific, intrageneric and super-generic level, the 16S rDNA similarities between strain YS19 and 17 other strains of Pantoea and 4 representatives of the closely related genera were analyzed. All the strains of Pantoea were clustered into 5 groups, and YS19 was clustered in a unique branch. The 16S rDNA similarity between YS19 and type strain JCM1236^T was 93.9%, much lower than the generally accepted value (=97%) for members of the same species, indicating that the 16S rDNA of YS19 has a distinct molecular characteristic.     

Production of the biocontrol agent Pantoea agglomerans strain CPA-2 using commercial products and by-products

The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strain CPA-2 of the biocontrol agent Pantoea agglomerans and minimum cost of media, whilst maintaining biocontrol efficacy. To reduce the cost of media, commercial products and by-products were tested. P. agglomerans can be produced using a combination of nitrogen sources such as yeast extract (5 g l(-1)) and dry beer yeast (10 g l(-1)) with inexpensive carbohydrates such as sucrose (10 g l(-1)) and molasses (20 g l(-1)), respectively, maintaining the efficacy of the biocontrol agent against Penicillium digitatum and P. italicum on oranges. The results obtained in this study could be used to provide a reliable basis for a scale-up of this fermentation process to an industrial level.     

成团泛菌低分子脂多糖的急性毒性、刺激性、致敏性及遗传毒性评估

本研究对成团泛菌低分子脂多糖(Pantoea agglomerans lipopolysaccharide,LPSp)的安全性进行初步评估.本研究采用一次限量法,用昆明种小鼠进行LPSp急性经口毒性试验,了解LPSp的急性毒性;采用新西兰兔分别进行LPSp急性和多次皮肤刺激性试验以及急性眼刺激性试验,了解LPSp的皮肤和粘膜刺激性;采用豚鼠进行LPSp皮肤变态反应试验,了解LPSp的致敏性;应用平板掺入法进行鼠伤寒沙门氏菌/回复突变试验和小鼠骨髓细胞微核试验考察LPSp的遗传危害.急性毒性试验结果显示,LPSp对小鼠经口一次灌胃的LD50大于5 000 mg/kg体重,属实际无毒级别;LPSp急性和多次皮肤刺激性试验以及急性眼刺激性试验结果显示,皮肤刺激和眼刺激积分均为0分,LPSp对皮肤无刺激性、对眼睛无急性刺激性;在皮肤变态反应试验中,LPSp在各观察时间点的皮肤变态反应积分均为0分,其致敏率均为0%,说明LPSp对豚鼠无致敏性; LPSp的鼠伤寒沙门氏菌/回复突变试验结果呈阴性(P>0.05);LPSp的小鼠骨髓细胞微核试验结果亦呈阴性,LPSp 各剂量组的微核发生率与阴性对照组未见统计学差异(P>0.05),而与阳性对照组有明显差异(P<0.01).本研究结果表明,在本实验剂量范围内,LPSp对小鼠经口毒性极低,属实际无毒级别,对家兔皮肤和眼睛无明显刺激性,对豚鼠无致敏性,对所试菌株和小鼠体细胞无诱变性和致突变性.