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.     

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:     

The purB gene controls rhizosphere colonization by Pantoea agglomerans

Aims: To isolate the rhizosphere competence‐defective transposon Tn5 mutant of Pantoea agglomerans NBRISRM (SRM) and to identify the gene causing defect in its root colonization ability. Methods and Results: From over 5000 clones containing Tn5, one mutant P. agglomerans NBRISRMT (SRMT) showing 6 log units less colonization when compared with SRM, after 30 days in sand‐nonsterilized soil assay system was selected for further work to determine the effects of the mutation on rhizosphere competence. Southern hybridization analysis of restricted genomic DNA of SRMT demonstrated that the mutant had a single Tn5 insert. SRM increased in titre to about 2 × 10⁸ CFU g^?1 root, compared with the indigenous bacterial population of heterotrophs of about 5 × 10⁷ CFU g^?1 root. In contrast, 30 days later, the titre value of SRMT was almost undetectable at 1 × 10² CFU g^?1 root, demonstrating its inability to survive and colonize the rhizosphere. Sequencing of the flanking region of the Tn5 mutant revealed that Tn5 disrupted the purB gene. Conclusions: A defect in the colonization phenotype of the SRMT was attributed to the disruption in adenylosuccinate lyase (EC 4.3.2.2) which is encoded by the pur B gene and is required for rhizosphere colonization in P. agglomerans. Significantly less exopolysaccharide and biofilm was formed by SRMT when compared to SRM, because of the disruption of the purB gene. Significance and Impact of the Study: This work provides the first evidence for a functional role of purB gene in rhizosphere competence and root colonization by any rhizobacteria.     

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.     

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.     

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.     

Secretion of anti-Plasmodium effector proteins from a natural Pantoea agglomerans isolate by using PelB and HlyA secretion signals

The insect-vectored disease malaria is a major world health problem. New control strategies are needed to supplement the current use of insecticides and medications. A genetic approach can be used to inhibit development of malaria parasites (Plasmodium spp.) in the mosquito host. We hypothesized that Pantoea agglomerans, a bacterial symbiont of Anopheles mosquitoes, could be engineered to express and secrete anti-Plasmodium effector proteins, a strategy termed paratransgenesis. To this end, plasmids that include the pelB or hlyA secretion signals from the genes of related species (pectate lyase from Erwinia carotovora and hemolysin A from Escherichia coli, respectively) were created and tested for their efficacy in secreting known anti-Plasmodium effector proteins (SM1, anti-Pbs21, and PLA2) in P. agglomerans and E. coli. P. agglomerans successfully secreted HlyA fusions of anti-Pbs21 and PLA2, and these strains are under evaluation for anti-Plasmodium activity in infected mosquitoes. Varied expression and/or secretion of the effector proteins was observed, suggesting that the individual characteristics of a particular effector may require empirical testing of several secretion signals. Importantly, those strains that secreted efficiently grew as well as wild-type strains under laboratory conditions and, thus, may be expected to be competitive with the native microbiota in the environment of the mosquito midgut.     

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.     

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.     

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.     

Solubilization of insoluble inorganic phosphates by a novel salt- and pH-tolerant Pantoea agglomerans R-42 isolated from soybean rhizosphere

To develop environment-friendly biofertilizer solubilizing insoluble phosphates, salt- and pH-tolerant, insoluble inorganic phosphate-solubilizing bacterium was isolated from soybean rhizosphere. On the basis of its physiological characteristics and Vitek analysis, this bacterium was identified as Pantoea agglomerans. The optimal medium composition and cultural conditions for the solubilization of insoluble phosphate by P. agglomerans R-42 were 3% (w/v) of glucose, 0.1% (w/v) of NH4NO3, 0.02% (w/v) of MgSO4 x 7H2O, and 0.06% (w/v) of CaCl2 x 2H2O along with initial pH 7.5 at 30 degree C. The soluble phosphate production under optimal condition was around 900 mg/l, which was approximately 4.6-fold higher than the yield in the MPVK medium. The solubilization of insoluble phosphate was associated with a drop in the pH of the culture medium. P. agglomerans R-42 showed resistance against different environmental stresses like 5-45 degrees C temperature, 1-5% salt concentration and 3-11 pH range. Insoluble phosphate solubilization was highest from CaHPO4 (1367 mg/l), hydroxyapatite (1357 mg/l) and Ca3(PO4)2 (1312 mg/l). However, the strain produced soluble phosphate to the culture broth with the concentrations of 28 mg/l against FePO4, and 19 mg/l against AlPO4, respectively.     

Role of glutamine synthetase in phenazine antibiotic production by Pantoea agglomerans Eh1087

Pantoea agglomerans strain Eh1087 produces the phenazine antibiotic D-alanylgriseoluteic acid. A glutamine auxotroph harboring an insertion in a putative glnA gene was obtained by transposon-mutagenesis of Eh1087 that produced less D-alanylgriseoluteic acid than the parental strain (strain Eh7.1). Cosmids encoding the Eh1087 glnA were isolated by their ability to complement the mutant for prototrophy. The role of the Eh1087 glnA locus was functionally confirmed by complementation of an Escherichia coli glnA mutant. Analysis of the nucleotide and deduced amino acid sequences of the Eh1087 glnA gene indicated a high degree of similarity to the glnA genes and glutamine synthetase enzymes of other Enterobacteriaceae. Isotopic labelling experiments with 15N-labelled ammonium sulfate demonstrated that wild-type Eh1087 incorporated 15N into griseoluteic acid more readily than the glnA mutant Eh7.1. We conclude that the 2 nitrogens in the phenazine nucleus originate from glutamine and the intracellular glutamine synthesized by Eh1087 is a source of the phenazine nucleus nitrogens even in glutamine-rich environments.     

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.     

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.     

Effect of leaf surface waxes on leaf colonization by Pantoea agglomerans and Clavibacter michiganensis

To evaluate the influence of leaf cuticular waxes on bacterial colonization of leaves, bacterial colonization patterns were examined on four glossy maize (Zea mays L.) mutants that were altered in their cuticular wax biosynthesis. Mutant gl3 was indistinguishable from the wild-type maize in its ability to foster colonization by the two bacterial species, Pantoea agglomerans and Clavibacter michiganensis subsp. nebraskensis. In contrast, the other three mutants supported the development of populations that significantly differed in size from those on the wild type. Mutant gl5 gl20 supported smaller populations of P. agglomerans, but not C. michiganensis, while mutant gl1 supported larger populations of C. michiganensis but not P. agglomerans. Mutant gl4 supported larger populations of both bacterial species. The exceptional ability of mutant gl4 to support bacterial colonization was hypothesized to result from the lower density of the crystalline waxes on gl4 than on the wild type, because a reduced crystal density could promote capillary water movement and water trapping among the wax crystals. This hypothesis was supported by the demonstration that the mechanical introduction of gaps among the wax crystals of the wild-type leaves resulted in the establishment of larger P. agglomerans populations on the altered leaves. These results provide the first direct evidence that leaf surface waxes affect bacterial leaf colonization at various stages of colonization and in a bacterial species-dependent manner.     

New blue pigment produced by Pantoea agglomerans and its production characteristics at various temperatures

A bacterium capable of producing a deep blue pigment was isolated from the environment and identified as Pantoea agglomerans. The pigment production characteristics of the bacterium under various conditions were studied. The optimal agar plate ingredients for pigment production by the bacterium were first studied: the optimal ingredients were 5 g/liter glucose, 10 g/liter tryptic soy broth, and 40 g/liter glycerol at pH 6.4. Bacterial cells grew on the agar plate during the incubation, while the pigment spread into the agar plate, meaning that it is water soluble. Pigment production was affected by the initial cell density. Namely, at higher initial cell densities ranging from 10(6.3) to 10(8.2) CFU/cm(2) on the agar plate, faster pigment production was observed, but no blue pigment was produced at a very high initial density of 10(9.1) CFU/cm(2). Thus, the cell population of 10(8.2) CFU/cm(2) was used for subsequent study. Although the bacterium was capable of growing at temperatures above and below 10°C, it could produce the pigment only at temperatures of ≥10°C. Moreover, the pigment production was faster at higher temperatures in the range of 10 to 20°C. Pigment production at various temperature patterns was well described by a new logistic model. These results suggested that the bacterium could be used in the development of a microbial temperature indicator for the low-temperature-storage management of foods and clinical materials. To our knowledge, there is no other P. agglomerans strain capable of producing a blue pigment and the pigment is a new one of microbial origin.