Purification, structural characterization and anti-UVB irradiation activity of an extracellular polysaccharide from Pantoea agglomerans.

Extracellular polysaccharide (EPS) produced by Pantoea agglomerans was purified for structural analysis and anti-UVB activity evaluation. The results showed that the EPS comprised of mannose and glucose in a ratio of 3:2. The molecular weight of EPS was estimated as 679 KDa. Fourier transform infrared spectrometry (FT-IR) showed that the EPS was composed of d-furanose without any substituent group. The structure of the EPS could be deduced as a repeated pentasaccharide arranged of three mannose molecules and two glucose molecules according to electrospray ionization mass spectrometry (ESI-MS) analysis and results mentioned above. The results also showed that the EPS attenuated UVB-induced cytotoxicity which was verified by morphology feature assay, down-regulated the expression of phosphor-JNK and active caspase3 significantly through MAPK pathway in UVB-irradiated HaCaT cells, and therefore protected the cells from apoptosis which could be identified by the lower apoptosis rate at low dose of UVB ray. In conclusion, the EPS as a macromolecule arranged by repeated pentasaccharide could protect HaCaT keratinocytes from UVB irradiation injury effectively, and the underlying mechanism may involve MAPK signaling pathway which contribute to apoptotic cell death. However, further studies on efficacy of the EPS in vivo are needed to determine.

Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions.

The ability to perform controlled experiments with bioaerosols is a fundamental enabler of many bioaerosol research disciplines. A practical alternative to using hazardous biothreat agents, e.g., for detection equipment development and testing, involves using appropriate model organisms (simulants). Several species of Gram-negative bacteria have been used or proposed as biothreat simulants. However, the appropriateness of different bacterial genera, species, and strains as simulants is still debated. Here, we report aerobiological stability characteristics of four species of Gram-negative bacteria (Pantoea agglomerans, Serratia marcescens, Escherichia coli, and Xanthomonas arboricola) in single-cell particles and cell clusters produced using four spray liquids (H2O, phosphate-buffered saline[PBS], spent culture medium[SCM], and a SCM-PBS mixture). E. coli showed higher stability in cell clusters from all spray liquids than the other species, but it showed similar or lower stability in single-cell particles. The overall stability was higher in cell clusters than in single-cell particles. The highest overall stability was observed for bioaerosols produced using SCM-containing spray liquids. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research.IMPORTANCE The outcome of this work contributes to improved selection of simulants, spray liquids, and particle size for use in bioaerosol research. Taken together, the results highlight the importance of careful and informed selection of Gram-negative bacterial biothreat simulants and also the accompanying particle size and composition. The results highlight how even moderate changes to one experimental parameter, e.g., bacterial species, spray liquid, or particle size, can strongly affect the aerobiological stability of Gram-negative bacteria. A key finding was the observation that stability differences caused by particle size or compositional changes frequently followed species-specific patterns.

Interactions Between Frankliniella fusca and Pantoea ananatis in the Center Rot Epidemic of Onion (Allium cepa).

An Enterobacteriaceae bacterium, Pantoea ananatis (Serrano) Mergaert, is the causal agent of an economically important disease of onion, center rot. P. ananatis is transmitted by an onion-infesting thrips, Frankliniella fusca (Hinds). However, interactions between F. fusca and P. ananatis as well as transmission mechanisms largely remain uncharacterized. This study investigated P. ananatis acquisition by thrips and transstadial persistence. Furthermore, the effects of bacterial acquisition on thrips fitness were also evaluated. When thrips larvae and adults were provided with acquisition access periods (AAP) on peanut leaflets contaminated with the bacterium, an exponentially positive relationship was observed between AAP and P. ananatis acquisition (R(2) ≥ 0.77, P = 0.01). P. ananatis persisted in thrips through several life stages (larvae, pupae, and adult). Despite the bacterial persistence, no significant effects on thrips fitness parameters such as fecundity and development were observed. Immunofluorescence microscopy of adult thrips with P. ananatis-specific antibody after 48 h AAP on contaminated food revealed that the bacterium was localized only in the gut. These results suggested that the pathogen is not circulative and could be transmitted through feces. Mechanical inoculation of onion seedlings with fecal rinsates produced center rot symptoms, whereas inoculation with rinsates potentially containing salivary secretions did not. These results provide evidence for stercorarian transmission (transmission through feces) of P. ananatis by F. fusca.

Habitat visualization and genomic analysis of "Candidatus Pantoea carbekii," the primary symbiont of the brown marmorated stink bug.

Phytophagous pentatomid insects can negatively impact agricultural productivity and the brown marmorated stink bug (Halyomorpha halys) is an emerging invasive pest responsible for damage to many fruit crops and ornamental plants in North America. Many phytophagous stink bugs, including H. halys, harbor gammaproteobacterial symbionts that likely contribute to host development, and characterization of symbiont transmission/acquisition and their contribution to host fitness may offer alternative strategies for managing pest species. "Candidatus Pantoea carbekii" is the primary occupant of gastric ceca lumina flanking the distal midgut of H. halys insects and it is acquired each generation when nymphs feed on maternal extrachorion secretions following hatching. Insects prevented from symbiont uptake exhibit developmental delays and aberrant behaviors. To infer contributions of Ca. P. carbekii to H. halys, the complete genome was sequenced and annotated from a North American H. halys population. Overall, the Ca. P. carbekii genome is nearly one-fourth (1.2 Mb) that of free-living congenerics, and retains genes encoding many functions that are potentially host-supportive. Gene content reflects patterns of gene loss/retention typical of intracellular mutualists of plant-feeding insects. Electron and fluorescence in situ microscopic imaging of H. halys egg surfaces revealed that maternal extrachorion secretions were populated with Ca. P. carbekii cells. The reported findings detail a transgenerational mode of symbiont transmission distinct from that observed for intracellular insect mutualists and illustrate the potential additive functions contributed by the bacterial symbiont to this important agricultural pest.

Transmission of Pantoea ananatis and P. agglomerans, causal agents of center rot of onion (Allium cepa), by onion thrips (Thrips tabaci) through feces.

Frankliniella fusca, the tobacco thrips, has been shown to acquire and transmit Pantoea ananatis, one of the causal agents of the center rot of onion. Although Thrips tabaci, the onion thrips, is a common pest of onions, its role as a vector of P. ananatis has been unknown. The bacterium, P. agglomerans, is also associated with the center rot of onion, but its transmission by thrips has not been previously investigated. In this study, we investigated the relationship of T. tabaci with P. ananatis and P. agglomerans. Surface-sterilized T. tabaci were provided with various acquisition access periods (AAP) on onion leaves inoculated with either P. ananatis or P. agglomerans. A positive exponential relationship was observed between thrips AAP duration and P. ananatis (R² = 0.967; P = 0.023) or P. agglomerans acquisition (R² = 0.958; P = 0.017). Transmission experiments conducted with T. tabaci adults indicated that 70% of the seedlings developed center rot symptoms 15 days after inoculation. Immunofluorescence microscopy with antibodies specific to P. ananatis revealed that the bacterium was localized only in the gut of T. tabaci adults. Mechanical inoculation of onion seedlings with fecal rinsates alone produced center rot but not with salivary secretions. Together these results suggested that T. tabaci could efficiently transmit P. ananatis and P. agglomerans.

Thermodynamic and kinetic controls on cotransport of Pantoea agglomerans cells and Zn through clean and iron oxide coated sand columns.

Recent observations that subsurface bacteria quickly adsorb metal contaminants raise concerns that they may enhance metal transport, given the high mobility of bacteria themselves. However, metal adsorption to bacteria is also reversible, suggesting that mobility within porous medium will depend on the interplay between adsorption-desorption kinetics and thermodynamic driving forces for adsorption. Till now there has been no systematic investigation of these important interactions. This study investigates the thermodynamic and kinetic controls of cotransport of Pantoea agglomerans cells and Zn in quartz and iron-oxide coated sand (IOCS) packed columns. Batch kinetic studies show that significant Zn sorption on IOCS takes place within two hours. Adsorption onto P. agglomerans surfaces reaches equilibrium within 30 min. Experiments in flow through quartz sand systems demonstrate that bacteria have negligible effect on zinc mobility, regardless of ionic strength and pH conditions. Zinc transport exhibits significant retardation in IOCS columns at high pH in the absence of cells. Yet, when mobile bacteria (non attached) are passed through simultaneously with zinc, no facilitated transport is observed. Adsorption onto cells becomes significant and plays a role in mobile metal speciation only once the IOCS is saturated with zinc. This suggests that IOCS exhibits stronger affinity for Zn than cell surfaces. However, when bacteria and Zn are preassociated on entering the column, zinc transport is initially facilitated. Subsequently, zinc partly desorbs from the cells and redistributes onto the IOCS as a result of the higher thermodynamic affinity for IOCS.

Variations in the molecular masses of the capsular exopolysaccharides amylovoran, pyrifolan and stewartan.

Erwinia amylovora, causing fire blight of apple, pear and some ornamentals, Erwinia pyrifoliae, causing Asian pear blight, and Pantoea stewartii, causing Stewart's wilt of sweet maize, synthesize capsular extracellular polysaccharides (EPSs) with a high molecular mass. The EPSs are virulence factors and form viscous aggregates, which participate in clogging vessels of infected plants and causing wilting. The sizes of EPSs produced under different environmental growth conditions were determined by analysis with large pore HPLC columns. Their molecular mass of ca. 5 MDa, when isolated from agar plates, decreases to ca. 1 MDa for E. amylovora amylovoran from freeze-dried supernatants from liquid cultures and to 2 MDa for freeze-dried preparations of P. stewartii stewartan. Size changes were also found following growth in various other media and for different strains. Stewartan, amylovoran and E. pyrifoliae pyrifolan were also shown to be completely degraded by a bacteriophage EPS depolymerase.

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.

Endophytic colonization ability of two deep-water rice endophytes, Pantoea sp. and Ochrobactrum sp. using green fluorescent protein reporter.

Colonization ability of the two endophytic bacteria, isolated from surface sterilized seeds of Jaisurya variety of deep-water rice viz., Pantoea sp. and Ochrobactrum sp., was compared after genetically tagging them with a constitutively expressing green fluorescent protein gene (gfp). Confocal laser scanning microscopy (CLSM) of hydroponically grown seedlings of Jaisurya rice, inoculated with gfp-tagged endophytes, revealed that both Pantoea sp. and Ochrobactrum sp. colonized the intercellular spaces in the root cortex when inoculated separately. Colonization by gfp-tagged Ochrobactrum sp. was severely inhibited when co-inoculated with an equal number (10(5) c.f.u. ml(-1)) of wild type Pantoea sp., but the converse was not true. Pantoea sp. was a more aggressive endophytic colonizer of its host than Ochrobactrum sp. The potential of using GFP reporter and CLSM as tools in evaluating competitive ability of colonization among endophytes is herewith demonstrated.

Structure of dried cellular alginate matrix containing fillers provides extra protection for microorganisms against UVC radiation.

Soil microorganisms in general and biocontrol agents in particular are very sensitive to UV light. The packaging of biocontrol microorganisms into cellular solids has been developed as a means of reducing loss caused by exposure to environmental UV radiation. The bacterial and fungal biocontrol agents Pantoea agglomerans and Trichoderma harzianum were immobilized in freeze-dried alginate beads containing fillers and subjected to 254 nm UV radiation (UVC). Immobilization of cells in freeze-dried alginate-glycerol beads resulted in greater survival after UV irradiation than for a free cell suspension. Adding chitin, bentonite or kaolin as fillers to the alginate-glycerol formulation significantly increased bacterial survival. Immobilization in alginate-glycerol-kaolin beads resulted in the highest levels of survival. The transmissive properties of the dried hydrocolloid cellular solid had a major influence on the amount of protection by the cell carrier. Dried alginate matrix (control) transmitted an average of 7.2% of the radiation. Filler incorporation into the matrix significantly reduced UV transmission: Alginate with kaolin, bentonite and chitin transmitted an average of 0.15, 0.38 and 3.4% of the radiation, respectively. In addition, the filler inclusion had a considerable effect on the bead's average wall thickness, resulting in a approximately 1.5- to threefold increase relative to beads based solely on alginate. These results suggest that the degree of protection of entrapped microorganisms against UVC radiation is determined by the UV-transmission properties of the dried matrix and the cellular solid's structure. It is concluded that for maximum protection against UV-radiation-induced cell loss, biocontrol microorganisms should be immobilized in alginate-glycerol beads containing kaolin.

Preservation of chitinolytic Pantoae agglomerans in a viable form by cellular dried alginate-based carriers.

Improved viability of Gram-negative bacteria during freeze-dehydration, storage, and soil inoculation is of crucial importance to their efficient application. The chitinolytic Pantoae (Enterobacter) agglomerans strain IC1270, a potential biocontrol agent of soil-borne plant-pathogenic fungi, was used as a model organism to study the efficacy of freeze-dried alginate-based beads (macrocapsules) as possible carriers for immobilized Gram-negative bacterial cells. These macrocapsules were produced by freeze-dehydration of alginate gel spherical beads, in which different amounts of bacteria, glycerol, and colloidal chitin were entrapped. Subsequent drying produced different unexpected structures, pore-size distributions, and changes in the outer and inner appearance of the resultant dried cellular solid. With increasing glycerol content, the proportion of larger pores increased. These structures can be related to changes in the slow-release properties of the dried beads. The amount of glycerol in the beads differed from that in the alginate solution as a result of leakage during the beads' preparation and dehydration. Entrapping 10(9) cells per bead produced from alginate solution containing 30% glycerol and 1% chitin resulted in improved (in comparison to other studies) survival prospects (95%) during freeze-drying. Moreover, immobilization of the bacterium sharply improved its survival in nonsterile irrigated and dry soils compared to bacteria in a water suspension. The results suggest that optimized conservation of Gram-negative bacteria in dry glycerol-containing alginate-based cellular solids is not only possible but applicable for a variety of uses.