Effects of single and co-inoculation with native phosphate solubilising strain Pantoea sp J49 and the symbiotic nitrogen fixing bacterium Bradyrhizobium sp SEMIA 6144 on peanut (Arachis hypogaea L.) growth

In the present study, attempts were made to analyze the effect of co-inoculation with an efficient phosphate solubilising native isolate Pantoea sp J49 and the symbiotic nitrogen fixing Bradyrhizobium sp SEMIA 6144 strain on Arachis hypogaea L. plants growth. Single and co-inoculation of peanut plants growing in plastic pots containing soil with low P content were developed. Plants were harvested at R1 and R4 growth stages and were analyzed in different growth parameters. Survival of strain Pantoea sp J49 was analyzed in soil samples and in root tissues. Plants inoculated only with Pantoea sp J49 showed the highest shoot and root weight in both reproductive growth stages evaluated. Plants co-inoculated with this strain and Bradyrhizobium sp SEMIA 6144 showed increase in aerial dry weight at R1 stage. Survival assays demonstrated that Pantoea sp J49 survives not only in the peanut rhizosphere but also inside plant tissues, including nodules formed when it was co-inoculated with Bradyrhizobium sp SEMIA 6144. Results obtained in this study confirm the great potential of the native Pantoea sp J49 isolate in the promotion of peanut plant growth, probably related with its capacity to solubilise phosphate.     

The ectomycorrhizae of <Emphasis Type="Italic">Lactarius rimosellus</Emphasis> and <Emphasis Type="Italic">Lactarius acatlanensis</Emphasis> with the endangered <Emphasis Type="Italic">Fagus grandifolia</Emphasis> var. <Emphasis Type="Italic">mexicana</Emphasis>

Gut bacterium Pantoea sp. is one of the predominant bacterial species in the larval gut of the diamondback moth, Plutella xylostella. The phenotypic characters of Pantoea sp. were investigated with BIOLOG phenotype MicroArray (PM) in this study. Totally 950 different metabolic phenotypes were tested using the PM plates 1–10. Results exhibited that Pantoea sp. was able to metabolize 37.37 % of the tested carbon sources, 91.32 % of nitrogen sources, 100 % of sulfur sources, and 98.31 % of phosphorus sources. Most informative utilization patterns for carbon sources of Pantoea sp. were organic acids and carbohydrates, and for nitrogen were various amino acids. The bacterium had 94 different biosynthetic pathways. It had a wide range of adaptabilities, and could still metabolize in osmolytes with up to 9 % sodium chloride, 6 % potassium chloride, 5 % sodium sulfate, 20 % ethylene glycol, 4 % sodium formate, 4 % urea, 5 % sodium lactate, 200 mmol/L sodium phosphate (pH 7.0), 100 mmol/L ammonium sulfate (pH 8.0), 100 mmol/L sodium nitrate, and 100 mmol/L sodium nitrite, respectively. It also exhibited active metabolism under pH values between 4.5 and 10. Pantoea sp. showed active decarboxylase activities while poor deaminase activities in the presence of various amino acids. The phenotypic characterization of Pantoea sp. increased our knowledge of the bacterium, in particular its interactions with insect hosts and the adaptability in gut environments, and showed us some possible approaches to controlling diamondback moth through decreasing Pantoea sp. density.     

Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1

Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest K_m values toward phytate, glucose-6-phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify dl-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features.     

Investigation of the internal bacterial flora of Eurygaster integriceps (Hemiptera: Scutelleridae) and pathogenicity of the flora members

Eurygaster integriceps (Hemiptera: Scutelleridae) is one of the most serious pest of wheat and other cereal crops throughout the Near and Middle East including Turkey. To contribute biological control of this pest, we investigated the culturable bacterial flora of E. integriceps and their virulence against it. We also tested different entomopathogenic fungi (Isaria fumosorosea ARSEF8356, Beauveria bassiana ARSEF8356, Metarhizium brunneum ARSEF8671 and Nomurae rileyi ARSEF1670) against this pest under laboratory conditions. Bacterial isolates were characterized based on morphological, biochemical, physiological and molecular characteristics (16S rRNA sequencing). The isolates were identified as Pantoea sp. (S1, S5, S7, S8, S10 and S11), P. agglomerans (S2, S3 and S4), Pseudomonas sp. S6 and Micrococcus luteus S9. The highest mortality within bacterial isolates was observed from Pantoea sp. S1, P. agglomerans S4 and Pantoea sp. S7 with 100%. Mortality of other bacterial species ranged from 33% to 88%. The highest mortality among the tested entomopathogenic fungi was obtained from Isaria fumosorosea ARSEF 8333 with 100%. Mortality of other fungi ranged from 33% to 50%. Consequently, Pantoea sp. S1, P. agglomerans S4, Pantoea sp. S7, Beauveria bassiana ARSEF 8356 and Isaria fumosorosea ARSEF 8356 seem to be promising candidates in the control of E. integriceps.     

<Emphasis Type="Italic">Rafflesia</Emphasis> spp.: propagation and conservation

Main Conclusion

The propagation of Rafflesia spp. is considered to be important for future development of ornamental and other applications. Thus far, the only successful propagation technique has been grafting. This mini-review succinctly emphasizes what is known about Rafflesia species.Members of the genus Rafflesia (Rafflesiaceae), which are holoparasitic plants known to grow on a host vine, Tetrastigma sp., are widely spread from the Malayan Peninsula to various islands throughout Indonesia. The plant’s geographical distribution as well as many other aspects pertaining to the basic biology of this genus have still not been studied. The young flower buds and flowers of wild Rafflesia hasseltii Suringar, Rafflesia keithii Meijer and Rafflesia cantleyi Solms-Laubach are used in local (Malaysia and Indonesia) traditional ethnomedicine as wound-healing agents, but currently no formal published research exists to validate this property. To maintain a balance between its ethnomedicinal and ornamental use, and conservation, Rafflesia spp. must be artificially cultivated to prevent overexploitation. A successful method of vegetative propagation is by host grafting using Rafflesia-impregnated Tetrastigma onto the stem of a normal Tetrastigma plant. Due to difficulties with culture contamination in vitro, callus induction was only accomplished in 2010 for the first time when picloram and 2,4-D were added to a basal Murashige and Skoog medium, and the tissue culture of holoparasitic plants continues to be extremely difficult. Seeds harvested from fertile fruit may serve as a possible method to propagate Rafflesia spp. This paper provides a brief synthesis on what is known about research related to Rafflesia spp. The objective is to further stimulate researchers to examine, through rigorous scientific discovery, the mechanisms underlying the ethnomedicinal properties, the flowering mechanisms, and suitable in vitro regeneration protocols that would allow for the fortification of germplasm conservation.

     

Loss of carotenoids from membranes of Pantoea sp. YR343 results in altered lipid composition and changes in membrane biophysical properties

Bacterial membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties that allows cells to respond to environmental conditions. Carotenoids are sterol analogs that are important for regulating membrane dynamics. The membrane of Pantoea sp. YR343 is characterized by the presence of the carotenoid zeaxanthin, and a carotenoid-deficient mutant, ΔcrtB, displays defects in root colonization, reduced secretion of indole-3-acetic acid, and defects in biofilm formation. Here we demonstrate that the loss of carotenoids results in changes to the membrane lipid composition in Pantoea sp. YR343, including increased amounts of unsaturated fatty acids in the ΔcrtB mutant membranes. These mutant cells displayed less fluid membranes in comparison to wild type cells as measured by fluorescence anisotropy of whole cells. Studies with artificial systems, however, have shown that carotenoids impart membrane rigidifying properties. Thus, we examined membrane fluidity using spheroplasts and vesicles composed of lipids extracted from either wild type or mutant cells. Interestingly, with the removal of the cell wall and membrane proteins, ΔcrtB vesicles were more fluid than vesicles made from lipids extracted from wild type cells. In addition, carotenoids appeared to stabilize membrane fluidity during rapidly changing temperatures. Taken together, these results suggest that Pantoea sp. YR343 compensates for the loss of carotenoids by changing lipid composition, which together with membrane proteins, results in reduced membrane fluidity. These changes may influence the abundance or function of membrane proteins that are responsible for the physiological changes observed in the ΔcrtB mutant cells.     

Biosurfactant Production by Antarctic Facultative Anaerobe <Emphasis Type="Italic">Pantoea</Emphasis> sp. During Growth on Hydrocarbons

The facultative anaerobe Pantoea sp. strain A-13, isolated from ornithogenic soil of Dewart Island (Frazier Islands), Antarctica, produced glycolipid biosurfactants when grown on n-paraffins or kerosene as the sole source of carbon and energy. Hemolysis of erythrocytes, growth inhibition of Bacillus subtilis, and thin-layer chromatography studies have suggested that the secreted glycolipids are rhamnolipids. Glycolipids produced by kerosene-grown cells decreased the surface tension at the air–water interface to 30 mN/m and possessed a low critical micelle concentration value of 40 mg/l, which indicated high surface activity. They efficiently emulsified aromatic hydrocarbons, kerosene, and n-paraffins. Biosurfactant production contributed to an increase in cell hydrophobicity, which correlated with increased growth of the strain on tested hydrocarbons. According to the results, the Antarctic biosurfactant-producing strain Pantoea sp. A-13 appears to be valuable source for application in accelerated environmental bioremediation.     

Improved Degradation of Monochlorophenols by a Constructed Strain

Pseudomonas sp. strain B13, a strain able to degrade 3-chlorobenzoate and, after prolonged adaptation (40 days), 4-chlorophenol, could transfer the ability to degrade chlorocatechols to a recipient, Alcaligenes sp. strain A7, which is able to grow with benzoate and phenol. Representative transconjugants, such as Alcaligenes sp. strain A7-2, were able to utilize all three isomeric chlorophenols; this property was not possessed by the donor or the recipient. The ability to grow readily with 4-chlorophenol may be attributable to a more rapid induction of phenol hydroxylase by Alcaligenes sp. strain A7-2 than by Pseudomonas sp. strain B13, a property which correlates with the greater level of resistance to chlorophenols shown by the transconjugant.     

Rhizoremediation of phenol and chromium by the synergistic combination of a native bacterial strain and Brassica napus hairy roots

A bacterial strain resistant to phenol and Cr (VI) was isolated from an industrial polluted soil of Córdoba province (Argentina), which was identified as Pantoea sp. FC 1. This microorganism was able to use phenol as sole carbon source. In addition it was capable of reducing Cr (VI) to Cr (III) in mineral and nutrient media. The isolated strain exhibited some properties as plant-growth promoting bacterium (PGPB), such as production of Indole Acetic Acid (IAA) and synthesis of siderophores, as well as being capable of solubilizing inorganic phosphates. A rhizoremediation system using the association Pantoea sp. FC 1-Brassica napus hairy roots (HRs) was tested for phenol and Cr (VI) removal in a hydroponic system. Microbial inoculation improved both phenol removal and chromium accumulation efficiency by HRs, showing a significant increase in Cr (III) accumulation compared to non-inoculated HRs, exceeding 1000 mg kg⁻¹. Cr (III) was detected in HR biomass and supernatants, suggesting a possible Cr (VI) reducing activity of B. napus HRs. Basic studies in plant model systems, such as HRs, provide additional useful information that could facilitate the transition of this technology into plants suitable for practical rhizoremediation applications.     

Occurrence of a Lysogenic Streptomyces sp. on the Nodule Surface of Black Gram (Vigna mungo (L.) Hepper)

A lysogenic Streptomyces sp., strain NS.A4, which was isolated from the nodule surface of black gram (Vigna mungo (L.) Hepper), was found to inhibit rhizobia of fast-and slow-growing strains of cowpeas and soybeans. It exhibited plaques when there was a change in cultural conditions. Repeated culturing of the organism in nutrient agar and broth confirmed the infection of Streptomyces sp. strain NS.A4 by an actinophage. Addition of the culture filtrate of Streptomyces sp. strain NS.A4 to shaken broth cultures of three other Streptomyces spp. resulted in phage infection.     

Cross-feeding niches among commensal leaf bacteria are shaped by the interaction of strain-level diversity and resource availability

Leaf microbiomes play crucial roles in plant health, making it important to understand the origins and functional relevance of their diversity. High strain-level leaf bacterial genetic diversity is known to be relevant for interactions with hosts, but little is known about its relevance for interactions with the multitude of diverse co-colonizing microorganisms. In leaves, nutrients like amino acids are major regulators of microbial growth and activity. Using metabolomics of leaf apoplast fluid, we found that different species of the plant genus Flaveria considerably differ in the concentrations of high-cost amino acids. We investigated how these differences affect bacterial community diversity and assembly by enriching leaf bacteria in vitro with only sucrose or sucrose + amino acids as possible carbon sources. Enrichments from F. robusta were dominated by Pantoea sp. and Pseudomonas sp., regardless of carbon source. The latter was unable to grow on sucrose alone but persisted in the sucrose-only enrichment thanks to exchange of diverse metabolites from Pantoea sp. Individual Pseudomonas strains in the enrichments had high genetic similarity but still displayed clear niche partitioning, enabling distinct strains to cross-feed in parallel. Pantoea strains were also closely related, but individuals enriched from F. trinervia fed Pseudomonas more poorly than those from F. robusta. This can be explained in part by the plant environment, since some cross-feeding interactions were selected for, when experimentally evolved in a poor (sucrose-only) environment but selected against in a rich (sucrose + amino acids) one. Together, our work shows that leaf bacterial diversity is functionally relevant in cross-feeding interactions and strongly suggests that the leaf resource environment can shape these interactions and thereby indirectly drive bacterial diversity.Subject terms: Microbiome, Plant ecology, Biodiversity, Microbial ecology, Bacterial genomics     

Biotransformation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) by a Rabbit Liver Cytochrome P450: Insight into the Mechanism of RDX Biodegradation by Rhodococcus sp. Strain DN22

A unique metabolite with a molecular mass of 119 Da (C₂H₅N₃O₃) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.     

Gut compartments and ovary bacterial symbionts of the Sunn pest

The Sunn pest, Eurygaster integriceps (Hemiptera: Scutelleridae), is the severe pest of cereals, especially of wheat in many parts of the world. Many insect species, including the Sunn pest that feed solely on nutritionally restricted diets, harbor symbiotic microorganisms. In the current study, we isolated and identified the Sunn pest bacterial symbionts of gut fractions and ovary. The phylogenetic analysis indicated that Sunn pest gut bacterial symbionts are polyphyletic and contained a taxonomic diversity belonging to three different phyla, including Firmicutes, Tenericutes, and Proteobacteria. Firmicutes was represented by Enterococcus, Proteobacteria by Pantoea and Acetobacteraceae, and Tenericutes by Spiroplasma. We isolated and identified Enterococcus, Acetobacteraceae, Spiroplasma and Pantoea from Sunn pest different gut compartments, and Pantoea from ovaries. There was considerable overlap between recognized symbionts from the 2nd and 3rd midgut sections (Acetobacteraceae), the 4th midgut section and hindgut (Spiroplasma), and 4th midgut section and ovary (Pantoea). Niche heterogeneity within a microbial habitat of gut fractions resulted in colonizing and adaptation of various communities of symbionts in each fraction. The Sunn pest gut compartments and ovary symbionts have been demonstrated to be of multiple evolutionary origins. This diversity may be of great importance to the Sunn pest fitness and survival in various overwintering niches.     

Stable Coexistence of Five Bacterial Strains as a Cellulose-Degrading Community

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, “knockout communities” in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.     

Growth in Coculture Stimulates Metabolism of the Phenylurea Herbicide Isoproturon by Sphingomonas sp. Strain SRS2

Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the β-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of ¹⁴C-labeled isoproturon to ¹⁴CO₂ and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.     

Karyotypic instability of endoprophase and mitotic cells of Amoeba sp. strain Cont from the “proteus-type” group (Amoebozoa,Euamoebida, Amoebidae)

We performed karyotyping of Amoeba sp. strain Cont. Based on the results of a cytological analysis, we concluded that the chromosome number of Amoeba sp. strain Cont in mitosis was unstable. In all cases they appeared to be hypergaploid (the basic chromosome number is 30), with monosomy of all chromosomes except four shortest ones. The presence of “extrachromosomes” in the nucleus could prolong until the beginning of the anaphase. It was only then that they were ejected from the nucleus and the euploidy (haploidy) was restored. The stage of endoprophase nucleus was revealed in the cell cycle of Amoeba sp. strain Cont. This stage has not yet been found in other amoebae from the “proteus-type” group that had been previously studied (A. proteus strain B and A. borokensis). The maximum number of endoreplication rounds in the strain Cont amoebae nuclear cycle was 4 or 5. The regular extrusion of chromosomes from the nucleus into the cytoplasm occurred in each of the endoreplication rounds. Comparative cytological analysis of A. proteus strain B, A. borokensis and Amoeba sp. strain Cont karyotypes indicated that strain Cont, though rather close to the former two amoebae, is actually a distinct species.     

Distribution and antimicrobial multiresistance in Gram-negative bacteria isolated from Turkish sea bass (Dicentrarchus labrax L., 1781) farm

In this study, the frequencies of 111 antibiotic-resistant Gram-negative bacteria obtained from cultured sea bass were investigated. All the strains were identified and tested whether they were resistive against ten different antibiotics or not. The results showed that most of the bacteria were resistant to trimethoprim-sulphamethoxazole, cephalotin, tetracycline and streptomycin. It was found multiple antibiotic resistance (MAR) index values ranging from 0.3–0.8 for the bacteria isolated from gill. A large number ofPseudomanas putida (25.2%),Moellerella wisconsensis (18%) andPseudomonas fluorescens (10.81%) were identified. Also, strains ofRalstonia pickettii (9%),Vibrio fluvialis (8.1%),Pantoea sp. (7.2%) andErwinia sp. (5.4%) were found. This study suggested that Turkish sea bass farms have antibacterial multiresistance bacteria and may play as a reservoirs response for disease pathogen bacteria.     

Diversity of sporadic symbionts and nonsymbiotic endophytic bacteria isolated from nodules of woody, shrub, and food legumes in Ethiopia

Fifty-five bacterial isolates were obtained from surface-sterilized nodules of woody and shrub legumes growing in Ethiopia: Crotalaria spp., Indigofera spp., and Erythrina brucei, and the food legumes soybean and common bean. Based on partial 16S rRNA gene sequence analysis, the majority of the isolates were identified as Gram-negative bacteria belonging to the genera Achromobacter, Agrobacterium, Burkholderia, Cronobacter, Enterobacter, Mesorhizobium, Novosphingobium, Pantoea, Pseudomonas, Rahnella, Rhizobium, Serratia, and Variovorax. Seven isolates were Gram-positive bacteria belonging to the genera Bacillus, Paenibacillus, Planomicrobium, and Rhodococcus. Amplified fragment length polymorphism (AFLP) fingerprinting showed that each strain was genetically distinct. According to phylogenetic analysis of recA, glnII, rpoB, and 16S rRNA gene sequences, Rhizobium, Mesorhizobium, and Agrobacterium were further classified into six different genospecies: Agrobacterium spp., Agrobacterium radiobacter, Rhizobium sp., Rhizobium phaseoli, Mesorhizobium sp., and putative new Rhizobium species. The strains from R. phaseoli, Rhizobium sp. IAR30, and Mesorhizobium sp. ERR6 induced nodules on their host plants. The other strains did not form nodules on their original host. Nine endophytic bacterial strains representing seven genera, Agrobacterium, Burkholderia, Paenibacillus, Pantoea, Pseudomonas, Rhizobium, and Serratia, were found to colonize nodules of Crotalaria incana and common bean on co-inoculation with symbiotic rhizobia. Four endophytic Rhizobium and two Agrobacterium strains had identical nifH gene sequences with symbiotic Rhizobium strains, suggesting horizontal gene transfer. Most symbiotic and nonsymbiotic endophytic bacteria showed plant growth-promoting properties in vitro, which indicate their potential role in the promotion of plant growth when colonizing plant roots and the rhizosphere.     

Diversity and phylogeny of bacteria on Zimbabwe tobacco leaves estimated by 16S rRNA sequence analysis

Microorganisms play important roles in the tobacco aging process. However, microbial communities on flue-cured tobacco leaves (FCTL) remain largely unknown. In this study, the total microbial genomic DNA of unaged and aging FCTL from Zimbabwe were isolated using a culture-independent method, and the bacterial communities were investigated through analyzing two 16S rRNA gene libraries. Eighty-four and 65 operational taxonomic units were obtained from the libraries of the unaged and aging FCTL, respectively. The following genera were represented more than 4% in both libraries (aging and unaged library): Sphingomonas (4.84%, 4.18%), Stenotrophomonas (4.84%, 5.23%), Erwinia (5.81%, 4.88%), Pantoea (19.35%, 18.47%), and Pseudomonas (21.29%, 24.04%). The dominant species varied between the two libraries. Specifically, several dominant species in unaged FCTL including Pseudomonas fulva, Pseudomonas sp. (AM909658), Klebsiella sp. (HM584796), and Pantoea sp. (AY501386) were not identified in aging FCTL, while several dominant species in aging FCTL such as Pantoea sp. (GU566350), Pseudomonas sp. (EF157292), and Buttiauxella izardii were not found in unaged FCTL. The phylogenetic analysis showed that bacteria from unaged and aging FCTL were divided into two clades, and two unique subclades were identified in aging FCTL. Our results revealed for the first time the bacterial diversities on Zimbabwe tobacco, and provided a basis for clarifying the roles of bacteria in aging process of FCTL.     

Identification of a Pantoea Biosynthetic Cluster That Directs the Synthesis of an Antimicrobial Natural Product

Fire Blight is a destructive disease of apple and pear caused by the enteric bacterial pathogen, Erwinia amylovora. E. amylovora initiates infection by colonizing the stigmata of apple and pear trees, and entering the plants through natural openings. Epiphytic populations of the related enteric bacterium, Pantoea, reduce the incidence of disease through competition and antibiotic production. In this study, we identify an antibiotic from Pantoea ananatis BRT175, which is effective against E. amylovora and select species of Pantoea. We used transposon mutagenesis to create a mutant library, screened approximately 5,000 mutants for loss of antibiotic production, and recovered 29 mutants. Sequencing of the transposon insertion sites of these mutants revealed multiple independent disruptions of an 8.2 kb cluster consisting of seven genes, which appear to be coregulated. An analysis of the distribution of this cluster revealed that it was not present in any other of our 115 Pantoea isolates, or in any of the fully sequenced Pantoea genomes, and is most closely related to antibiotic biosynthetic clusters found in three different species of Pseudomonas. This identification of this biosynthetic cluster highlights the diversity of natural products produced by Pantoea.