Isolation and characterization of bacteria from activated sludge capable of degrading 17α-ethinylestradiol, a contaminant of high environmental concern.

The compound 17α-ethinylestradiol (EE2) is a synthetic oestrogen which is classified as a group 1 carcinogen by the World Health Organization. Together with other endocrine disruptor compounds, EE2 has been included in the surface water Watch List by the European Commission, since it causes severe adverse effects in ecosystems. Thus, it became a high priority to find or improve processes such as biodegradation of EE2 to completely remove this drug from the wastewater treatment plants (WWTPs). The present study aimed at the isolation of bacteria capable of degrading EE2 using environmental samples, namely a sludge from the Faro Northwest WWTP. Four isolates with ability to grow in the presence of 50 mg l-1 EE2 were obtained. The analysis of 16SrRNA gene sequences identified the isolated bacteria as Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans and Shinella zoogloeoides. The results of biodegradation assays showed that Acinetobacter bouvetii, Acinetobacter kookii, Pantoea agglomerans and Shinella zoogloeoides were able to degrade 47±4 %, 55±3 %, 64±4% and 35±4 %, respectively of 13 mg l-1 EE2 after 168 h at 28 °C. To the best of our knowledge, these bacterial isolates were identified as EE2 degraders for the first time. In a preliminary experiment on the identification of metabolic products resulting from EE2 degradation products such as estrone (E1), γ-lactone compounds, 2-pentanedioic acid and 2-butenedioic acid an intermediate metabolite of the TCA cycle, were detected.

Ciceribacter ferrooxidans sp. nov., a nitrate-reducing Fe(II)-oxidizing bacterium isolated from ferrous ion-rich sediment.

A nitrate-reducing Fe(II)-oxidizing bacterial strain, F8825T, was isolated from the Fe(II)-rich sediment of an urban creek in Pearl River Delta, China. The strain was Gram-negative, facultative chemolithotrophic, facultative anaerobic, non-spore-forming, and rod-shaped with a single flagellum. Phy-logenetic analysis based on 16S rRNA gene sequencing indicated that it belongs to the genus Ciceribacter and is most closely related to C. lividus MSSRFBL1T (99.4%), followed by C. thiooxidans F43bT (98.8%) and C. azotifigens A.slu09T (98.0%). Fatty acid, polar lipid, respiratory quinone, and DNA G + C content analyses supported its classification in the genus Ciceribacter. Multilocus sequence analysis of concatenated 16S rRNA, atpD, glnII, gyrB, recA, and thrC suggested that the isolate was a novel species. DNA-DNA hybridization and genome sequence comparisons (90.88 and 89.86%, for values of ANIm and ANIb between strains F8825T with MSSRFBL1T, respectively) confirmed that strain F8825T was a novel species, different from C. lividus MSSRFBL1T, C. thiooxidans F43bT, and C. azotifigens A.slu09T. The physiological and biochemical properties of the strain, such as carbon source utilization, nitrate reduction, and ferrous ion oxidation, further supported that this is a novel species. Based on the polyphasic taxonomic results, strain F8825T was identified as a novel species in the genus Ciceribacter, for which the name Ciceribacter ferrooxidans sp. nov. is proposed. The type strain is F8825T (= CCTCC AB 2018196T = KCTC 62948T).

Canine olfactory detection of a vectored phytobacterial pathogen, Liberibacter asiaticus, and integration with disease control.

Early detection and rapid response are crucial to avoid severe epidemics of exotic pathogens. However, most detection methods (molecular, serological, chemical) are logistically limited for large-scale survey of outbreaks due to intrinsic sampling issues and laboratory throughput. Evaluation of 10 canines trained for detection of a severe exotic phytobacterial arboreal pathogen, Candidatus Liberibacter asiaticus (CLas), demonstrated 0.9905 accuracy, 0.8579 sensitivity, and 0.9961 specificity. In a longitudinal study, cryptic CLas infections that remained subclinical visually were detected within 2 wk postinfection compared with 1 to 32 mo for qPCR. When allowed to interrogate a diverse range of in vivo pathogens infecting an international citrus pathogen collection, canines only reacted to Liberibacter pathogens of citrus and not to other bacterial, viral, or spiroplasma pathogens. Canines trained to detect CLas-infected citrus also alerted on CLas-infected tobacco and periwinkle, CLas-bearing psyllid insect vectors, and CLas cocultured with other bacteria but at CLas titers below the level of molecular detection. All of these observations suggest that canines can detect CLas directly rather than only host volatiles produced by the infection. Detection in orchards and residential properties was real time, ∼2 s per tree. Spatiotemporal epidemic simulations demonstrated that control of pathogen prevalence was possible and economically sustainable when canine detection was followed by intervention (i.e., culling infected individuals), whereas current methods of molecular (qPCR) and visual detection failed to contribute to the suppression of an exponential trajectory of infection.

Allorhizobium terrae sp. nov., isolated from paddy soil, and reclassification of Rhizobium oryziradicis (Zhao et al. 2017) as Allorhizobium oryziradicis comb. nov.

A polyphasic taxonomic approach was used to characterize a nitrogen-fixing bacterium, designated strain CC-HIH110T, isolated from paddy soil in Taiwan. Cells of strain CC-HIH110T were Gram-stain-negative, rod-shaped, motile with polar flagella, catalase-positive and oxidase-positive. Optimal growth occurred at 30 °Ð¡, pH 7 and 1 % NaCl. Phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by strain CC-HIH110T associated with Rhizobium oryziradicis (98.4 % sequence identity), Allorhizobium vitis (97.8 %), Allorhizobium taibaishanense (97.7 %) and Allorhizobium undicola (96.0 %), and lower sequence similarity to other species. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain CC-HIH110T and the type strains of other closely related species were 71.5-88.6 % and 19.6-35.5 %, respectively. Strain CC-HIH110T contained C16 : 0 3-OH, C14 : 0 3-OH/iso C16 : 1 I and C18 : 1 ω7c/C18  : 1 ω6c as the predominant fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, phosphatidylcholine, three unknown aminophospholipids, two unknown phospholipids and an unknown lipid. The major polyamine was homospermidine. The DNA G+C content was 55.0 mol% and the predominant quinone was ubiquinone (Q-10). Based on its distinct phylogenetic, phenotypic and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence, ANI and dDDH analyses, strain CC-HIH110T is proposed to represent a novel Allorhizobium species, for which the name Allorhizobium terrae sp. nov. (type strain CC-HIH110T=BCRC 80932T=JCM 31228T). In addition, Rhizobium oryziradicis is reclassified as Allorhizobium oryziradicis (type strain N19T=ACCC 19962T=KCTC 52413T) comb. nov.

Neorhizobium tomejilense sp. nov., first non-symbiotic Neorhizobium species isolated from a dryland agricultural soil in southern Spain.

We describe for the first time a non-symbiotic species of the recently described genus Neorhizobium, lacking nodulation or nitrogen fixation genes. The strains were isolated from a dryland agricultural soil in southern Spain where no record of legume cultivation is available, thus we propose the name Neorhizobium tomejilense sp. nov. (type strain T17_20T, LMG 30623T and CECT 9621T). N. tomejilense exhibit a clear distinct lineage from the other Neorhizobium species, Neorhizobium galegae, Neorhizobium alkalisoli and Neorhizobium huautlense, based on polyphasic evidence. Phylogenetic marker analysis of 16S rDNA, atpD, glnII, recA, rpoB and thrC genes and genomic identity data derived from the draft genomic sequences showed that N. tomejilense strains clearly separated from the other Neorhizobium species and that N. galegae represents the closest species, with Average Nucleotide Identities (ANIb) ranging from 90% (for type strain HAMBI 540T) to just under 95.0% (for two N. galegae sv. officinalis strains). Genomes from N. galegae and N. tomejilense, however, clearly differed in important traits, such as the number of rRNA operon copies or the number of tRNAs. Phenotypic characterisation of N. tomejilense also displayed differences with the other Neorhizobium species. Whole-cell matrix-assisted laser-desorption time-of-flight mass spectrometry (WC MALDI-TOF-MS) fingerprint analysis and the dendrogram derived from the fingerprint profiles, showed a clearly distinct group formed by the three N. tomejilense isolates (T17_20T, T20_22 and T11_12) from the other Neorhizobium especies.

Shinella pollutisoli sp. nov., isolated from tetrabromobisphenol A-contaminated soil.

Strain AH-1T, a Gram-negative, aerobic, non-spore-forming, motile, rod-shaped bacterium, was isolated from tetrabromobisphenol A-contaminated soil in China. The taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain AH-1T was a member of the genus Shinella and showed the highest sequence similarity to Shinella fusca DC-196T (97.7 %), Shinella granuli Ch06T (97.3 %), Shinella daejeonensis MJ02T (97.1 %) and Shinella yambaruensis MS4T (96.8 %), and lower (<96.7 %) sequence similarity to other known Shinella species. Chemotaxonomic analysis revealed that strain AH-1T possessed Q-10 as the major isoprenoid quinone; and summed feature 8 (C18 : 1ω6c/C18 : 1ω7c), C16 : 0, C12 : 0 aldehyde, C18 : 0, C19 : 0 cyclo ω8c and C18 : 0 3-OH were the predominant fatty acids. Strain AH-1T showed low DNA-DNA relatedness to S. fusca DC-196T (28.6±5.7 %), S. granuli Ch06T (43.7±3.8 %) and S. daejeonensis MJ02T (48.1±2.6 %). The DNA G+C content was 68.2 mol%. Based on the phylogenetic and phenotypic characteristics, chemotaxonomic data and DNA-DNA hybridization, strain AH-1T is considered a novel species of the genus Shinella, for which the name Shinella pollutisoli sp. nov. (type strain AH-1T=KCTC 52677T=CCTCC AB 2017242T) is proposed.

Ciceribacter azotifigens sp. nov., a nitrogen-fixing bacterium isolated from activated sludge.

A Gram-reaction-negative, catalase- and oxidase-positive, aerobic, transparent, motile and rod-shaped bacterium that was capable of fixing dinitrogen (designated strain A.slu09T), isolated from activated sludge, was characterized by a polyphasic approach to clarify its taxonomic position. Strain A.slu09T was observed to grow optimally at 30 °C and at pH 7.0 on R2A agar medium. Strain A.slu09T showed ß-glucosidase activity, converting the major ginsenoside Rd to ginsenoside F2. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain A.slu09T belongs to the genus Ciceribacter of the family Rhizobiaceae and was most closely related to Ciceribacter lividus MSSRFBL1T (97.8 % similarity). The DNA G+C content was 67.2 mol%. The DNA-DNA hybridization value between strain A.slu09T and C. lividus KCTC 32403T was 16.9±1.17 %. The major polar lipids were phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, aminophospholipid and two glycolipids, and one unknown phospholipid as a minor lipid. The predominant quinone was ubiquinone-10 (Q-10). The major fatty acids were C19 : 0 cyclo ω8c, C18 : 1 ω7c and/or C18 : 1ω6c (summed feature 8) and C18 : 0, a profile that supported the affiliation of A.slu09T to the genus Ciceribacter. Moreover, the physiological and biochemical characteristics and low level of DNA-DNA relatedness allowed the phenotypic and genotypic differentiation of strain A.slu09T from the recognized species of the genus Ciceribacter. Therefore, strain A.slu09T represents a novel species of the genus Ciceribacter, for which the name Ciceribacter azotifigens sp. nov. is proposed. The type strain is A.slu09T (=KACC 19080T=LMG 29962T).

Ciceribacter thiooxidans sp. nov., a novel nitrate-reducing thiosulfate-oxidizing bacterium isolated from sulfide-rich anoxic sediment.

Two facultative chemolithotrophic, nitrate-reducing thiosulfate-oxidizing strains, F43bT and F21, were isolated from the sulfide-rich anoxic sediment of an urban creek in Pearl River Delta, China. Both strains were Gram-negative, facultatively anaerobic, non-spore-forming and rod-shaped with a flagellum. Phylogenetic analyses of 16S rRNA genes and the thrC, recA, glnII and atpD housekeeping genes revealed that the type strain shared high sequence similarities to Ciceribacter lividus MSSRFBL1T, with 98.8, 90.9, 94.8, 95.4 and 96.1 % identity, respectively. In addition, the major isoprenoid quinone (ubiquinone Q-10) and the DNA G+C content (66.0 mol%) of the type strain were similar to those of Ciceribacter lividus MSSRFBL1T. These results strongly support the classification of strains F43bT and F21 into the genus Ciceribacter. However, these strains diverged markedly from strain MSSRFBL1T with respect to several physiological and biochemical properties such as their semi-translucent colonies and nitrate-reducing and simultaneous thiosulfate-oxidizing respiration. Furthermore, the predominant fatty acids of strain F43bT were summed feature 2 (C18 : 1ω9t and/or C18 : 1ω9c and/or C18 : 1ω11t), C14 : 0 3-OH, C18 : 0 and C16 : 0, and its polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidymonomethylethanolamine and an unidentified glycolipid, which represented another two significant differences from strain MSSRFBL1T. Importantly, the DNA-DNA relatedness between strain F43bT and MSSRFBL1T was only 47.7 %. Based on the aforementioned polyphasic taxonomic results, the two isolates are suggested to represent a novel species of the genus Ciceribacter, for which the name Ciceribacterthiooxidans sp. nov. is proposed; the type strain is F43bT (=CCTCC AB 2016062T=KCTC 52231T).

Gellertiella hungarica gen. nov., sp. nov., a novel bacterium of the family Rhizobiaceae isolated from a spa in Budapest.

A novel alphaproteobacterium, strain RAM11T, belonging to the family Rhizobiaceae was isolated from the pool water of a thermal bath in Budapest, Hungary. Based on the 16S rRNA gene sequence strain RAM11T shows the highest sequence similarity values to Ensifer adhaerens Casida A (97.44 %), to Ensifer (syn. Sinorhizobium) americanus CFNEI 156T (96.87 %) and to Rhizobium azooxidifex Po 20/26T (96.76 %). The new bacterium is strictly aerobic, its optimum growth occurs at 20-37 °C, between pH 7 and 9 and without NaCl. It is motile due to a single polar flagellum, capable of budding and forms rosettes in liquid culture. The major isoprenoid quinone of strain RAM11T is Q-10, the major cellular fatty acids are C18 : 1ω7c and 11-MeC18 : 1ω7c. The polar lipid profile contains phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, an unidentified aminolipid and an unidentified phospholipid. The G+C content of DNA of the type strain is 62.9 mol%. Strain RAM11T (=DSM 29853T=NCAIM B.02618T) is proposed as type strain of a new genus and species with the proposed name Gellertiella hungarica gen. nov., sp. nov.

Pararhizobium antarcticum sp. nov., isolated from Antarctic water samples.

Two bacterial strains were isolated from sediments and microbial mats of Kingfisher Pond, Antarctica and characterized in a taxonomic study using a polyphasic approach. Cells were strictly aerobic, Gram-stain-negative, rod-shaped, motile (+50 flagellum-specific genes present in the genome sequence; motility observed under microscope) and formed creamy white, half-transparent colonies. Growth occurred at 4 to 28 °C with an optimum at 20 °C, with 0-5.0 % (w/v) NaCl (optimum at 0-1.0 %) and at pH 4.0-11.0 (optimum pH 7.0-9.0). The major fatty acid was C18 : 1ω7c. The respiratory quinone was ubiquinone 10 (Q-10). The DNA G+C content was 60.7 mol %. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmethanolamine in addition to three unidentified lipids, one unknown glycolipid and five unidentified phospholipids. Comparative analysis of 16S rRNA gene sequences showed highest sequence similarity (98.1 %) to Pararhizobium giardinii H152T, Pararhizobium herbae CCBAU 83011T, and 'Pararhizobium polonicum' F5.1. In silico average nucleotide identity (ANI) and genome-to-genome distance calculator (GGDC) showed 81.1 % identity (ANI) and 22.6 % identity (GGDC) to the closest relative, 'P. polonicum' F5.1. On the basis of phenotypic, phylogenetic, genomic and chemotaxonomic data, the two strains represent a novel species of the genus Pararhizobium, for which the name Pararhizobium antarcticum sp. nov. is proposed. The type strain is NAQVI 59T(=DSM 103442T=LMG 29675T).

Pararhizobium polonicum sp. nov. isolated from tumors on stone fruit rootstocks.

Five Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from galls on different stone fruit rootstocks in Poland: strains F5.1(T) and F5.3 from Prunus avium F12/1, strains CP3.5 and CP17.2.1 from Prunus avium and strain AL5.1.8 from Prunus cerasifera. On the basis of 16S rDNA phylogeny, the strains cluster together and belong to the genus Pararhizobium with type strain of Pararhizobium herbae (99.6-99.8%) as their closest relative. Phylogenetic analysis of the novel strains using housekeeping genes atpD, recA and rpoB revealed their distinct position separate from other known Rhizobium species and confirmed their relation to P. herbae. DNA-DNA hybridization of strains F5.1(T), with the type strain of P. herbae LMG 25718(T) and Pararhizobium giardinii R-4385(T) revealed 28.3% and 27.9% of DNA-DNA relatedness, respectively. Phenotypic and physiological properties differentiate the novel isolates from other closely related species. On the basis of the results obtained, the five isolates are considered to represent a novel species of the genus Pararhizobium, for which the name Pararhizobium polonicum sp. nov. (type strain F5.1(T)=LMG 28610(T)=CFBP 8359(T)) is proposed.

Genetic relatedness and recombination analysis of Allorhizobium vitis strains associated with grapevine crown gall outbreaks in Europe.

AIMS: To analyse genetic diversity and epidemiological relationships among 54 strains of Allorhizobium vitis isolated in Europe during an 8-year period and to assess the relative contribution of mutation and recombination in shaping their diversity. METHODS AND RESULTS: By using random amplified polymorphic DNA (RAPD) PCR, strains studied were distributed into 12 genetic groups. Sequence analysis of dnaK, gyrB and recA housekeeping genes was employed to characterize a representative subcollection of 28 strains. A total of 15 different haplotypes were found. Nucleotide sequence analysis suggested the presence of recombination events in A. vitis, particularly affecting dnaK locus. Although prevalence of mutation over recombination was found, impact of recombination was about two times greater than mutation in the evolution of the housekeeping genes analysed. CONCLUSIONS: The RAPD analysis indicated high degree of genetic diversity among the strains. However, the most abundant RAPD group was composed of 35 strains, which could lead to the conclusion that they share a common origin and were distributed by the movement of infected grapevine planting material as a most common way of crossing long distances. Furthermore, it seems that recombination is acting as an important driving force in the evolution of A. vitis. As no substantial evidence of recombination was detected within recA gene fragment, this phylogenetic marker could be reliable to characterize phylogenetic relationships among A. vitis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrated clear epidemiological relationship between majority of strains studied, suggesting a need for more stringent phytosanitary measures in international trade. Moreover, this is the first study to report recombination in A. vitis.

Isolation, transposon mutagenesis, and characterization of the novel nicotine-degrading strain Shinella sp. HZN7.

Nicotine is a significant toxic waste generated in tobacco manufacturing. Biological methods for the degradation of nicotine waste are in high demand. In this study, we report the identification and characterization of the novel nicotine-degrading strain Shinella sp. HZN7. This strain can degrade 500 mg/L nicotine completely within 3 h at 30 °C and pH values of 6.5 ∼ 8.0. The biodegradation of nicotine by Shinella sp. HZN7 involves five intermediate metabolites: 6-hydroxy-nicotine (6HN), 6-hydroxy-N-methylmyosmine, 6-hydroxypseudooxynicotine (6HPON), 6-hydroxy-3-succinoyl-pyridine (HSP), and 2,5-dihydroxypyridine, as detected by ultraviolet spectrophotometry, HPLC, and LC-MS. We generated three mutants, N7-W18, N7-X5, and N7-M17, by transposon mutagenesis, in which the nicotine-degrading pathway terminated at 6HN, 6HPON, and HSP, respectively. The production of the five intermediate metabolites and their order in the degradation pathway were confirmed in the three mutants, indicating that strain HZN7 degrades nicotine via a variant of the pyridine and pyrrolidine pathways. The mutant gene from strain N7-X5, orf2, was cloned by self-formed adaptor PCR, but the nucleotide and amino acid sequence showed no similarity to any gene or gene product with defined function. However, orf2 disruption and complementation suggested that the orf2 gene is essential for the conversion of 6HPON to HSP in strain HZN7. This is the first study to provide genetic evidence for this variant nicotine degradation pathway.

Phenotypic diversity and amylolytic activity of fast growing rhizobia from pigeonpea [Cajanus cajan (L) Millsp]

This study evaluated 26 pigeonpea rhizobial isolates according to their cultural characteristics, intrinsic antibiotic resistance, salt and temperature tolerance, carbon source utilization and amylolytic activity. The cultural characterization showed that the majority of them presented the ability to acidify the YMA. Among the 27 isolates evaluated, 25 were able to grow when incubated at 42° C and 11 showed tolerance to 3% (w/v) of NaCl in YMA medium. The patterns of carbon sources utilization was very diverse among the isolates. It was observed the capacity of three strains to metabolize all the carbon sources evaluated and a total of 42% of the bacterial isolates was able to grow in the culture medium supplemented with at least, six carbon sources. The carbon sources mannitol (control) and sucrose were metabilized by all isolates evaluated. The profile of intrinsic resistance to antibiotics showed that the isolates were mostly resistant to streptomycin and ampicillin, but susceptible to kanamycin and chloranphenicol. High amylolytic activity of, at least, four isolates was also demonstrated, especially for isolated 47.3b, which showed the highest enzymatic index. These results indicate the metabolic versatility of the pigeonpea rhizobia, and indicates the isolate 47.3b to further studies regarding the amylase production and characterization.

Nickel solubilizing capacity and characterization of rhizobacteria isolated from hyperaccumulating and non-hyperaccumulating subspecies of Alyssum serpyllifolium.

Bacterial strains were isolated from the rhizosphere of three populations of the Ni-hyperaccumulator Alyssum serpyllifolium subsp. lusitanicum (A. pintodasilvae; M, S, and L), one population of Ni-hyperaccumulator A. serpyllifolium subsp. malacitanum (A. malacitanum; SB), and one population of the non-hyperaccumulator A. serpyllifolium subsp. serpyllifolium (A. serpyllifolium; SN). Isolates were characterized genotypically by BOX-PCR genomic DNA fingerprinting and comparative sequence analysis of partial 16S rRNA gene, and phenotypically by their Ni tolerance (0-10 mM), presence of plant growth promoting traits (indoleacetic acid (IAA)-, siderophore-, or organic acid-production, and phosphate solubilization) or capacity to produce biosurfactants. Among the collection of rhizobacteria, 84 strains were selected (according to their BOX-PCR profiles and phenotypic characteristics) to assess their ability to modify Ni extractability from Ni-rich (serpentine) soils. Metabolites produced by 13 of the isolates mobilized soil Ni (originating from the rhizosphere of both Ni-hyperaccumulators and non-hyperaccumulator). In contrast, Ni extraction using culture medium filtrates which had supported the growth of 29 strains was significantly reduced. The remaining strains had no effect on Ni mobility. Bacterial induced Ni mobilization was not related to Ni resistance or the phenotypic traits tested. Isolates with potential use in phytoremediation techniques will be further studied in a plant-microorganism-soil system.

Biodegradation of nicotine by a novel Strain Shinella sp. HZN1 isolated from activated sludge.

The nicotine-degrading bacterium HZN1 was isolated from activated sludge and identified as Shinella sp. based on its physiological characteristics and analysis of 16S rDNA gene. Strain HZN1 is capable of using nicotine as the sole carbon source in the mineral salts medium. The optimum temperature and pH for strain HZN1 growth and nicotine degradation were 30°C and 7.0, respectively. It could degrade approximately 100 % of 0.5 g L(-1) of nicotine within 9 h. Three intermediate metabolites were produced by the strain HZN1 and identified as cotinine, myosmine and nicotyrine using gas chromatography-mass spectrometry. This is the first report of nicotine-degrading strain from the genus of Shinella. The results showed that strain HZN1 could be potentially employed in bioremediation of nicotine. Our findings would provide a new insight into the biodegradation of nicotine.

Characterization of root-nodulating bacteria associated to Prosopis farcta growing in the arid regions of Tunisia.

Diversity of 50 bacterial isolates recovered from root nodules of Prosopis farcta grown in different arid soils in Tunisia, was investigated. Characterization of isolates was assessed using a polyphasic approach including phenotypic characteristics, 16S rRNA gene PCR--RFLP and sequencing, nodA gene sequencing and MLSA. It was found that most of isolates are tolerant to high temperature (40°C) and salinity (3%). Genetic characterization emphasizes that isolates were assigned to the genus Ensifer (80%), Mesorhizobium (4%) and non-nodulating endophytic bacteria (16%). Forty isolates belonging to the genus Ensifer were affiliated to Ensifer meliloti, Ensifer xinjiangense/Ensifer fredii and Ensifer numidicus species. Two isolates belonged to the genus Mesorhizobium. Eight isolates failing to renodulate their host plant were endophytic bacteria and belonged to Bacillus, Paenibacillus and Acinetobacter genera. Symbiotic properties of nodulating isolates showed a diversity in their capacity to infect their host plant and fix atmospheric nitrogen. Isolate PG29 identified as Ensifer meliloti was the most effective one. Ability of Prosopis farcta to establish symbiosis with rhizobial species confers an important advantage for this species to be used in reforestation programs. This study offered the first systematic information about the diversity of microsymbionts nodulating Prosopis farcta in the arid regions of Tunisia.

Kaistia geumhonensis sp. nov. and Kaistia dalseonensis sp. nov., two members of the class Alphaproteobacteria.

Two strains, designated B1-1(T) and B6-8(T), were isolated from the Geumho River and the Dalseo Stream in Korea. Comparative 16S rRNA gene sequence analysis showed a clear affiliation of these two bacteria with the class Alphaproteobacteria, their closest relatives being Kaistia adipata KCTC 12095(T), Kaistia granuli KCTC 12575(T), Kaistia soli KACC 12605(T) and Kaistia terrae KACC 12910(T) with 16S rRNA gene sequence similarities of 95.3 -97.7 % to the two novel strains. Strains B1-1(T) and B6-8(T) shared a 16S rRNA gene sequence similarity value of 96.1 %. Cells of the two strains were Gram-reaction-negative, aerobic, non-motile, short rods or cocci. The predominant ubiquinone was Q-10. The major fatty acids were C(16 : 0,) C(18 : 1)ω7c, C(18 : 0) and C(19 : 0)ω8c cyclo for strain B1-1(T) and C(16 : 0,) C(18 : 1)ω7c, C(18 : 0), C(18 : 1) 2-OH, and C(19 : 0)ω8c cyclo for strain B6-8(T). The G+C contents of the genomic DNA of the strains B1-1(T) and B6-8(T) were 61.6 and 66.5 mol%, respectively. Based on the results of this polyphasic study, strains B1-1(T) ( = KCTC 12849(T) = DSM 18799(T)) and B6-8(T) ( = KCTC 12850(T) = DSM 18800(T)) represent two novel species of the genus Kaistia, for which the names Kaistia geumhonensis sp. nov. and Kaistia dalseonensis sp. nov. are proposed, respectively.

Shinella daejeonensis sp. nov., a nitrate-reducing bacterium isolated from sludge of a leachate treatment plant.

A Gram-negative-staining, motile, rod-shaped, aerobic bacterial strain, designated MJ02(T), was isolated from sludge of a leachate treatment plant in Daejeon (South Korea) and was characterized to determine its taxonomic position by using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain MJ02(T) belonged to the family Rhizobiaceae, class Alphaproteobacteria, and was most closely related to Shinella yambaruensis MS4(T) (97.6 % sequence similarity) and Shinella fusca DC-196(T) (97.5 %). The G+C content of the genomic DNA of strain MJ02(T) was 64.3 mol%. The detection of a quinone system with ubiquinone Q-10 as the predominant respiratory lipoquinone and a fatty acid profile with C18:1ω7c (45.8 %) and C16:0 (21.8 %) as the major components supported the affiliation of strain MJ02(T) to the genus Shinella. However, strain MJ02(T) exhibited relatively low levels of DNA-DNA relatedness with respect to S. fusca DSM 21319(T) (17±7 %) and S. yambaruensis KACC 14483(T) (12±6 %), showing clearly that the isolate constituted a new genospecies. Strain MJ02(T) could be clearly differentiated from its phylogenetic neighbours on the basis of several phenotypic, genotypic and chemotaxonomic features. Therefore, strain MJ02(T) is considered to represent a novel species of the genus Shinella, for which the name Shinella daejeonensis sp. nov. is proposed. The type strain is MJ02(T) ( = KCTC 22450(T) = JCM 16236(T)).

Comparative study of rhizobacterial community structure of plant species in oil-contaminated soil.

In this study, the identity and distribution of plants and the structure of their associated rhizobacterial communities were examined in an oil-contaminated site. The number of plant species that formed a community or were scattered was 24. The species living in soil highly contaminated with total petroleum hydrocarbon (TPH) (9,000-4,5000 mg/g-soil) were Cynodon dactylon, Persicaria lapathifolia, and Calystegia soldanella (a halophytic species). Among the 24 plant species, the following have been known to be effective for oil removal: C. dactylon, Digitaria sanguinalis, and Cyperus orthostachyus. Denaturing gradient gel electrophoresis (DGGE) profile analysis showed that the following pairs of plant species had highly similar (above 70%) rhizobacterial community structures: Artemisia princeps and Hemistepta lyrata; C. dactylon and P. lapathifolia; Carex kobomugi and Cardamine flexuosa; and Equisetum arvense and D. sanguinalis. The major groups of rhizobacteria were Betaproteobacteria, Gamma-proteobacteria, Chloroflexi, Actinobacteria, and unknown. Based on DGGE analysis, P. lapathifolia, found for the first time in this study growing in the presence of high TPH, may be a good species for phytoremediation of oil-contaminated soils and in particular, C. soldanella may be useful for soils with high TPH and salt concentrations. Overall, this study suggests that the plant roots, regardless of plant species, may have a similar influence on the bacterial community structure in oil-contaminated soil.