Phylogenomic analyses and reclassification of the Mesorhizobium complex: proposal for 9 novel genera and reclassification of 15 species.

BACKGROUD: The genus Mesorhizobium is shown by phylogenomics to be paraphyletic and forms part of a complex that includes the genera Aminobacter, Aquamicrobium, Pseudaminobacter and Tianweitania. The relationships for type strains belong to these genera need to be carefully re-evaluated. RESULTS: The relationships of Mesorhizobium complex are evaluated based on phylogenomic analyses and overall genome relatedness indices (OGRIs) of 61 type strains. According to the maximum likelihood phylogenetic tree based on concatenated sequences of 539 core proteins and the tree constructed using the bac120 bacterial marker set from Genome Taxonomy Database, 65 type strains were grouped into 9 clusters. Moreover, 10 subclusters were identified based on the OGRIs including average nucleotide identity (ANI), average amino acid identity (AAI) and core-proteome average amino acid identity (cAAI), with AAI and cAAI showing a clear intra- and inter-(sub)cluster gaps of 77.40-80.91% and 83.98-86.16%, respectively. Combined with the phylogenetic trees and OGRIs, the type strains were reclassified into 15 genera. This list includes five defined genera Mesorhizobium, Aquamicrobium, Pseudaminobacter, Aminobacterand Tianweitania, among which 40/41 Mesorhizobium species and one Aminobacter species are canonical legume microsymbionts. The other nine (sub)clusters are classified as novel genera. Cluster III, comprising symbiotic M. alhagi and M. camelthorni, is classified as Allomesorhizobium gen. nov. Cluster VI harbored a single symbiotic species M. albiziae and is classified as Neomesorhizobium gen. nov. The remaining seven non-symbiotic members were proposed as: Neoaquamicrobium gen. nov., Manganibacter gen. nov., Ollibium gen. nov., Terribium gen. nov., Kumtagia gen. nov., Borborobacter gen. nov., Aerobium gen. nov.. Furthermore, the genus Corticibacterium is restored and two species in Subcluster IX-1 are reclassified as the member of this genus. CONCLUSION: The Mesorhizobium complex are classified into 15 genera based on phylogenomic analyses and OGRIs of 65 type strains. This study resolved previously non-monophyletic genera in the Mesorhizobium complex.

Rhizobium hidalgonense and Rhizobium redzepovicii as faba bean (Vicia faba L.) microsymbionts in Mexican soils.

As a legume crop widely cultured in the world, faba bean (Vicia faba L.) forms root nodules with diverse Rhizobium species in different regions. However, the symbionts associated with this plant in Mexico have not been studied. To investigate the diversity and species/symbiovar affiliations of rhizobia associated with faba bean in Mexico, rhizobia were isolated from this plant grown in two Mexican sites in the present study. Based upon the analysis of recA gene phylogeny, two genotypes were distinguished among a total of 35 isolates, and they were identified as Rhizobium hidalgonense and Rhizobium redzepovicii, respectively, by the whole genomic sequence analysis. Both the species harbored identical nod gene cluster and the same phylogenetic positions of nodC and nifH. So, all of them were identified into the symbiovar viciae. As a minor group, R. hidalgonense was only isolated from slightly acid soil and R. redzepovicii was the dominant group in both the acid and neutral soils. In addition, several genes related to resistance to metals (zinc, copper etc.) and metalloids (arsenic) were detected in genomes of the reference isolates, which might offer them some adaptation benefits. As conclusion, the community composition of faba bean rhizobia in Mexico was different from those reported in other regions. Furthermore, our study identified sv. viciae as the second symbiovar in the species R. redzepovicii. These results added novel evidence about the co-evolution, diversification and biogeographic patterns of rhizobia in association with their host legumes in distinct geographic regions.

Microbacterium plantarum sp. nov. and Microbacterium thalli sp. nov., two endophytic metal-resistant bacteria isolated from Sphaeralcea angustifolia (Cav.) G. Don and Prosopis laevigata (Humb. et Bonpl. ex Willd) M.C. Johnston.

Four Gram-positive, aerobic, catalase- and oxidase-negative, rod-shaped, motile endophytic bacterial strains, designated NM3R9T, NE1TT3, NE2TL11 and NE2HP2T, were isolated from the inner tissues (leaf and stem) of Sphaeralcea angustifolia and roots of Prosopis laevigata. They were characterized using a polyphasic approach, which revealed that they represent two novel Microbacterium species. Phylogenetic analysis based on 16S rRNA gene sequencing showed that the species closest to NE2HP2T was Microbacterium arborescens DSM 20754T (99.6 %) and that closest to NM3R9T, NE2TL11 and NE2TT3 was Microbacterium oleivorans NBRC 103075T (97.4 %). The whole-genome average nucleotide identity value between strain NM3R9T and Microbacterium imperiale DSM 20530T was 90.91 %, and that between strain NE2HP2T and M. arborecens DSM 20754T was 91.03 %. Digital DNA-DNA hybridization showed values of less than 70 % with the type strains of related species. The polar lipids present in both strains included diphosphatidylglycerol, phosphatidylglycerol, glycolipids and unidentified lipids, whereas the major fatty acids included anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and C16 : 0. Whole-cell sugars included mannose, rhamnose and galactose. Strains NM3R9T and NE2HP2T showed physiological characteristics different from those present in closely related Microbacterium species. According to the taxonomic analysis, both strains belong to two novel species. The name Microbacterium plantarum sp. nov. is proposed for strain NE2HP2T (=LMG 30875T=CCBAU 101117T) and Microbacterium thalli sp. nov. for strains NM3R9T (=LMG 30873T=CCBAU 101116T), NE1TT3 (=CCBAU 101114) and NE2TL11 (=CCBAU 101115).

Metallophores production by bacteria isolated from heavy metal-contaminated soil and sediment at Lerma-Chapala Basin.

Environmental pollution as a result of heavy metals (HMs) is a worldwide problem and the implementation of eco-friendly remediation technologies is thus required. Metallophores, low molecular weight compounds, could have important biotechnological applications in the fields of agriculture, medicine, and bioremediation. This study aimed to isolate HM-resistant bacteria from soils and sediments of the Lerma-Chapala Basin and evaluated their abilities to produce metallophores and to promote plant growth. Bacteria from the Lerma-Chapala Basin produced metallophores for all the tested metal ions, presented a greater production of As3+ metallophores, and showed high HM resistance especially to Zn2+, As5+, and Ni2+. A total of 320 bacteria were isolated with 170 strains showing siderophores synthesis. Members of the Delftia and Pseudomonas genera showed above 92 percent siderophore units (psu) during siderophores production and hydroxamate proved to be the most common functional group among the analyzed siderophores. Our results provided evidence that Lerma-Chapala Basin bacteria and their metallophores could potentially be employed in bioremediation processes or may even have potential for applications in other biotechnological fields.

Heavy-metal resistance mechanisms developed by bacteria from Lerma-Chapala basin.

Heavy-metal (HM) contamination is a huge environmental problem in many countries including Mexico. Currently, microorganisms with multiple heavy-metal resistance and/or plant-promoting characteristics have been widely used for bioremediation of HM-contaminated soils. The aim of the study was isolated bacteria with multiple heavy-metal resistance and to determinate the resistance mechanism developed by these organisms. A total of 138 aerobic bacteria were isolated from soil and sediments surrounding the Lerma-Chapala basin located in the boundary of the States of Michoacán and Jalisco states of Mexico. One hundred and eight strains showed at least 1 plant growth-promoting features. The Lerma-Chapala basin bacteria were also resistant to high concentrations of HMs including the metalloid arsenic. Sequence analysis of 16S RNA genes reveled that these bacteria were mainly affiliated to the phyla Proteobacteria (38%), Firmicutes (31%) and Actinobacteria (25%), covering 21 genera with Bacillus as the most abundant one. Among them, at least 27 putative novel species were detected in the genera Acinetobacter, Arthrobacter, Bacillus, Agrobacterium, Dyadobacter, Enterobacter, Exiguobacterium, Kluyvera, Micrococcus, Microbacterium and Psychrobacter. In addition, these bacteria developed various heavy-metal-resistance mechanisms, such as biosorption/bioaccumulation, immobilization and detoxification. Therefore, the bacteria isolated from soils and sediments of Lerma-Chapala basin could be used in bioremediation strategies.

Paenibacillus glycinis sp. nov., an Endophytic Bacterium Isolated from the Nodules of Soybean (Glycine max (L.) Merr).

A Gram-negative-staining, endospore-forming, rod-shaped bacterium, designated T1T, was isolated from root nodules of soybean (Glycine max (L.) Merr) in Heilongjiang Province of China. The isolate was identified as a member of the genus Paenibacillus based on phenotypic and phylogenetic characteristics. The 16S rRNA sequence was closely related to that of Paenibacillus sacheonensis SY01T with a similarity of 98.4%. Average nucleotide identity and in silico DNA-DNA hybridization values between strain T1T and P. sacheonensis DSM 23054 T were 81.4% and 25.4%, respectively. The DNA G + C content of strain T1T was 58.2 mol%. meso-Diaminopimelic acid was detected in the cell-wall peptidoglycan. The major cellular fatty acids were anteiso-C15:0, iso-C16:0 and iso-C15:0. The predominant respiratory quinone was MK-7. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, five unidentified phospholipids, four unidentified aminophospholipids, an unidentified glycolipid and an unidentified lipid. Based on these results, T1T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus glycinis sp. nov. is proposed. The type strain is T1T (= CGMCC 1.18563 = KCTC43227).

Diverse Genomic Backgrounds Vs. Highly Conserved Symbiotic Genes in Sesbania-Nodulating Bacteria: Shaping of the Rhizobial Community by Host and Soil Properties.

Aiming at investigating the overall diversity, biogeography, and symbiosis gene evolutionary history of the Sesbania cannabina-nodulating rhizobia in China, a total of 874 rhizobial isolates originating from the root nodules of this plant grown at different sites were characterized and compared with those of some reference strains. All of the S. cannabina-nodulating rhizobia were classified into 16 (geno) species, including seven novel genospecies in the genera Ensifer, Rhizobium, Neorhizobium, and Agrobacterium, with Ensifer sesbaniae and Neorhizobium huautlense as the dominant and universal species. Ten of these species were found to nodulate other leguminous hosts or to lack nodulating abilities and were defined as symbiovar sesbania. Biogeographic patterns were observed, for which pH, TN, AK, and AP were the main determinants. The effects of pH were opposite to those of TN and AK, while AP presented effects independently of TN, AK, and pH. Symbiotic genes of these rhizobia showed a common origin, but nodA evolved faster than nifH. Point mutation is the main driving force in the evolution of both nodA and nifH, and lateral transfer of symbiotic genes might play an important role in the formation of diverse S. cannabina-nodulating rhizobial species. S. cannabina only nodulates with Sesbania rhizobia, demonstrating its severe selection on rhizobial symbiosis genes. Soil pH and physiochemical characteristics could affect rhizobial survival and competitive nodulation. This study provides insight into the community shifts and evolution of rhizobia in relation to their host and soil environments.

Mesorhizobium rhizophilum sp. nov., a 1-aminocyclopropane-1-carboxylate deaminase producing bacterium isolated from rhizosphere of maize in Northeast China.

A novel 1-aminocyclopropane-1-carboxylate deaminase producing bacterium, Gram- stain-negative, aerobic, motile, rod-shaped strain designated YM1C-6-2T was isolated from rhizosphere of maize grown in Northeast China. The 16S rRNA gene sequence analysis indicated that strain YM1C-6-2T belongs to the genus Mesorhizobium and is closely related to Mesorhizobium alhagi CCNWXJ12-2T and M. camelthorni CCNWXJ40-4T with sequence similarities of 98.4% and 97.9%, respectively. Multilocus sequence analysis of other housekeeping genes revealed that the new isolates YM1C-6-2T forms a phylogenetically group with some species in the genus Mesorhizobium. The genome size of strain YM1C-6-2T was 5.51 Mb, comprising 5378 predicted genes with a DNA G+C content of 64.5%. The average nucleotide identity and digital DNA-DNA hybridization comparisons between YM1C-6-2T and the most related type strains showed values below the accepted threshold for species discrimination. The major fatty acids of strain YM1C-6-2T were C19:0 cyclo ω8c (47.5%), summed feature 8 (C18:1ω7c and/or C18:1ω6c) (19.5%) and C16:0 (15.1%), which differed from the closely related reference strains in their relative abundance. The major polar lipids consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and an unidentified aminophospholipid. The predominant ubiquinone was identified as Quinone 10. Phenotypic and biochemical analysis results indicated that strain YM1C-6-2T can be distinguished from closely related type strains. Based on the above results, strain YM1C-6-2T represents a novel species of the genus Mesorhizobium, for which the name Mesorhizobium rhizophilum sp. nov. is proposed with YM1C-6-2T (= CGMCC 1.15487T = DSM 101712T) as the type strain.

Aeschynomene indica-Nodulating Rhizobia Lacking Nod Factor Synthesis Genes: Diversity and Evolution in Shandong Peninsula, China.

Aeschynomene indica is a semiaquatic legume that forms both stem and root nodules with rhizobia. Some A. indica rhizobia (AIRs) have been reported to nodulate the host using a Nod factor-independent pathway and possess photosynthetic abilities. To investigate the diversity and community structure of AIRs in China, a total of 300 rhizobial isolates were acquired from the root and stem nodules of A. indica grown at 4 sites in Shandong Peninsula, China. Nineteen representative strains were selected according to their recA phylogeny. With further classification in comparison with reference strains, 10 Bradyrhizobium genospecies were defined based on the 16S rRNA gene phylogeny and multilocus sequence analysis (MLSA) of housekeeping genes (HKGs) recA, atpD, glnII, dnaK, gyrB, and rpoB In addition, 6 genospecies were found only in China. No nodulation gene (nodA, nodB, nodC, or nodZ) was detected in the AIRs isolates by PCR amplification and Southern blotting. Phylogenetic analysis of nifH and the photosynthesis-related gene pufLM revealed their common origins. All representative strains formed root nodules, but only 9 representative strains for 4 genospecies formed stem nodules on A. indica, indicating that the stem nodulation process of A. indica is limited to some strains. The nucleotide diversity and recombination events of the HKGs, as well as nifH and pufLM genes, showed that mutation contributes more than recombination in evolution. The distribution of dominant AIR genospecies was mainly affected by available nitrogen, organic carbon, total nitrogen, and pH. Our study helps to characterize the diversity and evolution of AIRs.IMPORTANCEAeschynomene indica rhizobia (AIRs) can form both root and stem nodules via Nod factor-independent processes, which distinguishes them from other rhizobia. This study systematically uncovered the diversity and community composition of A. indica rhizobia distributed in eastern China. Our results reclassified all the A. indica rhizobia across the world and represent a useful contribution to evaluating the diversity and distribution of the symbiont. The presence of novel genospecies specifically distributed in China enriched the A. indica rhizobia resources and provided insight into the geographic distribution of rhizobia. The phylogenetic relationship between nifH and pufLM of A. indica rhizobia across the world provides insight into the evolution of their nitrogen fixation and photosynthetic abilities.

An endophytic Kocuria palustris strain harboring multiple arsenate reductase genes.

Aiming at revealing the arsenic (As) resistance of the endophytic Kocuria strains isolated from roots and stems of Sphaeralcea angustifolia grown at mine tailing, four strains belonging to different clades of Kocuria based upon the phylogeny of 16S rRNA genes were screened for minimum inhibitory concentration (MIC). Only the strain NE1RL3 was defined as an As-resistant bacterium with MICs of 14.4/0.0125 mM and 300/20.0 mM for As3+ and As5+, respectively, in LB/mineral media. This strain was identified as K. palustris based upon analyses of cellular chemical compositions (cellular fatty acids, isoprenoides, quinones, and sugars), patterns of carbon source, average nucleotide identity of genome and digital DNA-DNA relatedness. Six genes coding to enzymes or proteins for arsenate reduction and arsenite-bumping were detected in the genome, demonstrating that this strain is resistant to As possibly by reducing As5+ to As3+, and then bumping As3+ out of the cell. However, this estimation was not confirmed since no arsenate reduction was detected in a subsequent assay. This study reported for the first time the presence of phylogenetically distinct arsenate reductase genes in a Kocuria strain and evidenced the possible horizontal transfer of these genes among the endophytic bacteria.

Paenibacillus rhizophilus sp. nov., a nitrogen-fixing bacterium isolated from the rhizosphere of wheat (Triticum aestivum L.).

A novel Gram-stain-variable, endospore-forming, motile, rod-shaped, facultative aerobic bacterium, designated 7197T, was isolated from rhizosphere soil of wheat (Triticum aestivum L.) collected from Yakeshi County, Inner Mongolia, PR China. This isolate was found to have the highest 16S rRNA gene sequence similarity to Paenibacillussabinae T27T (98.0 %), followed by Paenibacillussophorae S27T (97.9 %) and Paenibacillusforsythiae T98T (97.7 %). To ascertain the genomic relatedness of this strain to its phylogenetic neighbours, its genome sequence was determined. The average nucleotide identity values of genome sequences between the novel isolate and the type strains of related species P. sabinae T27T, P. sophorae S27T and P. forsythiae T98T were 87.9 %, 85.8 and 83.9 %, respectively. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unidentified aminophospholipids and one unidentified aminolipid. The major cellular fatty acids were anteiso-C15 : 0 (56.3 %), C16 : 0 (15.7 %) and iso-C15 : 0 (14.1 %).The genome size of strain 7197T was 5.21 Mb, comprising 4879 predicted genes with a DNA G+C content of 51.9 mol%. Menaquinone-7 was reported as the major respiratory quinone. The diamino acid in the cell-wall peptidoglycan was found to be meso-diaminopimelic acid. Based on phylogenetic, genomic, chemotaxonomic and phenotypic characteristics, strain 7197T was classified as a novel species within the genus Paenibacillus, for which the name Paenibacillus rhizophilus sp. nov. is proposed. The type strain of Paenibacillus rhizophilus is 7197T (=DSM 103168T=CGMCC 1.15699T).

Nonspecific Symbiosis Between Sophora flavescens and Different Rhizobia.

We explored the genetic basis of the promiscuous symbiosis of Sophora flavescens with diverse rhizobia. To determine the impact of Nod factors (NFs) on the symbiosis of S. flavescens, nodulation-related gene mutants of representative rhizobial strains were generated. Strains with mutations in common nodulation genes (nodC, nodM, and nodE) failed to nodulate S. flavescens, indicating that the promiscuous nodulation of this plant is strictly dependent on the basic NF structure. Mutations of the NF decoration genes nodH, nodS, nodZ, and noeI did not affect the nodulation of S. flavescens, but these mutations affected the nitrogen-fixation efficiency of nodules. Wild-type Bradyrhizobium diazoefficiens USDA110 cannot nodulate S. flavescens, but we obtained 14 Tn5 mutants of B. diazoefficiens that nodulated S. flavescens. This suggested that the mutations had disrupted a negative regulator that prevents nodulation of S. flavescens, leading to nonspecific nodulation. For Ensifer fredii CCBAU 45436 mutants, the minimal NF structure was sufficient for nodulation of soybean and S. flavescens. In summary, the mechanism of promiscuous symbiosis of S. flavescens with rhizobia might be related to its nonspecific recognition of NF structures, and the host specificity of rhizobia may also be controlled by currently unknown nodulation-related genes.

Mechanism of arsenic resistance in endophytic bacteria isolated from endemic plant of mine tailings and their arsenophore production.

Arsenic contamination is an important environmental problem around the world since its high toxicity, and bacteria resist to this element serve as valuable resource for its bioremediation. Aiming at searching the arsenic-resistant bacteria and determining their resistant mechanism, a total of 27 strains isolated from roots of Prosopis laevigata and Spharealcea angustifolia grown in a heavy metal-contaminated region in Mexico were investigated. The minimum inhibitory concentration (MIC) and transformation abilities of arsenate (As5+) and arsenite (As3+), arsenophore synthesis, arsenate uptake, and cytoplasmatic arsenate reductase (arsC), and arsenite transporter (arsB) genes were studied for these strains. Based on these results and the 16S rDNA sequence analysis, these isolates were identified as arsenic-resistant endophytic bacteria (AREB) belonging to the genera Arthrobacter, Bacillus, Brevibacterium, Kocuria, Microbacterium, Micrococcus, Pseudomonas, and Staphylococcus. They could tolerate high concentrations of arsenic with MIC from 20 to > 100 mM for As5+ and 10-20 mM for As3+. Eleven isolates presented dual abilities of As5+ reduction and As3+ oxidation. As the most effective strains, Micrococcus luteus NE2E1 reduced 94% of the As5+ and Pseudomonas zhaodongensis NM2E7 oxidized 46% of As3+ under aerobic condition. About 70 and 44% of the test strains produced arsenophores to chelate As5+ and As3+, respectively. The AREB may absorb arsenate via the same receptor of phosphate uptake or via other way in some case. The cytoplasmic arsenate reductase and alternative arsenate reduction pathways exist in these AREB. Therefore, these AREB could be candidates for the bioremediation process.

Jeotgalibacillus proteolyticus sp. nov., a protease-producing bacterium isolated from ocean sediments.

A Gram-stain-positive, rod-shaped bacterial strain, 22-7T, was isolated from ocean sediment of Laizhou Bay, China, and was characterized by using a polyphasic approach. Optimal growth was observed at 33 °C on a 2216E agar plate of pH 7.5 and with 2 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences identified it as a member of the genus Jeotgalibacillus, most similar to Jeotgalibacillus campisalis SF-57T (98.7 % similarity), Jeotgalibacillus marinus DSM 1297T (98.2 %) and Jeotgalibacillus soli P9T (97.1 %). Average nucleotide identity values and digital DNA-DNA hybridization values were less than 74.2 and 18.1 %, respectively, between strain 22-7T and the type strains of closely related species. The major polar lipids were aminophospholipid, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major fatty acids (>10 %) were anteiso-C15 : 0 and iso-C15 : 0; and the major menaquinone was MK-7. The peptidoglycan type of the cell wall was A1α linked through l-lysine as the diamino acid. Combined data from phenotypic, chemotaxonomic and genotypic characterizations demonstrated that strain 22-7T represents a novel Jeotgalibacillus species, for which the name Jeotgalibacillus proteolyticus sp. nov. is proposed. The type strain is 22-7T(=MCCC 1H00228T=KCTC 33930T).

Mesorhizobium wenxiniae sp. nov., isolated from chickpea (Cicer arietinum L.) in China.

Three chickpea rhizobial strains (WYCCWR 10195T=S1-3-7, WYCCWR 10198=S1-4-3 and WYCCWR 10200=S1-5-1) isolated from Northwest China formed a group affiliated to Mesorhizobium based on 16S rRNA gene sequence comparison. To clarify their species status, multilocus sequence analysis and average nucleotide identity (ANI) values of whole genome sequences between the novel group and the type strains of the related species were further performed. Similarities of 95.7-96.6 % in the concatenated sequences of atpD-recA-glnII and 91.9-93.1 % of ANI values to the closest-related species Mesorhizobium muleiense, Mesorhizobium mediterraneum and Mesorhizobium temperatum demonstrated the novel group a unique genospecies. The most abundant fatty acid in cells of WYCCWR 10195T were C19 : 0 cyclo ω8c (51.4 %), followed by C18 : 1 ω7c 11-methyl (9.5 %) and C16 : 0 (9.3 %). Its genome size was 6.37 Mbp, comprising 6633 predicted genes with a DNA G+C content of 61.9 mol%. The similarities of 99.0-99.8 % for the nodC gene and 98.3-99.44 % for the nifH gene to those of the chickpea rhizobial species and nodulation with Cicer arietinum L. confirmed the strains of the new genospecies as symbiovar ciceri. The weak utilization of most of the tested sugars/organic acids and non-utilization of l(+)-rhamnose, l-cysteine and l-glycine as sole carbon source, tolerance to 1 % (w/v) NaCl, resistance to 5 µg ml-1 chloromycetin and non-hydrolysis of l-tyrosine distinguished the novel group from the related species and supported this group as a novel species, for which the name Mesorhizobium wenxiniae sp. nov. is proposed, with WYCCWR 10195T (=S1-3-7=HAMBI 3692T=LMG 30254T) as the type strain.

Rhizobium hidalgonense sp. nov., a nodule endophytic bacterium of Phaseolus vulgaris in acid soil.

One Gram-negative, aerobic, motile, rod-shaped bacterium, designated as FH14T, was isolated from nodules of Phaseolus vulgaris grown in Hidalgo State of Mexico. Results based upon 16S rRNA gene (≥99.8 % similarities to known species), concatenated sequence (recA, atpD and glnII) analysis of three housekeeping genes (≤93.4 % similarities to known species) and average nucleotide identity (ANI) values of genome sequence (ranged from 87.6 to 90.0 % to related species) indicated the distinct position of strain FH14T within the genus Rhizobium. In analyses of symbiotic genes, only nitrogen fixation gene nifH was amplified that had nucleotide sequence identical to those of the bean-nodulating strains in R. phaseoli and R. vallis, while nodulation gene nodC gene was not amplified. The failure of nodulation to its original host P. vulgaris and other legumes evidenced the loss of its nodulation capability. Strain FH14T contained summed feature 8 (C18:1 ω6c/C18:1 ω7c, 59.96 %), C16:0 (10.6 %) and summed feature 2 (C12:0 aldehyde/unknown 10.928, 10.24 %) as the major components of cellular fatty acids. Failure to utilize alaninamide, and utilizing L-alanine, L-asparagine and γ-amino butyric acid as carbon source, distinguished the strain FH14T from the type strains for the related species. The genome size and DNA G+C content of FH14T were 6.94 Mbp and 60.8 mol %, respectively. Based on those results, a novel specie in Rhizobium, named Rhizobium hidalgonense sp. nov., was proposed, with FH14T (=HAMBI 3636T = LMG 29288T) as the type strain.

Photobacterium proteolyticum sp. nov., a protease-producing bacterium isolated from ocean sediments of Laizhou Bay.

A protease-producing bacterial strain, 13-12T, was isolated from the ocean sediment of Laizhou Bay, PR China and systematically studied. The bacterium was Gram-stain negative, non spore-forming rods, which were motile with two flagella. It was positive for oxidase, the hydrolysis of starch, agar and gelatin, and for nitrate reduction. It was negative for catalase, esterase and the degradation of CM-cellulose. Optimum growth was observed at 28 °C, pH 6.5-7.0 and in the presence of 2-3 % (w/v) NaCl. Phylogenetic analysis of the 16S rRNA gene, and whole genome data, affiliated it to the genus Photobacterium. It was most closely related to Photobacterium jeanii R-40508T (96.7 % 16S rRNA gene similarity). Strain 13-12T was found to have less than 86.1 % similarities with the type strains of its most closely related species in multi-locus sequence analysis, less than 75.2 % using genome average nucleotide identities (ANI), and less than 18.5 % in DNA-DNA relatedness studies. Q8 was the predominant respiratory menaquinone. Phosphatidylethanolamine, phosphoaminolipid and phospholipid were the major polar phospholipids and summed feature 3 (48.2 %), C16 : 0 (18.4 %) and C18 : 1ω5c (14.1 %) the major fatty acids. The combined phenotypic, phylogenetic, genomic and chemotaxonomic data support this strain representing a novel species of the genus Photobacterium, for which the name Photobacterium proteolyticum sp. nov. is proposed, with 13-12T (=KCTC 42764T=CGMCC 1.14970) as the type strain. The genome size of 13-12T is 6.2 Mbp, comprising 5806 predicted genes and the DNA G+C content is 47.9 mol%.

Agrobacterium salinitolerans sp. nov., a saline-alkaline-tolerant bacterium isolated from root nodule of Sesbania cannabina.

Two Gram-staining-negative, aerobic bacteria (YIC 5082T and YIC4104) isolated from root nodules of Sesbania cannabina grown in a high-salt and alkaline environment were identified as a group in the genus Agrobacterium because they shared 100 and 99.7 % sequence similarities of 16S rRNA and recA+atpD genes, respectively. These two strains showed 99.2/100 % and 93.9/95.4 % 16S rRNA and recA+atpD gene sequence similarities to Agrobacterium radiobacter LMG140T and Agrobacterium. pusense NRCPB10T, respectively. The average nucleotide identities (ANI) of genome sequences were 89.95 % or lower between YIC 5082T and the species of the genus Agrobacterium examined. Moreover, these two test strains formed a unique nifH lineage deeply separated from other rhizobia. Although the nodC gene was not detected in YIC 5082T and YIC4104, they could form effective root nodules on S. cannabina plants. The main cellular fatty acids in YIC 5082T were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), C19 : 0cyclo ω8c, summed feature 2 (C12 : 0 aldehyde/unknown equivalent chain length 10.9525) and C16 : 0. The DNA G+C content of YIC 5082T was 59.3 mol%. The failure to utilize d-sorbitol as a carbon source distinguished YIC 5082T from the type strains of related species. YIC 5082T could grow in presence of 5.0 % (w/v) NaCl and at a pH of up to 10.0. Based on results regarding the genetic and phenotypic properties of YIC 5082T and YIC4104 the name Agrobacterium salinitolerans sp. nov. is proposed and YIC 5082T (=HAMBI 3646T=LMG 29287T) is designed as the type strain.

Cultivable endophytic bacteria from heavy metal(loid)-tolerant plants.

To evaluate the interactions among endophytes, plants and heavy metal/arsenic contamination, root endophytic bacteria of Prosopis laevigata (Humb and Bonpl. ex Willd) and Sphaeralcea angustifolia grown in a heavy metal(loid)-contaminated zone in San Luis Potosi, Mexico, were isolated and characterized. Greater abundance and species richness were found in Prosopis than in Sphaeralcea and in the nutrient Pb-Zn-rich hill than in the poor nutrient and As-Cu-rich mine tailing. The 25 species identified among the 60 isolates formed three groups in the correspondence analysis, relating to Prosopis/hill (11 species), Prosopis/mine tailing (4 species) and Sphaeralcea/hill (4 species), with six species ungrouped. Most of the isolates showed high or extremely high resistance to arsenic, such as ≥100 mM for As(V) and ≥20 mM for As(III), in mineral medium. These results demonstrated that the abundance and community composition of root endophytic bacteria were strongly affected by the concentration and type of the heavy metals and metalloids (arsenic), as well as the plant species.

Ensifer alkalisoli sp. nov. isolated from root nodules of Sesbania cannabina grown in saline-alkaline soils.

A group of Sesbania cannabina rhizobia belonging to four recA genotypes of a novel group was further characterized in comparison with the related Ensifer species. They showed 98.2 to 99.9 % similarities among themselves and 92.9 to 93.3 % similarities with the most related strain Ensifer sojae CCBAU 05684T in multilocus sequence analysis of recA, atpD and glnII. The genome average nucleotide identity values between representative strain YIC4027T and the type strains of its closely related species were 81.6 to 88.9 %. Identical symbiotic gene (nodA, nodC and nifH) sequences highly similar with those in other Sesbania-nodulating strains (Rhizobium sp. SIN-1, Neorhizobium huautlense S02T, Ensifer saheli ORS609T and Rhizobium sp. IRBG74) were detected. The representative strain YIC4027T could form effective nodules on its original host Sesbaniacannabina, but not on Sophora flavescens, Trifolium repens, Glycine max, Glycyrrhiza uralensis, Phaseolus vulgaris or Medicago sativa. The use of lactulose as sole carbon source, possession of C13 : 0 2-OH, C13 : 1 at 12-13, C15 : 1 iso ω9c, C17 : 0 anteiso and C18 : 0 iso and absence of C14 : 0 anteiso, C15 : 0 anteiso and C18 : 0 3-OH in fatty acids distinguished the strain YIC4027T from the type strains of its closely related species. Based on all the analyses mentioned above, we propose a novel species Ensifer alkalisoli sp. nov. and designate YIC4027T (=HAMBI 3655T=LMG 29286T) as the type strain. The genome size of YIC4027T is 5.97 Mbp, comprising 5588 predicted genes, and the DNA G+C content is 62.2 mol%.