Paraburkholderia translucens sp. nov. and Paraburkholderia sejongensis sp. nov., isolated from grassland soil and showing plant growth promoting potential.

Two Gram-negative bacterial strains designated MMS20-SJTN17T and MMS20-SJTR3T were isolated from a grassland soil sample, and taxonomically characterized using a polyphasic approach. The 16S rRNA gene sequence analysis indicates that both strains belong to the genus Paraburkholderia of the class Betaproteobacteria, with strain MMS20-SJTN17T being mostly related to Paraburkholderia sprentiae WSM5005T (96.45 % sequence similarity) and strain MMS20-SJTR3T to Paraburkholderia tuberum STM678T (98.59 % sequence similarity). MMS20-SJTN17T could grow at 15-40 °C (optimum, 25-30 °C) and at pH 6.0-8.0 (optimum, pH 6.0-7.0), whereas MMS20-SJTR3T could grow at 10-40 °C (optimum, 30-37 °C) and at pH 6.0-8.0 (optimum, pH 6.0). Both strains tolerated up to 1 % (w/v) NaCl (optimum, 0 %). The major fatty acids of MMS20-SJTN17T were C16 : 0 and C19 : 0 cyclo ω8c, and those of MMS20-SJTR3T were C17 : 0 cyclo and a summed feature comprising C18 : 1 ω7c and/or C18 : 1 ω6c. The major isoprenoid quinone of both strains was ubiquinone-8 and the diagnostic polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Regarding plant growth promoting potential, both strains were capable of producing indole acetic acid, siderophore and 1-aminocyclopropane-1-carboxylic acid deaminase, and also showed phosphate-solubilizing activity. A genome-based comparison using orthologous average nucleotide identity and digital DNA-DNA hybridization values indicates that strain MMS20-SJTN17T shares highest relatedness with Paraburkholderia monticola JC2948T and MMS20-SJTR3T with Paraburkholderia antibiotica G-4-1-8T, with values clearly below the cutoffs for species distinction. Examination of biosynthetic gene clusters responsible for secondary metabolite production reveals unique characteristics distinguishing each strain from closely related Paraburkholderia species. On the basis of genotypic, phenotypic, chemotaxonomic and phylogenomic data, each strain should be classified as a novel species of the genus Paraburkholderia, for which the names Paraburkholderia translucens sp. nov. (=MMS20-SJTN17T=LMG 32366T=KCTC 82783T) and Paraburkholderia sejongensis sp. nov. (=MMS20-SJTR3T=LMG 32367T=KCTC 82784T) are proposed.

Natural Chromosome-Chromid Fusion across rRNA Operons in a Burkholderiaceae Bacterium.

Chromids (secondary chromosomes) in bacterial genomes that are present in addition to the main chromosome appear to be evolutionarily conserved in some specific bacterial groups. In rare cases among these groups, a small number of strains from Rhizobiales and Vibrionales were shown to possess a naturally fused single chromosome that was reported to have been generated through intragenomic homologous recombination between repeated sequences on the chromosome and chromid. Similar examples have never been reported in the family Burkholderiaceae, a well-documented group that conserves chromids. Here, an in-depth genomic characterization was performed on a Burkholderiaceae bacterium that was isolated from a soil bacterial consortium maintained on diesel fuel and mutagenic benzo[a]pyrene. This organism, Cupriavidus necator strain KK10, was revealed to carry a single chromosome with unexpectedly large size (>6.6 Mb), and results of comparative genomics with the genome of C. necator N-1T indicated that the single chromosome of KK10 was generated through fusion of the prototypical chromosome and chromid at the rRNA operons. This fusion hypothetically occurred through homologous recombination with a crossover between repeated rRNA operons on the chromosome and chromid. Some metabolic functions that were likely expressed from genes on the prototypical chromid region were indicated to be retained. If this phenomenon-the bacterial chromosome-chromid fusion across the rRNA operons through homologous recombination-occurs universally in prokaryotes, the multiple rRNA operons in bacterial genomes may not only contribute to the robustness of ribosome function, but also provide more opportunities for genomic rearrangements through frequent recombination. IMPORTANCE A bacterial chromosome that was naturally fused with the secondary chromosome, or "chromid," and presented as an unexpectedly large single replicon was discovered in the genome of Cupriavidus necator strain KK10, a biotechnologically useful member of the family Burkholderiaceae. Although Burkholderiaceae is a well-documented group that conserves chromids in their genomes, this chromosomal fusion event has not been previously reported for this family. This fusion has hypothetically occurred through intragenomic homologous recombination between repeated rRNA operons and, if so, provides novel insight into the potential of multiple rRNA operons in bacterial genomes to lead to chromosome-chromid fusion. The harsh conditions under which strain KK10 was maintained-a genotoxic hydrocarbon-enriched milieu-may have provided this genotype with a niche in which to survive.

Capacity of soil bacteria to reach the phyllosphere and convergence of floral communities despite soil microbiota variation.

Leaves and flowers are colonized by diverse bacteria that impact plant fitness and evolution. Although the structure of these microbial communities is becoming well-characterized, various aspects of their environmental origin and selection by plants remain uncertain, such as the relative proportion of soilborne bacteria in phyllosphere communities. Here, to address this issue and to provide experimental support for bacteria being filtered by flowers, we conducted common-garden experiments outside and under gnotobiotic conditions. We grew Arabidopsis thaliana in a soil substitute and added two microbial communities from natural soils. We estimated that at least 25% of the phyllosphere bacteria collected from the plants grown in the open environment were also detected in the controlled conditions, in which bacteria could reach leaves and flowers only from the soil. These taxa represented more than 40% of the communities based on amplicon sequencing. Unsupervised hierarchical clustering approaches supported the convergence of all floral microbiota, and 24 of the 28 bacteria responsible for this pattern belonged to the Burkholderiaceae family, which includes known plant pathogens and plant growth-promoting members. We anticipate that our study will foster future investigations regarding the routes used by soil microbes to reach leaves and flowers, the ubiquity of the environmental filtering of Burkholderiaceae across plant species and environments, and the potential functional effects of the accumulation of these bacteria in the reproductive organs of flowering plants.

Description of antibiotic-producing novel bacteria Paraburkholderia antibiotica sp. nov. and Paraburkholderia polaris sp. nov.

Two white colony-forming, Gram-stain-negative, non-sporulating and motile bacteria, designated G-4-1-8T and RP-4-7T, were isolated from forest soil and Arctic soil, respectively. Both strains showed antimicrobial activity against Gram-negative pathogens (Pseudomonas aeruginosa and Escherichia coli) and could grow at a pH range of pH 4.0-11.0 (optimum, pH 7.0-9.0). Phylogenetic analyses based on their 16S rRNA gene sequences indicated that strains G-4-1-8T and RP-4-7T formed a lineage within the family Burkholderiaceae and were clustered as members of the genus Paraburkholderia. Strain G-4-1-8T showed the highest 16S rRNA sequence similarity to Paraburkholderia monticola JC2948T (98.1 %), while strain RP-4-7T showed the highest similarity to Paraburkholderia metrosideri DNBP6-1T (98.8 %). The only respiratory quinone in both strains was ubiquinone Q-8. Their principal cellular fatty acids were C16 : 0, cyclo-C17 : 0, summed feature 3 (iso-C15 :0 2-OH and/or C16 :1 ω7c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). Their major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and an unidentified aminophospholipid. The DNA G+C content of strains G-4-1-8T and RP-4-7T were 63.7 and 61.3 mol%, respectively, while their genome lengths were 7.44 and 9.67 Mb, respectively. The genomes of both strains showed at least 12 putative biosynthetic gene clusters. The average nucleotide identity and in silico DNA-DNA hybridization relatedness values between both strains and most closely related Paraburkholderia species were below the species threshold values. Based on a polyphasic study, these isolated strains represent novel species belonging to the genus Paraburkholderia, for which the names Paraburkholderia antibiotica sp. nov. (G-4-1-8T= KACC 21617T=NBRC 114603T) and Paraburkholderia polaris sp. nov. (RP-4-7T=KACC 21621T=NBRC 114605T) are proposed.

Metagenome-assembled genome of a Chitinophaga sp. and its potential in plant biomass degradation, as well of affiliated Pandoraea and Labrys species.

The prospection of new degrading enzymes of the plant cell wall has been the subject of many studies and is fundamental for industries, due to the great biotechnological importance of achieving a more efficient depolymerization conversion from plant polysaccharides to fermentable sugars, which are useful not only for biofuel production but also for various bioproducts. Thus, we explored the shotgun metagenome data of a bacterial community (CB10) isolated from sugarcane bagasse and recovered three metagenome-assembled genomes (MAGs). The genomic distance analyses, along with phylogenetic analysis, revealed the presence of a putative novel Chitinophaga species, a Pandoraea nosoerga, and Labrys sp. isolate. The isolation process for each one of these bacterial lineages from the community was carried out in order to relate them with the MAGs. The recovered draft genomes have reasonable completeness (72.67-100%) and contamination (0.26-2.66%) considering the respective marker lineage for Chitinophaga (Bacteroidetes), Pandoraea (Burkholderiales), and Labrys (Rhizobiales). The in-vitro assay detected cellulolytic activity (endoglucanases) only for the isolate Chitinophaga, and its genome analysis revealed 319 CAZymes, of which 115 are classified as plant cell wall degrading enzymes, which can act in fractions of hemicellulose and pectin. Our study highlights the potential of this Chitinophaga isolate provides several plant-polysaccharide-degrading enzymes.

Genomic Aromatic Compound Degradation Potential of Novel Paraburkholderia Species: Paraburkholderia domus sp. nov., Paraburkholderia haematera sp. nov. and Paraburkholderia nemoris sp. nov.

We performed a taxonomic and comparative genomics analysis of 67 novel Paraburkholderia isolates from forest soil. Phylogenetic analysis of the recA gene revealed that these isolates formed a coherent lineage within the genus Paraburkholderia that also included Paraburkholderiaaspalathi, Paraburkholderiamadseniana, Paraburkholderiasediminicola, Paraburkholderiacaffeinilytica, Paraburkholderiasolitsugae and Paraburkholderiaelongata and four unidentified soil isolates from earlier studies. A phylogenomic analysis, along with orthoANIu and digital DNA-DNA hybridization calculations revealed that they represented four different species including three novel species and P. aspalathi. Functional genome annotation of the strains revealed several pathways for aromatic compound degradation and the presence of mono- and dioxygenases involved in the degradation of the lignin-derived compounds ferulic acid and p-coumaric acid. This co-occurrence of multiple Paraburkholderia strains and species with the capacity to degrade aromatic compounds in pristine forest soil is likely caused by the abundant presence of aromatic compounds in decomposing plant litter and may highlight a diversity in micro-habitats or be indicative of synergistic relationships. We propose to classify the isolates representing novel species as Paraburkholderia domus with LMG 31832T (=CECT 30334) as the type strain, Paraburkholderia nemoris with LMG 31836T (=CECT 30335) as the type strain and Paraburkholderia haematera with LMG 31837T (=CECT 30336) as the type strain and provide an emended description of Paraburkholderia sediminicola Lim et al. 2008.

'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov., an endosymbiotic bacterium associated with nematode species of the genus Xiphinema (Nematoda, Longidoridae).

An intracellular bacterium, strain IAST, was observed to infect several species of the plant-parasitic nematode genus Xiphinema (Xiphinema astaregiense, Xiphinema incertum, Xiphinema madeirense, Xiphinema pachtaicum, Xiphinema parapachydermum and Xiphinema vallense). The bacterium could not be recovered on axenic medium. The 16S rRNA gene sequence of IAST was found to be new, being related to the family Burkholderiaceae, class Betaproteobacteria. Fungal endosymbionts Mycoavidus cysteinexigens B1-EBT (92.9 % sequence identity) and 'Candidatus Glomeribacter gigasporarum' BEG34 (89.8 % identity) are the closest taxa and form a separate phylogenetic clade inside Burkholderiaceae. Other genes (atpD, lepA and recA) also separated this species from its closest relatives using a multilocus sequence analysis approach. These genes were obtained using a partial genome of this bacterium. The localization of the bacterium (via light and fluorescence in situ hybridization microscopy) is in the X. pachtaicum females clustered around the developing oocytes, primarily found embedded inside the epithelial wall cells of the ovaries, from where they are dispersed in the intestine. Transmission electron microscopy (TEM) observations supported the presence of bacteria inside the nematode body, where they occupy ovaries and occur inside the intestinal epithelium. Ultrastructural analysis of the bacterium showed cells that appear as mostly irregular, slightly curved rods with rounded ends, 0.8-1.2 µm wide and 2.5-6.0 µm long, possessing a typical Gram-negative cell wall. The peptidoglycan layer is, however, evident only occasionally and not detectable by TEM in most cells. Another irregularly occurring shell surrounding the endosymbiont cells or the cell clusters was also revealed, probably originating from the host cell membrane. Flagella or spore-like cells do not occur and the nucleoid is diffusely distributed throughout the cell. This endosymbiont is transmitted vertically through nematode generations. These results support the proposal of IAST as a new species, although its obligate intracellular and obligate endosymbiont nature prevented isolation of a definitive type strain. Strain IAST is therefore proposed as representing 'Candidatus Xiphinematincola pachtaicus' gen. nov., sp. nov.

Phylogenomic analysis supports the reclassification of Burkholderia novacaledonica as Caballeronia novacaledonica comb. nov.

Burkholderia novacaledonica is a Betaproteobacterial species isolated from ultramafic soils in New Caledonia. The characterization and classification of this species into the Burkholderia genus was done simultaneously with the proposal of the new genus Caballeronia, initially composed of closely related Burkholderia glathei-like species. Thereafter, some reports based on the use of phylogenetic marker genes suggested that B. novacaledonica forms part of Caballeronia genus. Lacking a formal validation, and with the availability of its genome sequence, a genome-based phylogeny of B. novacaledonica was obtained to unravel its taxonomic position in Burkholderia sensu lato. A partial gyrB gene phylogeny, extended multilocus sequence typing on homologous protein sequences, and genomic distance-based phylogeny, all support the placement of this species in the Caballeronia genus. Therefore, the reclassification of B. novacaledonica to Caballeronia novacaledonica comb. nov. is proposed.

Paraburkholderia spp. are the main rhizobial microsymbionts of Mimosa tenuiflora (Willd.) Poir. in soils of the Brazilian tropical dry forests (Caatinga biome).

Mimosa tenuiflora (Willd.) Poir. is widespread in southern and central American drylands, but little information is available concerning its associated rhizobia. Therefore, this study aimed to characterize M. tenuiflora rhizobia from soils of the tropical dry forests (Caatinga) in Pernambuco State, Brazil, at the molecular and symbiotic levels. Soil samples of pristine Caatinga areas in four municipalities were used to grow M. tenuiflora. First, the bacteria from root nodules were subjected to nodC/nifH gene amplification, and the bacteria positive for both genes had the 16S rRNA gene sequenced. Then, ten strains were evaluated using recA, gyrB, and nodC gene sequences, and seven of them had their symbiotic efficiency assessed. Thirty-two strains were obtained and 22 of them were nodC/nifH positive. Twenty strains clustered within Paraburkholderia and two within Rhizobium by 16S rRNA gene sequencing. The beta-rhizobia were similar to P. phenoliruptrix (12) and P. diazotrophica (8). Both alpha-rhizobia were closely related to R. miluonense. The recA + gyrB phylogenetic analysis clustered four and five strains within the P. phenoliruptrix and P. diazotrophica branches, respectively, but they were somewhat divergent to the 16S rRNA phylogeny. For Rhizobium sp. ESA 637, the recA + gyrB phylogeny clustered the strain with R. jaguaris. The nodC phylogeny indicated that ESA 626, ESA 629, and ESA 630 probably represented a new symbiovar branch. The inoculation assay showed high symbiotic efficiency for all tested strains. The results indicated high genetic diversity and efficiency of M. tenuiflora rhizobia in Brazilian drylands and included P. phenoliruptrix-like bacteria in the list of efficient beta-rhizobia in the Caatinga biome.

Inoculation of Mimosa Pudica with Paraburkholderia phymatum Results in Changes to the Rhizoplane Microbial Community Structure.

Nitrogen fixing symbiosis between rhizobia and legumes contributes significant amounts of N to agricultural and natural environments. In natural soils, rhizobia compete with indigenous bacterial communities to colonize legume roots, which leads to symbiotic interactions. However, limited information is currently available on the effects of the rhizobial symbiont on the resident microbial community in the legume rhizosphere, rhizoplane, and endosphere, which is partly due to the presence of native nodulating rhizobial strains. In the present study, we used a symbiotic system comprised of Paraburkholderia phymatum and Mimosa pudica to examine the interaction of an inoculant strain with indigenous soil bacteria. The effects of a symbiont inoculation on the native bacterial community was investigated using high throughput sequencing and an analysis of 16S rRNA gene amplicons. The results obtained revealed that the inoculation induced significant alterations in the microbial community present in the rhizoplane+endosphere of the roots, with 13 different taxa showing significant changes in abundance. No significant changes were observed in the rhizospheric soil. The relative abundance of P. phymatum significantly increased in the rhizoplane+endosphere of the root, but significant decreased in the rhizospheric soil. While the rhizosphere, rhizoplane, and root endosphere contained a wide diversity of bacteria, the nodules were predominantly colonized by P. phymatum. A network analysis revealed that the operational taxonomic units of Streptomyces and Phycicoccus were positively associated with P. phymatum as potential keystone taxa. Collectively, these results suggest that the success of an inoculated symbiont depends on its ability to colonize the roots in the face of competition by other soil bacteria. A more detailed understanding of the mechanisms by which an inoculated strain colonizes its plant host is crucial for realizing the full potential of microbial inoculants in sustainable agriculture.

Paraburkholderia youngii sp. nov. and 'Paraburkholderia atlantica' - Brazilian and Mexican Mimosa-associated rhizobia that were previously known as Paraburkholderia tuberum sv. mimosae.

Previous studies have recognized South and Central/Latin American mimosoid legumes in the genera Mimosa, Piptadenia and Calliandra as hosts for various nodulating Paraburkholderia species. Several of these species have been validly named in the last two decades, e.g., P. nodosa, P. phymatum, P. diazotrophica, P. piptadeniae, P. ribeironis, P. sabiae and P. mimosarum. There are still, however, a number of diverse Paraburkholderia strains associated with these legumes that have an unclear taxonomic status. In this study, we focus on 30 of these strains which originate from the root nodules of Brazilian and Mexican Mimosa species. They were initially identified as P. tuberum and subsequently placed into a symbiovar (sv. mimosae) based on their host preferences. A polyphasic approach for the delineation of these strains was used, consisting of genealogical concordance analysis (using atpD, gyrB, acnA, pab and 16S rRNA gene sequences), together with comparisons of Average Nucleotide Identity (ANI), DNA G+C content ratios and phenotypic characteristics with those of the type strains of validly named Paraburkholderia species. Accordingly, these 30 strains were delineated into two distinct groups, of which one is conspecific with 'P. atlantica' CNPSo 3155T and the other new to Science. We propose the name Paraburkholderia youngii sp. nov. with type strain JPY169T (= LMG 31411T; SARCC751T) for this novel species.

Phylogeny of symbiotic genes reveals symbiovars within legume-nodulating Paraburkholderia species.

Bacteria belonging to the genus Paraburkholderia are capable of establishing symbiotic relationships with plants belonging to the Fabaceae (=Leguminosae) family and fixing the atmospheric nitrogen in specialized structures in the roots called nodules, in a process known as biological nitrogen fixation (BNF). In the nodulation and BNF processes several bacterial symbiotic genes are involved, but the relations between symbiotic, core genes and host specificity are still poorly studied and understood in Paraburkholderia. In this study, eight strains of nodulating nitrogen-fixing Paraburkholderia isolated in Brazil, together with described species and other reference strains were used to infer the relatedness between core (16S rDNA, recA) and symbiotic (nod, nif, fix) genes. The diversity of genes involved in the nodulation (nodAC) and nitrogen fixation (nifH) abilities was investigated. Only two groups, one containing three Paraburkholderia species symbionts of Mimosa, and another one with P. ribeironis strains presented similar phylogenetic patterns in the analysis of core and symbiotic genes. In three other groups events of horizontal gene transfer of symbiotic genes were detected. Paraburkholderia strains with available genomes were used in the complementary analysis of nifHDK and fixABC and confirmed well-defined phylogenetic positions of symbiotic genes. In all analyses of nod, nif and fix genes the strains were distributed into five clades with high bootstrap support, allowing the proposal of five symbiovars in nodulating nitrogen-fixing Paraburkholderia, designated as mimosae, africana, tropicalis, atlantica and piptadeniae. Phylogenetic inferences within each symbiovar are discussed.

Paraburkholderia lycopersici sp. nov., a nitrogen-fixing species isolated from rhizoplane of Lycopersicon esculentum Mill. var. Saladette in Mexico.

A survey of our in-house bacterial collection identified a group of six strains isolated from the tomato rhizoplane that possessed 16S rRNA gene sequences with 98.2% sequence similarity to Paraburkholderia pallida, suggesting that these strains represented a novel species. Multilocus sequence analysis using gltB, lepA and recA gene sequences showed the clustering of the strains and the BOX-PCR patterns were similar among these strains. The average nucleotide identity and the DNA-DNA virtual hybridization of strain TNe-862T was <89% and <34%, respectively, to the genomes of any sequenced Paraburkholderia species. The genome of strain TNe-862T possessed all the genes necessary for nitrogen fixation and biosynthesis of indoleacetic acid and antimicrobials terpenes, phosphonates and bacteriocins. It also contained genes for metal resistance, xenobiotic degradation, and hydrolytic enzymes such as a putative chitinase and isoamylase. Even though the strain contained potential genes for degradation of cellulose and starch, the bacterium was unable to utilize these substrates in culture medium. The genome encoded flagella and pili as well as multiple chemotaxis systems. In addition, genes encoding for the type I, II, IV, V and VI secretion systems were also present. The strains grow up to 42°C and 5% NaCl. The optimum growth pH was 8. The major cellular fatty acids were C16:0 and C18:1 ω7c. Based on this polyphasic analysis, these strains represent a novel species in the genus Paraburkholderia, for which the name Paraburkholderia lycopersici sp. nov. is proposed. The type strain is TNe-862T (=LMG 26415T=CIP 110323T).

Limnobacter alexandrii sp. nov., a thiosulfate-oxidizing, heterotrophic and EPS-bearing Burkholderiaceae isolated from cultivable phycosphere microbiota of toxic Alexandrium catenella LZT09.

A novel Gram-negative, aerobic, motile and short rod-shaped bacterium with exopolysaccharides production, designated as LZ-4T, was isolated from cultivable phycosphere microbiota of harmful algal blooms-causing marine dinoflagellate Alexandrium catenella LZT09 which produces paralytic shellfish poisoning toxins. Strain LZ-4T was able to use thiosulfate (optimum concentration 10 mM) as energy source for bacterial growth. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain LZ-4T belonged to the genus Limnobacter, showing high 16S rRNA gene sequences similarities with L. thiooxidans DSM 13612T (99.4%), L. humi NBRC 11650T (98.2%) and L. litoralis NBRC 105857T (97.2%), respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between LZ-4T and L. thiooxidans DSM 13612T were 78.9 and 21.9%, respectively. Both values were far lower than the thresholds (95-96% for ANI and 70% for dDDH) generally accepted for new species delineation. The respiratory quinone of strain LZ-4T was Q-8. The dominant cellular fatty acids were determined as summed feature 3 (C16:1 ω6c/ω7c), summed feature 8 (C18:1 ω6c/ω7c) and C16:0. Polar lipids profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and three unidentified polar lipids. The genomic DNA G+C content of strain LZ-4T was 52.5 mol%. Based on polyphasic characterization, strain LZ-4T represents a novel species of the genus Limnobacter, for which the name Limnobacter alexandrii sp. nov. is proposed. The type strain is LZ-4T (=CCTCC AB 2019004T =KCTC 72281T).

Paraburkholderia solitsugae sp. nov. and Paraburkholderia elongata sp. nov., phenolic acid-degrading bacteria isolated from forest soil and emended description of Paraburkholderia madseniana.

Two bacterial strains, 1NT and 5NT, were isolated from hemlock forest soil using a soluble organic matter enrichment. Cells of 1NT (0.65×1.85 µm) and 5NT (0.6×1.85 µm) are Gram-stain-negative, aerobic, motile, non-sporulating and exist as single rods, diplobacilli or in chains of varying length. During growth in dilute media (≤0.1× tryptic soy broth; TSB), cells are primarily motile with flagella. At higher concentrations (≥0.3× TSB), cells of both strains increasingly form non-motile chains, and cells of 5NT elongate (0.57×~7 µm) and form especially long filaments. Optimum growth of 1NT and 5NT occurred at 25-30 °C, pH 6.5-7.0 and <0.5% salinity. Results of comparative chemotaxonomic, genomic and phylogenetic analyses revealed that 1NT and 5NT were distinct from one another and their closest related type strains: Paraburkholderia madseniana RP11T, Paraburkholderia aspalathi LMG 27731T and Paraburkholderia caffeinilytica CF1T. The genomes of 1NT and 5NT had an average nucleotide identity (91.6 and 91.3%) and in silico DNA-DNA hybridization values (45.8%±2.6 and 45.5%±2.5) and differed in functional gene content from their closest related type strains. The composition of fatty acids and patterns of substrate use, including the catabolism of phenolic acids, also differentiated strains 1NT and 5NT from each other and their closest relatives. The only ubiquinone present in strains 1NT and 5NT was Q-8. The major cellular fatty acids were C16 : 0, 3OH-C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo ω8c and summed features 2 (3OH-C14 : 0 / C16 : 1 iso I), 3 (C16 : 1 ω6c/ω7c) and 8 (C18 : 1 ω7c/ω6c). A third bacterium, strain RL16-012-BIC-B, was isolated from soil associated with shallow roots and was determined to be a strain of P. madseniana (ANI, 98.8%; 16S rRNA gene similarity, 100%). Characterizations of strain RL16-012-BIC-B (DSM 110723=LMG 31706) led to proposed emendments to the species description of P. madseniana. Our polyphasic approach demonstrated that strains 1NT and 5NT represent novel species from the genus Paraburkholderia for which the names Paraburkholderia solitsugae sp. nov. (type strain 1NT=DSM 110721T=LMG 31704T) and Paraburkholderia elongata sp. nov. (type strain 5NT=DSM 110722T=LMG 31705T) are proposed.

Chitinimonas arctica sp. nov., isolated from Arctic tundra soil.

A Gram-stain-negative, rod-shaped, green-pigmented, aerobic and motile bacterium, strain R3-44T, was isolated from Arctic tundra soil. Stain R3-44T clustered closely with members of the genus Chitinimonas, which belongs to the family Burkholderiaceae, and showed the highest 16S rRNA sequence similarity to Chitinimonas naiadis AR2T (96.10%). Strain R3-44T grew optimally at pH 7.0, 28 °C and in the presence of 0-0.5 % (w/v) NaCl. The predominant respiratory isoprenoid quinone of strain R3-44T was identified as ubiquinone Q-8. The polar lipids consisted of phosphatidylglycerol, phosphatidylethanolamine, unidentified aminolipid and unidentified phospholipid. The main fatty acids were summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c, 40.6 %) and C16 : 0 (29.3 %). The DNA G+C content of strain R3-44T was 60.8 mol%. On the basis of the evidence presented in this study, strain R3-44T represents a novel species of the genus Chitinimonas, for which the name Chitinimonas arctica sp. nov. is proposed, with the type strain R3-44T (=CCTCC AB 2010422T=KCTC 72602T).

Paraburkholderia panacisoli sp. nov., a potentially antagonistic bacterium against the root rot fungal pathogen Cylindrocarpon destructans, isolated from ginseng cultivation soil.

A new bacterium, designated DCY113T, was isolated from ginseng cultivation soil in Gochang-gun, South Korea, and its taxonomic position identified by the polyphasic approach. 16S rRNA gene sequence analysis determined that this isolate belongs to the genus Paraburkholderia, and was closest to P. dipogonis DL7T (98.6%), P. phytofirmans PsJNT (98.5%), P. kirstenboschensis Kb15T (98.4%) and P. aromaticivorans BNT (98.1%). Strain DCY113T is Gram-reaction negative, strictly aerobic, rod-shaped, non-motile, and catalase and oxidase positive. The predominant isoprenoid quinone of DCY113T was ubiquinone Q-8. The major cellular fatty acids were C16:0, cyclo-C17:0 and the Summed feature 8 (C18:1ω7c and/or C18:1ω6c). The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and an unknown amino lipid (AL1). The G+C content of the genomic DNA was 62.2 mol%. Average nucleotide identity (ANI) between strain DCY113T and the related Paraburkholderia type strains were below the threshold value for species delineation. This low DNA relatedness in combination with phylogenetic and phenotypic tests indicates that strain DCY113T cannot be assigned to any recognized species. Strain DCY113T was also found to have antifungal activity against the pathogenic fungi Cylindrocarpon destructans. In conclusion, this study found DCY113T to be a novel species within the genus Paraburkholderia, for which the name P. panacisoli is proposed. The type strain is DCY113T (= KCTC 52951T = JCM 32098T).

Chitinasiproducens palmae gen. nov., sp. nov., a new member of the family Burkholderiaceae isolated from leaf tissues of oil palm (Elaeis guineensis Jacq.).

A Gram-stain-negative, aerobic, rod-shaped, leaf-associated bacterium, designated JS23T, was isolated from surface-sterilized leaf tissue of an oil palm grown in Singapore and was investigated by polyphasic taxonomy. Phylogenetic analyses based on 16S rRNA gene sequences and 180 conserved genes in the genome of several members of Burkholderiaceae revealed that strain JS23T formed a distinct evolutionary lineage independent of other taxa within the family Burkholderiaceae. The predominant ubiquinone was Q-8. The primary polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unidentified aminophospholipid. The major fatty acids were C16 : 0, summed feature 3 (C16 : 1 ω7c /C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c /C18 : 1 ω6c). The size of the genome is 5.36 Mbp with a DNA G+C content of 66.2 mol%. Genomic relatedness measurements such as average nucleotide identity, genome-to-genome distance and digital DNA-DNA hybridization clearly distinguished strain JS23T from the closely related genera Burkholderia, Caballeronia, Mycetohabitans, Mycoavidus, Pandoraea, Paraburkholderia, Robbsia and Trinickia. Furthermore, average amino acid identity values and the percentages of conserved proteins, 56.0-68.4 and 28.2-45.5, respectively, were well below threshold values for genus delineation and supported the assignment of JS23T to a novel genus. On the basis of the phylogenetic, biochemical, chemotaxonomic and phylogenomic evidence, strain JS23T is proposed to represent a novel species of a new genus within the family Burkholderiaceae, for which the name Chitinasiproducens palmae gen. nov., sp. nov., is proposed with the type strain of JS23T (= DSM 27307T=KACC 17592T).

Paraburkholderia atlantica sp. nov. and Paraburkholderia franconis sp. nov., two new nitrogen-fixing nodulating species isolated from Atlantic forest soils in Brazil.

A polyphasic study was conducted with 11 strains trapped by Mimosa pudica and Phaseolus vulgaris grown in soils of the Brazilian Atlantic Forest. In the phylogenetic analysis of the 16S rRNA gene, one clade of strains (Psp1) showed higher similarity with Paraburkholderia piptadeniae STM7183T (99.6%), whereas the second (Psp6) was closely related to Paraburkholderia tuberum STM678T (99%). An MLSA (multilocus sequence analysis) with four (recA, gyrB, trpB and gltB) housekeeping genes placed both Psp1 and Psp6 strains in new clades, and BOX-PCR profiles indicated high intraspecific genetic diversity within each clade. Values of digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) of the whole genome sequences were of 56.9 and 94.4% between the Psp1 strain CNPSo 3157T and P. piptadeniae; and of 49.7% and 92.7% between the Psp6 strain CNPSo 3155T and P. tuberum, below the threshold for species delimitation. In the nodC analysis, Psp1 strains clustered together with P. piptadeniae, while Psp6 did not group with any symbiotic Paraburkholderia. Other phenotypic, genotypic and symbiotic properties were evaluated. The polyphasic analysis supports that the strains represent two novel species, for which the names Paraburkholderia franconis sp. nov. with type strain CNPSo 3157T (= ABIP 241, = LMG 31644) and Paraburkholderia atlantica sp. nov. with type strain CNPSo 3155T (= ABIP 236, = LMG 31643) are proposed.

Paraburkholderia flava sp. nov., isolated from cool temperate forest soil.

A Gram-stain-negative, aerobic and short rod-shaped bacterial strain, designated LD6T, was isolated from a forest soil sample in Suwon, Gyeonggi-do, Republic of Korea. Strain LD6T grew at 10-37 °C (optimal temperature, 28 °C), and tolerated pH 8.0 and 2 % (w/v) NaCl. Strain LD6T was related most closely to members of the genus Paraburkholderia, namely Paraburkholderia azotifigens NF2-5-3T (98.2 % 16S rRNA gene sequence similarity), P. megapolitana A3T (97.9 %), P. ginsengiterrae DCY85T (97.9 %) and P. caribensis MWAP64T (97.7 %). The strain grew well on R2A agar, tryptone soya agar, Mueller-Hinton agar and nutrient agar. The major polar lipid profile comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and glycolipid. The major respiratory quinone was ubiquinone 8 (Q-8). The main fatty acids were C17 : 0 cyclo, C16 : 0, C16 : 0 3-OH, C19 : 0 cyclo ω8c and C12 : 0. The DNA G+C content of the isolated strain based on the whole genome sequence was 63.4 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain LD6T and its reference type strains ranged from 80.3 to 82.4%, and from 23.7 to 33.7%, respectively. Based on phenotypic, chemotypic and genotypic evidence, strain LD6T could be differentiated phylogenetically and phenotypically from the recognized species of the genus Paraburkholderia. Therefore, strain LD6T is considered to represent a novel species, for which the name Paraburkholderia flava sp. nov. is proposed. The type strain is LD6T (=KACC 21387T=JCM 33640T).