Massilia soli sp. nov., isolated from soil.

A Gram-stain-negative, catalase- and oxidase-positive and aerobic bacterium, designated strain R798T, was isolated from soil in South Korea. Cells were motile rods by means of a single polar flagellum. Growth of strain R798T was observed at 15-35 °C (optimum, 25-30 °C), pH 5.0-8.0 (optimum, 6.0) and 0-1.5 % NaCl (optimum, 0.3 %). Strain R798T contained ubiquinone-8 as the sole isoprenoid quinone, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and C16 : 0 as the major fatty acids and phosphatidylglycerol and phosphatidylethanolamine as the major polar lipids. The DNA G+C content of strain R798T calculated from the whole genome sequence was 63.3 mol%. Phylogenetic analyses based on the 16S rRNA gene and whole genome sequences revealed that strain R798T formed a distinct phyletic lineage within the genus Massilia. Strain R798T was most closely related to Massilia eurypsychrophila B528-3T with a 98.0 % 16S rRNA gene sequence similarity. Average nucleotide identity and digital DNA-DNA hybridization values between strain R798T and the type strain of M. eurypsychrophila were 79.2 and 22.7 %, respectively. Based on the phenotypic, chemotaxonomic and molecular analyses, strain R798T represents a novel species of the genus Massilia, for which the name Massilia soli sp. nov. is proposed. The type strain is R798T (=KACC 22114T=JCM 34601T).

Noviherbaspirillum aridicola sp. nov., isolated from an arid soil in Pakistan.

Strain NCCP-691T was isolated from a soil sample collected from an arid soil in Karak, Khyber Pakhtunkhwa, Pakistan. Phenotypically, the cells were Gram-stain-negative, aerobic and motile rods. The organism was able to grow between 20-40 °C (optimum at 30-37 °C), at pH 5.5-8.0 (optimum at pH 7.0-7.2) and tolerated 0-1.5% NaCl (w/v) (optimum at 0-0.5). Based on 16S rRNA gene sequences, strain NCCP-691T formed a distinct phylogenetic clade with Noviherbaspirillum arenae, N. agri, N. denitrificans and N. autotrophicum (having sequence similarities of 99.0; 98.1; 98.0 and 97.7% respectively). Phylogenetic analyses based on the whole genome sequences confirmed that strain NCCP-691T should be affiliated to the genus Noviherbaspirillum. The average nucleotide identity values compared to other species of Noviherbaspirillum were below 95-96 % and digital DNA-DNA hybridization values were less than 70 %. Chemotaxonomic analyses showed that the strain had ubiquinone-8, as the only respiratory quinine. The major cellular fatty acids were summed feature 3 (C16 : 1 ω 7 c/C16 : 1 ω 6 c, 35.9 %), summed feature 8 (C18 : 1 ω 7 c/C18 : 1 ω 6 c, 26.9 %) and C16 : 0 (22.9 %) and the polar lipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The genomic DNA G+C content was 65.5 mol% (from draft genome). Genome analyses showed that strain NCCP-691T had terpene and arylpolyene biosynthetic genes clusters and genes related to resistance against heavy metals. Based on phylogenetic analyses, phenotypic features and genomic comparison, it is proposed that strain NCCP-691T is a novel species of the genus Noviherbaspirillum and the name Noviherbaspirillum aridicola sp. nov. is proposed. Type strain is NCCP-691T (=KCTC 52721T=CGMCC 1.13600T).

Comparative Genomics Reveals Insights into Induction of Violacein Biosynthesis and Adaptive Evolution in Janthinobacterium.

Violacein has different bioactive properties conferring distinct selective advantages, such as defense from predation and interspecific competition. Adaptation of Janthinobacterium to diverse habitats likely leads to variation in violacein production among phylogenetically closely related species inhabiting different environments, yet genomic mechanisms and the influence of adaptive evolution underpinning violacein biosynthesis in Janthinobacterium are not clear. In this study, we performed genome sequencing, comparative genomic analysis, and phenotypic characterization to investigate genomic factors regulating violacein production in nine Janthinobacterium strains, including a type strain from soil and eight strains we isolated from terrestrial subsurface sediment and groundwater. Results show that although all nine Janthinobacterium strains are phylogenetically closely related and contain genes essential for violacein biosynthesis, they vary in carbon usage and violacein production. Sediment and groundwater strains are weak violacein producers and possess far fewer secondary metabolite biosynthesis genes, indicating genome adaptation compared to soil strains. Further examination suggests that quorum sensing (QS) may play an important role in regulating violacein in Janthinobacterium: the strains exhibiting strong potential in violacein production possess both N-acyl-homoserine lactone (AHL) QS and Janthinobacterium QS (JQS) systems in their genomes, while weaker violacein-producing strains harbor only the JQS system. Preliminary tests of spent media of two Janthinobacterium strains possessing both AHL QS and JQS systems support the potential role of AHLs in inducing violacein production in Janthinobacterium. Overall, results from this study reveal potential genomic mechanisms involved in violacein biosynthesis in Janthinobacterium and provide insights into evolution of Janthinobacterium for adaptation to oligotrophic terrestrial subsurface environment. IMPORTANCE Phylogenetically closely related bacteria can thrive in diverse environmental habitats due to adaptive evolution. Genomic changes resulting from adaptive evolution lead to variations in cellular function, metabolism, and secondary metabolite biosynthesis. The most well-known secondary metabolite produced by Janthinobacterium is the purple-violet pigment violacein. To date, the mechanisms of induction of violacein biosynthesis in Janthinobacterium is not clear. Comparative genome analysis of closely related Janthinobacterium strains isolated from different environmental habitats not only reveals potential mechanisms involved in induction of violacein production by Janthinobacterium but also provides insights into the survival strategy of Janthinobacterium for adaptation to oligotrophic terrestrial subsurface environment.

Collimonas silvisoli sp. nov. and Collimonas humicola sp. nov., two novel species isolated from forest soil.

Three aerobic, Gram-stain-negative, non-motile and rod-shaped bacteria, designated strains RXD178T, RXD172-2 and RLT1W51T, were isolated from two forest soil samples of Nanling National Nature Reserve in Guangdong Province, PR China. Phylogenetic analyses based on 16S rRNA gene sequences and 92 core genes showed that they belonged to the genus Collimonas, and were most closely related to four validly published species with similarities ranging from 99.4 to 98.2 %. The genomic DNA G+C contents of strains RXD178T, RXD172-2 and RLT1W51T were 57.1, 59.5 and 59.4 mol%, respectively. The genome-derived average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the novel strains and closely related type species were below 37.90 and 89.34 %, respectively. Meanwhile, the ANI and dDDH values between strains RXD172-2 and RLT1W51T were 98.27 and 83.50 %, respectively. The three novel strains contained C16 : 0, C17 : 0 cyclo and summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) as the major fatty acids, and summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) comprised a relative higher proportion in strain RXD178T than in other strains. Both strains RXD172-2 and RLT1W51T had phosphatidylglycerol (PG), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG) and an unidentified aminophospholipid (APL) as the main polar lipids while only PE and APL were detected in strain RXD178T. Ubiquinone 8 was the predominant quinone. Based on the phenotypic, chemotaxonomic, phylogenetic and genomic analyses, strain RXD178T should be considered as representing one novel species within the genus Collimonas and strains RXD172-2 and RLT1W51T as another one, for which the names Collimonas silvisoli sp. nov. and Collimonas humicola sp. nov. are proposed, with RXD178T (=GDMCC 1.1925T=KACC 21987T) and RLT1W51T (=GDMCC 1.1923T=KACC 21985T) as the type strains, respectively.

Undibacterium baiyunense sp. nov., Undibacterium curvum sp. nov., Undibacterium fentianense sp. nov., Undibacterium flavidum sp. nov., Undibacterium griseum sp. nov., Undibacterium hunanense sp. nov., Undibacterium luofuense sp. nov., Undibacterium nitidum sp. nov., Undibacterium rivi sp. nov., Undibacterium rugosum sp. nov. and Undibacterium umbellatum sp. nov., isolated from streams in China.

Twelve Gram-stain-negative, catalase- and oxidase-positive, rod-shaped and motile strains (CY7WT, CY18WT, CY22WT, FT31WT, FT137WT, FT147WT, BYS50W, BYS107WT, LFS511WT, LX15WT, LX22WT and NL8WT) were isolated from streams in China. Comparisons based on 16S rRNA gene sequences indicated that these strains take species of genus Undibacterium as close neighbours. The reconstructed phylogenetic and phylogenomic trees also showed that these strains cluster with species of genus Undibacterium together. The genome G+C contents of these strains were in the range of 45.3 to 53.3 mol%. The calculated pairwise OrthoANIu values and digital DNA-DNA hybridization values among these strains and related strains were in the range of 70.4 to 94.1% and 19.3 to 55.3% except that the values between strains CY7WT and BYS50W were 99.0 and 91.8 %, respectively. Q-8 was their predominant respiratory quinone. C16 : 1 ω7c and C16 : 0 were their major fatty acids. Their polar lipids profiles were similar, including phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and two kinds of unidentified aminolipids. Combining polyphasic taxonomic characteristics and phylogenetic relationships, twelve strains should represent eleven independent novel species of genus Undibacterium, for which the names Undibacterium baiyunense sp. nov. (type strain BYS107WT=GDMCC 1.2453T=KCTC 82653T), Undibacterium curvum sp. nov. (type strain CY22WT=GDMCC 1.1906T=KACC 21951T), Undibacterium fentianense sp. nov. (type strain FT137WT=GDMCC 1.2456T=KCTC 82656T), Undibacterium flavidum sp. nov. (type strain LX15WT=GDMCC 1.1910T=JCM 34286T), Undibacterium griseum sp. nov. (type strain FT31WT=GDMCC 1.1908T=KACC 21953T), Undibacterium hunanense sp. nov. (type strain CY18WT=GDMCC 1.1904T=KACC 21949T), Undibacterium luofuense sp. nov. (type strain LFS511WT=GDMCC 1.2458T=KCTC 82658T), Undibacterium nitidum sp. nov. (type strain LX22WT=GDMCC 1.1912T=KACC 21957T), Undibacterium rivi sp. nov. (type strain FT147WT=GDMCC 1.2457T=KCTC 82657T), Undibacterium rugosum sp. nov. (type strain CY7WT=GDMCC 1.1903T=KACC 21961T) and Undibacterium umbellatum sp. nov. (type strain NL8WT=GDMCC 1.1915T=KACC 21960T) are proposed.

Molecular signatures of Janthinobacterium lividum from Trinidad support high potential for crude oil metabolism.

BACKGROUND: Janthinobacterium lividum is considered to be a psychrotrophic bacterial species. For the first time in the literature, J. lividum strains were isolated from Trinidad presenting with atypical features - hydrocarbonoclastic and able to survive in a tropical environment. METHODS: Identification of the Trinidad strains was carried out through 16S rRNA phylogenetic analysis. Gene-specific primers were designed to target the VioA which encodes violacein pigment and the EstA/B gene which encodes secreted extracellular lipase. Bioinformatics analyses were carried out on the nucleotide and amino acid sequences of VioA and EstA/B genes of the Trinidad Janthinobacterium strains to assess functionality and phylogenetic relatedness to other Janthinobacterium sequences specifically and more broadly, to other members of the Oxalobacteraceae family of betaproteobacteria. RESULTS: 16S rRNA confirmed the identity of the Trinidad strains as J. lividum and resolved three of the Trinidad strains at the intra-specific level. Typical motility patterns of this species were recorded. VioAp sequences were highly conserved, however, synonymous substitutions located outside of the critical sites for enzyme function were detected for the Trinidad strains. Comparisons with PDB 6g2p model from aa231 to aa406 further indicated no functional disruption of the VioA gene of the Trinidad strains. Phylogeny of the VioA protein sequences inferred placement of all J. lividum taxa into a highly supported species-specific clade (bs = 98%). EstA/Bp sequences were highly conserved, however, synonymous substitutions were detected that were unique to the Trinidad strains. Phylogenetic inference positioned the Trinidad consensus VioA and EstA protein sequences in a clearly distinct branch. CONCLUSIONS: The findings showed that the primary sequence of VioAp and EstA/Bp were unique to the Trinidad strains and these molecular signatures were reflected in phylogenetic inference. Our results supported chemotaxis, possible elective inactivation of VioA gene expression and secreted lipase activity as survival mechanisms of the Trinidad strains in petrogenic conditions.

Massilia puerhi sp. nov., isolated from soil of Pu-erh tea cellar.

A Gram-reaction-negative, yellow-pigmented, non-spore-forming rod, aerobic, motile bacterium, designated SJY3T, was isolated from soil samples collected from a Pu-erh tea cellar in Bolian Pu-erh tea estate Co. Ltd. in Pu'er city, Yunnan, south-west China. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belonged to the genus Massilia. The closest phylogenetic relative was Massilia arenae CICC 24458T (99.5 %), followed by M. timonae CCUG45783T (97.9 %), M. oculi CCUG43427AT (97.8 %), and M. aurea DSM 18055T (97.8 %). The major fatty acids were C16 : 0 and C16 : 1 ω7c and/or C16 : 1 ω6c. The major respiratory quinone was ubiquinone Q-8 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Genome sequencing revealed a genome size of 5.97 M bp and a G+C content of 65.4 mol%. Pairwise determined whole genome average nucleotide identity (gANI) values and digital DNA-DNA hybridization (dDDH) values were all below the threshold. Although the 16S rRNA gene similarity of stain SJY3T and Massilia arenae CICC 24458T was more than 99 %, the gANI, dDDH values and genomic tree clearly indicated that they were not of the same species. In summary, strain SJY3T represents a new species, for which we propose the name Massilia puerhi sp. nov. with the type strain SJY3T (=CGMCC 1.17158T=KCTC 82193T).

Massilia rhizosphaerae sp. nov., a rice-associated rhizobacterium with antibacterial activity against Ralstonia solanacearum.

A novel rhizobacterium, designated strain NEAU-GH312T, with antibacterial activity against Ralstonia solanacearum was isolated from rhizosphere soil of rice (Heilongjiang Province, PR China) and characterized with a polyphasic approach. Cells of strain NEAU-GH312T were Gram-stain-negative, aerobic, non-spore-forming, motile with peritrichous flagella and rod-shaped. Colonies were light orange, convex and semi-translucent on Reasoner's 2A (R2A) agar after 2 days of incubation at 28 °C. Growth was observed on R2A agar at 10-40 °C, pH 4.0-8.0 and with 0-5 % (w/v) NaCl. The respiratory quinone was ubiquinone Q-8. The major cellular fatty acids of strain NEAU-GH312T were C16 : 1 ω7c and/or C16 : 1 ω6c, C16 : 0 and C18 : 1 ω7c and/or C18 : 1 ω6c. The main polar lipids were phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Phylogenetic analyses confirmed the well-supported affiliation of strain NEAU-GH312T within the genus Massilia, close to the type strains of Massilia arvi THG-RS2OT (98.7 %), Massilia norwichensis NS9T (98.7 %) and Massilia kyonggiensis TSA1T (98.6 %). Strain NEAU-GH312T had a genome size of 6.68 Mb and an average DNA G+C content of 66.3 mol%. Based on the genotypic, phenotypic and chemotaxonomic data obtained in this study, strain NEAU-GH312T could be classified as representative of a novel species of the genus Massilia, for which the name Massilia rhizosphaerae sp. nov. is proposed, with strain NEAU-GH312T (=DSM 109722T=CCTCC AB 2019142T) as the type strain.

Identification and Characterization of a Novel, Cold-Adapted d-Xylobiose- and d-Xylose-Releasing Endo-ß-1,4-xylanase from an Antarctic Soil Bacterium, Duganella sp. PAMC 27433.

Endo-ß-1,4-xylanase is a key enzyme in the degradation of ß-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-ß-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-ß-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg2+, Ca2+, or Cu2+ but also significantly suppressed by 1 mM Ni2+, Zn2+, or Fe2+. However, its enzyme activity was upregulated (>1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-ß-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-ß-1,4-xylanase.

Massilia horti sp. nov. and Noviherbaspirillum arenae sp. nov., two novel soil bacteria of the Oxalobacteraceae.

We isolated two new soil bacteria: ONC3T (from garden soil in NC, USA; LMG 31738T=NRRL B-65553T) and M1T (from farmed soil in MI, USA; NRRL B-65551T=ATCC TSD-197T=LMG 31739T) and characterized their metabolic phenotype based on Biolog, MALDI-TOF MS and fatty acid analyses, and compared 16S rRNA and whole genome sequences to other members of the Oxalobacteraceae after sequencing on an Illumina Nextera platform. Based on the results of 16S rRNA sequence analysis, ONC3T shows the highest sequence similarity to Massilia solisilvae J18T (97.8 %), Massilia terrae J11T (97.7 %) and Massilia agilis J9T (97.3 %). Strain M1T is most closely related to Noviherbaspirillum denitrificans TSA40T, Noviherbaspirillum agri K-1-15T and Noviherbaspirillum autotrophicum TSA66T (sequence identity of 98.2, 98.0 and 97.8 %, respectively). The whole genome of ONC3T has an assembled size of 5.62 Mbp, a G+C content of 63.8 mol% and contains 5104 protein-coding sequences, 56 tRNA genes and two rRNA operons. The genome of M1T has a length of 4.71 MBp, a G+C content of 63.81 mol% and includes 4967 protein-coding genes, two rRNA operons and 44 tRNA genes. Whole genome comparisons identified Massilia sp. WG5 with a 79.3 % average nucleotide identity (ANI) and 22.6 % digital DNA-DNA hybridization (dDDH), and Massilia sp. UBA11196 with 78.2 % average amino acid identity (AAI) as the most closely related species to ONC3T. M1T is most closely related to N. autotrophicum TSA66T with an ANI of 80.27 %, or N. denitrificans TSA40T with a dDDH of 22.3 %. The application of community-accepted standards such as <98.7 % in 16S sequence similarity and <95-96 % ANI or 70 % DDH support the classification of Massilia horti ONC3T and Noviherbaspirillum arenae M1T as novel species within the Oxalobacteraceae.

Undibacterium crateris sp. nov., isolated from water of crater lake.

A Gram-stain-negative, aerobic, flagellated, non-spore-forming, rod-shaped bacterium, named B2R-29T, was isolated from water collected from a crater lake on Da Hinggan mountain, PR China. Strain B2R-29T was oxidase- and catalase-positive. On the basis of the results of 16S rRNA gene sequence analyses, strain B2R-29T clearly belonged to the family Oxalobacteraceae, class Betaproteobacteria and showed the highest similarity to Undibacterium oligocarboniphilum EM1T (97.4 %) and to the other species of Undibacterium (less than 96.8 %). In the phylogenetic tree, strain B2R-29T formed a clade with U. oligocarboniphilum EM1T and Undibacterium squillarum CMJ-9T, indicating that is a member of the genus Undibacterium. Digital DNA-DNA hybridization and average nucleotide identity analyses were performed and the values between strain B2R-29T and its closely related Undibacterium species were less than 75.1 % and 16.9 %, respectively. The chemotaxonomic data of B2R-29T were as follows: major uniquinone, Q-8; predominant polar lipids, phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol; major fatty acids, C16 : 0 and summed feature 3 (C16 : 1 ω7c / C16 : 1 ω6c); predominant polyamines, putrescine, 2-hydroxyputrescine and spermidine. The DNA G+C content was 51.7 mol% from the genomic sequencing data. In accordance with the phenotypic, physiological and chemotaxonomic properties mentioned above, strain B2R-29T represents a novel species of the genus Undibacterium for which the name Undibacterium crateris sp. nov. is proposed. The type strain is B2R-29T (=CGMCC 1.13792T=KCTC 72018T).

Natural Bacterial Assemblages in Arabidopsis thaliana Tissues Become More Distinguishable and Diverse during Host Development.

To study the spatial and temporal dynamics of bacterial colonization under field conditions, we planted and sampled Arabidopsis thaliana during 2 years at two Michigan sites and surveyed colonists by sequencing 16S rRNA gene amplicons. Mosaic and dynamic assemblages revealed the plant as a patchwork of tissue habitats that differentiated with age. Although assemblages primarily varied between roots and shoots, amplicon sequence variants (ASVs) also differentiated phyllosphere tissues. Increasing assemblage diversity indicated that variants dispersed more widely over time, decreasing the importance of stochastic variation in early colonization relative to tissue differences. As tissues underwent developmental transitions, the root and phyllosphere assemblages became more distinct. This pattern was driven by common variants rather than those restricted to a particular tissue or transiently present at one developmental stage. Patterns also depended critically on fine phylogenetic resolution: when ASVs were grouped at coarse taxonomic levels, their associations with host tissue and age weakened. Thus, the observed spatial and temporal variation in colonization depended upon bacterial traits that were not broadly shared at the family level. Some colonists were consistently more successful at entering specific tissues, as evidenced by their repeatable spatial prevalence distributions across sites and years. However, these variants did not overtake plant assemblages, which instead became more even over time. Together, these results suggested that the increasing effect of tissue type was related to colonization bottlenecks for specific ASVs rather than to their ability to dominate other colonists once established.IMPORTANCE Developing synthetic microbial communities that can increase plant yield or deter pathogens requires basic research on several fronts, including the efficiency with which microbes colonize plant tissues, how plant genes shape the microbiome, and the microbe-microbe interactions involved in community assembly. Findings on each of these fronts depend upon the spatial and temporal scales at which plant microbiomes are surveyed. In our study, phyllosphere tissues housed increasingly distinct microbial assemblages as plants aged, indicating that plants can be considered collections of tissue habitats in which microbial colonists-natural or synthetic-are established with differing success. Relationships between host genes and community diversity might vary depending on when samples are collected, given that assemblages grew more diverse as plants aged. Both spatial and temporal trends weakened when colonists were grouped by family, suggesting that functional rather than taxonomic profiling will be necessary to understand the basis for differences in colonization success.

Duganella callida sp. nov., a novel addition to the Duganella genus, isolated from the soil of a cultivated maize field.

A Gram-negative, rod-shaped bacterium, strain Duganella callida DN04T, was isolated from the soil of a maize field in North Carolina, USA. Based on the 16S rRNA gene sequence, the most similar Duganella species are D. sacchari Sac-22T, D. ginsengisoli DCY83T, and D. radicis Sac-41T with a 97.8, 97.6, or 96.9 % sequence similarity, respectively. We compared the biochemical phenotype of DN04T to D. sacchari Sac-22T and D. zoogloeoides 115T and other reference strains from different genera within the Oxalobacteraceae and while the biochemical profile of DN04T is most similar to D. sacchari Sac-22T and other Duganella and Massilia strains, there are also distinct differences. DN04T can for example utilize turanose, N-acetyl-d-glucosamine, inosine, and l-pyroglutamic acid. The four fatty acids found in the highest percentages were C15 : 0 iso (24.6 %), C15 : 1 isoG (19.4 %), C17 : 0 iso3-OH (16.8 %), and summed feature 3 (C16:1 ⍵7c and/or C16:1 ⍵6c) (12.5 %). We also applied whole genome sequencing to determine if DN04T is a novel species. The most similar AAI (average amino acid identity) score was 70.8 % (Massilia plicata NZ CP038026T), and the most similar ANI (average nucleotide identity) score was 84.8 % (D. radicis KCTC 22382T), which indicates that DN04T is a novel species. The genome-to-genome-distance calculation (GGDC) revealed a DDH of 28.3 % to D. radicis KCTC 22382T, which is much lower than the new species threshold. Based on the morphological, phenotypic, and genomic differences, we propose Duganella callida sp. nov. as a novel species within the Duganella genus (type strain DN04T=NRRL B-65552T=LMG 31736T).

Noviherbaspirillum aerium sp. nov., isolated from air.

A Gram-stain-negative, strictly aerobic, non-spore-forming, rod-shaped, motile with polar flagella and pale-orange bacterium, designated strain 122213-3T, was isolated from air, collected at the foot of the Xiangshan Mountain, located in Beijing, PR China. Optimal growth occurred at 28 °C, at pH 7 and in the presence of 0-1 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that 122213-3T clustered with species of the genus Noviherbaspirillum and formed a distinct sublineage, showing highest similarities to Noviherbaspirillum malthae CC-AFH3T (96.88 %), Noviherbaspirillum massiliense JC206T (95.78 %) and Noviherbaspirillum aurantiacum SUEMI08T (95.78 %). The predominant cellular fatty acids were summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and C16 : 0. The predominant quinone was ubiquinone 8 (Q-8). The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol, unidentified phospholipid and two unidentified polar lipids. The polyamine pattern showed the presence of putrescine as the major polyamine, with minor amounts of 2-hydroxyputrescine. The DNA G+C content was 60.1 mol%. The phylogenetic analysis and physiological and biochemical data showed that strain 122213-3T should be classified as representing a novel species in the genus Noviherbaspirillum, for which the name Noviherbaspirillum aerium sp. nov. is proposed. The type strain of N. aerium is 122213-3T (=CFCC 14286T=LMG 30131T).

Description of Massilia rubra sp. nov., Massilia aquatica sp. nov., Massilia mucilaginosa sp. nov., Massilia frigida sp. nov., and one Massilia genomospecies isolated from Antarctic streams, lakes and regoliths.

Bacteria of the genus Massilia often colonize extreme ecosystems, however, a detailed study of the massilias from the Antarctic environment has not yet been performed. Here, sixty-four Gram-stain-negative, aerobic, motile rods isolated from different environmental samples on James Ross Island (Antarctica) were subjected to a polyphasic taxonomic study. The psychrophilic isolates exhibited slowly growing, moderately slimy colonies revealing bold pink-red pigmentation on R2A agar. The set of strains exhibited the highest 16S rRNA gene sequence similarities (99.5-99.9%) to Massilia violaceinigra B2T and Massilia atriviolacea SODT and formed several phylogenetic groups based on the analysis of gyrB and lepA genes. Phenotypic characteristics allowed four of them to be distinguished from each other and from their closest relatives. Compared to the nearest phylogenetic neighbours the set of six genome-sequenced representatives exhibited considerable phylogenetic distance at the whole-genome level. Bioinformatic analysis of the genomic sequences revealed a high number of putative genes involved in oxidative stress response, heavy-metal resistance, bacteriocin production, the presence of putative genes involved in nitrogen metabolism and auxin biosynthesis. The identification of putative genes encoding aromatic dioxygenases suggests the biotechnology potential of the strains. Based on these results four novel species and one genomospecies of the genus Massilia are described and named Massilia rubra sp. nov. (P3094T=CCM 8692T=LMG 31213T), Massilia aquatica sp. nov. (P3165T=CCM 8693T=LMG 31211T), Massilia mucilaginosa sp. nov. (P5902T=CCM 8733T=LMG 31210T), and Massilia frigida sp. nov. (P5534T=CCM 8695T=LMG 31212T).

Duganella lactea sp. nov., Duganella guangzhouensis sp. nov., Duganella flavida sp. nov. and Massilia rivuli sp. nov., isolated from a subtropical stream in PR China and proposal to reclassify Duganella ginsengisoli as Massilia ginsengisoli comb. nov.

Five Gram-stain-negative, catalase- and oxidase-positive, rod-shaped and motile strains (FT50WT, FT80WT, FT92WT, FT94W and FT135WT) were isolated from a subtropical stream in PR China. Comparisons based on 16S rRNA gene sequences showed that strains FT50WT, FT94W and FT135WT take strain Duganella sacchari Sac-22T, and strains FT80WT and FT92WT take strain Duganella ginsengisoli DCY83T as their closest neighbour in the phylogenetic trees, respectively. The G+C contents of strains FT50WT, FT80WT, FT92WT, FT94W and FT135WT were 63.3, 62.4, 62.8, 63.8 and 60.8 %, respectively. The reconstructed phylogenomic tree based on concatenated 92 core genes showed that strains FT50WT, FT80WT, FT94W and FT135WT clustered together with species of the genus Duganella, but strains FT92WT and D. ginsengisoli KCTC 42409T were located in the clades of the genus Massilia. The calculated pairwise average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among strains FT50WT, FT80WT, FT92WT, FT94W, FT135WT and related strains were in the ranges of 75.6-87.8% and 20.3-33.8% except that the values between strains FT50WT and FT94W were 98.7 and 89.2%, respectively. The respiratory quinone of these five strains was Q-8. The major fatty acids were C16 : 1 ω7c, C16 : 0, C18 : 1 ω7c and C12 : 0. The polar lipids included phosphatidylethanolamine, phosphatidylglycerol and one unidentified phospholipid. Considering the distinct phylogenetic relationships of D. ginsengisoli with species of the genus Massilia in the phylogenomic tree, it was reasonable to transfer D. ginsengisoli to the genus Massilia as Massilia ginsengisoli comb. nov. Combining the results of phylogenomic analysis, ANI and dDDH data, and a range of physiological and biochemical characteristics together, strains FT50WT and FT94W should belong to the same species and be assigned to genus Duganella with strains FT80WT and FT135WT together, and strain FT92WT should be assigned to the genus Massilia, for which the names Duganella lactea sp. nov. (type strain FT50WT=GDMCC 1.1674T=KACC 21466T), Duganella guangzhouensis sp. nov. (FT80WT=GDMCC 1.1678T=KACC 21470T), Duganella flavida sp. nov. (FT135WT=GDMCC 1.1745T=KACC 21659T) and Massilia rivuli sp. nov. (FT92WT=GDMCC 1.1682T=KACC 21474T) are proposed.

Massilia arenosa sp. nov., isolated from the soil of a cultivated maize field.

Strain MC02T, a Gram-stain-negative, rod-shaped bacterium, was isolated from field soil collected from California, USA. To examine if MC02T represents a novel species, we compared its colony morphology, 16S rRNA gene and whole genome sequence, and its metabolic phenotype using Biolog GenIII and MALDI-TOF analyses compared to reference strains. Based on 16S rRNA gene and whole genome sequencing, MC02T belongs to the genus Massilia and Massilia agri K-3-1T is the most similar strain with 96.97 % 16S rRNA gene sequence identity. MALDI-TOF analysis revealed that Massilia aerilata DSM19289T is the closest match, but the similarity score was much lower than the ≥1.7 threshold for a reliable identification at the genus level. The predominant fatty acids were summed feature 3 (C16 : 1⍵7c and/or C16 : 1⍵6c; 49.07 %) and C16 : 0 (30.01 %). The genome is 5.02 Mbp and the G+C content is 66.2 mol%. Whole genome comparisons to the closest related strains revealed an average amino acid identity value of 67.4 %, an OrthoANI similarity of 77.1 %, and a DNA-DNA-hybridization probability ≥70 %, confirming that MC02T represents a novel species. Strain MC02T can grow at pH 6 but not at pH 5, and a salt concentration of ≥1 % inhibits its growth. In contrast to other Massilia strains, MC02T can utilize turanose, inosine and l-serine. The genome of MC02T shows putative endophyte genes such as a nitrate reductase, several phosphatases, and biotin biosynthesis genes, 26 flagellar motility genes and 14 invasion and intracellular resistance genes. Based on its metabolic, physiological and genomic characteristics, we propose that strain MC02T (NRRL B-65554T=ATCC TSD-200T=LMG 31737T) represents a novel species of the genus Massilia with the name Massilia arenosa sp. nov.

Massilia arenae sp. nov., isolated from sand soil in the Qinghai-Tibetan Plateau.

A bacterial strain, designated GEM5T, was isolated from sand soil samples from the Qinghai-Tibet Plateau. The polyphasic study confirmed the affiliation of the isolate with the genus Massilia. GEM5T had Gram-stain-negative, non-spore-forming and rod-shaped cells and grew at 4-30 °C, pH 6-8 and with 0-2 % (w/v) NaCl. Its cell wall contained ribose. Q8 was the predominant respiratory quinone, and summed feature 3 (C16 : 1ω6c/ω7c) and C16 : 0 were the major components of the fatty acids. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unidentified phospholipid, an unidentified aminolipid and four unidentified lipids. The DNA G+C content was 65.1 mol%. The phylogenetic analysis based on the 16S rRNA gene showed a stable clade being formed by GEM5T, Massilia timonae CCUG 45783T (97.94 %) and Massilia oculi CCUG 43427AT (97.58 %). The average nucleotide identity (ANIb) values between GEM5T and M. timonae CCUG 45783T, M.oculi CCUG 43427AT were 91.3 and 91.7 %, respectively. On the basis of the morphological, physiological and chemotaxonomic pattern, it was proposed that strain GEM5T (=JCM 32744T=CICC 24458T) should be classified as representing a member of the genus Massilia with the name Massilia arenae sp. nov.

Duganella rivus sp. nov., Duganella fentianensis sp. nov., Duganella qianjiadongensis sp. nov. and Massilia guangdongensis sp. nov., isolated from subtropical streams in China and reclassification of all species within genus Pseudoduganella.

Four Gram-stain-negative, catalase-positive, rod-shaped and motile strains (FT55WT, FT93WT, CY13WT and DS3T) were isolated from subtropical streams in China. Comparisons based on 16S rRNA gene sequences indicated that strains FT55WT, FT93WT and CY13WT take strain Pseudoduganella danionis E3/2T, and strain DS3T takes strain Pseudoduganella eburnea 10R5-21T as their closest neighbour, respectively. The genome sizes of strains FT55WT, FT93WT, CY13WT and DS3T were 6.15, 5.10, 5.31 and 5.72 Mbp with G+C contents of 61.7, 60.9, 60.6 and 64.0%, respectively. The reconstructed phylogenomic tree based on concatenated 92 core genes showed that strain FT55WT clusters closely with Duganella radicis KCTC 22382T and Duganella sacchari Sac-22T, strains FT93WT and CY13WT form a distinct clade with P. danionis DSM 103461T and this clade clusters with the clades of genus Duganella together, and strain DS3T forms a distinct clade with P. eburnea JCM 31587T and Pseudoduganella violaceinigra DSM 15887T and this clade clusters closely with the clades of genus Massilia, respectively. The calculated pairwise OrthoANIu values and digital DNA-DNA hybridization (DDH) values among strains FT55WT, FT93WT, CY13WT, DS3T and related strains were in the ranges of 75.6-94.2% and 20.6-56.2%, respectively. Q-8 was the sole respiratory quinone of these four strains. The major fatty acids were C16:1ω7c, C16:0 and C12:0. The polar lipids included phosphatidylglycerol, phosphatidylethanolamine and one unidentified phospholipid. Considering the similar fatty acids and polar lipids profiles of species within genus Pseudoduganella, Massilia and Duganella, there is currently no justification for assigning the species of genus Pseudoduganella into the Massilia and Duganella clades in the phylogenomic tree. It is reasonable to transfer P. violaceinigra and P. eburnea to the genus Massilia as Massilia violaceinigrum comb. nov. and Massilia eburnea comb. nov., and transfer P. danionis to the genus Duganella as Duganella danionis comb. nov. Considering phylogenomic analysis, OrthoANIu data, digital DDH data and a range of physiological and biochemical characteristics, strains FT55WT, FT93WT and CY13WT should be assigned to genus Duganella, and strain DS3T should be classified as a novel species within genus Massilia, for which the names Duganella rivus sp. nov. (type strain FT55WT = GDMCC 1.1675T = KACC 21467T), Duganella fentianensis sp. nov. (type strain FT93WT = GDMCC 1.1683T = KACC 21475T), Duganella qianjiadongensis sp. nov. (type strain CY13WT = GDMCC 1.1669T = KACC 21461T) and Massilia guangdongensis sp. nov. (type strain DS3T = GDMCC 1.1636T = KACC 21312T) are proposed.

Rugamonas aquatica sp. nov. and Rugamonas rivuli sp. nov., isolated from a subtropical stream in PR China.

Two Gram-stain-negative, catalase- and oxidase-positive, rod-shaped, motile strains (FT29WT and FT103WT) were isolated from a subtropical stream in PR China. Comparisons based on 16S rRNA gene sequences showed that strains FT29WT and FT103WT showed 98.7 and 98.6 % 16S rRNA gene sequence similarities to Rugamonas rubra CCM 3730T as their closest neighbour, respectively. The calculated pairwise OrthoANIu values between strain R. rubra CCM 3730T and strains FT29WT and FT103WT were all 81.4 %. The respiratory quinone of strains FT29WT and FT103WT was determined to be Q-8. The major fatty acids were C16 : 1 ω7c, C16 : 0 and C12 : 0. The polar lipids of strain FT103WT included phosphatidylethanolamine, phosphatidylglycerol and one unidentified phospholipid, but strain FT29WT did not contain phosphatidylglycerol. The genome sizes of strains FT29WT and FT103WT were 7.23 and 6.84 Mbp with G+C contents of 63.8 and 63.9 %, respectively. Although the pairwise OrthoANIu value between strains FT29WT and FT103WT was 95.2 % which located in the transition region of species demarcation, the dissimilarities in the aspects of phenotypic, biochemical and genotypic characteristics supported these two strains should belong to the different species within genus Rugamonas, for which the names Rugamonas aquatica sp. nov. (type strain FT29WT=GDMCC 1.1643T=KACC 21316T) and Rugamonas rivuli sp. nov. (type strain FT103WT=GDMCC 1.1685T=KACC 21477T) are proposed.