Priority effects alter microbiome composition and increase abundance of probiotic taxa in treefrog tadpoles.

Host-associated microbial communities, like other ecological communities, may be impacted by the colonization order of taxa through priority effects. Developing embryos and their associated microbiomes are subject to stochasticity during colonization by bacteria. For amphibian embryos, often developing externally in bacteria-rich environments, this stochasticity may be particularly impactful. For example, the amphibian microbiome can mitigate lethal outcomes from disease for their hosts; however, this may depend on microbiome composition. Here, we examined the assembly of the bacterial community in spring peeper (Pseudacris crucifer) embryos and tadpoles. First, we reared embryos from identified mating pairs in either lab or field environments to examine the relative impact of environment and parentage on embryo and tadpole bacterial communities. Second, we experimentally inoculated embryos to determine if priority effects (i) could be used to increase the relative abundance of Janthinobacterium lividum, an amphibian-associated bacteria capable of preventing fungal infection, and (ii) would lead to observed differences in the relative abundances of two closely related bacteria from the genus Pseudomonas. Using 16S rRNA gene amplicon sequencing, we observed differences in community composition based on rearing location and parentage in embryos and tadpoles. In the inoculation experiment, we found that priority inoculation could increase the relative abundance of J. lividum, but did not find that either Pseudomonas isolate was able to prevent colonization by the other when given priority. These results highlight the importance of environmental source pools and parentage in determining microbiome composition, while also providing novel methods for the administration of a known amphibian probiotic. IMPORTANCE: Harnessing the functions of host-associated bacteria is a promising mechanism for managing disease outcomes across different host species. In the case of amphibians, certain frog-associated bacteria can mitigate lethal outcomes of infection by the fungal pathogen Batrachochytrium dendrobatidis. Successful probiotic applications require knowledge of community assembly and an understanding of the ecological mechanisms that structure these symbiotic bacterial communities. In our study, we show the importance of environment and parentage in determining bacterial community composition and that community composition can be influenced by priority effects. Further, we provide support for the use of bacterial priority effects as a mechanism to increase the relative abundance of target probiotic taxa in a developing host. While our results show that priority effects are not universally effective across all host-associated bacteria, our ability to increase the relative abundance of specific probiotic taxa may enhance conservation strategies that rely on captive rearing of endangered vertebrates.

Keguizhuia sedimenti gen. nov., sp. nov., isolated from river sediment represents a novel genus of the family Oxalobacteraceae.

A Gram-stain-negative bacterium, designated as R-40T, was isolated from sediment of the Mulong river in Mianyang city, Sichuan province, PR China. The cells of strain R-40T were aerobic non-motile and formed translucent white colonies on R2A agar. Growth occurred at 15-37 °C (optimum 30 °C), pH 5.0-9.0 (optimum 7.0) and salinities of 0-3.0 % (w/v, optimum 0 %). R-40T showed 95.2-96.6 % 16S rRNA gene sequence similarities with the type strains of species of the genera Oxalicibacterium, Herminiimonas, Lacisediminimonas, Paucimonas, Herbaspirillum and Noviherbaspirillum in the family Oxalobacteraceae. The results of phylogenetic analysis based on genome sequences indicated that the strain was clustered with type strains of species of the genera Oxalicibacterium and Herminiimonas in the family Oxalobacteraceae but formed a distinct lineage. The average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH) and average amino acid identity (AAI) values between R-40T and type strains of species of the genera Oxalicibacterium, Herminiimonas, Lacisediminimonas, Paucimonas, Herbaspirillum and Noviherbaspirillum ranged from 69.3 to 74.1 %, from 18.2 to 21.4 % and from 60.1 to 67.4 %, respectively. The major cellular fatty acids were C16 : 0, C17 : 0 cyclo and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The major quinone was ubiquinone-8 (Q-8). The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phospholipid and small amounts of glycophospholipids. The genome size of R-40T was 5.1 Mbp with 54.0 % DNA G+C content. On the basis of the evidence presented in this study, strain R-40T represents a novel species of a novel genus in the family Oxalobacteraceae, for which the name Keguizhuia sedimenti gen. nov., sp. nov. (type strain R-40T=MCCC 1K08818T=KCTC 8137T) is proposed.

Massilia litorea sp. nov., Marinobacter salinisoli sp. nov. and Rhodobacter xanthinilyticus sp. nov., isolated from coastal environments.

Three bacterial strains, namely LPB0304T, LPB0319T and LPB0142T, were isolated from coastal environments. The 16S rRNA gene sequences of the three isolates were found to show the highest sequence similarities to Massilia litorea (98.44 %), Marinobacter salinisoli (97.55 %) and Rhodobacter lacus (97.60 %), respectively. The low (<98.7 %) sequence similarities and tree topologies implied the novelty of the three isolates, representing novel genomic species of the genus Massilia, Marinobacter and Rhodobacter. Numerous biochemical and physiological features also supported the distinctiveness of the isolates from previously known species. Based on the phenotypic and phylogenetic data presented in this study, three novel species are suggested with the following names: Massilia litorea sp. nov. (LPB0304T=KACC 21523T=ATCC TSD-216T), Marinobacter salinisoli sp. nov. (LPB0319T=KACC 21522T=ATCC TSD-218T) and Rhodobacter xanthinilyticus sp. nov. (LPB0142T=KACC 18892T=JCM 31567T).

Bosea beijingensis sp. nov., Telluria beijingensis sp. nov. and Agrococcus beijingensis sp. nov., isolated from baijiu mash.

The baijiu fermentation environment hosts a variety of micro-organisms, some of which still remain uncultured and uncharacterized. In this study, the isolation, cultivation and characterization of three novel aerobic bacterial strains are described. The cells of strain REN20T were Gram-negative, strictly aerobic, motile and grew at 26-37 °C, at pH 6.0-9.0 and in the presence of 0-5.0   % (w/v) NaCl. The cells of strain REN29T were Gram-negative, strictly aerobic, motile and grew at 15-30 °C, at pH 6.0-9.0 and in the presence of 0-10.0   % (w/v) NaCl. The cells of strain REN33T were Gram-positive, strictly aerobic, motile and grew at 15-37 °C, at pH 5.0-10.0 and in the presence of 0-7.0   % (w/v) NaCl. The digital DNA-DNA hybridization and average nucleotide identity by orthology values between type strains in related genera and REN20T (20.3-36.8 % and 79.8-89.9  %), REN29T (20.3-36.8  % and 74.5-88.5  %) and REN33T (22.6-48.6  % and 75.8-84.2  %) were below the standard cut-off criteria for the delineation of bacterial species, respectively. Based on polyphasic taxonomy analysis, we propose three new species, Bosea beijingensis sp. nov. (=REN20T=GDMCC 1.2894T=JCM 35118T), Telluria beijingensis sp. nov. (=REN29T=GDMCC 1.2896T=JCM 35119T) and Agrococcus beijingensis sp. nov. (=REN33T=GDMCC 1.2898T=JCM 35164T), which were recovered during cultivation and isolation from baijiu mash.

Massilia agrisoli sp. nov., isolated from rhizospheric soil of banana.

A Gram-stain-negative, aerobic, short rod-shaped and motile novel bacterial strain, designated MAHUQ-52T, was isolated from the rhizospheric soil of a banana plant. Colonies grew at 10-35 °C (optimum, 28 °C), pH 6.0-9.5 (optimum, pH 7.0-7.5), and in the presence of 0-1.0 % NaCl (optimum 0 %). The strain was positive for catalase and oxidase tests, as well as hydrolysis of gelatin, casein, starch and Tween 20. Based on the results of phylogenetic analysis using 16S rRNA gene and genome sequences, strain MAHUQ-52T clustered together within the genus Massilia. Strain MAHUQ-52T was closely related to Massilia soli R798T (98.6 %) and Massilia polaris RP-1-19T (98.3 %). The novel strain MAHUQ-52T has a draft genome size of 4 677 454 bp (25 contigs), annotated with 4193 protein-coding genes, 64 tRNA and 19 rRNA genes. The genomic DNA G+C content was 63.0 %. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain MAHUQ-52T and closely related type strains were ≤88.4 and 35.8 %, respectively. The only respiratory quinone was ubiquinone-8. The major fatty acids were identified as C16 : 0 and summed feature 3 (C15 : 0 iso 2-OH and/or C16 : 1 ω7c). Strain MAHUQ-52T contained phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol as the major polar lipids. On the basis of dDDH and ANI values, as well as genotypic, chemotaxonomic and physiological data, strain MAHUQ-52T represents a novel species within the genus Massilia, for which the name Massilia agrisoli sp. nov. is proposed, with MAHUQ-52T (=KACC 21999T=CGMCC 1.18577T) as the type strain.

Roseateles albus sp. nov., Roseateles koreensis sp. nov. and Janthinobacterium fluminis sp. nov., isolated from freshwater at Jucheon River, and emended description of Roseateles aquaticus comb. nov.

Three Gram-stain-negative, facultatively anaerobic, rod-shaped, catalase-positive, oxidase-negative bacterial strains were designated as hw1T, hw8T and hw3T. Strains hw1T, hw8T and hw3T grew at 15-28 °C (optimum, 25 °C), 15-35 °C (optimum, 30 °C) and 4-28 °C (optimum, 20 °C), respectively, and at pH 7.0-12.0 (optimum, pH 9.0), pH 6.0-11.0 (optimum, pH 9.0) and 5.0-12.0 (optimum, pH 7.0), respectively. Additionally, strains hw1T and hw8T only grew when the NaCl concentration was 0 %, while strain hw3T grew at between 0 and 0.5 % (w/v; optimum, 0 %). The average nucleotide identity (ANI) values between strains hw1T, hw8T and the Roseateles type strains ranged from 73.8 to 84.2 %, while the digital DNA-DNA hybridization (dDDH) values ranged from 19.7 to 27.5 %. The ANI values between strain hw3T and the Janthinobacterium type strains ranged from 78.7 to 80.7 %, while dDDH values ranged from 22.3 to 23.0 %. The draft genomes of strains hw1T, hw8T and hw3T consisted of 5.5, 4.4 and 5.9 Mbp, with DNA G+C contents of 61.7, 61.8 and 66.0 mol%, respectively. The results of the dDDH, ANI, phylogenetic, biochemical and physiological analyses indicated that the novel strains were distinct from other members of their genera. Thus, we proposed the names Roseateles albus sp. nov. (type strain hw1T= KACC 22887T= TBRC 16613T), Roseateles koreensis sp. nov. (type strain hw8T= KACC 22885T= TBRC 16614T) and Janthinobacterium fluminis sp. nov. (type strain hw3T= KACC 22886T= TBRC 16615T).

The mineral weathering ability of Collimonas pratensis PMB3(1) involves a Malleobactin-mediated iron acquisition system.

Mineral weathering by microorganisms is considered to occur through a succession of mechanisms based on acidification and chelation. While the role of acidification is established, the role of siderophores is difficult to disentangle from the effect of the acidification. We took advantage of the ability of strain Collimonas pratensis PMB3(1) to weather minerals but not to acidify depending on the carbon source to address the role of siderophores in mineral weathering. We identified a single non-ribosomal peptide synthetase (NRPS) responsible for siderophore biosynthesis in the PMB3(1) genome. By combining iron-chelating assays, targeted mutagenesis and chemical analyses (HPLC and LC-ESI-HRMS), we identified the siderophore produced as malleobactin X and how its production depends on the concentration of available iron. Comparison with the genome sequences of other collimonads evidenced that malleobactin production seems to be a relatively conserved functional trait, though some collimonads harboured other siderophore synthesis systems. We also revealed by comparing the wild-type strain and its mutant impaired in the production of malleobactin that the ability to produce this siderophore is essential to allow the dissolution of hematite under non-acidifying conditions. This study represents the first characterization of the siderophore produced by collimonads and its role in mineral weathering.

Functional characterization of the methylarsenite-inducible arsRM operon from Noviherbaspirillum denitrificans HC18.

Microbial arsenic methylation by arsenite (As(III)) S-adenosylmethionine methyltransferases (ArsMs) can produce the intermediate methylarsenite (MAs(III)), which is highly toxic and is used by some microbes as an antibiotic. Other microbes have evolved mechanisms to detoxify MAs(III). In this study, an arsRM operon was identified in the genome of an MAs(III)-methylation strain Noviherbaspirillum denitrificans HC18. The arsM gene (NdarsM) is located downstream of an open reading frame encoding an MAs(III)-responsive transcriptional regulator (NdArsR). The N. denitrificans arsRM genes are co-transcribed whose expression is significantly induced by MAs(III), likely by alleviating the repressive effect of ArsR on arsRM transcription. Both in vivo and in vitro assays showed that NdArsM methylates MAs(III) to dimethyl- and trimethyl-arsenicals but does not methylate As(III). Heterologous expression of NdarsM in arsenic-sensitive Escherichia coli AW3110 conferred resistance to MAs(III) but not As(III). NdArsM has the four conserved cysteine residues present in most ArsMs, but only two of them are essential for MAs(III) methylation. The ability to methylate MAs(III) by enzymes such as NdArsM may be an evolutionary step originated from enzymes capable of methylating As(III). This finding reveals a mechanism employed by microbes such as N. denitrificans HC18 to detoxify MAs(III) by further methylation.

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).

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).

Functional characterization of a novel violacein biosynthesis operon from Janthinobacterium sp. B9-8.

Violacein is a secondary metabolite mainly produced by Gram-negative bacteria that is formed from tryptophan by five enzymes encoded by a single operon. It is a broad-spectrum antibacterial pigment with various important biological activities such as anti-tumor, antiviral, and antioxidative effects. The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8. The key enzyme-encoding genes were cloned to construct the multigene coexpression plasmids pET-vioAB and pRSF-vioCDE. The violacein biosynthesis pathway was heterologously introduced into engineered Escherichia coli VioABCDE and VioABCDE-SD. The factors affecting violacein production, including temperature, pH, inoculum size, carbon and nitrogen source, precursor, and inducers were investigated. The violacein titer of VioABCDE-SD reached 107 mg/L in a two-stage fermentation process, representing a 454.4% increase over the original strain. The violacein operon from B9-8 provides a new microbial gene source for the analysis of the violacein synthesis mechanism, and the constructed engineering E. coli strains lay a foundation for the efficient and rapid synthesis of other natural products.Key points• The newly discovered violacein operon vioABCDE was identified in the genome of the extremophile Janthinobacterium sp. B9-8.• The violacein synthesis pathway was reconstructed in E. coli using two compatible plasmids.• A two-stage fermentation process was optimized for improved violacein accumulation.

Genome Analysis of the Janthinobacterium sp. Strain SLB01 from the Diseased Sponge of the Lubomirskia baicalensis.

The strain Janthinobacterium sp. SLB01 was isolated from the diseased freshwater sponge Lubomirskia baicalensis (Pallas, 1776) and the draft genome was published previously. The aim of this work is to analyze the genome of the Janthinobacterium sp. SLB01 to search for pathogenicity factors for Baikal sponges. We performed genomic analysis to determine virulence factors, comparing the genome of the strain SLB01 with genomes of other related J. lividum strains from the environment. The strain Janthinobacterium sp. SLB01 contained genes encoding violacein, alpha-amylases, phospholipases, chitinases, collagenases, hemolysin, and a type VI secretion system. In addition, the presence of conservative clusters of genes for the biosynthesis of secondary metabolites of tropodithietic acid and marinocine was found. We present genes for antibiotic resistance, including five genes encoding various lactamases and eight genes for penicillin-binding proteins, which are conserved in all analyzed strains. Major differences were found between the Janthinobacterium sp. SLB01 and J. lividum strains in the spectra of genes for glycosyltransferases and glycoside hydrolases, serine hydrolases, and trypsin-like peptidase, as well as some TonB-dependent siderophore receptors. Thus, the study of the analysis of the genome of the strain SLB01 allows us to conclude that the strain may be one of the pathogens of freshwater sponges.

Repeated Exposure of Aspergillus niger Spores to the Antifungal Bacterium Collimonas fungivorans Ter331 Selects for Delayed Spore Germination.

The bacterial strain Collimonas fungivorans Ter331 (CfTer331) inhibits mycelial growth and spore germination in Aspergillus niger N402 (AnN402). The mechanisms underlying this antagonistic bacterial-fungal interaction have been extensively studied, but knowledge on the long-term outcome of this interaction is currently lacking. Here, we used experimental evolution to explore the dynamics of fungal adaptation to recurrent exposure to CfTer331. Specifically, five single-spore isolates (SSIs) of AnN402 were evolved under three selection scenarios in liquid culture, i.e., (i) in the presence of CfTer331 for 80 growth cycles, (ii) in the absence of the bacterium for 80 cycles, and (iii) in the presence of CfTer331 for 40 cycles and then in its absence for 40 cycles. The evolved SSI lineages were then evaluated for phenotypic changes from the founder fungal strain, such as germinability with or without CfTer331. The analysis showed that recurrent exposure to CfTer331 selected for fungal lineages with reduced germinability and slower germination, even in the absence of CfTer331. In contrast, when AnN402 evolved in the absence of the bacteria, lineages with increased germinability and faster germination were favored. SSIs that were first evolved in the presence of CfTer331 and then in its absence showed intermediate phenotypes but overall were more similar to SSIs that evolved in the absence of CfTer331 for 80 cycles. This suggests that traits acquired from exposure to CfTer331 were reversible upon removal of the selection pressure. Overall, our study provides insights into the effects on fungi from the long-term coculture with bacteria. IMPORTANCE The use of antagonistic bacteria for managing fungal diseases is becoming increasingly popular, and thus there is a need to understand the implications of their long-term use against fungi. Most efforts have so far focused on characterizing the antifungal properties and mode of action of the bacterial antagonists, but the possible outcomes of the persisting interaction between antagonistic bacteria and fungi are not well understood. In this study, we used experimental evolution in order to explore the evolutionary aspects of an antagonistic bacterial-fungal interaction, using the antifungal bacterium Collimonas fungivorans and the fungus Aspergillus niger as a model system. We show that evolution in the presence or absence of the bacteria selects for fungal lineages with opposing and conditionally beneficial traits, such as slow and fast spore germination, respectively. Overall, our studies reveal that fungal responses to biotic factors related to antagonism could be to some extent predictable and reversible.

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.

Duganella aceris sp. nov., isolated from tree sap and proposal to transfer of Rugamonas aquatica and Rugamonas rivuli to the genus Duganella as Duganella aquatica comb. nov., with the emended description of the genus Rugamonas.

A Gram-reaction-negative, strictly aerobic, betaproteobacterial strain, designated SAP-35T, was isolated from sap extracted from Acer pictum in Mt. Halla in Jeju, Republic of Korea, and its taxonomic status was examined by a polyphasic approach. Cells of the organism were non-sporulating, motile rods and grew at 4-30 °C, pH 6-7 and in the absence of NaCl. 16S rRNA gene- and whole genome-based phylogenetic analyses showed that strain SAP-35T belonged to the family Oxalobacteraceae and was closely related to Rugamonas rivuli (98.9% 16S rRNA gene sequence similarity) and Rugamonas aquatica (98.4%). The phylogenomic clustering and average amino acid identity values supported that strain SAP-35T belonged to the genus Duganella and two Rugamonas species should be transferred to the genus Duganella. The major isoprenoid quinone of the isolate was Q-8. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and an unidentified aminophospholipid. The predominant fatty acids were summed feature 3, C16:0 and C17:0 cyclo. The G + C content of genome was 64.9%. The average nucleotide identity and dDDH values between strain SAP-35T and the members of the genera Rugamonas and Duganella were < 85.1% and < 49%, respectively. Based on the combined data presented here, strain SAP-35T (= KCTC 72227T = NBRC 113903T) represents a novel species of the genus Duganella, for which the name Duganella aceris sp. nov. is proposed. Also, Rugamonas aquatica Lu et al. (Int J Syst Evol Microbiol 70: 3328-3334, 2020) and Rugamonas aquatica Lu et al. 2020 are reclassified as Duganella aquatica comb. nov., with the emended description of the genus Rugamonas.

Noviherbaspirillum galbum sp. nov., a bacterium isolated from soil.

A Gram-stain-negative, aerobic, non-motile and yellow-colored bacterium, strain 17J57-3 T, was isolated from soil collected in Pyeongchang city, Korea. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 17J57-3 T formed a distinct lineage within the family Oxalobacteraceae (order Burkholderiales, class Betaproteobacteria). Strain 17J57-3 T was the most closely related to Noviherbaspirillum humi U15T (96.4% 16S rRNA gene sequence similarity) and Noviherbaspirillum massiliense JC206T (96.2%). The draft genome size of strain 17J57-3 T was 6,117,206 bp. Optimal growth occurred at 30 °C, pH 7.0 without NaCl. The predominant cellular fatty acids were summed feature 3 (C16:1 ω6c/C16:1 ω7c) and C16:0. The major respiratory quinone was Q-8. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Biochemical, chemotaxonomic and phylogenetic analyses indicated that strain 17J57-3 T represents a novel bacterial species within the genus Noviherbaspirillum, for which the name Noviherbaspirillum galbum is proposed. The type strain of Noviherbaspirillum galbum is 17J57-3 T (= KCTC 62213 T = NBRC 114384 T).

Noviherbaspirillum pedocola sp. nov., isolated from oil-contaminated experimental soil.

An orange-coloured, rod-shaped, and aerobic bacterial strain DKR-6 T was isolated from oil-contaminated experimental soil. The strain was Gram-stain-negative, catalase and oxidase positive, and grew at temperature 10-42 °C, at pH 5.5-9.5, and at 0-3.0% (w/v) NaCl concentration. The phylogenetic analysis and 16S rRNA gene sequence analysis suggested that the strain DKR-6 T was affiliated to the genus Noviherbaspirillum, with the closest species being Noviherbaspirillum massiliense JC206T (96.3% sequence similarity). The chemotaxonomic profiles revealed the presence of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylcholine as the principal polar lipids; C16:0, C17:0 cyclo, summed feature 3 (C16:1ω7c and/or C16: 1ω6c), and summed feature 8 (C18:1ω7c/or C18:1ω6c) as the main fatty acids; and Q-8 as a sole ubiquinone. The DNA G + C content was 61.6%. The polyphasic taxonomic features illustrated in this study clearly implied that strain DKR-6 T represents a novel species in the genus Noviherbaspirillum, for which the name Noviherbaspirillum pedocola sp. nov. is proposed with the type strain DKR-6 T (= KACC 22074 T = NBRC 114727 T).

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.

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).

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.