Morphology, physiology and biochemical analysis
Cells of the strain KVB23T were facultative anaerobic, Gram-stain-negative, rod-shaped, oxidase-negative, catalase-positive and devoid of flagella. Colonies were white, convex and circular with entire edges. The colour of colonies were white after culture for 5 days and it became slightly brown at the center of the colonies after ten days of incubation at 30℃ (Fig.1). The strain grew in the presence of 0-2% (w/v) NaCl (optimum 0%), at 7-35℃ (optimum 30℃) and at pH 6.5–7.5 (optimum pH 7.0). It did not show photoautotrophic and photoheterotrophic growth under anaerobic conditions. No absorption maxima were detected at 377, 590, 803 and 860 nm confirmig that the strain KVB23T did not contain any photosynthetic pigments. In addition, the genome of strain KVB23T did not have photosynthetic genes. Growth occurred at temperatures ranging from 7-35℃ (optimum, 30℃), at pH 6.0-8.0 (optimum, pH 7.0) and in the presence of 0-03% NaCl (w/v: optimum, 0%). The new isolated was unable to perform anoxygenic photosynthesis or to grow phototrophically under anoxic conditions, and this character can clearly distinguish strain KVB23T from the species of genus Tabrizicola and Rhodobacter.Growth of strain KVB23T was not occured in Jensen’s nitrogen free medium. Occurrence of halo zone around the colonies in phosphate-solubilizing agar showed the solubilizing ability of strain KVB23T (Fig. S1).In the API ZYM system, the strain KVB23T displayed positive results for alkaline phosphatase, esterase, esterase lipase, leucine arylamidase, valine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and α-glucosidase activities however it displayed negative results for lipase, cystine arylamidase, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase activities.In the API 20NE system, the strain did not reduce nitrate and did not produce indole. Moreover, it did not ferment glucose and did not hydrolyse arginine and gelatin. It only assimilated malic acid but did not assimilate D-glucose, L-arabinose, D-mannose, N-Acetyl-D-glucosamine, D-maltose, potassium gluconate, capric acid, adipic acid, trisodium citrate and phenylacetic acid. KVB23T could be differentiated from other closely related members by using several phenotypic and biochemical properties, such as able to grow in low temperature of 7℃, could grow in anaerobic condition and able to hydrolyse esculin. Most importantly, strain KVB23T showed brown pigmentation at the center of the colony after ten days of incubation but the close relatives were not shown the pigmentation.The differentiatingcharacteristics between strain KVB23T and the members of its related genera within the family Rhodobacteriaceae and also the reference strains were presented in Table 1.
Phylogenetic and genome analysis
16S rRNA gene sequence of strain KVB23T indicated that strain was most closely related to Tabrizicola fusiformis SY72T (96.5%), Rhodobacter thermarum YIM 73036T (96.2%) and Tabrizicola alkalilacus DJCT (96.0%). Phylogenetic analysis based on neighbour-joining tree (Fig. 2)further revealed that the novel strain KVB23T, formed a distinct lineage within the family Rhodobacteriaceae, clustering with the clade comprising phototrophic species belonging to the genus Tabrizicola and Rhodobacter, and non-phototrophic species belonging to Falsirhodobacter and Xinfangfangia. Similar results were obtained in both maximum-likelihood (Fig. S2) and maximum parsimony trees (Fig. S3). The topologies of all tree trees were almost same and available in the online version of this article.Phylogenomic tree also gave the similar results (Fig. S4).
The genome size of strain KVB23Twas found to be 3,808,773 bp. The complete genome of strain KVB23T consisted of 3,720 coding genes, 3 rRNA and 46 tRNA genes and 32 pseudogenes. The number of contigs was 21 and N50 value was 446,226. The G + C content of genomic DNA is 63.1 % which is within the range for the members of the family Rhodobacteriaceae. The ANI values between strain KVB23T and the closely related reference strains T. fusiformis KCTC 62105T, R. thermarum KCTC 52712T and F. deserti KCTC 32408T were 78.2, 74.4 and 72.9% and the corresponding dDDH values were 21, 19.2 and 19.4%. These ANI and dDDH values are clearly below the species-delineating thresholds (95% and 70%, respectively) (Chun et al. 2018; Richter and Rossello-Mora 2009; Stackebrandt and Ebers 2006), supporting the conclusion that strain KVB23T represents a novel genus in the family Rhodobacteriaceae. The antiSMASH server revealed eight gene clusters for the biosynthesis of several secondary metabolites; one gene cluster each for redox-cofactor, terpene, Type I polyketide synthase (T1PKS), non-ribosomal peptide synthetase, three gene clusters for hserlactone, and one gene cluster for RRE-element.The presence of three striking hserlactone gene clusters may have potential ecological roles, which may be related to the communication between fungi and bacteria (Shiner et al. 2005).The comparison of biosynthetic gene clusters between strain KVB23T and its reference strains is provided in Table S1. According to RAST annotation, 1455 protein encoding genes in whole genome of strain KVB23T were classified into 27 functional categories (Table 2). Interesting point was all strains have genes for motility (Flagellar) and chemotaxis except F. deserti KCTC 32408T. However, all strains were found to be non-flagellated. Strain KVB23T, also had five gene clusters for motility and chemotaxis however it was not flagellated.The genome of strain KVB23T was compared with those of phylogenetically related species belonging to the family Rhodobacteriaceae. Four gene clusters for auxin biosynthesis were also annotated in the genome of strain KVB23T. The main precursor for the synthesis of IAA is typtophan, four genes encoding for tryptophan biosynthesis were also found: tryptophan-rich sensory protein (JAESVP010000001.1), tryptophan 2, 3-dioxygenase (kynA; JAESVP010000002.1), tryptophan synthase subunitbeta (trpB; JAESVP010000003.1) and tryptophan synthase subunit alpha (JAESVP010000005.1).Cobalamin has been suggested to stimulate plant development and could be synthesized either via de novo or salvage pathways. Five gene clusters for cobalamin biosynthesis were also found in the genome of strain KVB23T. Based on Venn diagrams of protein clusters, Fig S5 showed the number of orthologous clusters shared among strain KVB23T and other closely related members.
The major respiratory quinone of the strain KVB23T was ubiquinone Q-10, which is common in Rhodobacteriaceae family.The polar lipid of strain KVB23T were diphosphatidylglycerol (DPG), phoshatidylethanolamine (PE), seven unidentified phosphoglycolipid (PGL), two unidentified aminophosphoglycolipid (APGL), one unidentified glycolipid (GL) and four unidentified lipids (L) which characteristically differentiated KVB23T from other recognized families (Fig. S6).The fatty acid profiles of strain KVB23T and its closely related members were presented in Table 3. The drastic difference in the major fatty acids also differentiate strain KVB23T from other close relatives. Difference in the percentage of major fatty acid C16:0, C18:0 and summed feature 8 (comprising C18:1 ω7c and / or C18:1 ω6c) distinguish the strain KVB23T from its close relatives. Absence of anteiso C15:0, iso C16:0, presence of C18:1 ω7c and some other qualitative and quantitative differences in the fatty acid composition between the novel strain KVB23T and other members of the family Rhodobacteriaceaecould considered as a distinguishing characteristics for the novel genus.
Strain KVB23T has the ability to solubilize phosphate and the genome of strain KVB23T contains striking genes that may contribute to rice plant growth stimulation and has good application potential in sustainable agriculture.Based on theaforementioned distinct phylogenetic, phenotypic, biochemical, chemotaxonomic and genomic data the novel isolate KVB23T cannot be assigned to any previously recognized bacterial taxa and therefore, we propose that strain KVB23T represents a novel species belonging to a novel genus Fuscibacteroryzaegen. nov., sp. nov., within a novel family, Rhodobacteriaceae fam. Nov.
Description ofFuscibacteroryzaegen. nov.
Fuscibacter gen. nov. (Fus.ci.bac'ter. L. adj. fuscus, brown; L. n. bacter (from Gr. bakterion) a rod; N.L. masc. n. Fuscibacter, a brown rod).
Cells are Gram-stain-negative, facultative anaerobic, catalase- and oxidase-negative, asporogenous, short-rod shaped, non-flagellated and non-motile. Flexirubin-type pigments are absent. They contained Q-10 as the sole respiratory quinone. The main cellular fatty acids are C16:0, C18:0 and summed feature 8 (comprising C18:1 ω7c and / or C18:1 ω6).The polar lipids include diphosphatidylglycerol, phoshatidylethanolamine, seven unidentified phosphoglycolipid, two unidentified aminophosphoglycolipid, one unidentified glycolipid and four unidentified lipids. The DNA G+C content of the type strain of the type species is 63.1%. Based on phylogenetic analysis, the genus belongs to the family Rhodobacteriaceae within the phylum Proteobacteria. The type species is Fuscibacteroryzae.
Description of Fuscibacteroryzaesp. nov
F. oryzaesp. nov. (o.ry'zae. L. fem. n.oryzae, of rice, referring to the isolation of the type strain from the root of a rice plant)
Cells are Gram-stain negative, aerobic, ovoid to rod-shaped, non-motile, asporogenous, 0.4-0.5μm long and 0.8-1.4μm wide after 3 days of culture on R2A. Colonies on R2A agar are white to brown pigmented, smooth, circular, convex and have an entire margin. Cells are non-motile, multiply by binary fissionand negative for catalase and oxidase activities. Growth occurs at 7-35 ˚C (optimum 25-30 ˚C) and pH 6.0-8.0 (optimum 6.5-7.0). Hardly tolerates NaCl (w/v) upto 2% (optimum 0% NaCl). Good growth occurs on R2A agar and NA, weak growth on TSA and no growth on MA and LB. Strain KVBT was able to grow in the absence of oxygen and showed negative activities for catalase and oxidase reaction.Strain KVB23T able to hydrolyze esculin and Tween 20, but not Tween 40 and 60, starch, casein and CM-cellulose. It does not have a vesicular photosynthetic membrane. Moreover, it does not contain bacteriochlorophyll a, carotenoids and flexirubin. Photoautotrophic and photoheterotrophic growth does not occur. Furthermore, photosynthetic pigments are not produced and apparently photosynthetic genes were also not found in the genome of strain KVB23T. Strain KVB23T was able to dissolve phosphate when grown in Pikovakaya’s medium. The predominant respiratory quinone is ubiquinone Q-10 and the G + C content of the genomic DNA of the type strain is 63.1%. The main cellular fatty acids are C16:0, C18:0 and summed feature 8 (comprising C18:1 ω7c and / or C18:1 ω6).The polar lipids include diphosphatidylglycerol, phoshatidylethanolamine, seven unidentified phosphoglycolipid, two unidentified aminophosphoglycolipid, one unidentified glycolipid and four unidentified lipids.
The type strain KVB23T (=KACC 21711T =NBRC 114716T) was isolated from root of rice plant collectedfromrice field near Ilsan, South Korea. The GenBank accession number of the 16S rRNA gene sequence of the strain KVB23T is MN955430 and its draft genome sequence accession number is JAESVP000000000.