16S rRNA gene sequence similarities and& phylogenetic analysis
An almost full-length sequence of the 16S rRNA gene (1484 bp) was determined for strain RG28T, which has been stored in the GenBank databases under the accession number MW386408. The full length of the 16S rRNA gene of strain RG28T& showed the highest sequence similarity to G. acidiceleris CBD 119T& (98.6%), G. solisilvae LMG 18422T (98.5%) and G. luciferensis LMG 18422T (98.4%). Sequence similarities to all other species of the genus Bacillus, Cytobacillus, Neobacillus, Sutcliffiella and Metabacillus were below 95.2%. The NJ phylogenetic analysis revealed that strain RG28T formed a separate cluster with G. acidiceleris CBD 119T, G. solisilvae LMG 18422T and G. luciferensis LMG 18422T with high topology& which was also supported by the trees reconstructed using the ML, ME and MP methods (Fig. 1, Fig S1, Fig S2 and Fig S3). The position of strain RG28T did not vary with the tree reconstruction method used. Reference strains Gottfriedia acidiceleris KEMB 1602-188T, G. luciferensis KEMB 7305-013T and G. solisilvae DSM 100485T obtained from Korean Environmental Microorganisms Bank (KEMB) and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) were used for subsequent comparison.
Genome sequencing, assembly and annotation&
The draft genome of strain RG28T was 4,081,222 bp long with a G+C content of 33.5 mol% and consisted of 34 contigs. The N50 length was 362,561 bp. A total of 3,940 genes were predicted with 3,802 protein-coding genes and 15 RNAs (seven 5S rRNAs, four 16S rRNAs, four 23S rRNAs), 73 tRNAs and three ncRNAs. The ANI value between strain RG28T and& G. acidiceleris KEMB 1602-188T, G. solisilvae DSM 100485T and G. luciferensis KEMB 7305-013T were 74.7, 74.8 and 74.9%, respectively, clearly below the recommended cut-off value of 95–96% for species identification (Goris et al. 2007). The estimated dDDH value between strain RG28T and& G. acidiceleris KEMB 1602-188T, G. solisilvae DSM 100485T and G. luciferensis KEMB 7305-013T were 21.6, 21.8 and 21.9%, respectively, which was well below the 70% threshold described by Chun et al. (2018). These results indicated that strain RG28T represents a novel species of the genus& Gottfriedia.& The overall comparison analysis result is displayed as Venn diagram in Fig. 2. A total of 2299 orthologous genes were shared among all the compared species, of which 79 were shared only between strain RG28T and& G. acidiceleris KEMB 1602-188T, 77 between strain RG28T and& G. solisilvae DSM 100485T and 71 between strain RG28T and& G. luciferensis KEMB 7305-013T.& According to the genome-based phylogeny, strain RG28T formed a clade with& G. acidiceleris KEMB 1602-188T, G. solisilvae DSM 100485T and G. luciferensis KEMB 7305-013T in the genus& Gottfriedia with bootstrap support of 92%, confirming the topology determined by 16S rRNA gene sequencing. Furthermore, four& Gottfriedia species formed a monophyletic cluster and were clearly separated from other species of the genus Bacillus, Neobacillus and Cytobacillus (Fig S4).& AntiSMASH analysis results showed four gene clusters within the genome of strain& RG28T, namely two gene cluster for terpene, one gene cluster for thiopeptides and one gene cluster for linear azol (in) e-containing peptide (LAP). When the results of& secondary metabolic gene clusters were compared between strain RG28T and its closest relatives, the gene cluster for thiopeptide was only found in strain RG28T which distinguish the novel isolates from its close relatives. In addition, large number of genes involve in sporulation, spore formation were also detected in the genome of strain RG28T (Table S1), which is consistent with our results showing the production of endospores.& The genome of strain RG28T contained four genes related to tryptophan biosynthesis: tryptophan synthase subunit beta (trpB) (JAGIYQ010000010), tryptophan--tRNA ligase (trpS) (JAGIYQ010000003), tryptophan 2,3-dioxygenase (JAGIYQ010000006) and anthranilate synthase component I (JAGIYQ010000021)& which indicates that strain RG28T could contribute to the plant growth-promoting activity in rice plants. When we compared the plant growth promoting rhizobacteria (PGPR) genes of strain RG28T with its close strains, interestingly the reference strains had more genes related to tryptophan biosynthesis. In addition, the reference strains had genes for siderophore and indole biosynthesis which were not found in the genome of strain RG28T (Table 2). These genomic features indicate that strain RG28T and its reference strains could be a PGPR candidate.
Strain RG28T was Gram-stain-positive, catalase-positive and oxidase-negative. Colonies grown on R2A plates were circular, white, smooth and 1–3 mm in diameter after three days of culture. Cells of the isolate were motile with peritrichous flagella (Fig. S5). Cells were able to grow at 10–50°C and pH 5.0–10.0, with optimal growth at 25–30°C and pH 7.0. The growth occured in 0-5 % NaCl (w/v) with optimum 0%. The strain showed no anaerobic growth on R2A plates. Endospores were produced at the termini in non-swollen sporangia (Fig. S6). Strain RG28T grew well on R2A, NA, TSA and LB but grew only moderately on MA. Strain RG28T was tolerant up to 5% NaCl, while& G. solisilvae LMG 18422T tolerated to 4%,& G. acidiceleris CBD 119T& to 2 % and& G. luciferensis LMG 18422T to 1% of NaCl in culture media. Cell morphology, colony colour, presence of flagella, oxidase activity, temperature and pH range for growth, differentiates the strain RG28T from its close strains. The detailed physiological and biochemical characteristics of strain RG28T are summarized in the species description and characteristics that differentiated strain RG28T from its closest related type strains are listed in Table 1.
The whole-cell fatty acid profile of strain RG28T contained large amounts of C16:0 (25.5%), iso-C15:0& (26.4%) and anteiso-C15:0& (33.4%).& A comparison of strain RG28T with closely related members of the genus& Gottfriedia is presented in Table S2. Strain RG28T had a similar fatty acid profile to& G. acidiceleris KEMB 1602-188T,& G. solisilvae DSM 100485T and& G. luciferensis KEMB 7305-013T, but the absence of C18:0& and iso-C13:0& in strain RG28T distinguish it from its close relatives. The peptidodoglycan of strain RG28T contained meso-DAP as the diagnostic diamino acid. The predominant menaquinone was MK-7 (57 %), followed by MK-6 (43 %). The quinone and peptidoglycan diamino acid of strain RG28T as found in all known members of the genus& Gottfriedia.& However, strain RG28T could be distinguished from the reference strains with the proportions of MK-7 and MK-6. In addition,& G. acidiceleris CBD 119T has MK-8, which is not found in strain RG28T and other two reference strains, which clearly differentiates them from each other (Table 2).& The polar lipid profile of strain RG28T consist of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) , four unidentified aminophosphoglycolipids (APGL1-4), four unidentified aminophospholipids (APL1-4), two unidentified glycolipids (GL1-2), one unidentified aminoglycolipid (AGL) and four unidentified lipids (L1-4) (Fig. S7). The major polar lipid profile was same with other three reference strains however the presence of other minor lipids distinguish the strain RG28T from other closely related strains (Pan et al. 2017).
Indole acetic acid (IAA) production and quantification
Change in color showed that strain RG28T and its reference strains G. acidiceleris KEMB 1602-188T, G. solisilvae DSM 100485T and G. luciferensis KEMB 7305-013T showed the ability to synthesize IAA only in the presence of the precursor L-tryptophan and could produce 40.5, 55.2, 56.8 and 50.5 µg/ml IAA, respectively (Fig. 3).& To the best of our knowledge, no reports are available for plant growth activities of genus& Gottfriedia so far, in this study we found that all strain of genus& Gottfriedia including novel strain RG28T were able to produce IAA in sufficient amounts.& Genome annotation also revealed the number of genes associated with& tryptophan biosynthesis which is consistent with our results which& suggest a potential use of& Gottfriedia species as& biofertilizer.
Phylogenetic and phylogenomic analysis indicated that the strain RG28T formed a different cluster with the three members of genus& Gottfriedia& with high topology. Based on the above polyphasic taxonomic analysis, strain RG28T was confirmed as a novel species in the genus& Gottfriedia. Therefore, the name& Gottfriedia endophyticus sp. nov. is proposed.
Description of Gottfriedia endophyticus sp. nov.
Gottfriedia endophyticus& (en.do.phy′ti.cus. Gr. pref. Endo within; G. n. phyton plant; L. masc. suff. -icus adjectival suffix used with the sense of belonging to; N.L. masc. adj. endophyticus within plant, endophytic, pertaining to the original isolation from plant tissues).
Cells are short to long-rods, Gram-positive, aerobic, capable of forming ellipsoidal endospores and motile by flagella. Colonies on R2A agar are moist, flat, white and undulate in margins after three days of incubation at 30°C.& Growth occurs at 10–50°C& (optimum, 30°C) and pH of 5.0–10.0 (optimum, pH 7.0) and tolerates upto 5.0% NaCl. NaCl is not required for growth. Cells were positive for hydrolysis of casein, starch, CM-cellulose and Tween 80 but negative for chitin hydrolysis. Cells are positive for catalase and negative for oxidase activity.& According to the API ZYM system, cells were positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, α-chymotrypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase, β-glucuronidase, β-glucosidase. In the API 20NE system, all phenotypic characteristics are negative except for indole production, arginine dihydrolase, esculin hydrolysis and β-galactosidase. In 50CH, following compounds are utilized as sole source of carbon and energy: D-ribose, D-glucose, N-acetylglucosamine, esculin, slicine, D-maltose and 5-keto-gluconate. Menaquinone-7 (MK-7) and meso-diaminopimelic acid (meso-DAP) are the major respiratory quinone and the diagnostic amino acid of the cell-wall peptidoglycan, respectively.& The main components of the whole-cell fatty acids (10 %) are& C16:0, iso-C15:0 and anteiso-C15:0.& The major polar lipids consisted of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, four unidentified aminophosphoglycolipids, four unidentified aminophospholipids, two unidentified glycolipids, one unidentified aminoglycolipid and four unidentified lipids.& & & & & &
The type strain is RG28T& (=KCTC 43327T=TBRC 15151T) which was isolated from the roots of rice plant collected from Goyang, South Korea. The DNA G+C content of the type strain is 33.5%. The GenBank accession number for the 16S rRNA gene sequence is MW386408, and the genome accession number is JAGIYQ000000000.&