Genomic characteristics, BLAST analysis and phylogenetic inference
Genome size of the strains JC665Tand JC747 are 8.05 Mb and 8.04 Mb with an N50 value of 238,467 and 226,135, respectively. Genome of the strain JC665T has 6,431genes of which 6,307 are protein coding genes, 80 genes code for RNAs (3 genes for encoding r-RNAs, 74 genes for t-RNAs and 3 for other RNAs) and 44 genes are pseudogenes (Table S1). Genome of the strain JC747 has 6,420 genes of which 6,299 are protein coding genes, 80 genes code for RNAs (3 genes for encoding r-RNAs, 74 genes for t-RNAs and 3 for other RNAs) and 41 genes are pseudogenes (Table. S1). The organization of orthologous clusters among the strains will provide a better understanding in genome structure and gene/protein function. We could predict from the genome wide annotation, 7106, 7102, 7373 and 6643 proteins for strains JC665T, JC747, “P. soli” JC670T and P. borealis PX4T respectively. The predicted proteins of strains JC665 and JC747 showed 6973, 6969 orthologous clusters and 103 singletons for which no orthologs were found in other species (Fig. S1A). “P. soli” JC670T showed 4612 clusters and 2464 singletons. P. borealis PX4T showed 4268 clusters and 2160 single tons (Fig S1A). The comparative analysis of orthologous gene clusters performed shows that these species formed 7499 clusters, 4032 orthologous clusters (at least contains two species) and 3467 single-copy gene clusters (Fig. S1B). A total of 14738 proteins were present in orthologous clusters found in all the strains whereas the strain JC670 and PX4 showed 314, 190 proteins in the clusters specific to them (Fig. S1C). Further comparison of shared orthologous gene clusters showed that 3556 clusters were observed in all the strains, no unique clusters were observed in the strains JC665T and JC747 whereas “P. soli” JC670T and P. borealis PX4T showed 127 and 73 unique clusters respectively (Fig. S1D).
Genomic DNA G + C content of both strains (JC665T, JC747) is 66.4 mol% (Table S1). The 16S rRNA gene sequences of the strains JC665Tand JC747 extracted from the genomes have sequence length of 1521 nt. BLAST analysis of 16S rRNA gene sequence of strains JC665T and strain JC747 in EzBioCloud server showed identity of 94.6% and 96.7% with P. borealis PX4T and “P. soli” JC670T, respectively (Fig. 1). Comparison of dDDH, gANI, and AAI values of strains JC665T and JC747 with Paludisphaera spp. yielded similarity of (19.4–20.3) %, (62.4–68.6) %, and (75.1–77.9) %, respectively. AAI, OrthoANI and dDDH values fell well below the recommended cut-off of 80%, 95–96% and 70%, respectively for prokaryotic species delineation (Rodriguez and Konstantinidis 2014; Meier-Kolthoff et al. 2014; Chun et al. 2018). Thus, both the newly isolated strains represent a novel species of the genus Paludisphaera. However, high values of 16s rRNA (100%), dDDH (100%), gANI (100%), and AAI (99.9%) between strains JC665Tand JC747 suggest these strains to be same species from two different and distantly located ecosystems.The 16S rRNA gene sequence based phylogenetic tree with combined bootstrap values obtained from NJ, ME, ML trees (Fig. 2) and 92 core genes based phylogenomic tree (Fig. 3) confirmed the distinct monophyletic clustering of strains JC665T and JC747 with Paludisphaera members within the family Isosphaeraceae and suggest a novel species within the genus Paludisphaera.
In-silico metabolic characterisation
In order to have better understanding on metabolic functions, the COGs annotation was performed. The results showed that both strains, JC665T and JC747 showed similar results as that of the other members of the genus Paludisphaera. Most of the genes predicted were of unknown function and later followed by genes involved in energy production and conversion (Fig. S2). The CAZy annotation of genomes shows that the strains JC665T and JC747 contains more genes encoding glycoside hydrolases followed by glycosyl transferases compared to that of other members of the genus Paludisphaera. The analysis also shows that 80–90% of the enzymes belonged to families of glycoside hydrolases and glycosyl transferases (Fig. S3). The presence of higher number of carbohydrate active enzymes with respect to bacterial metabolism needs further studies. In silico metabolic characterisation showed that strain JC665T and JC747 have the 2-C-methyl-Derythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway for the biosynthesis of five carbon isoprene units (isopentenyl pyrophosphate), that acts as a precursor for the synthesis of carotenoids and quinones. The putative genes encoding for enzymes like 15-cis-phytoene synthase [EC:184.108.40.206], phytoene desaturase [EC:220.127.116.11 18.104.22.168 22.214.171.124 126.96.36.199] etc which helps in formation of lycopene as an end product with intermediary products like phytoene, zeta carotene and neurosporene were predicted in the genomes of both strains. The genes for assimilatory nitrate reduction were observed only in P. borealis PX4T. The putative genes for CAM (Crassulacean acid metabolism), was observed in JC665T and JC747 only. All the strains belonging to genus Paludisphaera along with JC665T and JC747 showed putative gene clusters for synthesis of Type I, Type III polyketide synthases and terpene biosynthesis. However, genes for production of indole were predicted exclusively in JC665T and JC747 only (Fig. S4). In-silico genome analysis of all the four strains of Paludisphaera also showed the presence of the putative hopanoid biosynthesis pathway genes like squalene synthase (hpnC), squalene/phytoene desaturase (hopC), squalene hopene cyclase (Shc; codes for the key enzyme of hopanoid biosynthesis), radical S-adenosyl-L-methionine (SAMe) required for addition of adenosyl group to hopane skeleton (hpnH), acetylornithine aminotransferase/amino-bacteriohopanetriol synthase (hpnO), hopanoid associated sugar epimerase (hpnA) and sterol desaturase family protein (erg32).
Morphological and physiological analysis
SEM image show that cells of strain JC665T and JC747 are spherical to oval shaped (1.7-1.8x1.3-1.5µm; Fig. 4A) and have well distribution of crateriform structures (CR) all over the cell surface. TEM image of the cells shows the presence of cytoplasmic membrane (CM), outer membrane (OM), invagination of cytoplasmic membrane (ICM), cytoplasm (CP), nucleoid region (N), Ribosomes (RB) and cell reproduction by budding (BD) where daughter cell is protruding from mother cell (Fig. 4B).
NAG is not obligate for the growth of strains JC665T, JC747, “P. soli” JC670T and P. borealis DSM 28747T. Strains JC665T, JC747, “P. soli” JC670T and P. borealis DSM 28747T utilizes following organic carbon sources: α-D-glucose, sucrose, Na-pyruvate, D-galactose, mannose, rhamnose, and trehalose. Neither of the strains utilizes following organic carbon sources: starch, ascorbate, acetate, mannitol, malate, inulin, benzoate, Na-succinate and citrate. Lactose and maltose are utilized by the strains JC665T, JC747, and P. borealis DSM 28747T. Fructose and D-xylose are utilized by the strains “P. soli” JC670T and P. borealis DSM 28747T. Cellobiose and ribose are exclusively utilized by the strain P. borealis DSM 28747T. Fumarate and propionate are exclusive for the strains JC665T (including JC747) and “P. soli” JC670T, respectively. All the strains utilizes the following nitrogen sources for their growth: ammonium sulphate, peptone, yeast extract, DL-alanine, L-arginine, casamino acid and sodium nitrate. Neither of the strains utilizes following nitrogen sources: L-aspartic acid, urea and valine. The following nitrogen sources are exclusively utilized by the strains JC665T, JC747 and “P. soli” JC670T: L-glycine, L-phenylalanine, L-lysine, L-glutamine, L-proline, L-isoleucine, L-leucine, DL-ornithine and DL-threonine. However, L-methionine and cysteine are exclusively utilized by the strains JC665T and JC747. L-serine and L-tyrosine is exclusive for the strain “P. soli” JC670T (Table 2). Strain JC665T can hydrolyse phytagel (Fig. S5) only in the absence of N-acetylglucosamine in the medium, as also observed previously for P. borealis PX4 T and “P. soli” JC670T (Kaushik et al.2020; Kulichevskayaet al.2016).
All the four strains showed positive for esterase (C4), leucine arylamidase, and valine arylamidase. However, all the strains show negative for lipase (C14), cysteine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-glucosidase, β- glucosidase, α-mannosidase, and α-fucosidase. Alkaline phosphatase, esterase lipase (C8), and acid phosphatase are exclusively positive for the strains “P. soli” JC670T and P. borealis DSM 28747T. Naphthol-AS-BI-phosphohydrolase show positive for the strains JC665T, JC747 and “P. soli” JC670T. β-galactosidase and N-acetyl- β-glucosaminidase, are exclusively positive for the strain P. borealis DSM 28747T only.
The major fatty acids in strains JC665T, JC747, “P. soli” JC670T and P. borealis DSM 28747T are C18:1ω9c, C18:0 and C16:0. In terms of fatty acids composition, significant differences were found among all the strains (Table. S2). The polar lipids of strains JC665T and JC747 are; phosphatidylcholine (PC), two unidentified glycolipids (GL1, 2), six unidentified lipids (UL1-7) and two unidentified phospholipid (PL1, 2) (Fig. S6A). The polar lipids of strain P. borealis DSM 28747T compose of phosphatidylcholine (PC), phosphatidylethanolamine (PE), one unidentified choline lipid (CL1), two unidentified glycolipids (GL1, 2), two unidentified lipids (UL1, 2), two unidentified amino lipids (AL1, 2) and four unidentified phospholipids (PL3-6) (Fig. S6B) and is not found in congruence of earlier study, as different method was adopted for the identification of polar lipids (Kulichevskaya et al. 2016). The polar lipids of strain “P. soli” JC670T include phosphatidylcholine, two unidentified phospholipids and six unidentified lipids (Fig. S6C) and is found in congruence of earlier study (Kaushik et al. 2020). Polyamines of the strains JC665T and “P. soli” JC670T include sym-homospermidine and putrescine. Polyamine of the strain JC747 include spermidine and two unidentified polyamines (1, 3). Polyamine of the strain P. borealis DSM 28747T include spermidine and two unidentified polyamines (2, 3) (Fig. S7). MK6 is the predominant quinone for all the strains.
Proposal of strain JC665 T as a new species of the genus Paludisphaera
Strains JC665T and JC747 have clear phylo-genomic differences with “P. soli” JC670T and P. borealis DSM 28747T but between themselves they are similar species (Fig. 1,2,3; Fig. S1, Table. S1). The phylo-genomic differences are well supported by chemotaxonomic and phenotypic differences (Table. 1), which support strain JC665T as a novel species of the genus Paludisphaera. For this, we propose the name of the type strain JC665T as Paludisphaera rhizosphaerae and strain JC747 as its non-type strain.
Descriptions of Paludisphaera rhizosphaerae sp. nov.
Paludisphaera rhizosphaerae (rhi.zo.sphae'rae. Gr. n. rhiza, root; L. n. sphaera, sphere; N.L. gen. n. rhizosphaerae, from the rhizosphere)
Color of chemotrophically grown culture is pale pink. Cells are spherical to oval shaped, and are strictly aerobic. Cell division is through budding. NaCl is not obligate for growth and can tolerates up to 2% (w/v). Optimum pH and temperature for growth are 7.0 (range 6.0–9.0) and 25ºC (range 4–34ºC) respectively. N-acetylglucosamine (NAG) is not obligate for the growth. As an organic carbon substrate, D-glucose, Sucrose, Pyruvate, D-galactose, Mannose, Rhamnose, rhamnose, inositol, fumarate, lactose, maltose, sorbitol, and trehalose are utilized. Fructose, Na-propionate, D-xylose, starch, ascorbate, acetate, mannitol, malic acid, inulin, succinate, benzoic acid and citrate are not utilized. Ammonium sulphate, peptone, casamino acid, yeast extract sodium nitrate, L-cysteine, L-methionine, L-histidine, L-glutamic acid, L-arginine, DL- alanine, l-glycine, L-glutamine, L-proline, L-isoleucine, L-ornithine and DL-threonine are utilized as nitrogen source. L-serine, L-tyrosine, L-aspartic acid, L-tryptophan, Urea and L-valine are not utilized as nitrogen source. Hydrolyse phytagel. Major fatty acids are C18:1ω9c, C16:0, and C18:0. Minor fatty acids include anteiso-C11:0, anteiso-C12:0, C13:0, CI4:0, anteiso-C15:0, C15:2OH, C15:1ω5c, C17:0, C17:1ω8c, anteiso-C17:0, anteiso-C17:0 and C18:3ω6c,9c,12c. Putrescine and sym- homospermidine are the major polyamines. The polar lipids phosphatidylcholine, two unidentified glycolipids (GL1, 2), seven unidentified lipids (UL1-7) and two unidentified phospholipid (PL1, 2). MK6 is the only quinone. Nitrate is not reduced. API ZYM shows positive for esterase (C4), leucine arylamidase, and valine arylamidase and Naphthol-AS-BI-phosphohydrolase. Negative for lipase (C14), cysteine arylamidase, trypsin, α-chymotrypsin, α-galactosidase, β-glucuronidase, α-glucosidase, β- glucosidase, α-mannosidase, α-fucosidase. Alkaline phosphatase, esterase lipase (C8), acid phosphatase, β-galactosidase and N-acetyl- β-glucosaminidase. The type strain JC665T (= NBRC 114305 = KCTC 72671T) was isolated from the rhizosphere soil of Erianthus ravennae (commonly known as “Plume grass”) collected from Loktak lake located in the Northeast part of India, Manipur (exact location: 24°30’21” N 93°47’43” E). JC747 is an additional strain isolated from a wetland located (village: Pallikkara) in the southwest part of India, Kerala (12° 23' 02'' N 75° 02' 33'' E). The GenBank accession numbers of the 16S rRNA gene sequence and genome sequence of strain JC65T and JC747 are LR746340, OU374731 and JAALCR000000000 and JAHPZK000000000, respectively.