Morphological, physiological and biochemical characteristics
Morphologically, colonies of strain UniB2T were mucoid, brownish-yellow in colour with an entire margin and convex elevation. In contrast, colonies of strain UniB3T were off-white, circular, entire in the margin, flat opaque. The strain UniB2T and UniB3T were found motile, rod in shape with 0.8 to 1.0 μM width; however, their length varied from 2 to 3.5 and 2 to 4.5 μM, respectively. Both the strains showed varied gram character. The strains were able to grow on pH 6- 8 (Optimum 7.0; 5.0 -12.0 pH range in increment of 1 pH unit), salinity 0.5- 2 % (Optimum 0.5 %; 0.5 – 4 % range in increment of 0.5 %).
According to API 20NE tests, strain UniB2T showed positive results for hydrolysis of aesculin and P-nitrophenyl-β-D-galactopyranoside and assimilation of glucose, mannitol, N-acetyl-glucosamine, maltose, whereas it showed negative results for arabinose, gluconate and malate. Among three, only Paenibacillus pasadenensis NBRC 101214T showed a positive result for assimilation of arabinose and malate. For gluconate assimilation, strain UniB2T alone showed a negative result. By API ZYM tests, the strain UniB2T was positive for ά-galactosidase, ß-galactosidase, ά glucosidase, ß glucosidase activity. The strain UniB2T tested negative for esterase activity, while its phylogenetically closest neighbours (Paenibacillus humicus NBRC 102415T and Paenibacillus pasadenensis NBRC 101214T) tested positive. Sugar utilization patterns were checked by Biolog GEN III microbial identification system. Strain UniB2T did not utilize N-acetyl-D-galactosamine, glycerol, methyl pyruvate, L-malic acid but NBRC 102415T and NBRC 101214 T were tested positive for utilizing these sugars. The strain NBRC 102415T and NBRC 101214T were tested negative for utilization of 3-methyl glucose, D-fucose, L-Rhamnose, L-arginine, L-aspartic acid, whereas strain UniB2T was able to utilize those sugars. The strain UniB2T and NBRC 101214T were positive for utilizing gentiobiose, α-D-glucose, L-fucose, D-fructose-6-Phosphate, D-galacturonic acid, β-hydroxy-D, L-butyric acid, acetic acid, whereas strain NBRC 102415T was tested negative for these sugars. Strain UniB2T and NBRC 102415T were tested negative for L-lactic acid, D-malic acid, bromo-succinic acid, whereas strain Paenibacillus pasadenensis NBRC 101214T showed positive results for utilization of these sugars. Strain UniB2T was sensitive to rifamycin SV, lithium chloride, whereas NBRC 102415T and NBRC 101214T were insensitive to these chemicals. Both NBRC 102415T and NBRC 101214T were sensitive to D-serine, guanidine-HCl whereas strain UniB2T was found resistant to these chemicals. The morphological, cultural, physiological and biochemical characteristics of strain UniB2T are given in Table 1.
Strain UniB3T showed positive results for hydrolysis of P-nitrophenyl-βD-galactopyranoside and assimilation of arabinose, mannose, N-acetyl- glucosamine, gluconate, malate and citrate. At the same time, its closest relatives Niallia nealsonii DSM 15077T, showed a negative result for hydrolysis of P-nitrophenyl-β-D-galactopyranoside and assimilation of arabinose, gluconate, malate and citrate. In comparison, Niallia circulans DSM 11T showed a negative result for assimilation of arabinose mannose, N- acetyl glucosamine and malate, according to API 20NE tests. According to the API ZYM test, Strain UniB3T showed naphthol AS-BI-phosphohydrolase, ß-galactosidase, ά-glucosidase enzyme activity whereas both the closest neighbours were negative for ß-galactosidase, ά-glucosidase activity and showed positive activity for naphthol AS-BI-phosphohydrolase similar to strain UniB3T. Sugar utilization pattern was tested by Biolog GEN III microbial identification system; strain UniB3T and DSM 11T showed a positive result for utilization of dextrin, D-maltose, D-cellobiose, D-turanose, stachyose, D-raffinose, α-D-lactose, β-methyl-D-glucoside, D-salicin, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, inosine, D-sorbitol, D-mannitol, glycerol, D-gluconic acid, methyl pyruvate, tween 40, acetoacetic acid, acetic acid whereas the strain DSM 15077T showed a negative result for these sugars. The strain DSM 15077T showed a positive sugar utilization reaction for N-acetyl-D-galactosamine, 3-methyl glucose, D-fucose, D-galacturonic acid, Mucic acid, β-hydroxy-D, L-butyric acid and other both the strains were negative for these sugars. Strain UniB3T and DSM 15077T showed a positive result for the utilization of L-fucose, L-rhamnose, D-glucuronic acid, and strain DSM 11T showed negative utilization results for these sugars. The strain DSM 11T showed positive results for Myo-inositol, L-alanine, L-malic acid, whereas strain UniB3T and DSM 15077T showed negative results for these sugars. Strain UniB3T was not observed utilizing bromo-succinic acid, D-glucose-6-Phosphate, while both the strains in the comparative study were observed to use these sugars. Strain UniB3T was found sensitive to troleandomycin and rifamycin SV antibiotics, while both the other strains were not found susceptible to both antibiotics. Strain UniB3T and DSM 11T were found sensitive to tetrazolium blue, 1 % sodium lactate, nalidixic lithium chloride, Lithium chloride, aztreonam, sodium bromate, whereas strain DSM 15077T was not found sensitive to these chemicals (Table 2).
Chemotaxonomic characterization
The major fatty acids in strain UniB2T were C16:00 (13.9 %), C15:0 anteiso (39.7 %), C17:0 anteiso (15.5 %). In Strain UniB3T, major fatty acids were C16:0 (13.54 %), C15:0 anteiso (40.09 %) and C17:0 anteiso (16.03 %). All data related to the fatty acid composition of UniB2T and UniB3T are presented in Tables 3 and 4, respectively.
Major Polar lipids of strain UniB2T were Diphosphotidylglycerol and Phosphatidylethanolamine and possessed unknown phospholipid (PL1, PL2, and PL3) and unknown amino phospholipids (APL 1, APL2, and APL3). APL4 was not observed in Strain UniB2T but detected in phylogenetically closest neighbours of strain UniB2T, whereas PL2 and APL3 were not detected in strain NBRC 102415T and PL1, PL2 and APL3 were not detected in strain NBRC 101214T. Diphosphotidylglycerol and Phosphatidylethanolamine were the major polar lipids found in strain UniB3T. This strain possessed unknown phospholipid PL1. Phospholipid PL1, PL2 and PL3 were not detected in strain DSM 15077T, and PL4 was not detected in strain UniB3T but detected in DSM 11T and DSM 15077T (Fig. S2).
Phylogenetic and genotypic analysis
The 16S rRNA gene sequence of strain UniB2T (MK751590) and UniB3T (MK751589) were generated and deposited at NCBI GenBank. The strain UniB2T showed the closest match with Paenibacillus humicus NBRC 102415T (Vaz-Moreira et al. 2007) (98.30 %, BIMD01000104) followed by Paenibacillus pasadenensis NBRC 101214T (Osman et al. 2006) (98.06 %, AY167820). The strain Unib3T showed the closest match with Niallia circulans DSM 11T (Jordan 1890; Gupta et al. 2020) (98.95 %, AY724690), followed by Niallia nealsonii DSM 15077T (Venkateswaran et al. 2003; Gupta et al. 2020) (98.47 %, EU656111).
Sequencing of UniB2T and UniB3T on Illumina MiSeq platform generated 4,373,992 reads and 3,315,487 reads (150×2 chemistry), respectively. ONT sequencing generated 159,965 reads and 165,209 reads respectively for UniB2T and UniB3T. The final UniB2T assembly contained one scaffold corresponding to 5,168,992 bp. This genome was found to be 99.03 % complete with 248× depth coverage. The final UniB3T assembly had two scaffolds corresponding to 5,241,236 bp. This genome was found to be 92.78 % complete with 184× depth coverage. The DNA G+C contents were 63.74 mol % and 35.6 mol % for UniB2T and UniB3T. The strain UniB2T was predicted to have 4434 protein-coding genes, 82 tRNA and 24 rRNA genes, whereas strain UniB3T was predicted to have 4574 protein-coding genes, 83 tRNA and 33 rRNA genes (Zhao et al. 2011).
The strain UniB2T clustered in a monophyletic clade with P. pasadenensis NBRC 101214T and P. humicus NBRC 102415T in the phylogenetic tree drawn using the 16S rRNA gene sequences (Fig. S3). Similarly, the strain UniB3T clustered in a monophyletic clade with Niallia circulans DSM 11T (Fig. S4). This analysis was supported by the core-genome phylogenetic tree constructed using BPGA and UBCG
The orthoANI value for strain UniB2T against NBRC 101214T was 89.55 %, with NBRC 102415T, it was 77.72 %, and with "P. herberti" R33, it was 76.2 %. The digital DNA–DNA hybridization (dDDH) values for strain UniB2T against NBRC 101214T, NBRC 102415T and R33 were 38.3, 22.6 and 21.2 %, respectively. The orthoANI value for strain UniB3T against DSM 11T was 84.62 %, and with DSM 15077T, it was 74.2 %. The dDDH values for strain UniB3T against DSM 11T and DSM 15077T were 29.1 and 22.2 %, respectively. The orthoANI and dDDH values computed for both the strains strongly support them to be novel species. Therefore, based on physiological, chemotaxonomic characters, phylogenetic and genomics indices, we propose strains UniB2T and UniB3T represent two novel species named Paenibacillus albicerus sp. nov. and Niallia alba sp. nov. are proposed, respectively.
Description of Paenibacillus albicerus sp. nov.
Paenibacillus albicerus (albi'ce.rus L. masc. adj. albicerus, pale yellow, the colour of colonies.)
Aerobic, catalase and oxidase-positive and Gram-Stain variable in nature. Spore-forming, motile rod with 0.8 to 1.0 μM width and 2 to 3.5 um in length. Colonies are mucoid, brownish-yellow in colour with an entire margin and convex elevation. The colony size on solid media (Nutrient agar) is 2-3 mm. Cells can grow on 5 – 40 0C temperature range (Optimum 30 0 C) on NA medium and able to grow on pH ranges from 6.0-8.0 (Optimum 7.0) unit and salinity 0.5 - 2.0 % (Optimum 0.5 %). Cells show positive results for hydrolysis of aesculin and P-nitrophenyl-βD-galactopyranoside and assimilation of glucose, mannitol, N-acetyl-glucosamine, maltose and shows negative results for gluconate assimilation. Cells show esterase lipase, naphthol AS-BI-phosphohydrolase, ά-galactosidase, ß-galactosidase, ά-glucosidase and ß-glucosidase activity. It utilizes D-sorbitol, 3-methyl glucose, D-fucose, L-Rhamnose, L-arginine, L-aspartic acid, gentiobiose, α-D-glucose, L-fucose, D-fructose-6-Phosphate, D-galacturonic acid, β-hydroxy-D, L-butyric acid, acetic acid. It shows sensitivity towards nalidixic lithium chloride, lithium chloride, rifamycin SV. C16:00 (13.9 %), C15:0 anteiso (39.7 %) and C17:0 anteiso (15.5 %) as major fatty acids and Diphosphotidylglycerol and Phosphatidylethanolamine as major polar lipids. The G+C content of type strain UniB2T is 63.74 mol %.
The type strain UniB2T (= MCC 3997 = KCTC 43095=JCM 34513) was isolated from digestive syrup from Pune, India.
Description of Niallia alba sp. nov.
Niallia alba (al'ba L. fem. adj. alba, white, the colour of colonies).
The cell is rod-shaped motile, aerobic, catalase and oxidase-positive, Gram-Stain variable in nature. The colonies are off white, circular, entire in the margin, flat and non-transparent. Rods are 0.8 to 1 μM in width and 2 to 4.5 μM in length. The optimum temperature for growth is 30 0C and can grow in a temperature range of 5 – 400C on a Nutrient Agar medium. Able to grow on pH ranges from 6.0-8.0 (Optimum 7.0) unit and salinity 0.5 - 2.0 % (Optimum 0.5 %). The cell is negative for glucose fermentation. Cells can hydrolyze P-nitrophenyl-βD-galactopyranoside and assimilates arabinose, mannose, N-acetyl, glucosamine, gluconate, malate, citrate. It shows naphthol AS-BI-phosphohydrolase, ß-galactosidase, ά-glucosidase enzyme activities. Strain UniB3T can utilize a variety of sugars and chemicals like dextrin, D-maltose, D-cellobiose, D-turanose, stachyose, D-raffinose, α-D-lactose, β-methyl-D-glucoside, D-salicin, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, Inosine, D-sorbitol, D-mannitol, glycerol, D-gluconic Acid, methyl pyruvate, tween 40, acetoacetic acid, acetic acid. Sensitive to antibiotics and chemicals like troleandomycin, rifamycin SV, tetrazolium Blue, 1 % sodium lactate, nalidixic, lithium chloride, lithium chloride, aztreonam, sodium bromate. Major fatty acids were C16:0 (13.54 %), C15:0 anteiso (40.09 %) and C17:0 anteiso (16.03 %). Diphosphotidylglycerol and Phosphatidylethanolamine as major polar lipids. The G+C content of the type strain UniB3T is 35.6 mol %.
The type strain UniB3T (=MCC 3998 =KCTC 43235 =JCM 34492) was isolated from digestive syrup from Pune, India.