Description of Paenibacillus albicerus sp. nov. and Niallia alba sp. nov., isolated from digestive syrup.

Two aerobic, Gram-stain variable, catalase and oxidase-positive, spore-forming, motile rods of strain UniB2 T and UniB3 T, were isolated from digestive syrup containing fungal diastase (10 mg/ml), pepsin (2 mg/ml) and sugar base containing polyethene glycol. Based on 16S rRNA gene sequence analysis, strain UniB2 T has the highest sequence similarity with Paenibacillus humicus NBRC 102415 T (98.30 %) and strain UniB3 T showed the highest sequence similarity with Niallia circulans DSM 11 T (98.95 %). The DNA G+C content of UniB2 T was 63.74 mol %. The dDDH and ANI values between the strain UniB2 T and its phylogenetically close relative were <38.3 % and <89.55 %, respectively. The major fatty acids of the strain UniB2 T were C16:00 (13.9 %), C 15:0 anteiso (39.7 %), C 17:0 anteiso (15.5 %). The DNA G+C content of UniB3 T was 35.6 mol%. The dDDH and ANI values between the strain UniB3 T and its close relatives were <29.1 % and 84.62%, respectively. The major fatty acids of strain UniB3 T were C16:0 (13.54 %), C15:0 anteiso (40.09 %) and C17:0 anteiso (16.03 %). Major polar lipids for both strains were Diphosphotidylglycerol and Phosphatidylethanolamine. Additionally, both the strains showed unique carbon utilization and assimilation pattern that differentiated them from their phylogenetically related neighbours. These phenotypic, genotypic and chemotaxonomic characters indicated the strains UniB2 T and UniB3 T represent two novel species for which the names Paenibacillus albicerus sp. nov. (Type strain UniB2 T =MCC 3997 T =KCTC 43095 T =JCM34513 T ) and Niallia alba sp. nov. (Type strain UniB3 T = 3998 T =KCTC 43235 T =JCM 34492 T ) are proposed.


Introduction
Genus Paenibacillus was reclassi ed and separated from the Bacillus genus in 1993 (Ash et al. 1993). The name Paenibacillus came into existence from the Latin word 'Paene' means 'almost', so Paenibacillus means 'almost Bacilli'. The genus Paenibacillus was initially placed in a former 'group 3' of genus Bacillus. Comparative 16S rRNA sequence analyses revealed that group 3 bacilli were phylogenetically distinct and sparsely related to type species Bacillus subtilis. Strains belonging to genus Paenibacillus have been reported from various environments like soil, rhizosphere, water, plant, animal, and human clinical samples (Yoon et al. 2005;Roux et al. 2008;Zhang et al. 2016;Hwang and Ghim 2017;Simon et al. 2017;Yun et al. 2017). The genus is composed of more than 320 validly published species based on the List of Prokaryotic Names with Standing in Nomenclature (Parte et al. 2020); some of these species exhibits various activities like nitrogen xation , phosphorous solubilization (Singh and Singh 1993), antibiotic production (Piuri et al. 1998) and production of chitinase .
Genus Niallia has been recently proposed and separated from the genus Bacillus (Gupta et al. 2020). Early classification of Bacillus species were based on two characteristics: aerobic growth and endospore formation (Logan and De Vos 2009;). There were more than 570 species reported in this genus to date. Bacillus has long appeared as a heterogeneous phylogenetic group (Parte et al. 2020). Most of the species of this genus show minimal characters and exhibits substantial polyphyly. Gupta et al. (2020) identi ed conserved signature indels (CSIs) speci c for each identi ed clade and proposed reclassi cation of 148 Bacillus species into 17 novel genera and 11 existing genera. They identi ed 128 CSIs, which exhibit uniqueness to the speci c clade and provide reliable means for their distinction (Gupta et al. 2020). This report describes two novel species belonging to the genus Paenibacillus and Niallia isolated from the digestive syrup, closely related to the Paenibacillus humicus (Vaz-Moreira et al. 2007) and Niallia circulans (Gupta et al. 2020).

Methods And Materials
Bacterial isolation and culture conditions Strain UniB2 T and UniB3 T were isolated from the digestive syrup containing digestive enzymes [diastase; isolated from Aspergillus niger (10 mg/ml) and pepsin (2 mg/ml)] used in digestive troubles. The contaminated syrup was centrifuged, and the pellet was serially diluted and spread on a Nutrient agar (NA) medium (M001; Himedia, India), and cultures were grown at 30 0 C for 48 hours.

Morphological, physiological and biochemical characteristics
The size and shape of the cells were determined by scanning electron microscopy (Zeiss, EVO 18, Version 6.02), as seen in supplementary Figure S1. The Gram character for both the strains was determined by Gram staining kit (K001-KT; Himedia, India) and observed by light microscopy (Model BX53; Olympus, USA). Motility was analyzed by the hanging drop method under a light microscope (Model BX53; Olympus, USA). Oxidase and catalase activity were investigated using oxidase discs (DD018; Himedia, India) and bubble production in 3 % (v/v) H2O2, respectively. The strains were optimally grown on NA at 30 0 C (5 -40 0 C range) in 96 hours. The pH range for growth was determined at pH 5-12 with increments of 1 pH unit with different buffer systems like acetate (4 to 5 unit), phosphate (6 to 8 units) and glycinesodium hydroxide (9 to 12 units). Growth of strains was detected using a Bioscreen C microbiology reader (Oy Growth Curves AB Inc., Finland). Salt tolerance for growth was examined in Nutrient broth (NB) with 0-4 % NaCl with an increment of 0.5 % at 30 °C for ve days, and the growth was observed by a 'Bioscreen C Microbiology' reader. The pH, salinity, and temperature optimization were carried out on NB for ve days. Biochemical characteristics like utilization, assimilation of different carbon sources and enzyme activity against different substrates were tested by analytical pro le index, biomerieux (API 20NE, API ZYM) and Biolog GEN III Microplate assay according to the instructions of the manufacturers.

Chemotaxonomic characterization
The whole-cell fatty acids (Fatty Acid Methyl ester, FAME) and polar lipids were determined for chemotaxonomic analyses. Forty-eight hours old cultures, grown on tryptic soy agar at pH 7.3±0.2 (M1968; Himedia) of strain UniB2 T , NBRC 102415 T , NBRC 101214 T , UniB3 T , DSM 11 T and DSM 15077 T were harvested for cell mass and extracted total cell fatty acids were analyzed by RTSBA6 library on Sherlock Microbial Identi cation System (Version 6.1; MIDI) (Sasser 2001). For polar lipid extraction, cell mass was harvested from cultures in the logarithmic phase (Bligh and Dyer 1959;Card 1973;Minnikin et al. 1984).

Phylogenetic and genotypic analysis
All obtained colonies were subjected to genomic DNA extraction (Ausubel et al. 1994), followed by their identi cation by 16S rRNA gene sequencing. The 16S rRNA gene PCR was done with universal 16S rRNA gene primers (Sambrook and Russell 2001), and the EzBioCloud database (Yoon et al. 2017) was used to identify the closest type relative. The genomic DNA of the strains UniB2 T and UniB3 T were sequenced on the Illumina MiSeq and Oxford Nanopore Technology (ONT) platforms. The reads were quality checked using FastQC version 0.10.1 (Brown et al. 2017). The ONT sequencing data were base-called with quality ltering (>Q7) using Guppy version 3.5.4. All QC passed Illumina reads (>Q30), and ONT reads were used to generate a hybrid assembly in Unicycler version 0.4.8 (Wick et al., 2017). The genome quality was assessed using QUAST (v5.0.2) (Gurevich et al. 2013) and CheckM (v1.1.3) (Parks et al. 2015). The genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). 16S rRNA gene phylogeny was constructed using the software packages MEGA version 7.0 (Kumar et al. 2016) with three algorithms, the neighbour-joining (Saitoh 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony (Fitch 1971) methods. Further, to support the taxonomic position of strain UniB2 T and UniB3 T , a core-genome phylogenetic tree was constructed using BPGA, version 1.3.0 (Chaudhari et al. 2016) and UBCG, version 3.0 (Na et al. 2018) tools for both strains ( Fig. 1 and 2). The BPGA tool searched for core genes from the genomes of 17 members phylogenetically close to strain UniB2 T and among 20 members related to UniB3 T . The BPGA pipeline generated orthologous amino acid sequences clusters using the integrated USEARCH algorithm (Edgar 2010). Among these, 20 random clusters were aligned and concatenated. Finally, the phylogenetic tree was reconstructed using the BPGA concatenated sequences by the neighbour-joining method in MEGA 7. A total of 12,595 and 14,771 (UniB2 T and UniB3 T ) amino acid positions in BPGA nal dataset were used for the tree generation. The length of the UBCG concatenated alignment was 86,964 bp containing 92 marker genes for UniB2 T and 85,746 bp containing 92 marker genes for UniB3 T identi ed using HMMER (Potter et al. 2018) and predicted using Prodigal (Hyatt et al. 2010) search.

Results And Discussion
Morphological, physiological and biochemical characteristics Morphologically, colonies of strain UniB2 T were mucoid, brownish-yellow in colour with an entire margin and convex elevation. In contrast, colonies of strain UniB3 T were off-white, circular, entire in the margin, at opaque. The strain UniB2 T and UniB3 T 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 UniB2 T showed positive results for hydrolysis of aesculin and Pnitrophenyl-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 101214 T showed a positive result for assimilation of arabinose and malate. For gluconate assimilation, strain UniB2 T alone showed a negative result. By API ZYM tests, the strain UniB2 T was positive for α-galactosidase, ß-galactosidase, ά glucosidase, ß glucosidase activity. The strain UniB2 T tested negative for esterase activity, while its phylogenetically closest neighbours (Paenibacillus humicus NBRC 102415 T and Paenibacillus pasadenensis NBRC 101214 T ) tested positive. Sugar utilization patterns were checked by Biolog GEN III microbial identi cation system. Strain UniB2 T did not utilize N-acetyl-D-galactosamine, glycerol, methyl pyruvate, L-malic acid but NBRC 102415 T and NBRC 101214 T were tested positive for utilizing these sugars. The strain NBRC 102415 T and NBRC 101214 T were tested negative for utilization of 3-methyl glucose, D-fucose, L-Rhamnose, Larginine, L-aspartic acid, whereas strain UniB2 T was able to utilize those sugars. The strain UniB2 T and NBRC 101214 T 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 102415 T was tested negative for these sugars. Strain UniB2 T and NBRC 102415 T were tested negative for L-lactic acid, D-malic acid, bromo-succinic acid, whereas strain Paenibacillus pasadenensis NBRC 101214 T showed positive results for utilization of these sugars. Strain UniB2 T was sensitive to rifamycin SV, lithium chloride, whereas NBRC 102415 T and NBRC 101214 T were insensitive to these chemicals.
Both NBRC 102415 T and NBRC 101214 T were sensitive to D-serine, guanidine-HCl whereas strain UniB2 T was found resistant to these chemicals. The morphological, cultural, physiological and biochemical characteristics of strain UniB2 T are given in Table 1.
Strain UniB3 T 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 15077 T , showed a negative result for hydrolysis of Pnitrophenyl-β-D-galactopyranoside and assimilation of arabinose, gluconate, malate and citrate. In comparison, Niallia circulans DSM 11 T 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 UniB3 T showed naphthol AS-BI-phosphohydrolase, ß-galactosidase, α-glucosidase enzyme activity whereas both the closest neighbours were negative for ß-galactosidase, α-glucosidase activity and showed positive activity for naphthol AS-BI-phosphohydrolase similar to strain UniB3 T . Sugar utilization pattern was tested by Biolog GEN III microbial identi cation system; strain UniB3 T and DSM 11 T showed a positive result for utilization of dextrin, D-maltose, D-cellobiose, D-turanose, stachyose, D-ra nose, α-D-lactose, β-methyl-D-glucoside, D-salicin, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, inosine, Dsorbitol, D-mannitol, glycerol, D-gluconic acid, methyl pyruvate, tween 40, acetoacetic acid, acetic acid whereas the strain DSM 15077 T showed a negative result for these sugars. The strain DSM 15077 T showed a positive sugar utilization reaction for N-acetyl-D-galactosamine, 3-methyl glucose, Dfucose, D-galacturonic acid, Mucic acid, β-hydroxy-D, L-butyric acid and other both the strains were negative for these sugars. Strain UniB3 T and DSM 15077 T showed a positive result for the utilization of Lfucose, L-rhamnose, D-glucuronic acid, and strain DSM 11 T showed negative utilization results for these sugars. The strain DSM 11 T showed positive results for Myo-inositol, L-alanine, L-malic acid, whereas strain UniB3 T and DSM 15077 T showed negative results for these sugars. Strain UniB3 T 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 UniB3 T was found sensitive to troleandomycin and rifamycin SV antibiotics, while both the other strains were not found susceptible to both antibiotics. Strain UniB3 T and DSM 11 T were found sensitive to tetrazolium blue, 1 % sodium lactate, nalidixic lithium chloride, Lithium chloride, aztreonam, sodium bromate, whereas strain DSM 15077 T was not found sensitive to these chemicals (Table 2).
Major Polar lipids of strain UniB2 T 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 UniB2 T but detected in phylogenetically closest neighbours of strain UniB2 T , whereas PL2 and APL3 were not detected in strain NBRC 102415 T and PL1, PL2 and APL3 were not detected in strain NBRC 101214 T . Diphosphotidylglycerol and Phosphatidylethanolamine were the major polar lipids found in strain UniB3 T . This strain possessed unknown phospholipid PL1.
Phospholipid PL1, PL2 and PL3 were not detected in strain DSM 15077 T , and PL4 was not detected in strain UniB3 T but detected in DSM 11 T and DSM 15077 T (Fig. S2). Sequencing of UniB2 T and UniB3 T 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 UniB2 T and UniB3 T . The nal UniB2 T assembly contained one scaffold corresponding to 5,168,992 bp. This genome was found to be 99.03 % complete with 248× depth coverage. The nal UniB3 T 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 UniB2 T and UniB3 T . The strain UniB2 T was predicted to have 4434 protein-coding genes, 82 tRNA and 24 rRNA genes, whereas strain UniB3 T was predicted to have 4574 protein-coding genes, 83 tRNA and 33 rRNA genes (Zhao et al. 2011).
The strain UniB2 T clustered in a monophyletic clade with P. pasadenensis NBRC 101214 T and P. humicus NBRC 102415 T in the phylogenetic tree drawn using the 16S rRNA gene sequences (Fig. S3). Similarly, the strain UniB3 T clustered in a monophyletic clade with Niallia circulans DSM 11 T (Fig. S4). This analysis was supported by the core-genome phylogenetic tree constructed using BPGA and UBCG The orthoANI value for strain UniB2 T against NBRC 101214 T was 89.55 %, with NBRC 102415 T , it was 77.72 %, and with "P. herberti" R33, it was 76.2 %. The digital DNA-DNA hybridization (dDDH) values for strain UniB2 T against NBRC 101214 T , NBRC 102415 T and R33 were 38.3, 22.6 and 21.2 %, respectively.
The orthoANI value for strain UniB3 T against DSM 11 T was 84.62 %, and with DSM 15077 T , it was 74.2 %.
The dDDH values for strain UniB3 T against DSM 11 T and DSM 15077 T 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 UniB2 T and UniB3 T represent two novel species named Paenibacillus albicerus sp. nov. and Niallia alba sp. nov. are proposed, respectively.
Description of Niallia alba sp. nov.
The type strain UniB3 T (=MCC 3998 =KCTC 43235 =JCM 34492) was isolated from digestive syrup from Pune, India. Author contributions VT, KK, PD and PK carried out the polyphasic taxonomy experiments; VT, KK, and BT did the phenotypic and genome data analysis and wrote the rst draft of the manuscript. YS and SS provided the project funding and in house facilities. TL and AY did the formal analysis, reviewed, edited and nalized the manuscript. AY conceptualized, lead the investigation and provided the funding for the study. All the authors reviewed and approved the nal version of the paper.
Compliance with ethical standards

Con icts of interest
The authors declare that there are no con icts of interest.

Ethical statement
No human or animal subjects were recruited in this study.       Figure 1 The combined pan-genome phylogenetic tree of strain UniB2T based on orthologous protein and gene sequences derived using BPGA and UBCG tools. The BPGA-and UBCG-based phylogenetic trees were reconstructed in MEGA 7 using the neighbour-joining method and maximum likelihood methods, respectively, at 1000 bootstrap replicates. Figures at branch points are bootstrap values seen in trees obtained using BPGA and UBCG, respectively. The respective sequences of Niallia circulans ATCC 4513 (BCVE00000000) were used as an outgroup. Bar indicates the number of substitutions per site.

Figure 2
The combined pan-genome phylogenetic tree of strain UniB3T based on orthologous protein and gene sequences derived using BPGA and UBCG tools. The BPGA-and UBCG-based phylogenetic trees were reconstructed in MEGA 7 using the neighbour-joining method and maximum likelihood methods, respectively, at 1000 bootstrap replicates.