Alkalihalobacterium elongatum gen. nov. sp. nov.: An Antibiotic-Producing Bacterium Isolated From Lonar Lake and Reclassification of the Genus Alkalihalobacillus Into Seven Novel Genera

A Gram-stain positive, long, rod-shaped, motile, and spore-forming bacterium (MEB199T) was isolated from a sediment sample collected from Lonar Lake, India. The strain was oxidase and catalase positive. The strain grew optimally at pH 10, NaCl concentration of 3.5% at 37°C. The major fatty acids were iso-C15:0, iso-C16:0, anteiso-C15:0, and iso-C17:0. The peptidoglycan contained meso-diaminopimelic acid (meso-DAP). Phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol were the major polar lipids of MEB199T. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain MEB199T belonged to the family Bacillaceae and exhibited a distinctive position among the members of the genus Alkalihalobacillus (Ahb.). Strain MEB199T shared the highest 16S rRNA gene sequence similarity with Alkalihalobacillus alkalinitrilicus ANL-iso4T (98.36%), whereas with type species Ahb. alcalophilus DSM 485T, it is 94.91%, indicating that strain MEB199T is distinctly related to the genus Alkalihalobacillus. The G + C content of genomic DNA was 36.47 mol%. The digital DNA–DNA hybridization (dDDH) (23.6%) and average nucleotide identity (ANI) (81%) values between strain MEB199T and Ahb. alkalinitrilicus ANL-iso4T confirmed the novelty of this new species. The pairwise identity based on the 16S rRNA gene sequence between the species of genus Alkalihalobacillus ranges from 87.4 to 99.81% indicating the heterogeneity in the genus. The different phylogenetic analysis based on the genome showed that the members of the genus Alkalihalobacillus separated into eight distinct clades. The intra-clade average amino acid identity (AAI) and percentage of conserved proteins (POCP) range from 52 to 68% and 37 to 59%, respectively, which are interspersed on the intra-genera cutoff values; therefore, we reassess the taxonomy of genus Alkalihalobacillus. The phenotypic analysis also corroborated the differentiation between these clades. Based on the phylogenetic analysis, genomic indices, and phenotypic traits, we propose the reclassification of the genus Alkalihalobacillus into seven new genera for which the names Alkalihalobacterium gen. nov., Halalkalibacterium gen. nov., Halalkalibacter gen. nov., Shouchella gen. nov., Pseudalkalibacillus gen. nov., Alkalicoccobacillus gen. nov., and Alkalihalophilus gen. nov. are proposed and provide an emended description of Alkalihalobacillus sensu stricto. Also, we propose the Ahb. okuhidensis as a heterotypic synonym of Alkalihalobacillus halodurans. Based on the polyphasic taxonomic analysis, strain MEB199T represents a novel species of newly proposed genus for which the name Alkalihalobacterium elongatum gen. nov. sp. nov. is proposed. The type strain is MEB199T (= MCC 2982T, = JCM 33704T, = NBRC 114256T, = CGMCC 1.17254T).

A Gram-stain positive, long, rod-shaped, motile, and spore-forming bacterium (MEB199 T ) was isolated from a sediment sample collected from Lonar Lake, India. The strain was oxidase and catalase positive. The strain grew optimally at pH 10, NaCl concentration of 3.5% at 37 • C. The major fatty acids were iso-C 15:0 , iso-C 16:0 , anteiso-C 15:0 , and iso-C 17:0 . The peptidoglycan contained meso-diaminopimelic acid (meso-DAP). Phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol were the major polar lipids of MEB199 T . Phylogenetic analysis based on 16S rRNA gene sequence showed that strain MEB199 T belonged to the family Bacillaceae and exhibited a distinctive position among the members of the genus Alkalihalobacillus (Ahb.). Strain MEB199 T shared the highest 16S rRNA gene sequence similarity with Alkalihalobacillus alkalinitrilicus ANL-iso4 T (98.36%), whereas with type species Ahb. alcalophilus DSM 485 T , it is 94.91%, indicating that strain MEB199 T is distinctly related to the genus Alkalihalobacillus. The G + C content of genomic DNA was 36.47 mol%. The digital DNA-DNA hybridization (dDDH) (23.6%) and average nucleotide identity (ANI) (81%) values between strain MEB199 T and Ahb. alkalinitrilicus ANL-iso4 T confirmed the novelty of this new species. The pairwise identity based on the 16S rRNA gene sequence between the species of genus Alkalihalobacillus ranges from 87.4 to 99.81% indicating the heterogeneity in the genus. The different phylogenetic analysis based on the genome showed that the members of the genus Alkalihalobacillus separated into eight distinct clades. The intra-clade average amino acid identity (AAI) and percentage of conserved proteins (POCP) range from 52 to 68% and 37 to 59%, respectively, which are interspersed on the intra-genera cutoff values; therefore, we reassess the taxonomy of genus Alkalihalobacillus. The phenotypic analysis also corroborated the differentiation between these clades. Based on the phylogenetic analysis, genomic indices, and phenotypic traits, we propose the reclassification of the genus Alkalihalobacillus into seven new genera for which the names Alkalihalobacterium gen. nov., Halalkalibacterium gen. nov., Halalkalibacter gen. nov., Shouchella gen. nov., Pseudalkalibacillus gen. nov.,

INTRODUCTION
The genus Bacillus is an extremely diverse group of bacteria within the phylum Firmicutes whose members currently exhibit great phylogenetic and phenotypic diversity. Numerous species that are part of this genus are unrelated to the type species as they do not share a common evolutionary history (La Duc et al., 2004). Recently, using phylogenomic approaches resolved the issue of the phylogenetic heterogeneity of the genus Bacillus by reclassifying existing species into novel genera, such as Alkalihalobacillus (Ahb.), Cytobacillus, Mesobacillus, Metabacillus, Neobacillus, and Peribacillus . Among all these genera, the genus Alkalihalobacillus consists of rod-shaped, endospore-forming, and Gram-stainvariable bacteria included in the family Bacillaceae with the type species Alkalihalobacillus alcalophilus. Based on phylogenomic studies, it was proposed that most of the members of the genus Alkalihalobacillus exclusively shared 10 CSIs found in the different proteins . The genus Alkalihalobacillus contains 39 species. 1 Most species of this genus are aerobic, but some members are facultative anaerobic and anaerobic. Species are found to be motile by peritrichous flagella, while a few members are non-motile. Members of genus Alkalihalobacillus were isolated from diverse environments including Soda lake soil/sediment, saltpan, hypersaline lake, mushroom compost, seawater, sea urchin, guts of larvae, feces, rhizosphere soil, non-saline forest soil, mud goldmine, mangrove sediment, mural paintings, etc., (Vedder, 1934;Nielsen et al., 1995;Li et al., 2002;Heyrman et al., 2003;Yumoto et al., 2003Yumoto et al., , 2005Ivanova et al., 2004;Santini et al., 2004;Yoon et al., 2004;Nogi et al., 2005;Olivera et al., 2005;Vargas et al., 2005;Nowlan et al., 2006;Borchert et al., 2007;Ghosh et al., 2007;Sorokin et al., 2008;Aizawa et al., 2010;Denizci et al., 2010;Borsodi et al., 2011Borsodi et al., , 2017Chen et al., 2011a,b;Madhaiyan et al., 2011;Zhang et al., 2011;Nedashkovskaya et al., 2012;Lei et al., 2014;Zhu et al., 2014;Reddy et al., 2015;Dou et al., 2016;Song et al., 2016;Singh et al., 2018;Liu et al., 2019;Gupta et al., 2020;Mo et al., 2020;Patel and Gupta, 2020;Shin et al., 2020). The majority of species from this genus are alkaliphilic and can grow in the pH range of 6-11 with optimum growth at pH 9-10. Some of the members are found to be obligately alkaliphilic in nature. The members are halotolerant or halophilic in nature as they grow in the presence of 1-5% w/v NaCl concentration. Members of this genus are mesophilic and grow at a temperature from 4 to 45 • C with optimum growth at 25-37 • C. Several species from this genus are of considerable industrial interest due to the production of enzymes such as cellulases, xylanases, proteases, and cyclodextrin glucanotransferase. Ahb. rhizosphaerae are diazotrophic, while Ahb. clausii exhibit probiotic activity (Nielsen et al., 1995;Madhaiyan et al., 2011).
While exploring the bacterial diversity of alkaline Lonar Lake, an antimicrobial compound producing alkaliphilic, moderately halophilic bacterial strain designated as MEB199 T was isolated from the sediment sample. Its taxonomic position was determined by employing a polyphasic taxonomic approach including whole genome-based analysis. During the assessment of the taxonomic status of the strain MEB199 T , it was observed that the genome-based phylogenetic analysis and overall genome relatedness index (OGRI) indicated that the genus Alkalihalobacillus is composed of heterogeneous members, and its reclassification is required. Apart from having phylogenetic differences, members of the genus Alkalihalobacillus also differ in phenotypic characters such as morphology, growth requirement, polar lipids, and fatty acid composition. Based on phenotypic characteristics, phylogenetic analysis, and OGRI, we propose the reclassification of genus Alkalihalobacillus into seven new genera and provide an emended description of the genus Alkalihalobacillus sensu stricto. Similarly, the combined phenotypic and genotypic analysis indicate that the strain MEB199 T represents a novel species of the newly proposed genus Alkalihalobacterium gen. nov., for which the name Alkalihalobacterium elongatum gen. nov. sp. nov. is proposed. Based on digital DNA-DNA hybridization (dDDH) and ANI value, it was noticed that Ahb. halodurans DSM 497 T and Ahb. okuhidensis DSM 13666 T belong to the same species. Therefore, we propose Ahb. okuhidensis as a heterotypic synonym of Ahb. halodurans.

Sample Collection and Bacterial Isolation
Strain MEB199 T was isolated from a sediment sample collected from Lonar, an Indian soda lake situated at Buldhana District, Maharashtra, India, at a depth of 0.46 m (1.5 ft) on October 27, 2010. At the time of sampling, the pH of the sample was found to be 9.8 and temperature was 28 • C. The sediment sample was serially diluted, spread on nutrient agar (pH 9.8; HiMedia, catalog no. M001), and incubated aerobically at 30 • C. Bacterial colonies were observed after 2 days, which were purified after three successive transfers to a fresh medium. Ahb. alkalinitrilicus DSM 22532 T was procured from DSMZ German Collection of Microorganisms and Cell Cultures GmbH. Desertibacillus haloalkaliphilus KJ1-10-99 T was shared with us by Dr. Hitarth B. Bhatt, Saurashtra University, Rajkot, Gujarat, India, as a gratis. All strains were grown on nutrient agar (pH 9.8) and preserved as glycerol (20% v/v) stock, which was stored at −80 • C and in liquid nitrogen.

Phenotypic, Physiological, and Biochemical Characterization
The phenotypic characterization of MEB199 T and Ahb. alkalinitrilicus DSM 22532 T was carried out under the same laboratory conditions. Morphological characteristics were studied following the growth on nutrient agar (pH 9.8) media (HiMedia, catalog no. M001) plates incubated at 37 • C for 48-72 h. Gram staining and spore staining were performed following standard procedures. Catalase and oxidase tests was carried as described earlier (Smibert and Krieg, 1994). Motility was checked by the hanging drop method. Hydrolysis of casein, starch, gelatin, nitrate, and nitrite were tested separately as reported previously (Smibert and Krieg, 1994). API 20E, API 20NE, API 50CH, API ZYM strips (bioMérieux, France), and BIOLOG GEN III plate were used to study the activities of constitutive enzymes, fermentation/oxidation profile, acid production, and substrate utilization as sole carbon and energy sources at 37 • C for 48 h according to the instructions of the manufacturers. The temperature range for growth was determined on nutrient agar (pH 10) plates by incubating cultures at 4-45 • C (4, 10, 20, 28, 37, and 45 • C) for 72-96 h. Tolerance to various NaCl concentrations and pH were investigated using salt basal medium (SBM) as described earlier (Dimitriu et al., 2005) by measuring the optical densities (wavelength 600 nm) at 37 • C up to 96 h. Tolerance to NaCl was tested using SBM with various NaCl concentrations (0-10%, w/v, at intervals of 0.5%). Growth was assessed in SBM adjusted to pH 7-11 at intervals of 0.5 pH unit by KH 2 PO 4 /K 2 HPO 4 or Na 2 CO 3 /NaHCO 3 buffer system. All parameters (temperature, NaCl concentration, and pH of the medium) were tested in triplicate.

Chemotaxonomic Analyses
For cellular fatty acid analysis, strain MEB199 T and Ahb. alkalinitrilicus DSM 22532 T were grown on nutrient agar (pH 10) plates at 37 • C for 16 h and collected at the same physiological age (at a logarithmic phase of growth). Cellular fatty acid methyl esters (FAMEs) were obtained from cells by saponification, methylation, and extraction following the protocol of MIDI. Cellular FAMEs were separated by gas chromatography (7890N, Agilent Technologies) and analyzed using the Sherlock Microbial Identification System (MIDI with database RTSBA6) according to the protocol described by the Sherlock Microbial Identification System. Cell wall samples were prepared from approximately 3 g of wet cells. Whole-cell hydrolyzates were prepared (6 M HCl, 100 • C, 18 h) and examined by thin layer chromatography (TLC) on cellulose plates using n-butanol:water:acetic acid (50:25:25, v/v) as the solvent system. Polar lipids were extracted from both the strains and analyzed. The cultures were harvested at a logarithmic phase, and the pellet was used for polar lipid extraction with methanol/chloroform/0.3% sodium chloride (2:1:0.8, by vol.) as described by Bligh and Dyer (1959) considering the modifications of Card (1973). Lipids were separated using silica gel TLC (Kieselgel 60 F254; Merck) by twodimensional chromatography using chloroform:methanol:water (65:25:4 by vol.) in the first dimension and chloroform:acetic acid:methanol:water (40:7.5:6:2, by vol.) in the second dimension (Minnikin et al., 1984). The dried plates were subjected to spraying with 5% ethanolic phosphomolybdic acid for total lipids and further characterized by spraying with ninhydrin (specific for amino groups), molybdenum blue (specific for phosphates), Dragendorff (quaternary nitrogen), or α-naphthol (specific for sugars).

Genomic DNA Isolation and 16S rRNA Gene Sequence Analysis
For DNA extraction, strain MEB199 T and Desertibacillus haloalkaliphilus KJ1-10-99 T were grown on nutrient agar (pH 10) medium and incubated at 37 • C for 48-96 h. Genomic DNA was extracted as described earlier, and 16S rRNA genes were amplified using the universal primer set 27F (5 -AGAGTTTGATCMTGGCTCAG-3 ) and 1488R (5 -CGGTTACCTTGTTACGACTTCACC-3 ) (Brosius et al., 1978). Purified PCR products were sequenced on both strands on an ABI 3730 xl DNA analyzer using the Big Dye terminator kit (Applied Biosystems). The sequence was compared with the 16S rRNA gene sequence available at the EzBioCloud database (Yoon et al., 2017).

Genome Sequencing and Annotations
The genomes of MEB199 T and Desertibacillus haloalkaliphilus KJ1-10-99 T were sequenced on the Illumina MiSeq (250 × 2 chemistry) platform. The reads were assembled using SPAdes (version 3.1.3.0), and the quality of the assembly was checked using QUAST (version 5.0.2). The obtained genome sequence was subsequently deposited in NCBI. The Gene prediction was performed using GeneMarkS and validated with the prokaryotic annotation pipeline of NCBI, PGAP. The Rapid Annotations using Subsystem Technology (RAST) server was used for the genome annotations. 2 The GenBank accession numbers for the 16S rRNA gene sequence and draft genome sequence of strain MEB199 T are KX171019 and WMKZ00000000, respectively.

Genome Sequences Used in This Study and Pathway Analysis
Type strains of 41 species of the genus Alkalihalobacillus (28 type strains), Desertibacillus, and Anaerobacillus and type species of the different genera of the family Bacilla, whose genomes were available in the public database, were considered in this study. Fifty genomes of non-type strains of genus Alkalihalobacillus were also included in this study. Streptococcus gordonii ATCC 10558 T and Streptococcus agalactiae ATCC 13813 T genomes were used to root (outgroup) the phylogenetic tree. The genome of strain MEB199 T and Desertibacillus haloalkaliphilus KJ1-10-99 T were sequenced under this study, and all the other genomes were downloaded from the PATRIC database (Supplementary Table 1). The functional annotations of each genome was carried out using EggNOG-mapper v2 (Cantalapiedra et al., 2021). The pathway was mapped using the KEGG Orthology (KO) Database 3 for all the selected genomes. Heatmap to visualize the distribution of pathways across all the members of the genus Alkalihalobacillus was constructed using heatmapper. 4

Production of Secondary Metabolites
The antiSMASH bacterial version 6.0 was used to understand the secondary metabolite biosynthetic gene clusters in strain MEB199 T (Blin et al., 2021). To check the antibacterial activity of the compound produced by MEB199 T , the aqueous extract of strain MEB199 T was tested for its activity against four multidrugresistant (MDR, resistant to three or more antimicrobial classes) pathogens: Acinetobacter baumannii BAC01, Escherichia coli BAC03, Staphylococcus aureus MCC 2043 T , and Klebsiella pneumoniae BAC02 using the agar overlay method. All the above pathogens used in the present study are clinical isolates resistant to more than six antibiotics. The strain MEB199 T was grown aerobically in nutrient broth (pH 10) medium under shaking conditions (150 rev min −1 ) for 96 h at 37 • C. After incubation, the culture broth was centrifuged at 16,770 × g for 30 min. The supernatant was filtered through a filter of 0.22-µm pore size. The filtrate was concentrated 10-fold by lyophilization. The antimicrobial activity of the extract was carried out using a well diffusion method where 50 µl of the concentrated filtrate was added to wells (6-mm diameter) in Mueller-Hinton agar plates containing pathogenic indicator strains and incubated at 37 • C for 96 h. The inhibition of growth was expressed as the diameter of the zone of inhibition around the well. All tests were carried out in triplicate.

Phylogenetic Analysis
The 16S rRNA gene sequences of all the Alkalihalobacillus spp. and related members were retrieved from the NCBI database. Using the Up-to-date bacterial core gene (UBCG) tool, 92 core genes were extracted from all the genomes including two outgroups i.e., Streptococcus gordonii ATCC 10558 T and Streptococcus agalactiae ATCC 13813 T (Na et al., 2018). A concatenated sequence was used to construct the phylogenetic tree using MEGA7. The distance was calculated with Kimura two-parameter as a model of nucleotide substitution, in pairwise deletion procedure, Poisson model. The phylogenetic tree was constructed using the neighbor-joining (NJ), maximumparsimony (MP), and maximum-likelihood (ML) methods with bootstrap analysis of 1,000 resamplings in the MEGA7 software package (Kumar et al., 2016). To assess the taxonomic position of the strain MEB199 T , a codon tree based on 500 single-copy genes was reconstructed using amino acid and nucleotide sequences as described in Suresh et al. (2019). The Genome Taxonomy Database toolkit (GTDB-Tk) was used to construct the phylogenetic tree from the genome sequences (Chaumeil et al., 2019).

Pan-Genome, Conserved Signature Indels, and Genomic Indices
The pan-genome of the species of the genus Alkalihalobacillus was analyzed by the Bacterial Pan Genome Analysis (BPGA) software (Chaudhari et al., 2016). To understand the interspecies variation and core genome, BPGA was used at its default parameters. Similarly, the pan-genome of the proposed genera was analyzed independently. Conserved signature indels (CSIs) were identified using protein sequences from the core proteins present in the members of the genus Alkalihalobacillus as described by Gupta (2014). Multiple sequence alignments were performed using Clustal Omega. 5 The alignments were visually inspected for sequence gaps (insertion or deletion) of fixed lengths. Average nucleotide identity (ANI) analysis between the species of the genus Alkalihalobacillus was performed by using the ANI calculator. 6 The dDDH was calculated using the genometo-genome distance calculator using the HSP length, and formula 2 values were considered in this analysis (Goris et al., 2007). The percentage of conserved proteins (POCP) and average amino acid identity (AAI) for the genus level delineation were calculated. The POCP was calculated as described by Qin et al. (2014), and the AAI was computed using an online ANI/AAI-Matrix calculator. 7

Isolation of Strain MEB199 T and Morphology
During the exploration of bacterial diversity of the alkaline saline Lonar Lake, a strain MEB199 T was isolated in nutrient agar (pH 10) from a sediment sample. Cells of strain MEB199 T showed 2-to 5-mm, cream-colored, flat, and dry colonies with irregular margins on nutrient agar (pH 10) medium after 48 h at 37 • C. The cells of the strain MEB199 T was Gram stain positive, motile, long thick rods (6.4-16.5 × 0.6-2.4 µm), and spore forming. The strain was oxidase and catalase positive. The strain MEB199 T is alkaliphilic and halophilic, grew optimally at pH 10, at an NaCl concentration of 3.5%.

Physiological Characterization of Strain MEB199 T
The strain MEB199 T and Ahb. alkalinitrilicus DSM 22532 T were tested for utilization and assimilation of various carbon sources and enzyme activity against different substrates by API (bioMérieux, France) and BIOLOG GEN III plate. The differential morphological, physiological, and biochemical characteristics of strain MEB199 T and Ahb. alkalinitrilicus DSM 22532 T are given in Table 1. In the BIOLOG GEN III plate, MEB199 T was positive for various substrates like gentiobiose, D-melibiose, α-D-glucose, D-fructose, D-galactose, 3-methyl glucose, D-fucose, L-fucose, L-rhamnose, D-fructose-6-PO 4 , Dgalacturonic acid, D-glucuronic acid, glucuronamid, and sodium butyrate. The strain is negative for acetoacetic acid and acetic acid, while its closest phylogenetic neighbor, Ahb. alkalinitrilicus DSM 22532 T , was positive for those substrates. The strain MEB199 T showed a weak positive reaction for propionic acid, while Ahb. alkalinitrilicus DSM 22532 T showed negative activity for that substrate. In the API ZYM system, both the strains under study tested positive for the production of enzymes like leucine arylamidase, valine arylamidase, α-chymotrypsin, napthol AS-BI-phosphohydrolase, and α-glucosidase. Ahb. alkalinitrilicus DSM 22532 T could be able to produce enzymes like esterase (C4) and esterase lipase (C8), while MEB199 T could not. Acid phosphatases and ß-glucosidase were produced by MEB199 T and not found in Ahb. alkalinitrilicus DSM 22532 T ; this differentiated the novel strain from the type strain DSM 22532 T . In the API 20E system, MEB199 T showed negative results for Voges Proskauer and sucrose fermentation, while Ahb. alkalinitrilicus showed positive results for both tests. Strain MEB199 T reduced nitrate to nitrite, hydrolyze esculin, and could assimilate mannitol and malate, while Ahb. alkalinitrilicus showed negative results for all those substrates but could assimilate maltose in the API 20 NE system. The physiological characteristics using BIOLOG GEN III and API analyses provided further support to investigate strain MEB199 T for its unique taxonomic position.

16S rRNA Sequence and Analysis
The complete (1,551 bp) 16S rRNA gene sequence of strain MEB199 T was used for sequence and phylogenetic analysis. Based on EzTaxon-e search analysis, the closest phylogenetic neighbor of strain MEB199 T is Ahb. alkalinitrilicus DSM 22532 T , with which it shared 98.36% sequence similarity, followed by Desertibacillus haloalkaliphilus KJ1-10-99 T (97.10%), Anaerobacillus alkaliphilus B16-10 T (96.72%), and Anaerobacillus isosaccharinicus NB2006 T (96.59%). It showed a similarity of <96% with other species. However, the pairwise sequence identity level between strain MEB199 T and Ahb. alcalophilus DSM485 T , the type species of the genus Alkalihalobacillus, was 94.91%, which indicates that strain MEB199 T might not be a member of the genus Alkalihalobacillus.

16S rRNA Phylogeny
The phylogenetic tree based on the 16S rRNA gene placed strain MEB199 T and a closely related Ahb. alkalinitrilicus DSM 22532 T in a separate clade (Figure 1). The phylogenetic trees constructed using MP and ML methods revealed a similar tree topology with the common node with Ahb. alkalinitrilicus DSM 22532 T , which confirmed a close similarity between these two strains. The genus Alkalihalobacillus was separated into eight different clades, which were referred to as Clade I, Clade II, Clade III, Clade IV, Clade V, Clade VI, Clade VII, and Clade VIII in the subsequent discussion (Figure 1). It is interesting to point out that the 16S rRNA gene sequence similarities between species of the genus Alkalihalobacillus ranged from 87.40 to 99.81% (Supplementary Table 2). The pairwise distance of 16S rRNA gene sequence identity value of <95 indicates affiliation with different genera (Rosselló-Móra and Amann, 2015). This wide range (87.40-99.81%) of 16S rRNA gene sequence similarity indicates heterogeneity in the genus Alkalihalobacillus and signposts the need to reassess the taxonomy of the genus. The pairwise sequence identity among the Alkalihalobacillus species showed that 16S rRNA gene sequences have limited power, and to better resolve the taxonomic affiliation in the members of the genus Alkalihalobacillus, other approaches available in the genomic era have to be investigated.

Whole-Genome Phylogeny
Phylogenomic analysis based on the core genome made up of 92 genes and codon tree of the recently described genera by Patel and Gupta (2020) as well as the remaining species in the genus Alkalihalobacillus formed well-supported eight clades (Figure 2 and Supplementary Figure 2). The phylogenomic tree based on 120 ubiquitous single-copy proteins was also constructed using GTDB-Tk, which also separates the genus Alkalihalobacillus into eight clades (Supplementary Figure 3).  (Figure 2).

Analysis of Core and Pan-Genome of the Genus Alkalihalobacillus
Bacterial pan-genome analysis was carried out between the type strains of the genus Alkalihalobacillus. In the 28 analyzed species of the genus Alkalihalobacillus, 598 genes were identified as core genes indicating that members of the genus Alkalihalobacillus share a very small number of core genes. The core genes encode for the shikimate pathway, isoprenoid biosynthesis (nonmevalonate pathway), thiamine biosynthesis, tetrahydrofolate biosynthesis, pantothenate biosynthesis, lysine biosynthesis, riboflavin biosynthesis, inosine monophosphate biosynthesis, uridine monophosphate biosynthesis, F-type ATPase, heme biosynthesis, pyrimidine deoxyribonucleotide biosynthesis, citrate cycle (TCA cycle, Krebs cycle), lysine biosynthesis, coenzyme A biosynthesis, glycolysis (Embden-Meyerhof pathway), pantothenate biosynthesis, gluconeogenesis, glycolysis, pyruvate oxidation, adenine ribonucleotide biosynthesis, NAD biosynthesis, guanine ribonucleotide biosynthesis, UDP-N-acetyl-D-glucosamine biosynthesis, and dicarboxylatehydroxybutyrate cycle. The core genome analysis was also carried out for the individual clade formed in the phylogenomic tree. Clade I shares 1,991 as core genes with 3,024 genes as accessory genomes. Clade II shares 1,873 core genes with 3,128 accessory    genes (Figure 3 and Table 3). Clade III shares 2,070 core genes and 1,942 as accessory genes. Clade IV shares 1,482 as core genes, and Clade V shares 1,195 core genes. Clade VI consists of only two members of the genus Alkalihalobacillus, which shares 2,233 core genes between them (Figure 3 and Table 3).
In the clade-wise pan-genome analysis, there was an increase in the number of core genes, which showed the divergence in inter-clade genomes.
FIGURE 4 | Heatmap showing functional potential of all the members of the genus Alkalihalobacillus. Functional annotations were performed using EggNOG, and pathways were reconstructed using the KEGG Orthology (KO) Database server, and heatmap was generated by the Heatmapper using average linkage clustering method and Spearman rank correlation distance measurement method. The color variations depict the relative abundance of genes in the pathways wherein red denotes the maximally abundant pathways, and green represents the least abundant pathways.

Functional Analysis and Significance of Genus Alkalihalobacillus
The members of the genus Alkalihalobacillus are an industrially important group of bacteria, which have been isolated from diverse ecological niches (Jones et al., 1998;Grant, 2003). They are likely to play an important but yet unexplored role in the functional stability and maintenance of the ecosystem. Several species from this genus are of considerable industrial interest due to their production of enzymes such as cellulases, proteases for inclusion in laundry detergents, xylanases for use in the pulp paper industry, and cyclodextrin glucanotransferase for manufacture of cyclodextrin from starch (Horikoshi, 2006). The genus Alkalihalobacillus is attracting interest because its members have a great biotechnological potential for producing compatible solutes or hydrolytic enzymes (Horikoshi, 1999;Margesin and Schinner, 2001;Arahal and Ventosa, 2002;Krulwich et al., 2007). Some of these bacterial species are believed to have industrial potential as a source of alkali-stable enzymes (Gessesse and Gashe, 1997). Ahb. patagoniensis, Ahb. lehensis, and Ahb. marmarensis are producers of alkaline proteases, while Ahb. lonarensis and Ahb. oshimensis could produce various protease, lipase, and xylanase enzymes. Obligately alkaliphilic species, Ahb. krulwichiae, can degrade aromatic compounds in alkaline conditions, while Ahb. ligniniphilus, a halotolerant alkaliphilic bacterium, is used to degrade lignin (Zhu et al., 2017). Ahb. rhizosphaerae, which is diazotrophic and can fix atmospheric nitrogen to ammonia and other species such as Alkalihalobacillus clausii, exhibits probiotic activity due to the production of antimicrobial compounds (Nielsen et al., 1995;Madhaiyan et al., 2011). The strain MEB199 T also showed an antimicrobial compound-producing activity. The heatmap clustering based on the distributions of metabolic pathways in the genomes is shown in Figure 4. The clusters formed in the heatmap corroborate the clades in phylogenomic analysis except Ahb. ligniniphilus L1 T , Ahb. hemicellulosilyticus DSM 16731 T , and Ahb. lonarensis 25nlg T . The genome mining showed that the 11 subunits of NADH:quinone oxidoreductase (nuoA, nuoB, nuoC, nuoD, nuoH, nuoI, nuoJ, nuoK, nuoL, nuoM, and nuoN) was exclusively present in the members of Clade I, Clade VI, and Alkalihalobacillus ligniniphilus L1 T and absent in all the other members of genus Alkalihalobacillus. The genetic capability to synthesize the isoprenoid compounds and terpenoid was screened in the genomes. All the members of the genus Alkalihalobacillus harbors methylerythritol phosphate (MEP) i.e., non-mevalonate pathway for the synthesis of isoprenoid precursors isopentenyl pyrophosphate (IPP). IPP is the precursor for the various isoprenoid molecules playing diverse roles in different processes in the bacterial cell. Menaquinones, a lipid-soluble quinone, are isoprenoid compounds that participate in the electron transport chain. All the members of the genus Alkalihalobacillus has the menaquinone (MK) biosynthesis pathway, but an alternative futalosine pathway to convert chorismate to 1,4-dihydroxy-6naphthoate requires four enzymes encoded by mqnABCD, which are absent in Clade VI (Ahb. macyae DSM 16346 T and Ahb. caeni HB172195 T ) and Ahb. murimartini LMG 21005 T . The classical MK pathway is exclusively present in all aerobic and facultatively anaerobic bacteria in contrast to the futalosine pathway, which is present in aerobic and anaerobic bacteria. All the Clade I, Clade VI, and VII members have sporulenol synthase gene, which gives the genetic capability to cyclize tetraprenyl beta-curcumene into sporulenol (C35 terpenes), a pentacyclic sesquarterpene (Sato et al., 2011). Sporulenol, produced during the sporulation, is present in the spores and increases the resistance to reactive oxygen species (Kontnik et al., 2008).

Description of the new combinations in the genus
The description of this taxon is as given by Nogi et al. (2005) N-1 T (= DSM 2521 = JCM 9140) Halalkalibacter okhensis comb. nov. (ok.hen'sis. N.L. masc. adj. okhensis pertaining to Port Okha, a port of the Dwarka region in India, where the type strain was isolated) Alkalihalobacillus okhensis (Nowlan et al., 2006) Patel and Gupta, 2020 The description of this taxon is as given by Nowlan et al. (2006) GMBE 72 T (= DSM 21297 = JCM 15719) Halalkalibacter akibai comb. nov. (a.ki.ba'i. N.L. gen. n. akibai of Akiba, named after the Japanese microbiologist Teruhiko Akiba, who made fundamental contributions to the study of alkaliphilic bacteria) Alkalihalobacillus akibai (Nogi et al., 2005) Patel and Gupta, 2020 The description of this taxon is as given by Nogi et al. (2005) 1139  Alkalihalobacillus clausii (Nielsen et al., 1995) Patel and Gupta, 2020 The description of this taxon is as given by Nielsen et al. (1995) PN Alkalihalobacillus patagoniensis (Olivera et al., 2005) Patel and Gupta, 2020 The description of this taxon is as given by Olivera et al. (2005) PAT 5  Alkalihalobacillus plakortidis (Borchert et al., 2007) Patel and  The description of this taxon is as given by Borchert et al. The description of this taxon is as given by Lei et al. (2014) HNA-14 T (= DSM 26902 = KCTC 33145) Shouchella lonarensis comb. nov. (lo.nar.en'sis. N.L. fem. adj. lonarensis of or belonging to Lonar lake, India, from where the type strain was isolated) Alkalihalobacillus lonarensis (Reddy et al., 2015) Patel and Gupta, 2020 The description of this taxon is as given by Reddy et al., 2015 LMG 27974 T (= CGMCC 1.12817 = KCTC 33413 = 25 nlg) Shouchella hunanensis comb. nov. (hu.nan.en'sis. N.L. fem. adj. hunanensis, pertaining to Hunan Province, PR China, the source of the sample from which the type strain was isolated)

Description of the new combinations in the genus Pseudalkalibacillus
Pseudalkalibacillus decolorationis comb. nov.
(Type species of this genus) (de.co.lo.ra.ti.o'nis. L. gen. n. decolorationis of discoloration) Alkalihalobacillus decolorationis (Heyrman et al., 2003) Patel and Gupta, 2020 The description of this taxon is as given by Heyrman et al.

Description of the new combinations in the genus Alkalihalophilus
Alkalihalophilus pseudofirmus comb. nov.

Use of Average Nucleotide Identity and Digital DNA-DNA Hybridization for Species Delineation
The ANI value between MEB199 T and Ahb. alkalinitrilicus strain DSM 22532 T was 81%. The in silico dDDH was carried out using the genome-to-genome distance calculator 8 between the strain MEB199 T and Ahb. alkalinitrilicus DSM 22532 T . for the type species of the genus Alkalihalobacillus as well as between the newly described genera in the family Bacillaceae (Figure 5 and Supplementary Table 5). Consequently, strain MEB199 T , together with its close phylogenetic neighbors, i.e., Ahb. alkalinitrilicus DSM 22532 T and Ahb. bogoriensis ATCC BAA-922 T , are considered to represent a novel genus within the family Bacillaceae.
To confirm whether the clades observed in the phylogenomic tree might represent different genera, the genomic indices POCP and AAI were also calculated with the species of genus Alkalihalobacillus (Supplementary Table 5). Considering recent work on genus delineation based on mean protein sequence similarity of all protein-coding genes, members of the family Bacillaceae can be distinguished by 65-70% AAI value at the genus level (Aliyu et al., 2016). The inter-clade AAI values ranged from 52 to 68% indicating that the genus Alkalihalobacillus is divergent and polyphyletic (Figure 5 and Supplementary  Table 5). Although Qin et al. (2014) proposed <50% POCP to delineate the genera, POCP values between different newly proposed Bacillus genera ranged from 34.8 to 69.8% (Aliyu et al., 2016;Patel and Gupta, 2020). The inter-clade POCP values ranged from 37 to 68% (Figure 5 and Supplementary Table 5). The AAI and POCP values also indicate that the members of the genus Alkalihalobacillus are divergent, and there is a need for reclassification of the genus Alkalihalobacillus. Each clade in phylogenomic analysis represents a novel genus. A detailed survey of the phenotypic characters was carried out to determine if the description of new taxa at the genus level is possible, or such clades were only clusters or genomovars within the genus Alkalihalobacillus. Because the genomic analysis of 52 Arcobacter cryaerophilus strains indicate four different genomospecies, but the phenotypic study failed to delineate the species, therefore, they were considered genomovars of the same species (Pérez-Cataluña et al., 2018).

Conserved Signature Indels Specific for Different Monophyletic Clades of Alkalihalobacillus Species
Phylogenetic analysis (16S rRNA gene-based and genomebased) indicated the existence of polyphyletic clades of genus Alkalihalobacillus. The conserved signature insertions and deletions (CSIs) in the proteins are the rare genetic changes that are exclusively shared by evolutionary linked organisms. CSIs are useful molecular signatures for evolutionarily and taxonomic studies. Therefore, the CSIs specific for the novel clades of members of the Alkalihalobacillus was studied. Patel and Gupta have reported that six CSIs are the signature of the genus Alkalihalobacillus , whereas four CSI signatures in protein transcription-repair coupling factor, tRNA uridine-5-carboxymethylaminomethyl (34) synthesis enzyme (mnmG), 50S ribosomal protein L11 methyltransferase, and homoserine kinase were exclusively shared by all the members of the genus Alkalihalobacillus except Ahb. alkalinitrilicus and Ahb. bogoriensis. These results separate Clade I from the rest of the members of the genus Alkalihalobacillus. In the present study, CSIs that are distinctive characteristics of other clades were identified. The species of Clade II harbor the CSI of a two-amino acid insertion in the protein translocase subunit (secD) protein, which is absent in other members of the genus Alkalihalobacillus (Supplementary Figure 5). Members of Clade III consist of the CSI of a two-amino acid insertion in the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2, 6-diaminopimelate ligase (murE) protein, which is absent in other members of the genus Alkalihalobacillus (Supplementary Figure 6). Two CSIs exclusively present in the members of Clade IV and absent in other species of the genus Alkalihalobacillus were identified. Insertion of two amino acids in DNA mismatch repair (mutL) protein and one amino acid deletion in ATP-dependent protease ATPase subunit (hslU) protein were observed in the species of Clade IV (Supplementary Figures 7, 8). Similarly, two CSIs were exclusively present in the species of Clade V, which are fiveamino acid insertion in the protein translocase subunit (secY) protein and one-amino acid insertion in ATP-dependent protease ATPase subunit (hslU) protein (Supplementary Figures 9, 10). These CSIs in Clades I, II, III, IV, and V also showed the genetic distinctness of these clades formed in phylogenetic analysis and support the reclassification. We were not able to identify any CSIs for Clades VI, VII, and VIII.

Phenotypes to Support Reclassifications
Phylogenomic analysis as well as genomic indices, indicated that Alkalihalobacillus species are too divergent to be placed into the same genus. To support this further, phenotypic and chemotaxonomic markers were also surveyed to further understand its taxonomic position. The comparison of phenotypic characters between the clades and nearest genera is given in Table 4. The phenotypic characters are also able to distinguish these clades, as the members of Clade I are isolated from a soda lake, alkaliphilic, long thick rods, Gram-stain positive, G + C content range from 35.1 to 37.5%, and strictly aerobic in nature, whereas members of Clade II are facultative anaerobes, Gram-stain variable, G + C content range from 40.76 to 43.9 mol%, and motile (Table 4; Nielsen et al., 1995;Li et al., 2002;Vargas et al., 2005;Sorokin et al., 2008;Zhu et al., 2014). Members of Clade IV are alkaliphilic, quinone (MK-5, MK-6, or MK-7), the optimum temperature is 37 • C, oxidase negative, and contain glycolipid, which makes it unique from the rest of the members of the genus Alkalihalobacillus (Yumoto et al., 2003;Nogi et al., 2005;Nowlan et al., 2006;Borsodi et al., 2011Borsodi et al., , 2017Zhang et al., 2011;Song et al., 2016;Liu et al., 2019;Gupta et al., 2020). Clade V is composed of mesophilic and neutrophilic organisms that differentiate them from the rest of the Alkalihalobacillus species (Nielsen et al., 1995;Olivera et al., 2005;Yumoto et al., 2005;Ghosh et al., 2007;Chen et al., 2011a;Madhaiyan et al., 2011;Lei et al., 2014;Reddy et al., 2015;Singh et al., 2018;Gupta et al., 2020;Shin et al., 2020). Members of Clade VI can grow both aerobically and anaerobically and alkalitolerant with optimum growth at pH 7, whereas other members are alkaliphilic in nature (Heyrman et al., 2003;Ivanova et al., 2004;Santini et al., 2004;Yoon et al., 2004;Chen et al., 2011b;Nedashkovskaya et al., 2012;Mo et al., 2020). Ahb. murimartini LMG 21005 T is a neutrophilic, coccoidal-shaped bacterium that separates it from the rest of the members of the genus Alkalihalobacillus (Borchert et al., 2007). These phenotypic differences also indicate that there is a need for reclassification of the genus Alkalihalobacillus.

CONCLUSION
Based on phenotypic, genomic, phylogenetic, and chemotaxonomic characteristics, we propose the reclassification of genus Alkalihalobacillus into seven new genera with an emended description of the genus Alkalihalobacillus sensu stricto. We propose members of Clade I to be classified into a new genus for which we propose the name Alkalihalobacterium gen. nov. Though genomic indices showed that Ahb. bogoriensis is distinctly related to members of Clade I, we prefer to transfer Ahb. bogoriensis into Alkalihalobacterium gen. nov. for practical reasons to separate new genera until more strains related to this taxon become available. Therefore, Alkalihalobacterium alkalinitrilicus comb. nov. is proposed as type species of the newly proposed genus Alkalihalobacterium gen. and other members of the Clade IV ranges from 63 to 68%, which indicates that it belongs to a distinct genus. Therefore, we propose members of Clade VIII as a novel genus for which name Alkalihalophilus gen. nov. is proposed. The description of the newly proposed genera is given below, and a description of all names and new combinations is given in Table 5.

Emended Description of the Genus
Alkalihalobacillus  Members can be isolated from the guts of larvae, soil, and feces. Cells are rod shaped, Gram-stain positive, endospore forming, aerobic, and motile, and tolerate NaCl concentration up to 5-10% with optimum growth at 2% w/v. Growth occurs in the range 10-40 • C, with optimum growth at 30 • C; alkaliphilic with growth in the range of pH 8-10 with optimum growth at pH 9. The major isoprenoid quinone is MK-7. The major fatty acids are iso-C 15:0 , anteiso-C 15:0 , anteiso-C 17:0 , and iso-C 17:0 . The polar lipid profile contains diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and unidentified phospholipids. meso-DAP is cell wall diamino acid. The DNA G + C content is 36.2-39.0 mol%.
The type species is Alkalihalobacillus alcalophilus.
Taxonomic Note on Alkalihalobacillus okuhidensis (Li et al., 2002;Patel and Gupta, 2020) Based on ANI and dDDH analysis, Alkalihalobacillus okuhidensis is not a distinct species as the differences between Ahb. halodurans and Ahb. okuhidensis represent intra-species divergence. It should be noted that Ahb. halodurans and Ahb. okuhidensis differ with regard to fatty acid content, G + C content, growth at pH 6 and 11, utilization of carbon sources, hydrolysis of hippurate, tween 20, tween 40, and tween 60 (Li et al., 2002). Though there are minor differences in Ahb. halodurans and Ahb. okuhidensis but not enough for delineating two species. Therefore, based on the physiological, chemotaxonomic, and genotypic analysis, we propose Ahb. okuhidensis as a heterotypic synonym of Alkalihalobacillus halodurans.
Description of Alkalihalobacterium gen. nov.
The type species is Alkalihalobacterium alkalinitrilicum.
The type strain, MEB199 T (= MCC 2982 T = CGMCC 1.17254 T = JCM 33704), was isolated from a sediment sample collected from alkaline Lonar Lake, India. The DNA G + C content of the type strain is 36.47 mol%. The GenBank accession numbers for the 16S rRNA gene and draft genome sequence of strain MEB199 T are KX171019 and WMKZ00000000, respectively.
Description of Halalkalibacterium gen. nov.
Rod shaped, Gram-stain-variable. Members are aerobic or facultative anaerobes; endospore forming; have been isolated from sediments of the South China Sea/hot spa area, Motile, Tolerate NaCl concentration up to 12% with optimum growth at 2% (w/v). Growth occurs in the range 15-60 • C, with optimum growth temperature in the range 30-40 • C. All members of this genus are alkaliphilic with growth in the range of pH 6-11 with optimum growth at pH (9-10). The predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The major isoprenoid quinone is MK-7. The major fatty acids are iso-C 14:0 and anteiso-C 15:0 . meso-DAP is cell wall diamino acid. The DNA G + C content is 40.76-43.9 mol%.
The type species is Halalkalibacterium halodurans.
Description of Halalkalibacter gen. nov.
Halalkalibacter (Hal.al.ka.li.bac'ter. Gr. masc. n. hals, salt; Arabic n. al-qalyi, soda ash; N.L. masc. n. bacter, rod; N.L. masc. n. Halalkalibacter briny and alkaline media-loving rodshaped cells). Cells are rod shaped, Gram-stain-variable, endospore forming, motile or non-motile, Aerobic or facultative anaerobic in nature, have been isolated from mushroom compost, sediment sample from the sea, seawater, soda pond, saltpan/soil, tolerate NaCl concentration up to 12% with optimum growth at 3-7% characterisation of novel strain and maintained the bacterial cultures. TL carried out the genomic data retrieval from databases, phylogenomic, phylogenetic data analysis, and calculated the genomic indices. NJ carried out the FAME analysis. PK did the polar lipids profiling. All authors contributed to writing the manuscript and accepted it for publication.