Promotion of Bacillus Subtilis Subsp. Inaquosorum, Bacillus Subtilis Subsp

Antonie van Leeuwenhoek (2020) 113:1–12

https://doi.org/10.1007/s10482-019-01354-9 (0123456789().,-volV)( 0123456789().,-volV)

ORIGINAL PAPER

Promotion of Bacillus subtilis subsp. inaquosorum, Bacillus subtilis subsp. spizizenii and Bacillus subtilis subsp. stercoris to species status

Christopher A. Dunlap . Michael J. Bowman . Daniel R. Zeigler

Received: 17 October 2019 / Accepted: 26 October 2019 / Published online: 12 November 2019 Ó This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2019

Abstract Bacillus subtilis currently encompasses phenotypes. The results show that one difference four subspecies, Bacillus subtilis subsp. subtilis, among the subspecies is they produce different Bacillus subtilis subsp. inaquosorum, Bacillus subtilis bioactive secondary metabolites. B. subtilis subsp. subsp. spizizenii and Bacillus subtilis subsp. stercoris. spizizenii is shown conserve the genes to produce Several studies based on genomic comparisons have mycosubtilin, bacillaene and 3,30-neotrehalosadi- suggested these subspecies should be promoted to amine. B. subtilis subsp. inaquosorum is shown species status. Previously, one of the main reasons for conserve the genes to produce bacillomycin F, leaving them as subspecies was the lack of distin- fengycin and an unknown PKS/NRPS cluster. B. sub- guishing phenotypes. In this study, we used compar- tilis subsp. stercoris is shown conserve the genes to ative genomics to determine the genes unique to each produce fengycin and an unknown PKS/NRPS cluster. subspecies and used these to lead us to the unique While B. subtilis subsp. subtilis is shown to conserve the genes to produce 3,30-neotrehalosadiamine. In addition, we update the chemotaxonomy and pheno- Electronic supplementary material The online version of typing to support their promotion to species status. this article (https://doi.org/10.1007/s10482-019-01354-9) contains supplementary material, which is available to authorized Keywords Core genome Á Secondary metabolites Á users. Antifungal Á Antibiotic Á Lipopeptide Á Surfactin Á C. A. Dunlap (&) Bacilysin Á Subtilosin Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, IL, USA e-mail: [email protected]; Introduction [email protected] Bacillus subtilis was ﬁrst divided into two subspecies M. J. Bowman by Nakamura et al. (1999), based on a DNA related- Bioenergy Research Unit, National Center for Agricultural Utilization Research, Agricultural Research ness of 60–70% between the two sub-groups. In Service, United States Department of Agriculture, 1815 addition, cell walls of Bacillus subtilis subsp. spiz- North University Street, Peoria, IL, USA izenii were reported to contain ribitol or anhydroribi- tiol, while strains Bacillus subtilis subsp. subtilis did D. R. Zeigler Bacillus Genetic Stock Center, The Ohio State University, not (Nakamura et al. 1999). A third subspecies, Columbus, OH, USA 123 2 Antonie van Leeuwenhoek (2020) 113:1–12

Bacillus subtilis subsp. inaquosorum, was described strains. The core genome determination and subse- by Rooney et al. (2009) after identifying a unique quent alignments were produced for all the type strains clade during multilocus sequence analysis of the in the group with BIGSdb software (Jolley and Maiden B. subtilis species complex. B subtilis subsp. inaqu- 2010) and consists of 3501 genes. The phylogenetic osorum was reported to contain a unique MALDI-TOF tree was constructed using MEGA X software (Kumar MS biomarker at m/z 1120 as a distinguishing et al. 2018). The neighbor-joining tree was determined phenotype among the subspecies. A fourth subspecies, using the Tamura-Nei model (0.40, gamma distributed Bacillus subtilis subsp. stercoris was described by with invariant sites) based on model testing under Adelskov et al. (2016), based on an average nucleotide MEGA X (Kumar et al. 2018). Measures of bootstrap identity of 95.6% between the genomes of B. subtilis support for internal branches were obtained from 1000 subsp. subtilis and B. subtilis subsp. stercoris (Adel- pseudoreplicates. Determination of the core genomes skov and Patel 2017). No distinguishing phenotypes and related comparison were generated with the were reported for B. subtilis subsp. stercoris (Adel- genome comparator function implemented under skov and Patel 2016). BIGSdb software (Jolley and Maiden 2010). A In recent years, the subspecies status of these strains complete genome served as a reference strain for each has come under question by several studies on the subspecies and was used to BLAST all genomes in the basis of average nucleotide identity of their genomes set. The determination of genes gained or lost by the (Brito et al. 2018; Dunlap et al. 2019; Knight et al. different groups was based on a 90/10 comparison. For 2018; Yi et al. 2014). One of the reasons previously example, the change in genes was determined at each cited for not deﬁning species was the lack of distin- branch point in the tree, the change is reported as the guishing phenotypes (Rooney et al. 2009). Our number of genes found in 90% of the genomes of the laboratories recently identiﬁed differences in iturinic branch, but only in 10% of the genomes from the lipopeptides as unique phenotypes for these sub- opposite branch. The average nucleotide identity species (Dunlap et al. 2019). This motivated us to re- (ANI) of the genomes was determined using examine these subspecies and perform comparative OrthoANI software (Lee et al. 2016). genomics to identify distinguishing phenotypes. Fatty acid analysis

Materials and methods Total cellular fatty acid content was measured using the MIDI protocol (Microbial Identiﬁcation Inc. Genomes Newark, DE) and analyzed on an Agilent 7890 Gas Chromatograph. The strains were grown for 24 h at All available genomes categorised as B. subtilis or 28 °C on TSA and prepared using the standard MIDI Bacillus sp. were downloaded from GenBank on May protocol for extraction and production of fatty acid 1, 2019 and combined with an in-house database of methyl esters. Bacillus genomes. A 6 gene MLSA was used to assign the genomes to the correct species and subspecies Morphology and physiology taxonomy (Rooney et al. 2009). Genomes of strains belonging to B. subtilis were used for further analysis. We tested the growth temperature range at 10, 14, 28, This set included genomes of B. subtilis subsp. subtilis 37, 45, 50 and 55 °C using tryptone-glucose-yeast- (n = 224), B. subtilis subsp. spizizenii (n = 27), B extract media (TGY, Difco). The pH range for growth subtilis subsp. inaquosorum (n = 27), B. subtilis was determined from pH 3.0 to pH 12.0 in steps of 0.5 subsp. stercoris (n = 7). pH unit in TGY broth buffered and adjusted with phosphate buffer, Tris/HCl buffer, HCl or NaOH Genome-based phylogeny and comparative (Breznak and Costilow 1994)at28 °C. NaCl tolerance genomics of Bacillus subtilis strains was investigated by using TGY broth supplemented with 0–12% (w/v) NaCl, in 1% increments at 28 °C. A core genome phylogeny of the strains was per- Growth under anaerobic conditions was determined on formed to show the taxonomic relationship of these anaerobic agar (Difco) at 30 °C using a GasPak jar 123 Antonie van Leeuwenhoek (2020) 113:1–12 3

(\ 1% O2; C 13% CO2) (Merck) for 7 days (Vos et al. Results and discussion 2009). Spore morphology was determined on 1/10

TSB ? 50 mg/L MnSO4 from samples grown at Genome-based phylogeny of Bacillus subtilis 28 °C for 48 h. The endospores were heat ﬁxed and strains imaged using a phase contrast microscope. Carbon source utilisation was tested using the OmniLogÒ Data The core genome phylogeny (Fig. 1) shows each Collection system (Biolog Inc, Hayward, CA). Strains species or subspecies forms a distinct clade which selected for characterization were cultured overnight contains all members of the group. The results also on Biolog universal growth plates and prepared show B. subtilis and its subspecies are not a mono- according to manufacturer’s instructions for the phyletic clade with Bacillus tequilensis and Bacillus GEN III MicroPlateTM test panel using protocol A vallismortis also falling within the clade. This analysis (Biolog Inc, Hayward, CA) at 33 °C. An OmniLogÒ is based on 3501 genes that comprise the core genome Data Collection instrument (Biolog Inc, Hayward, (genes found in 90% of the genomes) in this dataset, CA) was used to collect data in 15 min increments for which is somewhat biased towards B. subtilis subsp. 22 h. Catalase and oxidase activities were examined subtilis, since it has the most genomes (n = 224). In using 3% (v/v) hydrogen peroxide solution and 1% (w/ other core-genome analyses with fewer genes (991 v) tetramethyl-p-phenylenediamine dihydrochloride genes) spanning the B. subtilis group species complex, (Difco), respectively. we found B. subtilis and its subspecies to be a monophyletic clade (Dunlap et al. 2019). In addition to Mass spectrometry the genome phylogeny we conducted a 16S rRNA phylogeny to meet the minimum standards to describe Strains were grown in 5 ml TGY media at 37 °C until new species (Logan et al. 2009). During this analysis the late stationary phase (* 72 h). The culture media we discovered the previously released 16S rRNA of was centrifuged at 13,0009g for 10 min and the B. subtilis subsp. stercoris (JHCA01000027) was only supernatant removed. Mass spectrometry of the 1268 bp, so we submitted a new genome-derived full supernatant samples (25 lL injections) were collected length 16S rRNA (MN536904). We also noticed the by LC–MS (Thermo Acella HPLC) through a narrow- 16S rRNA sequence used at EzBioCloud (Yoon et al. bore (2.1 lmm 9 150 mm, 3 lm particle size) C18 2017)ofB. subtilis subsp. spizizenii differed by one column (Inertsil, GL Sciences, Inc., Torrance, CA) nucleotide, so we submitted an updated version of this running a gradient elution of 95% A:5% B (eluent A sequence (MN536905). The previously reported 16S 18 MX water/0.1% formic acid, eluent B 100% rRNA sequence for B. subtilis subsp. inaquosorum methanol/0.1% formic acid) to 5% A:95% B over (AMXN01000021) is the same as we determined. 65 min at a ﬂow rate of 250 lL/min, followed by a 5 min B washout and 10 min re-equilibration, while Average nucleotide identity maintaining a constant column temperature of 30 °C. Electrospray positive mode ionization data were The average nucleotide identity (ANI) of the type collected with a linear ion trap-Orbitrap mass spec- strains of these strains is reported in Fig. 2. The trometer (Thermo LTQ-Orbitrap Discovery) under pairwise comparisons of the four subspecies of Xcalibur 2.1 control. Prior to LC–MSn experiments B. subtilis varied between (91.3 and 95.4%), with the the instrument was tuned and calibrated using the LTQ two closest subspecies being B. subtilis subsp. subtilis tune mix. Masses corresponding to the metabolites of and B. subtilis subsp. stercoris. The results below or interest were used to limit the collection of MS2 data to borderline on the 95–96% threshold for species only those metabolites (Fig. 3). Tandem mass spectral delineation (Richter and Rossello´-Mo´ra 2009). In the data of target metabolites was collected using colli- Bacillus genus, recent species descriptions have used sion-induced dissociation (CID, collision energy the 96% threshold for species delineation (Dunlap (CE) = 25 and 35) in the LTQ and Higher-energy et al. 2015, 2016, 2017; Liu et al. 2017). These results collision dissociation (HCD, CE = 35 and 45) in the are consistent with the four subspecies of B. subtilis Orbitrap analyzer. being four separate species. Interestingly, the ANI-

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Fig. 1 Neighbor-joining phylogenomic tree reconstructed from on 1000 pseudoreplicates are indicated on branch points. The the core genome of strains in the B. subtilis dataset for this study scale bar corresponds to 0.010 nucleotide substitutions per site. and closely related type strains. Bootstrap values [ 50%, based B. amyloliquefaciens was used as an outgroup and not shown based phylogenetic tree shows show B. subtilis and its neighbor difference is due to the gain of a gene by subspecies are a monophyletic clade. horizontal gene transfer (HGT) or its loss by deletion. Of the 376 genes identified, 73 are present in both of Comparative genomics two non-neighboring clades, suggesting that some orthologues may have a complex history within the Core genome analysis can identify sets of genes that group, with multiple independent HGT/deletion differ between neighboring clades, uncovering gen- events required to explain their present-day occur- ome changes that have accompanied speciation within rence. For example, a five-gene cluster for the the group. A total of 376 gene differences have been synthesis of a nonreducing disaccharide (3,30-neotre- identified, using as a benchmark the presence of an halosadiamine) with antimicrobial properties (Vetter orthologue in at least 90% of the genomes on one side et al. 2013), is present in the B. subtilis subsp. of a phylogenetic node, but the absence of an spizizenii and B. subtilis subsp. subtilis clades but not orthologue in at least 90% of the genomes on the in either of their nearest neighbors. Conversely, 14 other side of the node (summarised in Fig. 3, with genes that appear to encode biosynthetic enzymes for details provided in Table S(I-VI) in the supplementary producing an uncharacterised nonribosomal peptide information accompanying this article). This type of and a polyketide are present in a 42.6 kb cluster in the analysis cannot distinguish whether a nearest-

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Fig. 2 Average nucleotide identity of subspecies of B. subtilis and closely related type strains determined using OrthoANI (Lee et al. 2016)

Fig. 3 Changes in genes at each branch point for subspecies of and \ 10% of the genomes on the opposite side. Secondary B. subtilis. The change in genes signiﬁes the genes that are metabolites and suspected function are highlighted under the found in [ 90% of the genomes on that side of the branch branch

B. subtilis subsp. inaquosorum and B. subtilis subsp. intensively studied since the late 1950s (Zeigler stercoris clades only. et al. 2008). It has all the hallmarks of a model Closer analysis of these differences between phy- organism, including an accurate genome sequence logenetic neighbors is greatly facilitated by compar- with meticulously curated, continuously updated isons with the genome of B. subtilis 168. An annotations (Borriss et al. 2018; Zhu and Stulke auxotrophic mutant of ATCC 6051T with improved 2018), a well-inventoried transcriptome studied under transformation competence, strain 168 has been a wide range of expression conditions (Nicolas et al. 123 6 Antonie van Leeuwenhoek (2020) 113:1–12

2012), and a sophisticated suite of genetic tools, and bacillaene (Butcher et al. 2007). B. subtilis subsp. including single-gene knockout and knockdown inaquosorum also contains three additional biosyn- libraries for every coding sequence in the genome thetic clusters with genes to produce the iturinic (Koo et al. 2017; Peters et al. 2016). Comparison of the lipopeptide, bacillomycin F (Dunlap et al. 2019; clade-specific genes with their orthologues in strain Mhammedi et al. 1982), the antifungal fengycin 168, then, allows for several observations. Of the (Steller et al. 1999) and an unknown metabolite genes present in B. subtilis subsp. subtilis but absent produced by a NRPS/PKS cluster (Table SIV). from B. subtilis subsp. stercoris, over half (26/48) are associated with post-exponential phase developmental MS/MS analysis of secondary metabolites events in response to nutrient limitation. In strain 168, these genes are differentially expressed during spore Mass spectrometry was used to confirm the presence formation or germination or during biofilm formation of the metabolites in the type strains that were in late stationary phase. A similar proportion of spore identified in the comparative genomics (Fig. 3). The or biofilm formation genes (15/26) are found among results confirm most of the metabolites are observed in sequences specific for the subtilis-stercoris clade. standard culturing conditions in the late stationary Another subset of clade-associated genes is linked phase (Fig. 4). We observe surfactin and subtilosin A with cellular responses to toxic compounds or other in all four strains. Bacilysin was not produced in harsh environmental conditions. Of the 74 genes sufficient quantities in any of the strains to be detected, associated with the subtilis or subtilis/stercoris clades, even though they contained the gene cluster to produce 7 are known members of regulons associated with it. Bacillibactin was only observed in trace amounts general or cell envelope stress in strain 168, and 7 under these conditions (data not shown); its production others are known to be induced by heat, cold, is usually induced by iron starvation (Miethke et al. electrophilic compounds, ethanol, or high salt. Addi- 2006). The distinguishing lipopeptides are observed as tional clade-associated genes are present to help deal expected: B. subtilis subsp. spizizenii (Fig. 4A. myco- with specific challenges, such as the Czr system for subtilin), B. subtilis subsp. inaquosorum (Fig. 4C. exporting toxic metal ions (Moore et al. 2005). bacillomycin F and fengycin) and B. subtilis subsp. Scattered among the clades are at least five genes that stercoris (Fig. 4D. fengycin). Bacillaene was not are predicted to specify resistance to antimicrobials observed in B. subtilis subsp. spizizenii under these compounds such as streptothricin, oxacillin, conditions; previously we only observed bacillaene methylenomycin A, chloramphenicol, and fusaric production in the exponential growth phase and with acid. Relatively few of the identified genes can be negative mode MS detection for other Bacillus species associated with an expanded metabolic capacity. One (Dunlap et al. 2016). 3,30-Neotrehalosadiamine was example, however, is a 13-gene, 11.1 kb cluster not detected for the two strains with the gene cluster; encoding transporters and enzymes for converting however, the setup of this analytical instrumentation S-methyl-cysteine to cysteine—a pathway suggested was not optimized for that class of analyte. to be present in plant-associated microbes (Chan et al. 2014). Chemotaxonomy In addition to the stress resistance genes identified, a key difference in the subspecies is the bioactive In order to promote these subspecies to species status, secondary metabolites they can produce. The analysis it is necessary to bring their descriptions in line with identified that all four subspecies contained the genes the minimal standards for describing new species to produce the antibiotic subtilosin (Babasaki et al. (Logan et al. 2009). The total cellular fatty acid 1985), the antibiotic bacilysin (Steinborn et al. 2005), profiles showed a large amount of branched fatty the siderophore bacillibactin (May et al. 2001), and acids; the major components (\ 5.0%) were anteiso- surfactin (Cosmina et al. 1993). B. subtilis subsp. C15 : 0, iso-C15 : 0, iso-C17 : 0, anteiso-C17 : 0 (Table 1). spizizenii contains three additional biosynthetic clus- The profiles were consistent with previously reported ters with genes to produce the iturinic lipopeptide profiles for these subspecies (Rooney et al. 2009). No mycosubtilin (Duitman et al. 1999; Dunlap et al. novel or uniquely identifying fatty acids were 2019), 3,30-neotrehalosadiamine (Vetter et al. 2013) observed. The cell-wall peptidoglycan for B. subtilis 123 Antonie van Leeuwenhoek (2020) 113:1–12 7

Fig. 4 Extracted ion-chromatograms of secondary metabolites corresponding to [M ? 2H]2? and [M ? H]? fengycins of bacillus strain analyzed by LC-MS. m/z 1022–1023; (green). m/z 1135–1136, 1701–1702, corresponding to Sub- 1036–1037; and 1044–1045, corresponding to surfactin tilosin [M ? 3H]3? and [M ? 2H]2? (blue). A B. subtilis subsp. [M ? H]? components (black). m/z 1085–1086; 1099–1100, spizizenii. B B. subtilis subsp. subtilis. C B. subtilis subsp. corresponding to Iturinic lipopeptides [M ? H]? (BacF and inaquosorum. D B. subtilis subsp. stercoris mycosubtilin). m/z 746–747; 753–754; 1491–1492, 1506–1507, is meso-diaminopimelic acid (Schleifer and Kandler diaminopimelic acid containing peptidoglycans, in 1972). Genome analysis of conﬁrms all subspecies agreement with a recent study on cell wall variations contain the same pathways for synthesis of meso- in B. subtilis (Ahn et al. 2018). 123 8 Antonie van Leeuwenhoek (2020) 113:1–12

Table 1 Fatty acid proﬁles Fatty acida Bacillus subtilis susbp. of the subspecies of Bacillus subtilis subtilis spizizenii inaquosorum stercoris NRRL NRS-744T NRRL B-23049T NRRL B-23052T JCM 30051T

15:0 iso 22.2 23.7 21.3 26.5 15:0 anteiso 41.5 40.2 44.6 41.8 16:0 iso 1.5 2.2 2.4 1.5 16:1 x11c 1.1 0.5 0.8 0.8 16:0 2.2 3.9 2.7 2.9 17:1 x10c 1.0 0.4 0.6 0.6 17:0 iso 15.7 13.8 14.5 12.1 a Fatty acids greater than 17:0 anteiso 13.8 14.5 12.6 13.3 1.0% of total fatty acids

Morphology and physiology macroscopic morphology between the type strains (Figure S1), but it is unclear if these differences A summary of morphological and physiological encompass all representative strains of these species. characters of the four type strains is provided in Sporangia morphology is cylindrical-ellipsoidal, cen- Table 2. The results are consistent with historical tral-paracentral, non-swollen for all strains (Fig- reports of no conventional phenotypic differences ure S2). There were no observed differences in between the strains. There are minor differences in growth temperature range, NaCl or pH tolerance in

Table 2 Summary of phenotypic properties Trait Bacillus subtilis susbp. subtilis spizizenii inaquosorum stercoris NRRL NRS-744T NRRL B-23049T NRRL B-23052T JCM 30051T

Sporangia Ellipsoidal, central- Ellipsoidal, central- Ellipsoidal, central- Ellipsoidal, central- paracentral, non-swollen paracentral, non-swollen paracentral, non-swollen paracentral, non-swollen Cell size 0.5 9 2–3 lm 0.5 9 2–3 lm 0.5 9 2–3 lm 0.5 9 2–3 lm Growth at: 14–50 °C 14–50 °C 14–50 °C 14–50 °C NaCl 0–10% 0–10% 0–10% 0–10% tolerance pH tolerance 5–9 5–9 5–9 5–9 Anaerobic Facultative Facultative Facultative Facultative Catalase ???? Urease --w ? Nitrate ???? reduction Hydrolysis of: Casein ???? Gelatin ???? Starch ???? Citrate ???? utilization Acid from ???? glucose

123 Antonie van Leeuwenhoek (2020) 113:1–12 9 direct comparison assays. Urease activity was a Vegetative cells are rods that measure differential phenotype amongst the type strains, 0.5 9 2–3 lm and occur either singly or in chains. Interestingly, all four type strains contain the ureABC Motile. Cylindrical-ellipsoidal spores form centrally urease cluster, while B. subtilis subsp. stercoris, or paracentrally in non-swollen sporangia. Faculta- B. subtilis subsp. spizizenii, and B subtilis subsp. tively anaerobic on anaerobic agar. Catalase-positive. inaquosorum also contain a urea ABC transporter Grows at 14–50 °C, with optimum growth at cluster (urtABCDE). Using Christensen’s Urease agar, 28–30 °C. Growth in 7 % (w/v) NaCl occurs after only B. subtilis subsp. stercoris gives a strong positive 72 h. The major fatty acids are were anteiso-C15 : 0, result after 48 h, while B subtilis subsp. inaquosorum iso-C15 : 0, iso-C17 : 0, anteiso-C17 : 0. Hydrolyses starch provides a weak result at 72 h (Figure S3) and and casein; reduces nitrate to nitrite. Citrate is utilized. B. subtilis subsp. spizizenii and B. subtilis subsp. Contains the genes to produce the antibiotic subtilosin subtilis only grow on the agar without giving a and bacilysin, the lipopeptides mycosubtilin and detectable pH change. Biolog assays for the four type surfactin, and the antibiotic 3,30-neotrehalosadiamine. strains were nearly identical with the strains showing The average genome size is approximately 4.1 Mbp the ability to utilise: dextrin, D-maltose, D-trehalose, D- with an average G ? C mole content of 43.8%. T T- cellobiose, gentiobiose, sucrose, D-turanose, D-rafﬁ- The type strain is TU-B-10 (= NRRL B-23049 T T T nose, D-melbiose, b-methyl-D-glucoside, D-salicin, a- = LMG 19156 = DSM 15029 = BCRC 17366 ). D-glucose, D-mannose, D-fructose, D-sorbitol, D-man- The 16S rRNA sequence is available at GenBank itol, myo-insoitol, glycerol, L-alanine, L-aspartic acid, MN536905. L-histidine, pectin, D-galacturonic acid, L-galactonic acid, D-gluconic, D-glucuronic, glucuronamide, quinic Description of Bacillus inaquosorum sp. nov acid, methyl pyruvate, L-lactic acid, citric acid and D- malic acid. One notable difference under these con- Bacillus inaquosorum [in.a.quo.so0rum. L. adj. inaqu- ditions was B. subtilis subsp. spizizenii was susceptible osus poor of water, L. gen. pl. n. (solium) inaquosorum to rifampycin SV, while the others were not. Interest- from (soils) poor of water (desert soils) from which ingly, it has been shown that mutations in the RNA this organism was isolated]. polymerase associated with rifampycin resistance Vegetative cells are rods that measure have been shown to regulate the production of the 0.5 9 2–3 lm and occur either singly or in chains. antibiotic 3,30-neotrehalosadiamine in B. subtilis Motile. Cylindrical-ellipsoidal spores form centrally (Inaoka and Ochi 2011). Future studies should explore or paracentrally in non-swollen sporangia. Faculta- the regulation of 3,30-neotrehalosadiamine in B. sub- tively anaerobic on anaerobic agar. Catalase-positive. tilis subsp. spizizenii and B. subtilis subsp. subtilis to Grows at 14–55 °C, with optimum growth at understand the evolution of these genes. 28–30 °C. Growth in 10 % (w/v) NaCl occurs after 72 h. Growth in the presence of 0.001 % (w/v) lysozyme is variable. Produces acetyl-methylcarbinol Conclusions (Voges–Proskauer test) at pH 5.5–5.7. The major fatty

acids are were anteiso-C15 : 0, iso-C15 : 0, iso-C17 : 0, Based on the phylogeny, comparative genomics and anteiso-C17 : 0. Hydrolyses starch and casein; reduces distinguishing phenotypes of these strains, they should nitrate to nitrite. Citrate is utilized. Contains the genes be promoted to species status. to produce the antibiotic subtilosin and bacilysin, the lipopeptides bacillomycin F, surfactin and fengycin. Description of Bacillus spizizenii sp. nov The average genome size is approximately 4.2 Mbp with an average G ? C mole content of 43.8%. Bacillus spizizenii [spi.zi.ze´ni.i. L. gen. n. spizizenii The type strain is DV7-B-4T (= NRRL B-23052T- named after the American bacteriologist J. Spizizen, = KCTC 13429T = BGSC 3A28T = DSM 22148T- whose seminal studies of Bacillus subtilis made this = BCRC 17984T). The 16S rRNA sequence is species a model organism of bacterial genetics]. available at GenBank AMXN01000021.

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Description of Bacillus stercoris sp. nov References

Bacillus stercoris [ster’co.ris. L. gen. n. stercoris,of Adelskov J, Patel BKC (2016) A molecular phylogenetic compost, from which the strain was isolated]. framework for Bacillus subtilis using genome sequences and its application to Bacillus subtilis subspecies stecoris Vegetative cells are rods that measure 0.5 9 2–3 strain D7XPN1, an isolate from a commercial food-waste lm and occur either singly or in chains. Motile. degrading bioreactor. 3 Biotech 6:96. https://doi.org/10. Cylindrical-ellipsoidal spores form centrally or para- 1007/s13205-016-0408-8 centrally in non-swollen sporangia. Facultatively Adelskov J, Patel BKC (2017) Erratum to: A molecular phylogenetic framework for Bacillus subtilis using genome anaerobic on anaerobic agar. Catalase-positive. Grows sequences and its application to Bacillus subtilis sub- at 14–50 °C, with optimum growth at 45 °C. Growth species stecoris strain D7XPN1, an isolate from a com- in 7 % (w/v) NaCl occurs after 72 h. The major fatty mercial food-waste degrading bioreactor. 3 Biotech 7:142. acids are were anteiso-C , iso-C , iso-C , https://doi.org/10.1007/s13205-017-0747-0 15 : 0 15 : 0 17 : 0 Ahn S, Jun S, Ro HJ, Kim JH, Kim S (2018) Complete genome anteiso-C17 : 0. Hydrolyses starch and casein; reduces of Bacillus subtilis subsp. subtilis KCTC 3135(T) and nitrate to nitrite. Citrate is utilized. Contains the genes variation in cell wall genes of B. subtilis strains. J Micro- to produce the antibiotic subtilosin and bacilysin, the biol Biotechnol 28:1760–1768. https://doi.org/10.4014/ lipopeptides fengycin and surfactin. jmb,1712.12006 Babasaki K, Takao T, Shimonishi Y, Kurahashi K (1985) The average genome size is approximately 4.1 Mbp Subtilosin A, a new antibiotic peptide produced by Bacillus with an average G ? C mole content of 43.8%. subtilis 168: isolation, structural analysis, and biogenesis. The type strain is D7XPN1T (= KCTC 33554T- J Biochem 98:585–603. https://doi.org/10.1093/ = JCM 30051T). The 16S rRNA sequence is available oxfordjournals.jbchem.a135315 Borriss R, Danchin A, Harwood CR, Medigue C, Rocha EPC, at GenBank MN536904. Sekowska A, Vallenet D (2018) Bacillus subtilis, the model Gram-positive bacterium: 20 years of annotation Acknowledgements The authors would like to thank Heather refinement. Microb Biotechnol 11:3–17. https://doi.org/10. Walker for her technical expertise in genome sequencing and 1111/1751-7915.13043 phenotype analysis. Any opinions, findings, conclusions, or Breznak JA, Costilow RN (1994) Physicochemical factors in recommendations expressed in this publication are those of the growth. In: Methods for general and molecular bacteriol- author(s) and do not necessarily reflect the view of the U.S. ogy, pp 137–154 Department of Agriculture. The mention of firm names or trade Brito PH, Chevreux B, Serra CR, Schyns G, Henriques AO, products does not imply that they are endorsed or recommended Pereira-Leal JB (2018) Genetic competence drives genome by the USDA over other firms or similar products not diversity in Bacillus subtilis. Genome Biol Evolut mentioned. USDA is an equal opportunity provider and 10:108–124. https://doi.org/10.1093/gbe/evx270 employer. This material is also based in part upon work Butcher RA, Schroeder FC, Fischbach MA, Straight PD, Kolter supported by the National Science Foundation under Grant No. R, Walsh CT, Clardy J (2007) The identification of bacil- 1756219. laene, the product of the PksX megacomplex in Bacillus subtilis. Proc Natl Acad Sci USA 104:1506–1509. https:// Author contributions The study conception, design, doi.org/10.1073/pnas.0610503104 comparative genomics and interpretation was performed by Chan CM, Danchin A, Marliere P, Sekowska A (2014) Paralo- Christopher Dunlap. Mass spectroscopy and analysis was gous metabolism: S-alkyl-cysteine degradation in Bacillus performed by Michael Bowman. Correlation of the genes to subtilis. Environ Microbiol 16:101–117. https://doi.org/10. the Bacillus subtilis orthologs and associated interpretation the 1111/1462-2920.12210 results was performed by Daniel Zeigler. The first draft of the Cosmina P, Rodriguez F, de Ferra F, Grandi G, Perego M, manuscript was written by Christopher Dunlap and all authors Venema G, van Sinderen D (1993) Sequence and analysis commented on previous versions of the manuscript. All authors of the genetic locus responsible for surfactin synthesis in read and approved the final manuscript. Bacillus subtilis. Mol Microbiol 8:821–831. https://doi. org/10.1111/j.1365-2958.1993.tb01629.x Compliance with ethical standards Duitman EH, Hamoen LW, Rembold M, Venema G, Seitz H, Saenger W, Bernhard F, Reinhardt R, Schmidt M, Ullrich Conflict of interest The authors declare no conflicts of C, Stein T, Leenders F, Vater J (1999) The mycosubtilin interest. synthetase of Bacillus subtilis ATCC6633: a multifunc- tional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proc Natl Acad Sci Research involving human participants and/or animals USA 96:13294–13299. https://doi.org/10.1073/pnas.96. This article does not contain any studies with human participants 23.13294 or animals performed by the author. Dunlap CA, Kwon S-W, Rooney AP, Kim S-J (2015) Bacillus paralicheniformis sp. nov., isolated from fermented

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