ORIGINAL PAPER

Promotion of Bacillus subtilis subsp. inaquosorum, Bacillussubtilis subsp. spizizenii and Bacillus subtilis subsp. stercoristo species status

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

Received: 17 October 2019 /Accepted: 26 October 2019 / Published online: 12 November 2019

� 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

four subspecies, Bacillus subtilis subsp. subtilis,

Bacillus subtilis subsp. inaquosorum, Bacillus subtilis

subsp. spizizenii and Bacillus subtilis subsp. stercoris.

Several studies based on genomic comparisons have

suggested these subspecies should be promoted to

species status. Previously, one of the main reasons for

leaving them as subspecies was the lack of distin-

guishing phenotypes. In this study, we used compar-

ative genomics to determine the genes unique to each

subspecies and used these to lead us to the unique

phenotypes. The results show that one difference

among the subspecies is they produce different

bioactive secondary metabolites. B. subtilis subsp.

spizizenii is shown conserve the genes to produce

mycosubtilin, bacillaene and 3,30-neotrehalosadi-amine. B. subtilis subsp. inaquosorum is shown

conserve the genes to produce bacillomycin F,

fengycin and an unknown PKS/NRPS cluster. B. sub-

tilis subsp. stercoris is shown conserve the genes to

produce fengycin and an unknown PKS/NRPS cluster.

While B. subtilis subsp. subtilis is shown to conserve

the genes to produce 3,30-neotrehalosadiamine. In

addition, we update the chemotaxonomy and pheno-

typing to support their promotion to species status.

Keywords Core genome � Secondary metabolites �Antifungal � Antibiotic � Lipopeptide � Surfactin �Bacilysin � Subtilosin

Introduction

Bacillus subtilis was first divided into two subspecies

by Nakamura et al. (1999), based on a DNA related-

ness of 60–70% between the two sub-groups. In

addition, cell walls of Bacillus subtilis subsp. spiz-

izenii were reported to contain ribitol or anhydroribi-

tiol, while strains Bacillus subtilis subsp. subtilis did

not (Nakamura et al. 1999). A third subspecies,

Electronic supplementary material The online version ofthis article (https://doi.org/10.1007/s10482-019-01354-9) con-tains supplementary material, which is available to authorizedusers.

C. A. Dunlap (&)

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: christopher.dunlap@ars.usda.gov;

Christopher.dunlap@usda.gov

M. J. Bowman

Bioenergy Research Unit, National Center for

Agricultural Utilization Research, Agricultural Research

Service, United States Department of Agriculture, 1815

North University Street, Peoria, IL, USA

D. R. Zeigler

Bacillus Genetic Stock Center, The Ohio State University,

Columbus, OH, USA

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Antonie van Leeuwenhoek (2020) 113:1–12

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

Bacillus subtilis subsp. inaquosorum, was described

by Rooney et al. (2009) after identifying a unique

clade during multilocus sequence analysis of the

B. subtilis species complex. B subtilis subsp. inaqu-

osorumwas reported to contain a uniqueMALDI-TOF

MS biomarker at m/z 1120 as a distinguishing

phenotype among the subspecies. A fourth subspecies,

Bacillus subtilis subsp. stercoris was described by

Adelskov et al. (2016), based on an average nucleotide

identity of 95.6% between the genomes of B. subtilis

subsp. subtilis and B. subtilis subsp. stercoris (Adel-

skov and Patel 2017). No distinguishing phenotypes

were reported for B. subtilis subsp. stercoris (Adel-

skov and Patel 2016).

In recent years, the subspecies status of these strains

has come under question by several studies on the

basis of average nucleotide identity of their genomes

(Brito et al. 2018; Dunlap et al. 2019; Knight et al.

2018; Yi et al. 2014). One of the reasons previously

cited for not defining species was the lack of distin-

guishing phenotypes (Rooney et al. 2009). Our

laboratories recently identified differences in iturinic

lipopeptides as unique phenotypes for these sub-

species (Dunlap et al. 2019). This motivated us to re-

examine these subspecies and perform comparative

genomics to identify distinguishing phenotypes.

Materials and methods

Genomes

All available genomes categorised as B. subtilis or

Bacillus sp. were downloaded from GenBank on May

1, 2019 and combined with an in-house database of

Bacillus genomes. A 6 gene MLSA was used to assign

the genomes to the correct species and subspecies

taxonomy (Rooney et al. 2009). Genomes of strains

belonging to B. subtilis were used for further analysis.

This set included genomes of B. subtilis subsp. subtilis

(n = 224), B. subtilis subsp. spizizenii (n = 27), B

subtilis subsp. inaquosorum (n = 27), B. subtilis

subsp. stercoris (n = 7).

Genome-based phylogeny and comparative

genomics of Bacillus subtilis strains

A core genome phylogeny of the strains was per-

formed to show the taxonomic relationship of these

strains. The core genome determination and subse-

quent alignments were produced for all the type strains

in the group with BIGSdb software (Jolley andMaiden

2010) and consists of 3501 genes. The phylogenetic

tree was constructed using MEGA X software (Kumar

et al. 2018). The neighbor-joining tree was determined

using the Tamura-Nei model (0.40, gamma distributed

with invariant sites) based on model testing under

MEGA X (Kumar et al. 2018). Measures of bootstrap

support for internal branches were obtained from 1000

pseudoreplicates. Determination of the core genomes

and related comparison were generated with the

genome comparator function implemented under

BIGSdb software (Jolley and Maiden 2010). A

complete genome served as a reference strain for each

subspecies and was used to BLAST all genomes in the

set. The determination of genes gained or lost by the

different groups was based on a 90/10 comparison. For

example, the change in genes was determined at each

branch point in the tree, the change is reported as the

number of genes found in 90% of the genomes of the

branch, but only in 10% of the genomes from the

opposite branch. The average nucleotide identity

(ANI) of the genomes was determined using

OrthoANI software (Lee et al. 2016).

Fatty acid analysis

Total cellular fatty acid content was measured using

the MIDI protocol (Microbial Identification Inc.

Newark, DE) and analyzed on an Agilent 7890 Gas

Chromatograph. The strains were grown for 24 h at

28 �C on TSA and prepared using the standard MIDI

protocol for extraction and production of fatty acid

methyl esters.

Morphology and physiology

We tested the growth temperature range at 10, 14, 28,

37, 45, 50 and 55 �C using tryptone-glucose-yeast-

extract media (TGY, Difco). The pH range for growth

was determined from pH 3.0 to pH 12.0 in steps of 0.5

pH unit in TGY broth buffered and adjusted with

phosphate buffer, Tris/HCl buffer, HCl or NaOH

(Breznak and Costilow 1994) at 28 �C. NaCl tolerancewas investigated by using TGY broth supplemented

with 0–12% (w/v) NaCl, in 1% increments at 28 �C.Growth under anaerobic conditions was determined on

anaerobic agar (Difco) at 30 �C using a GasPak jar

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2 Antonie van Leeuwenhoek (2020) 113:1–12

(\ 1%O2; C 13%CO2) (Merck) for 7 days (Vos et al.

2009). Spore morphology was determined on 1/10

TSB ? 50 mg/L MnSO4 from samples grown at

28 �C for 48 h. The endospores were heat fixed and

imaged using a phase contrast microscope. Carbon

source utilisation was tested using the OmniLog�Data

Collection system (Biolog Inc, Hayward, CA). Strains

selected for characterization were cultured overnight

on Biolog universal growth plates and prepared

according to manufacturer’s instructions for the

GEN III MicroPlateTM test panel using protocol A

(Biolog Inc, Hayward, CA) at 33 �C. An OmniLog�

Data Collection instrument (Biolog Inc, Hayward,

CA) was used to collect data in 15 min increments for

22 h. Catalase and oxidase activities were examined

using 3% (v/v) hydrogen peroxide solution and 1% (w/

v) tetramethyl-p-phenylenediamine dihydrochloride

(Difco), respectively.

Mass spectrometry

Strains were grown in 5 ml TGY media at 37 �C until

the late stationary phase (* 72 h). The culture media

was centrifuged at 13,0009g for 10 min and the

supernatant removed. Mass spectrometry of the

supernatant samples (25 lL injections) were collected

by LC–MS (Thermo Acella HPLC) through a narrow-

bore (2.1 lmm 9 150 mm, 3 lm particle size) C18

column (Inertsil, GL Sciences, Inc., Torrance, CA)

running a gradient elution of 95% A:5% B (eluent A

18 MX water/0.1% formic acid, eluent B 100%

methanol/0.1% formic acid) to 5% A:95% B over

65 min at a flow rate of 250 lL/min, followed by a

5 min B washout and 10 min re-equilibration, while

maintaining a constant column temperature of 30 �C.Electrospray positive mode ionization data were

collected with a linear ion trap-Orbitrap mass spec-

trometer (Thermo LTQ-Orbitrap Discovery) under

Xcalibur 2.1 control. Prior to LC–MSn experiments

the instrument was tuned and calibrated using the LTQ

tune mix. Masses corresponding to the metabolites of

interest were used to limit the collection ofMS2 data to

only those metabolites (Fig. 3). Tandem mass spectral

data of target metabolites was collected using colli-

sion-induced dissociation (CID, collision energy

(CE) = 25 and 35) in the LTQ and Higher-energy

collision dissociation (HCD, CE = 35 and 45) in the

Orbitrap analyzer.

Results and discussion

Genome-based phylogeny of Bacillus subtilis

strains

The core genome phylogeny (Fig. 1) shows each

species or subspecies forms a distinct clade which

contains all members of the group. The results also

show B. subtilis and its subspecies are not a mono-

phyletic clade with Bacillus tequilensis and Bacillus

vallismortis also falling within the clade. This analysis

is based on 3501 genes that comprise the core genome

(genes found in 90% of the genomes) in this dataset,

which is somewhat biased towards B. subtilis subsp.

subtilis, since it has the most genomes (n = 224). In

other core-genome analyses with fewer genes (991

genes) spanning the B. subtilis group species complex,

we found B. subtilis and its subspecies to be a

monophyletic clade (Dunlap et al. 2019). In addition to

the genome phylogeny we conducted a 16S rRNA

phylogeny to meet the minimum standards to describe

new species (Logan et al. 2009). During this analysis

we discovered the previously released 16S rRNA of

B. subtilis subsp. stercoris (JHCA01000027) was only

1268 bp, so we submitted a new genome-derived full

length 16S rRNA (MN536904). We also noticed the

16S rRNA sequence used at EzBioCloud (Yoon et al.

2017) of B. subtilis subsp. spizizenii differed by one

nucleotide, so we submitted an updated version of this

sequence (MN536905). The previously reported 16S

rRNA sequence for B. subtilis subsp. inaquosorum

(AMXN01000021) is the same as we determined.

Average nucleotide identity

The average nucleotide identity (ANI) of the type

strains of these strains is reported in Fig. 2. The

pairwise comparisons of the four subspecies of

B. subtilis varied between (91.3 and 95.4%), with the

two closest subspecies being B. subtilis subsp. subtilis

and B. subtilis subsp. stercoris. The results below or

borderline on the 95–96% threshold for species

delineation (Richter and Rossello-Mora 2009). In the

Bacillus genus, recent species descriptions have used

the 96% threshold for species delineation (Dunlap

et al. 2015, 2016, 2017; Liu et al. 2017). These results

are consistent with the four subspecies of B. subtilis

being four separate species. Interestingly, the ANI-

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Antonie van Leeuwenhoek (2020) 113:1–12 3

based phylogenetic tree shows show B. subtilis and its

subspecies are a monophyletic clade.

Comparative genomics

Core genome analysis can identify sets of genes that

differ between neighboring clades, uncovering gen-

ome changes that have accompanied speciation within

the group. A total of 376 gene differences have been

identified, using as a benchmark the presence of an

orthologue in at least 90% of the genomes on one side

of a phylogenetic node, but the absence of an

orthologue in at least 90% of the genomes on the

other side of the node (summarised in Fig. 3, with

details provided in Table S(I-VI) in the supplementary

information accompanying this article). This type of

analysis cannot distinguish whether a nearest-

neighbor difference is due to the gain of a gene by

horizontal gene transfer (HGT) or its loss by deletion.

Of the 376 genes identified, 73 are present in both of

two non-neighboring clades, suggesting that some

orthologues may have a complex history within the

group, with multiple independent HGT/deletion

events required to explain their present-day occur-

rence. For example, a five-gene cluster for the

synthesis of a nonreducing disaccharide (3,30-neotre-halosadiamine) with antimicrobial properties (Vetter

et al. 2013), is present in the B. subtilis subsp.

spizizenii and B. subtilis subsp. subtilis clades but not

in either of their nearest neighbors. Conversely, 14

genes that appear to encode biosynthetic enzymes for

producing an uncharacterised nonribosomal peptide

and a polyketide are present in a 42.6 kb cluster in the

Fig. 1 Neighbor-joining phylogenomic tree reconstructed from

the core genome of strains in the B. subtilis dataset for this study

and closely related type strains. Bootstrap values[ 50%, based

on 1000 pseudoreplicates are indicated on branch points. The

scale bar corresponds to 0.010 nucleotide substitutions per site.

B. amyloliquefaciens was used as an outgroup and not shown

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4 Antonie van Leeuwenhoek (2020) 113:1–12

B. subtilis subsp. inaquosorum and B. subtilis subsp.

stercoris clades only.

Closer analysis of these differences between phy-

logenetic neighbors is greatly facilitated by compar-

isons with the genome of B. subtilis 168. An

auxotrophic mutant of ATCC 6051T with improved

transformation competence, strain 168 has been

intensively studied since the late 1950s (Zeigler

et al. 2008). It has all the hallmarks of a model

organism, including an accurate genome sequence

with meticulously curated, continuously updated

annotations (Borriss et al. 2018; Zhu and Stulke

2018), a well-inventoried transcriptome studied under

a wide range of expression conditions (Nicolas et al.

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

B. subtilis. The change in genes signifies the genes that are

found in[ 90% of the genomes on that side of the branch

and\ 10% of the genomes on the opposite side. Secondary

metabolites and suspected function are highlighted under the

branch

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Antonie van Leeuwenhoek (2020) 113:1–12 5

2012), and a sophisticated suite of genetic tools,

including single-gene knockout and knockdown

libraries for every coding sequence in the genome

(Koo et al. 2017; Peters et al. 2016). Comparison of the

clade-specific genes with their orthologues in strain

168, then, allows for several observations. Of the

genes present in B. subtilis subsp. subtilis but absent

from B. subtilis subsp. stercoris, over half (26/48) are

associated with post-exponential phase developmental

events in response to nutrient limitation. In strain 168,

these genes are differentially expressed during spore

formation or germination or during biofilm formation

in late stationary phase. A similar proportion of spore

or biofilm formation genes (15/26) are found among

sequences specific for the subtilis-stercoris clade.

Another subset of clade-associated genes is linked

with cellular responses to toxic compounds or other

harsh environmental conditions. Of the 74 genes

associated with the subtilis or subtilis/stercoris clades,

7 are known members of regulons associated with

general or cell envelope stress in strain 168, and 7

others are known to be induced by heat, cold,

electrophilic compounds, ethanol, or high salt. Addi-

tional clade-associated genes are present to help deal

with specific challenges, such as the Czr system for

exporting toxic metal ions (Moore et al. 2005).

Scattered among the clades are at least five genes that

are predicted to specify resistance to antimicrobials

compounds such as streptothricin, oxacillin,

methylenomycin A, chloramphenicol, and fusaric

acid. Relatively few of the identified genes can be

associated with an expanded metabolic capacity. One

example, however, is a 13-gene, 11.1 kb cluster

encoding transporters and enzymes for converting

S-methyl-cysteine to cysteine—a pathway suggested

to be present in plant-associated microbes (Chan et al.

2014).

In addition to the stress resistance genes identified,

a key difference in the subspecies is the bioactive

secondary metabolites they can produce. The analysis

identified that all four subspecies contained the genes

to produce the antibiotic subtilosin (Babasaki et al.

1985), the antibiotic bacilysin (Steinborn et al. 2005),

the siderophore bacillibactin (May et al. 2001), and

surfactin (Cosmina et al. 1993). B. subtilis subsp.

spizizenii contains three additional biosynthetic clus-

ters with genes to produce the iturinic lipopeptide

mycosubtilin (Duitman et al. 1999; Dunlap et al.

2019), 3,30-neotrehalosadiamine (Vetter et al. 2013)

and bacillaene (Butcher et al. 2007). B. subtilis subsp.

inaquosorum also contains three additional biosyn-

thetic clusters with genes to produce the iturinic

lipopeptide, bacillomycin F (Dunlap et al. 2019;

Mhammedi et al. 1982), the antifungal fengycin

(Steller et al. 1999) and an unknown metabolite

produced by a NRPS/PKS cluster (Table SIV).

MS/MS analysis of secondary metabolites

Mass spectrometry was used to confirm the presence

of the metabolites in the type strains that were

identified in the comparative genomics (Fig. 3). The

results confirmmost of the metabolites are observed in

standard culturing conditions in the late stationary

phase (Fig. 4). We observe surfactin and subtilosin A

in all four strains. Bacilysin was not produced in

sufficient quantities in any of the strains to be detected,

even though they contained the gene cluster to produce

it. Bacillibactin was only observed in trace amounts

under these conditions (data not shown); its production

is usually induced by iron starvation (Miethke et al.

2006). The distinguishing lipopeptides are observed as

expected: B. subtilis subsp. spizizenii (Fig. 4A. myco-

subtilin), B. subtilis subsp. inaquosorum (Fig. 4C.

bacillomycin F and fengycin) and B. subtilis subsp.

stercoris (Fig. 4D. fengycin). Bacillaene was not

observed in B. subtilis subsp. spizizenii under these

conditions; previously we only observed bacillaene

production in the exponential growth phase and with

negative mode MS detection for other Bacillus species

(Dunlap et al. 2016). 3,30-Neotrehalosadiamine was

not detected for the two strains with the gene cluster;

however, the setup of this analytical instrumentation

was not optimized for that class of analyte.

Chemotaxonomy

In order to promote these subspecies to species status,

it is necessary to bring their descriptions in line with

the minimal standards for describing new species

(Logan et al. 2009). The total cellular fatty acid

profiles showed a large amount of branched fatty

acids; the major components (\ 5.0%) were anteiso-

C15 : 0, iso-C15 : 0, iso-C17 : 0, anteiso-C17 : 0 (Table 1).

The profiles were consistent with previously reported

profiles for these subspecies (Rooney et al. 2009). No

novel or uniquely identifying fatty acids were

observed. The cell-wall peptidoglycan for B. subtilis

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6 Antonie van Leeuwenhoek (2020) 113:1–12

is meso-diaminopimelic acid (Schleifer and Kandler

1972). Genome analysis of confirms all subspecies

contain the same pathways for synthesis of meso-

diaminopimelic acid containing peptidoglycans, in

agreement with a recent study on cell wall variations

in B. subtilis (Ahn et al. 2018).

Fig. 4 Extracted ion-chromatograms of secondary metabolites

of bacillus strain analyzed by LC-MS. m/z 1022–1023;

1036–1037; and 1044–1045, corresponding to surfactin

[M ? H]? components (black). m/z 1085–1086; 1099–1100,

corresponding to Iturinic lipopeptides [M ? H]? (BacF and

mycosubtilin). m/z 746–747; 753–754; 1491–1492, 1506–1507,

corresponding to [M ? 2H]2? and [M ? H]? fengycins

(green). m/z 1135–1136, 1701–1702, corresponding to Sub-

tilosin [M ? 3H]3? and [M ? 2H]2? (blue).A B. subtilis subsp.

spizizenii. B B. subtilis subsp. subtilis. C B. subtilis subsp.

inaquosorum. D B. subtilis subsp. stercoris

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Antonie van Leeuwenhoek (2020) 113:1–12 7

Morphology and physiology

A summary of morphological and physiological

characters of the four type strains is provided in

Table 2. The results are consistent with historical

reports of no conventional phenotypic differences

between the strains. There are minor differences in

macroscopic morphology between the type strains

(Figure S1), but it is unclear if these differences

encompass all representative strains of these species.

Sporangia morphology is cylindrical-ellipsoidal, cen-

tral-paracentral, non-swollen for all strains (Fig-

ure S2). There were no observed differences in

growth temperature range, NaCl or pH tolerance in

Table 1 Fatty acid profiles

of the subspecies of

Bacillus subtilis

aFatty acids greater than

1.0% of total fatty acids

Fatty acida Bacillus subtilis susbp.

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

17:0 anteiso 13.8 14.5 12.6 13.3

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-

paracentral, non-swollen

Ellipsoidal, central-

paracentral, non-swollen

Ellipsoidal, central-

paracentral, non-swollen

Ellipsoidal, central-

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 �CNaCl

tolerance

0–10% 0–10% 0–10% 0–10%

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

? ? ? ?

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8 Antonie van Leeuwenhoek (2020) 113:1–12

direct comparison assays. Urease activity was a

differential phenotype amongst the type strains,

Interestingly, all four type strains contain the ureABC

urease cluster, while B. subtilis subsp. stercoris,

B. subtilis subsp. spizizenii, and B subtilis subsp.

inaquosorum also contain a urea ABC transporter

cluster (urtABCDE). Using Christensen’s Urease agar,

only B. subtilis subsp. stercoris gives a strong positive

result after 48 h, while B subtilis subsp. inaquosorum

provides a weak result at 72 h (Figure S3) and

B. subtilis subsp. spizizenii and B. subtilis subsp.

subtilis only grow on the agar without giving a

detectable pH change. Biolog assays for the four type

strains were nearly identical with the strains showing

the ability to utilise: dextrin, D-maltose, D-trehalose, D-

cellobiose, gentiobiose, sucrose, D-turanose, D-raffi-

nose, D-melbiose, b-methyl-D-glucoside, D-salicin, a-D-glucose, D-mannose, D-fructose, D-sorbitol, D-man-

itol, myo-insoitol, glycerol, L-alanine, L-aspartic acid,

L-histidine, pectin, D-galacturonic acid, L-galactonic

acid, D-gluconic, D-glucuronic, glucuronamide, quinic

acid, methyl pyruvate, L-lactic acid, citric acid and D-

malic acid. One notable difference under these con-

ditions was B. subtilis subsp. spizizeniiwas susceptible

to rifampycin SV, while the others were not. Interest-

ingly, it has been shown that mutations in the RNA

polymerase associated with rifampycin resistance

have been shown to regulate the production of the

antibiotic 3,30-neotrehalosadiamine in B. subtilis

(Inaoka and Ochi 2011). Future studies should explore

the regulation of 3,30-neotrehalosadiamine in B. sub-

tilis subsp. spizizenii and B. subtilis subsp. subtilis to

understand the evolution of these genes.

Conclusions

Based on the phylogeny, comparative genomics and

distinguishing phenotypes of these strains, they should

be promoted to species status.

Description of Bacillus spizizenii sp. nov

Bacillus spizizenii [spi.zi.zeni.i. L. gen. n. spizizenii

named after the American bacteriologist J. Spizizen,

whose seminal studies of Bacillus subtilis made this

species a model organism of bacterial genetics].

Vegetative cells are rods that measure

0.5 9 2–3 lm and occur either singly or in chains.

Motile. Cylindrical-ellipsoidal spores form centrally

or paracentrally in non-swollen sporangia. Faculta-

tively anaerobic on anaerobic agar. Catalase-positive.

Grows at 14–50 �C, with optimum growth at

28–30 �C. Growth in 7 % (w/v) NaCl occurs after

72 h. The major fatty acids are were anteiso-C15 : 0,

iso-C15 : 0, iso-C17 : 0, anteiso-C17 : 0. Hydrolyses starch

and casein; reduces nitrate to nitrite. Citrate is utilized.

Contains the genes to produce the antibiotic subtilosin

and bacilysin, the lipopeptides mycosubtilin and

surfactin, and the antibiotic 3,30-neotrehalosadiamine.

The average genome size is approximately 4.1 Mbp

with an average G ? C mole content of 43.8%.

The type strain is TU-B-10T (= NRRL B-23049T-

= LMG 19156T = DSM 15029T = BCRC 17366T).

The 16S rRNA sequence is available at GenBank

MN536905.

Description of Bacillus inaquosorum sp. nov

Bacillus inaquosorum [in.a.quo.so0rum. L. adj. inaqu-

osus poor of water, L. gen. pl. n. (solium) inaquosorum

from (soils) poor of water (desert soils) from which

this organism was isolated].

Vegetative cells are rods that measure

0.5 9 2–3 lm and occur either singly or in chains.

Motile. Cylindrical-ellipsoidal spores form centrally

or paracentrally in non-swollen sporangia. Faculta-

tively anaerobic on anaerobic agar. Catalase-positive.

Grows at 14–55 �C, with optimum growth at

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

(Voges–Proskauer test) at pH 5.5–5.7. The major fatty

acids are were anteiso-C15 : 0, iso-C15 : 0, iso-C17 : 0,

anteiso-C17 : 0. Hydrolyses starch and casein; reduces

nitrate to nitrite. Citrate is utilized. Contains the genes

to produce the antibiotic subtilosin and bacilysin, the

lipopeptides bacillomycin F, surfactin and fengycin.

The average genome size is approximately 4.2 Mbp

with an average G ? C mole content of 43.8%.

The type strain is DV7-B-4T (= NRRL B-23052T-

= KCTC 13429T = BGSC 3A28T = DSM 22148T-

= BCRC 17984T). The 16S rRNA sequence is

available at GenBank AMXN01000021.

123

Antonie van Leeuwenhoek (2020) 113:1–12 9

Description of Bacillus stercoris sp. nov

Bacillus stercoris [ster’co.ris. L. gen. n. stercoris, of

compost, from which the strain was isolated].

Vegetative cells are rods that measure 0.5 9 2–3

lm and occur either singly or in chains. Motile.

Cylindrical-ellipsoidal spores form centrally or para-

centrally in non-swollen sporangia. Facultatively

anaerobic on anaerobic agar. Catalase-positive. Grows

at 14–50 �C, with optimum growth at 45 �C. Growthin 7 % (w/v) NaCl occurs after 72 h. The major fatty

acids are were anteiso-C15 : 0, iso-C15 : 0, iso-C17 : 0,

anteiso-C17 : 0. Hydrolyses starch and casein; reduces

nitrate to nitrite. Citrate is utilized. Contains the genes

to produce the antibiotic subtilosin and bacilysin, the

lipopeptides fengycin and surfactin.

The average genome size is approximately 4.1 Mbp

with an average G ? C mole content of 43.8%.

The type strain is D7XPN1T (= KCTC 33554T-

= JCM 30051T). The 16S rRNA sequence is available

at GenBank MN536904.

Acknowledgements The authors would like to thank Heather

Walker for her technical expertise in genome sequencing and

phenotype analysis. Any opinions, findings, conclusions, or

recommendations expressed in this publication are those of the

author(s) and do not necessarily reflect the view of the U.S.

Department of Agriculture. The mention of firm names or trade

products does not imply that they are endorsed or recommended

by the USDA over other firms or similar products not

mentioned. USDA is an equal opportunity provider and

employer. This material is also based in part upon work

supported by the National Science Foundation under Grant No.

1756219.

Author contributions The study conception, design,

comparative genomics and interpretation was performed by

Christopher Dunlap. Mass spectroscopy and analysis was

performed by Michael Bowman. Correlation of the genes to

the Bacillus subtilis orthologs and associated interpretation the

results was performed by Daniel Zeigler. The first draft of the

manuscript was written by Christopher Dunlap and all authors

commented on previous versions of the manuscript. All authors

read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest The authors declare no conflicts of

interest.

Research involving human participants and/or animalsThis article does not contain any studies with human participants

or animals performed by the author.

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