1
Solibacillus kalamii sp. nov., isolated from the International Space Station HEPA
2
filter system
3
Aleksandra Checinska1#, Rajendran Mathan Kumar2#, Deepika Pal2, Shanmugam
4
Mayilraj2, and Kasthuri Venkateswaran1*
5
1
6
2
7
Technology (CSIR-IMTECH), Chandigarh, India.
8
#
9
*Corresponding author:
Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA
MTCC- Microbial Type Culture Collection & Gene Bank, CSIR- Institute of Microbial
both authors have contributed equally
10
Dr. Kasthuri Venkateswaran
11
Senior Research Scientist California Institute of Technology
12
Jet Propulsion Laboratory, Biotechnology and Planetary Protection Group
13
M/S 89-2
14
4800 Oak Grove Dr., Pasadena, CA 91109
15
Tel: (818) 393-1481; Fax: (818) 3934176
16
E-mail: kjvenkat@jpl.nasa.gov
17
Running title: Solibacillus kalamii sp. nov.
18
Subject category: New taxa - Firmicutes and related organisms
19
Key words: Solibacillus, Endospores, International Space Station, HEPA, 16S rRNA
20
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain
21
ISSFR-015T is KT763359.
22
1
23
Abstract
24
A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain
25
designated as ISSFR-015T,isolated from the International Space Station (ISS) high-
26
efficiency particulate arrestance (HEPA) filter, was characterized by polyphasic
27
taxonomy. A comparative analysis of the 16S rRNA gene sequence (1,494 bp) of the
28
strain ISSFR-015T showed the highest similarity to Solibacillus isronensis B3W22T
29
( 98.9%), followed by Solibacillus silvestris HR3-23T (98.6%), and Bacillus cecembensis
30
PN5T (96.7%). DNA-DNA hybridization (DDH) analysis revealed the DNA relatedness
31
values of strain ISSFR-015T with other closely related species to be in the range of 41 to
32
47% (S. silvestris MTCC 10789T [47%], S. isronensis MTCC 7902T [41%], and B.
33
cecembensis MTCC 9127T [43%]). The DNA G+C content of the strain ISSFR-015T was
34
45.4 mol%. The major fatty acids were iso-C15:0 (45.2%) and C17:1ω10c (12.1%). The
35
polar
36
phosphatidylethanolamine, phosphatidylserine, and one unknown phospholipid. The
37
isoprenoid quinones present in the strain ISSFR-015T were MK-7 (86.8 %), MK-6 (11.6
38
%), and MK-8 (1.0 %). The peptidoglycan type of the cell wall was A4α L-Lys-D-Glu.
39
Based on phylogenetic analysis, strain ISSFR-015T belongs to the genus Solibacillus. The
40
polyphasic taxonomy data, including low DNA-DNA hybridization values and the
41
chemotaxonomic analysis, confirmed that the strain ISSFR-015T represents a novel
42
species, for which the name Solibacillus kalamii sp. nov. is proposed. The type strain for
43
this proposed species is ISSFR-015T (=NRRL B-65388T= DSM 101595T).
lipid
profile
contained
diphosphatidylglycerol,
phosphatidylglycerol,
44
2
45
Solibacillus silvestris was originally described as Bacillus silvestris, and the phylogenetic
46
analysis classified this species within Bacillus RNA Group 2 (Rheims et al., 1999),
47
followed by reclassification to novel genus Solibacillus (Krishnamurthi et al., 2009).
48
Despite the typical Bacillus morphology, i.e., rod cells and endospore formation, S.
49
silvestris lacks some Bacillus “core” characteristics (Kämpfer et al., 2006). The members
50
of Solibacillus genus are positive for catalase, but negative for oxidase, Voges-Proskauer,
51
nitrate reduction, and indole production, and they produce round endospores in terminally
52
swollen sporangia. Recently, Mual et al. (2016) reported an emended description of the
53
genus Solibacillus including cell wall as A4αL-Lys, D-Glu peptidoglycan type. The
54
major menaquinone is MK-7 with small amounts of MK-5, -6 and -8. Major fatty acids
55
are
56
diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine
57
(PE), and phosphatidylserine (PS). Until recently S. silvestris was the only representative
58
of this genus, however, the taxonomy of Bacillus isronensis was revisited, and the species
59
was reclassified as Solibacillus isronensis (Mual et al., 2016).
60
Microbial surveillance of the International Space Station (ISS) has revealed that bacteria
61
and fungi inhabit this unique, closed life-support system orbiting the Earth, although
62
microbial species found on the ISS have not yet been thoroughly characterized (Pierson
63
et al., 2012). The environmental control system aboard the ISS includes a distributed
64
ventilation system that contains high-efficiency particulate arrestance (HEPA) filter
65
elements to remove suspended particulate matter from the cabin atmosphere and protect
66
humidity-control and air-purification equipment from debris accumulation and biofouling
67
(Perry, 2005). The HEPA filter element analyzed during this study was manufactured in
iso-C15:0,
C16:1ω7c,
and
iso-C17:1ω7c.
The
major
polar
lipids
are
3
68
September 1998, installed in the ISS in January 2008, and returned aboard space shuttle
69
flight STS-134/ULF6 in late May 2011. This filter had been installed in ISS Node 2 and
70
was in service for 40 months at this location. The HEPA filter remained untouched in its
71
shipment packaging from the time it was removed from the ISS until particulates were
72
recovered at Jet Propulsion Laboratory for microbial characterization in September 2013
73
(Venkateswaran et al., 2014).
74
The HEPA filter elements were divided into small pieces, and particulates associated
75
with the pieces were aseptically collected using sterile scalpels before being
76
quantitatively measured. Approximately 1 g of HEPA-filter-associated particles were
77
weighed and placed into a sterile tube containing 25 mL of sterile phosphate-buffered
78
saline (PBS) and vortexed for 1 min. After vigorous mixing, large particles were allowed
79
to settle, and aliquots of samples were carefully siphoned and allocated for culture-based
80
and culture-independent analyses.
81
For estimating bacterial populations, after suitable serial ten-fold dilution in sterile PBS,
82
100 μL aliquots of the sample suspension were spread onto two plates of R2A medium
83
(BD Difco, Franklin Lakes, NJ, USA) and incubated at 25°C for 2 to 7 days. Several
84
strains of distinctive morphologies were isolated and archived for identification
85
(Venkateswaran, et al., 2014). In an earlier study examining the HEPA filter element
86
used in the ISS for microbial diversity, 41 bacterial strains were isolated and
87
phylogenetically analyzed for their taxonomical affiliations (Checinska et al., 2015).
88
While analyzing the 16S rRNA gene sequences of ~200 ISS strains that include 41 ISS-
89
HEPA strains, some were shown to exhibit no clear phylogenic affiliation to any of the
4
90
species. Polyphasic taxonomic data enabled description of one of the ISS-HEPA strain
91
ISSFR-015T as a novel species of the genus Solibacillus.
92
Strain ISSFR-015T and the closely related type strains S. isronensis MTCC 7902T and S.
93
silvestris MTCC 10789T) were grown on trypticase soy agar (TSA; BD Difco) at a range
94
of temperatures: 12, 25, 30, 37 and 42°C. The pH range and pH optimum were tested at
95
values of 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, adjusted with biological buffers (Xu et al.,
96
2005). Growth at various NaCl concentrations (0, 2, 5, 7, 10, and 12%) was tested in
97
trypticase soy broth (TSB; BD Difco). Cell morphology was observed by light
98
microscopy (Zeiss) at ×1000.Endospore formation was determined as described by
99
Smibert et al.(1994) and observed by phase contrast microscopy. The Gram reaction was
100
determined using the commercially available kit (HiMedia, India) according to the
101
manufacturer's instructions. Motility was checked using the method described by
102
Skerman (1967). Other polyphasic taxonomic characteristic features were tested
103
according to the proposed minimal standards for describing new taxa of Gram-stain-
104
positive endospore forming bacteria (Logan et al., 2009). Tests for the following
105
reactions were performed as described previously (Barrow & Feltham, 1993; Murray et
106
al., 1994): catalase, oxidase, and hydrogen sulfide production; starch, casein, and gelatin
107
hydrolysis; indole formation; citrate utilization; nitrate reduction; methyl red and Voges-
108
Proskauer reaction tests. Oxidation of different substrates and enzymatic activity were
109
determined using Biolog GP and VITEK 2GP cards according to the manufacturer’s
110
instructions. Acid production from different carbohydrates was tested on minimal
111
medium (ammonium sulfate 0.2%, potassium hydrogen phosphate 0.024%, magnesium
5
112
sulfate 0.024%, potassium chloride 0.01%, yeast extract 0.01%, and bromocresol purple
113
0.2% as acid–base indicator).
114
Cells of the strain ISSFR-015T are Gram-stain-positive, motile, and rod-shaped;
115
endospores are round and sub-terminally positioned in swollen sporangia (Supplementary
116
Fig. S1). Colonies are beige colour with circular and smooth edges. Strain ISSFR-015T
117
grows at temperatures between 12 and 37˚C (with optimum growth at 30˚C), at a pH
118
range between 6.0 and 10.0 (with optimum at 8.0), and in the presence of 0–5% NaCl.
119
The ISSFR-015T strain is positive for oxidase and catalase, and it liquefies gelatin. The
120
strain is negative for hydrolysis of starch and casein, production of indole, and utilization
121
of citrate. It also exhibits negative methyl red and Voges-Proskauer reactions; produces
122
urease and hydrolyses of Tween 80. Nitrate was not reduced to nitrite, and hydrogen
123
sulfide was not produced. The phenotypic differentiation of the strain ISSFR-015T from
124
other closely related reference species is shown in Table 1. Production of tyrosine
125
arylamidase and hydrolysis of gelatin and utilization of glycyl-L-proline, L-arginine, L-
126
aspartic acid, guadinine HCl, and quinic acid as sole carbon substrates are unique to the
127
strain ISSFR-015T.
128
Genomic DNA extraction and 16S rRNA gene amplification were performed as
129
described by Mayilraj et al. (2006). Identification of phylogenetic neighbours and the
130
calculation of pairwise 16S rRNA gene sequence similarities were achieved using the
131
EzTaxon-e server (Kim et al., 2012) and aligned using MEGA version 6.0 (Tamura et al.,
132
2013).
133
The comparative analysis of the 16 rRNA gene sequence (1,494 bp) revealed that the
134
strain ISSFR-015T has the highest 16S rRNA gene sequence similarity to S. isronensis
6
135
B3W22T ( 98.9%), followed by S. silvestris HR3-23T (98.6%), and B. cecembensis PN5T
136
(96.7%). The phylogenetic tree was constructed by neighbour-joining (NJ) algorithm
137
(Saitou & Nei, 1987) as well as by maximum-parsimony (MP), and maximum-likelihood
138
(ML) in MEGA 6.0 (Tamura, et al., 2013). The evolutionary distances were computed
139
using the neighbour-joining method. The bootstrap test was based on 1,000 replicates,and
140
the values are shown in figures next to the branches (Felsenstein, 1985). A combined
141
phylogenetic tree shows that strain ISSFR-015T forms a separate branch, along with S.
142
isronensis B3W22T and S. silvestris HR3-23T (Fig. 1). The same figure also shows
143
branches recovered in additional models (MP and ML).
144
DNA–DNA hybridization (DDH) was repeated three times by the membrane filter
145
method (Tourova et al., 1987), using freshly isolated genomic DNA in each iteration. The
146
DNA-DNA hybridization analysis showed that the ISSFR-015T strain yields relatedness
147
values of  47% with other closely related species (S. silvestris MTCC 10789T [47%], S.
148
isronensis MTCC 7902T [41%], and B. cecembensis MTCC 9127T [43%]). The values of
149
DDH obtained with respect to the most closely related species (>97 % 16S rRNA
150
similarity) clearly support the proposal for a new species (Stackebrandt & Goebel,1994).
151
The G+C content of genomic DNA was determined spectrophotometrically (Lambda 35,
152
Perkin Elmer, Waltham, MA, USA) using the thermal denaturation method (Mandel &
153
Marmur, 1968). The DNA G+C content of the strain ISSFR-015T is 45.4 mol%, which is
154
slightly higher than the values for the reference species [S. isronensis MTCC 7902T
155
40.1mol% (40.0mol % (Shivaji et al., 2009) and S. silvestris MTCC 10789T 43.1 mol%
156
(39.3 mol %; (Krishnamurthi, et al., 2009)].
157
7
158
Freeze-dried cells for chemotaxonomic analysis (except for fatty acid analysis) were
159
prepared by harvesting the bacterial cells in the late exponential phase following their
160
growth in TSB (HiMedia, India) at 30˚C. For the determination of cellular fatty acids,
161
strain ISSFR-015T and the reference strains were grown on TSA medium at 30˚C for 30
162
hours before collecting biomass. Cellular fatty acids were extracted, methylated,and
163
analyzed by gas chromatography according to the instructions of the Sherlock Microbial
164
Identification System (MIDI, USA Version 4.0) as described previously (Müller et al.,
165
1998; Pandey et al., 2002). Menaquinones and phospholipids were extracted and
166
analyzed using the methods described by Minnikin et al. (1984). Two-dimensional thin-
167
layer chromatography (TLC) was carried out for identification of polar lipids according
168
to established procedures (Komagata & Suzuki, 1987). Lipid spots were detected by
169
spraying molybdophosphoric acid (5% w/v) prepared in absolute ethanol. Hydrolysis was
170
performed (4.0 N HCl, 100 ˚C, 16 h) to measure peptidoglycan andtwo-dimensional
171
ascending TLC as described by Schumann (2011) was followed to separate amino acids
172
and peptides. The results were compared with the information available on the DSMZ
173
website: www.peptidoglycan-info.com.
174
The detailed fatty acid profiles are given in Supplementary Table S1. Major fatty acids
175
(higher than 10% of the total) are iso-C15:0 (45.2%) and C17:1ω10c (12.1%). The
176
intermediate fatty acids (5–10% of the total) are iso-C16:0 (8.1%), iso-C17:0 (7.9%),
177
andC16:1ω7c alcohol (9.1%). The fatty acid composition of ISSFR-015T is not
178
significantly different from those of S. isronensis MTCC 7902T and S. silvestris MTCC
179
10789T, but it differs vastly from B. subtilis MTCC 121T (Supplementary Table S1). The
8
180
only exception is C16:0, whichis much lower in strain ISSFR-015T when compared to the
181
other two closely related species.
182
The characteristic respiratory quinones of the genus Solibacillus (Rheims, et al., 1999),
183
are MK7, MK6 and MK8; the same pattern was also observed in ISSFR-015T [MK-7
184
(86.8%), MK-6 (11.6%), and MK-8 (1.0%)]. The polar lipid profile of ISSFR-015T
185
consists
186
phosphatidylethanolamine
187
phospholipid (PL) (Total lipids and glycolipids; Supplementary Fig. S2). Similar polar
188
lipid profiles were also observed in S. silvestris MTCC 10789T as reported elsewhere
189
(Rheims, et al., 1999). The peptidoglycan type of the strain ISSFR-015Tis A4 α L-Lys ←
190
D-Glu (Supplementary Fig.S3), the same pattern was observed in the two closely related
191
species S. isronensis and S. silvestris [peptidoglycan type A4 α L-Lys ← D-Glu (type
192
A11.33 according to http://www.peptidoglycan-types.info/) Mual et al., 2016].
193
Comparing 16S rRNA sequences revealed that ISSFR-015T is the most closely related to
194
S. isronensis and S. silvestris, and DDH analysis also proved that the ISSFR-015T strain is
195
distinct sincethe DDH values are below the 70% threshold level. Furthermore, the lipid
196
profile of the ISSFR-015T strain is comprised of DPG, PG, PE, and PS. The Solibacillus
197
species and S. kalamii strain contain MK-7 as their major menaquinone, but they also
198
have low amounts of MK-6 and MK-8. The fatty acid composition profile is very similar
199
to that of S. silvestris, and the signature fatty acids for Solibacillus, such as iso-C15:0 (45-
200
47.4% of total), are dominant in the ISSFR-015T strain (Supplementary Table S1).
201
Chemotaxonomic features such as polar lipid profile, fatty acid compositions,
202
physiological and biochemical characteristic features, and phylogenetic analysis strongly
of
diphosphatidylglycerol
(PE),
(DPG),
phosphatidyl
serine
phosphatidylglycerol
(PS),
and
one
(PG),
unknown
9
203
support that the strain ISSFR-015T should be classified as a novel species of the genus
204
Solibacillus. Several differences in biochemical profiles as well as DDH analysis results
205
from other Solibacillus species, clearly show that the strain ISSFR-015T can be
206
distinguished as a novel species within the genus Solibacillus, a species for which we
207
propose the name Solibacillus kalamii sp. nov.
208
209
Description of Solibacillus kalamii sp.nov.
210
Solibacillus kalamii (ka.lam'i.i.N.L. gen. n. kalamii referring to Abdul Kalam, a well-
211
known scientist who advanced space research in India).
212
Cells are Gram-stain-positive, strictly aerobic, motile rods (0.4–0.7×1.1–3.6 μm) and
213
form endospores (round endospores in subterminal sporangia). Colonies are circular
214
andbeige after 2 days of incubation on TSA at 30 °C. Cells grow at 12- 37 ˚C (optimum
215
at 30 ˚C) and at pH range of 6.0–10.0 (optimum pH at 8.0), and tolerate NaCl
216
concentration in a range of 0-5 % (w/v). Positive for catalase and oxidase. Nitrate is not
217
reduced to nitrite, and hydrogen sulfide is not produced. Negative for hydrolysis of casein
218
and starch, production of indole and utilization of citrate. Positive for hydrolysis of
219
gelatin and Tween 80, and for urease production. Negative for methyl red and Voges-
220
Proskauer reactions. Acid is not produced from arabinose, adonitol, dulcitol, mannitol,
221
mannose, and xylose. Positive for arginine dihydrolase 1, arginine dihydrolase 2
222
production and L-lactate alkalinization; Positive for growth at 1% sodium lactate;
223
oxidation of L-pyroglutamic acid, glucuronamide; negative for oxidation of D-
224
amygdalin, phosphatidylinositol phospholipase C, D-xylose, Ala-Phe-Pro arylamidase, α-
225
glucosidase,
β-glucosidase
cyclodextrin,
β-galactopyranosidase,
α-mannosidase,
10
226
phosphatase, α-galactosidase, L-proline arylamidase, β-glucuronidase, β-galactosidase,
227
D-ribose, lactose, N-acetyl-D-glucosamine, D-mannitol, D-manose, Methyl-B-D-
228
Glucopyranoside, pullulan, D-raffinose.; negative for utilization of sole carbon and
229
energy source of D-maltose, D-trehalose, D-cellobiose, gentiobiose, sucrose, D-turanose,
230
D-lactose, D-melibiose, methyl-D-glucoside, D-salicin, N-acetyl-D-mannosamine, N-
231
acetyl-D-galactosamine, D-fructose, D-galactose, 3-methyl glucose, D-fucose, L-
232
rhamnose, inosine, fusidic acid, D-sorbitol, D-arabitol, myo-inositol, D-aspartic acid, D-
233
serine, pectin, D-gluconic acid, mucic acid, D-saccharic acid, p-hydroxy-phenylacetic
234
acid, D-lactic acid methyl ester, citric acid, keto-glutaric acid, amino-butryric acid,
235
propionic acid and formic acid. Major cellular fatty acids (>10 %) are iso-C15:0 and
236
C17:1ω10c. The major menaquinone is MK-7. The main polar lipids are DPG, PG, PE, PS,
237
and one unknown phospholipid. The DNA G+C content of the strain ISSFR-015T is 45.4
238
mol%. The type strain is ISSFR-015T(=NRRL B-65388T= DSM 101595T) isolated from a
239
HEPA filter element used in the ISS.
240
Acknowledgements
241
Part of the research described in this publication was carried out at the Jet Propulsion
242
Laboratory, California Institute of Technology, under a contract with NASA. This
243
research was funded by a 2012 Space Biology NNH12ZTT001N grant no. 19-12829-26
244
under Task Order NNN13D111T award to K. Venkateswaran. We acknowledge Dr. Jay
245
Perry form Marshall Space Flight Center and Dr. David Eisenman for coordinating with
246
the ISS program to deliver the ISS HEPA filters intact. We appreciate technical help from
247
Dr. Parag Vaishampayan of JPL and Mr. Malkit Singh of the Institute of Microbial
248
Technology (IMTECH). This work is partly supported by a project, "Expansion and
11
249
Modernization of Microbial Type Culture Collection and Gene Bank (MTCC),” jointly
250
supported by the Council of Scientific and Industrial Research (CSIR) Grant No.
251
BSC0402 and Department of Biotechnology (DBT) Govt. of India Grant No.
252
BT/PR7368/INF/22/177/2012." © 2016 California Institute of Technology. Government
253
sponsorship acknowledged.
254
255
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Figure Legends
347
Fig. 1. Phylogenetic tree based upon neighbour-joining algorithm showing the
348
relationship between S. kalamii sp. nov. ISSFR-015T, other closely related Solibacillus
349
species and members of non-Solibacillus genera of rRNA group. Sequence from
350
Paenibacillus polymyxa IAM 13419T was used as outgroup. Bar, 0.01 substitutions per
351
site. Dots indicate branches that were also recovered using the maximum-parsimony and
352
maximum-likelihood approaches.
353
354
355
356
357
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359
360
361
362
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365
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Table 1. Differential characteristics features of strains 1. ISSFR-015T, 2. Solibacillus
369
silvestris HR3-23T, and 3. S. isronensis B3W22T.
370
All the data are from present study unless otherwise indicated.
Characteristics features
1
2
3
Growth at 42°C
-
-
+
Fructose
-
+
-
Trehalose
-
-
+
l-pyrrolidonyl-arylamidase
+
+
-
Phenylalanine arylamidase
-
+
+
Leucine arylamidase
-
+
-
Ellman test for thiol group reduction
-
+
-
Tyrosine arylamidase
+
-
-
-
+
-
Acid production from:
Enzyme activity using VITEK® 2-GP cards
Utilization of D-ribose
Oxidation of compounds using Biolog GP2 MicroPlates
Dextrin
-
+
+
Stachyose
-
-
+
D-serine
-
-
+
Glycerol
-
+
-
Gelatin
+
-
-
Glycyl-L-proline
+
-
-
L-alanine
-
-
+
18
L-arginine
+
-
-
L-aspartic acid
+
-
-
L-glutamic acid
-
+
+
L-histidine
-
-
+
L-serine
-
+
-
Guanidine HCL
+
-
-
D-galacturonic acid
+
-
+
D-glucuronic acid
+
+
-
Quinic acid
+
-
-
Tetrazolium violet
-
+
+
Tetrazolium blue
-
+
+
L-lactic acid
-
+
-
D-malic acid
-
-
+
L-malic acid
+
-
+
Bromosuccinc acid
-
-
+
Tween 40
-
+
-
Hydroxy-butyric acid
+
+
-
β -hydroxy-D-L-butyric acid
+
+
-
Acetoacetic acid
+
-
+
45.4
43.1 (39.3$)
40.1 (40.0*)
DNA G+C content mol %
371
DNA G+C content mol %; $Solibacillus silvestris HR3-23T (Shivaji, et al., 2009),
372
*Solibacillus isronensis B3W22T (Krishnamurthi, et al., 2009), data taken from the
373
published manuscript.
374
19