2
Supplementary document
3
4
5
6
7
8
Trehalose/2-sulfotrehalose biosynthesis and glycine-betaine uptake are widely
spread mechanisms for osmoadaptation in the Halobacteriales
Authors: Noha H. Youssef, Kristen N. Savage-Ashlock, Alexandra L. McCully, Brandon
Luedtke, Edward I. Shaw, Wouter D. Hoff, and Mostafa S. Elshahed
9
10
1
11
12
Detailed Materials and Methods
13
Archaeal strains. Haladaptatus paucihalophilus strain DX253T (DSM18195) was
14
isolated from Zodletone spring, a sulfide- and sulfur-rich spring in southwestern
15
Oklahoma (Savage et al 2007), and maintained in our laboratory. Natrialba magadii
16
DSM3394, Natrinema pellirubrum DSM15624, Natronobacterium gregorii DSM3393,
17
Natronococcus occultus DSM3396, Halalkalicoccus jeotgali DSM18796, and
18
Haloterrigena turkmenica DSM5511 were kindly provided by Drs. Jonathan A. Eisen
19
and Marc T. Facciotti (University of California Davis). All other strains were purchased
20
from the German Collection of Microorganisms (DSMZ, Braunschweig, Germany). All
21
cultures were maintained in DSMZ-recommended media (Table S1) on sealed agar plates
22
or agar slants and stored at 4ºC.
23
Genome of Haladaptatus paucihalophilus strain DX253T. DNA was extracted from
24
100 mls of a late exponential phase culture using MoBio Powersoil DNA Extraction kit
25
(MO BIO Laboratories, Carlsbad, California). The high-quality extracted DNA was
26
sequenced using the service of a commercial sequencing provider (Engencore, now Selah
27
Genomics, Columbia, South Carolina) using 454 FLX technology. The genome was
28
assembled using Newbler assembler into 32 Contigs (N50 = 267, 250 bp, N90 = 100,497
29
bp). Gene calling, annotation, and metabolic construction was conducted using the
30
Department of Energy Integrated Microbial Genomes (IMG) platform (Markowitz et al
31
2012).
32
Growth media and Experimental setup. Haladaptatus paucihalophilus was grown in a
33
defined media containing (g.l-1): MgCl2.6H2O (20), K2SO4 (5), CaCl2.2H2O (0.1), NH4Cl
34
(0.5), KH2PO4 (0.05), and sodium pyruvate (0.5). The medium was buffered to a pH of
2
35
6.8 using 25mM HEPES. After autoclaving, 1 ml of modified Wolin’s metal solution
36
containing (g.l-1): EDTA, 0.5; MnSO4·H2O, 3; NaCl, 1; CaCl2·2H2O, 0.1; ZnSO4·7H2O,
37
0.1; FeSO4·7H2O, 0.1; CuSO4·5H2O, 0.01; AlK(SO4)2, 0.01; Na2MoO4·2H2O, 0.01; boric
38
acid, 0.01; Na2SeO4, 0.005;NiCl2·6H2O, 0.003, and 10 ml of vitamin solution (Widdel
39
and Pfennig 1981) were added to 1 l of the above medium. This medium has a similar
40
composition to DSMZ 1125, but with no yeast extract and with pyruvate rather than
41
sucrose as a carbon source. This was done to ensure that any compatible solutes detected
42
in cell extracts are synthesized de novo. NaCl concentrations were varied between 75-275
43
g.l-1 in different experiments. All other strains were cultured in their DSMZ media at
44
three different salt concentrations (Table S1) that are close to their lower, optimum, and
45
higher NaCl growth range. In experiments assessing glycine betaine uptake in
46
Haladaptatus paucihalophilus, glycine betaine (10 g.l-1, Sigma-Aldrich, St. Louis, MO)
47
was added to the growth medium described above. For all other microorganisms, no
48
glycine betaine was added since they were grown in complex media containing yeast
49
extract, where glycine betaine is one of its components (estimated at 110 μg/g yeast
50
extract (Dulaney et al 1968)). Approximately, 1.5x108 cells in the exponential phase were
51
used as an inoculum for 250 ml cultures in all experiments. Cultures were grown to late
52
exponential phase (O.D. ~0.5), harvested, and used for the quantification of various
53
intracellular solutes as described below.
54
Analytical methods.
55
Identification of compatible solutes in cell-free ethanol extracts. The presence and
56
identity of compatible solutes in cell-free extracts were initially screened using 1H-
57
nuclear magnetic resonance spectroscopy (1H-NMR), and high performance liquid
3
58
chromatography (HPLC). Cell pellets were extracted using a modified ethanol extraction
59
method (Falicia Goh et al 2011). Cell pellets were washed three times in a sterile isotonic
60
NaCl solution, followed by lyophilization. The lyophilized cell pellets were extracted
61
with 70% ethanol, vortexed for 5 min, then centrifuged at 10,000 xg for 10 min. Ethanol
62
extraction was repeated 2 more times and the combined ethanol extracts were evaporated
63
overnight at 37ºC. For HPLC analysis, dried ethanol extracts were re-dissolved in water
64
and 20 µl were analyzed on an Agilent 1100 Series (Santa Clara, CA) HPLC with a
65
refractive index detector and an Aminex HPX-87P column. Analysis was conducted at
66
85°C using a mobile phase of deionized water pumped at 0.6 mL/min for 30 min (Sluiter
67
et al 2008). Trehalose was identified in the cell-free extracts by comparison to a trehalose
68
standard (Sigma-Aldrich). For 1H-NMR analysis, dried ethanol extracts were dissolved in
69
0.5 ml of D2O and 0.09 mM solution of 2,2-Dimethyl-2-Silapentane-5-Sulfonate-d6
70
sodium salt (DSS) in D2O. Spectra were taken at 25oC with a one-pulse sequence with
71
water pre-saturation on a Varian Inova 600 NMR spectrometer with a 5 mm indirect
72
detection probe, using a 1.5 second pre-saturation delay between pulses and 6.6
73
microsecond 90 degree pulse. The spectra were referenced to DSS at 0 ppm, with error in
74
ppm values of +/- 0.05 ppm. Spectra were transformed with MNova software (Masterlab
75
research, Santiago de Compostela, Spain), using solvent signal suppression to remove the
76
residual water resonance. Reference trehalose was used for comparison.
77
Quantification of ions and compatible solutes. Cell pellets were washed in sterile
78
isotonic NaCl solution. After removal of all traces of medium, the pellet was frozen at -
79
20ºC overnight followed by thawing in 10 ml solution of 10% perchloric acid (PCA).
80
Thawed pellets were vortexed for 5 min, followed by incubation at room temperature for
4
81
2 hours during which cells lysed and proteins precipitated. Extracts were then centrifuged
82
at 10,000 xg for 15 min. The pellet containing the precipitated proteins was suspended in
83
0.85% saline solution and used for protein quantification using total protein kit (Micro
84
Lowry kit, Onishi and Barr Modification by Sigma Aldrich). The supernatant, containing
85
the cell-free, protein-free, extract was used for quantification of intracellular solutes and
86
ions as described below.
87
Quantification of trehalose in cell-free extracts. The concentration of trehalose in cell-
88
free PCA extracts was determined enzymatically (Neves et al 1994) using a trehalase
89
assay kit (Megazyme®, Wicklow, Ireland). In addition to trehalose, this enzymatic assay
90
is suitable for the quantification of 2-sulfotrehalose, a putative compatible solute
91
previously identified in a few alkaliphilic Halobacteriales genera (Natrialba magadii,
92
Natronobacterium gregorii, and Natronococcus occultus (Desmarais et al 1997)). This is
93
due to the fact that trehalase enzyme requires only one free hydroxyl group at the 2′
94
position for hydrolytic activity (Mori et al 2009, Ocon et al 2007), and hence the presence
95
of a sulfonate group at one of the 2′ positions should not hinder its activity.
96
Therefore, to differentiate trehalose from 2-sulfotrehalose, we devised a fast
97
method to detect the presence of a 2′-O-sulfonate group based on the action of abalone
98
snail sulfatase (Sigma-Aldrich), shown to be specific for cleaving the 2′-O-sulfonate
99
group of trehalose (Uzawa et al 2004), followed by quantification of the released
100
inorganic sulfate turbidimetrically (Lundquist et al 1980). PCA extracts positive for
101
trehalose were assayed for the presence of free inorganic sulfate before and after
102
treatment with abalone snail sulfatase (1 U per 1.5 µmole trehalose for 75 hours at 37ºC).
103
Differences in inorganic sulfate content were converted to µmole sulfate and compared to
5
104
the total amount of trehalose (µmoles) in the extracts. The sulfate:trehalose ratio obtained
105
for Natronococcus occultus, previously shown to produce 2-sulfotrehalose (Desmarais et
106
al 1997), was 0.44±0.26. This value is in accordance with the previously reported yield of
107
abalone snail sulfatase of 28% on p-nitrophenyl-2-O-sulfo-glycoside (Uzawa et al 2004).
108
Sulfate:trehalose ratios > 0.1 were deemed indicative of 2-sulfotrehalose production. By
109
comparison, the sulfate:trehelose ratio for Haladaptatus paucihalophilus, shown by 1H-
110
NMR to produce trehalose rather than 2-sulfotrehalose, never exceeded 0.01.
111
Quantification of glycine betaine in cell-free extracts. Glycine betaine was quantified in
112
PCA extracts using a colorimetric assay for quaternary ammonium compounds (QACs)
113
as described before (Grieve and Grattan 1983). The assay is based on the precipitation of
114
QACs as their periodide salts. Under acidic conditions (pH 0-1) and at low temperatures
115
(0-4ºC), betaine precipitates, when reacted with a KI-I2 reagent, as glycine betaine-
116
periodide complex, which has a characteristic absorbance at 365 nm when dissolved in
117
1,2-dichloroethane. Concentrations of glycine betaine in PCA extracts were calculated
118
using a glycine betaine standard curve (0-200 µg/ml).
119
Quantification of intracellular potassium in cell-free extracts. Intracellular K+ levels
120
were quantified in cell free extracts of various Halobacteriales species
121
spectophotmetrically using an approach based on the formation of K+-K+-specific
122
ionophore complex in the presence of a dye anion followed by the extraction of the K+-
123
K+-specific ionophore complex using an organic solvent and measuring the increase in
124
the absorbance of the organic layer (Takagi et al 1981). PCA extracts were mixed with
125
the K+-specific ionophore, Kryptofix 221 (Sigma Aldrich) in the presence of the anionic
126
dye picrate. The K+-Kryptofix 221 complex was then extracted by vortexing with
6
127
toluene. The increase in the absorbance of the organic layer, proportional to the K+
128
concentration in the PCA extracts, was measured at 360 nm against toluene.
129
Quantification of intracellular free amino acids in cell-free extracts. PCA extracts were
130
assayed for free amino acids using the services of the Protein Chemistry Lab of Texas
131
A&M University. Free Amino acids are derivatized with o-phthalaldehyde (OPA) and 9-
132
fluoromethyl-chloroformate (FMOC). OPA reacts with all amino acids except proline,
133
while FMOC reacts with proline. The derivatized amino acids are separated by reverse
134
phase HPLC on an Agilent 1260 liquid chromatograph using a 5 µm reverse phase
135
column and a gradient elution using 100% solvent A (20mM Na acetate buffer with
136
0.018% v/v triethylamine, 0.05mM EDTA, and 0.3% tetrahydrofuran, pH 7.2) at 0 min,
137
going to 60% Solvent B (100 mM Na acetate buffer: acetonitrile: methanol (1:2:2 v/v))
138
over 17 min with flow rate of 0.45 ml/min. Amino acids tagged with OPA are detected at
139
338/390 nm by the Variable Wavelength (UV) detector (G1365D), while proline (tagged
140
with FMOC) is detected at 266/324 nm.
141
RNA extraction and gene expression. Total RNA was extracted from triplicate
142
Haladaptatus paucihalophilus cultures grown in defined medium described above in
143
presence of multiple salt concentrations (75, 125, 175, 225, and 275 g.l-1) using Tri
144
Reagent (Ambion®, Austin, TX) (Morgan et al 2010). Cell pellets from late exponential
145
phase cultures were solubilized in TRI Reagent® Solution (Ambion®), and processed
146
according to the manufacturer’s instruction. Contaminating DNA was removed from all
147
RNA preparations using RQ1 DNase (Promega®), followed by phenol/chloroform
148
extraction, and ethanol precipitation. To check for the complete removal of DNA, the
149
RNA preparation was used as a template in a PCR reaction conducted in a 50-μl volume
7
150
mixture containing the following (final concentrations are given): 2 μl of extracted RNA,
151
1× PCR buffer (Promega), 2.5 mM MgSO4, 0.2 mM dNTPs mixture, 2.5 U of GoTaq
152
Flexi DNA polymerase (Promega), and 0.5 µM each of H. paucihalophilus-specific 16S
153
rRNA gene primers F: 5′-GCTTTTGAGAGGAGGTGCAT-3′, and R: 5′-
154
TAATGTACCCAGCCACCACA-3′, corresponding to bases 974-993, and 1078-1097,
155
respectively of H. paucihalophilus 16S rRNA gene (GenBank accession number
156
NR_043744). PCR amplification was carried out according to the following protocol:
157
initial denaturation for 5 min at 95°C, followed by 30 cycles of denaturation at 95°C for
158
45 s, annealing at 55°C for 45 s, and elongation at 72°C for 30 sec. A final elongation
159
step at 72°C for 5 min was included. If needed, the DNase treatment was repeated until
160
the RNA was free of any contaminating DNA as confirmed by PCR. DNA-free RNA was
161
then precipitated using Lithium Chloride Precipitation Solution (Ambion, Carlsbad, CA)
162
according to manufacturer’s instructions to remove any contaminating small RNAs.
163
High-quality RNA preparations were stored at -80ºC until used. Total RNA reverse
164
transcription (cDNA construction) was conducted on 1 µg of RNA preparations using
165
Superscript III first strand synthesis kit (Invitrogen) with random hexamers (Invitrogen).
166
To study the expression levels of genes involved in trehalose synthesis in
167
Haladaptatus paucihalophilus, Reverse Transcription qPCR (RT-qPCR) was conducted
168
on the cDNA obtained using primers shown in Table S2. Levels of expression of
169
different genes were compared to Glyceraldehyde-3-Phosphate dehydrogenase gene
170
expression. qPCR was conducted using a MyIQ thermocycler (Bio-Rad Laboratories,
171
Hercules, CA) and Sybr GreenER qPCR mix (Invitrogen). The 20-µl reaction contained 2
172
µl of cDNA, 0.5 µM each of the forward and reverse primers, and 10 µl of the qPCR mix.
8
173
The reactions were heated at 50°C for 2 min, followed by heating at 95ºC for 8.5 min.
174
This was followed by 65 cycles, with one cycle consisting of 15 sec at 95°C, 60 sec at
175
52°C, and 30 sec at 72°C.
176
Phylogenetic analysis. RpoB′ protein sequences from publicly available Halobacteriales
177
genomes (n=82) were aligned in ClustalX (Larkin et al 2007 ) and used to construct a
178
maximum likelihood tree in Mega (Tamura et al 2011) using a WAG model with Γ
179
distribution of rates among sites as previously suggested (Minegishi et al 2010). Support
180
values were obtained using 1000 bootstrapping events. To examine the evolutionary
181
history of trehalose synthesis, Halobacteriales OtsA (trehalose-6-phosphate synthase),
182
and OtsB (trehalose-6-phosphatase) protein sequences, as well as those from reference
183
bacterial and archaeal taxa were aligned using ClustalX (Larkin et al 2007 ). The
184
alignment was used to predict the best protein substitution model in Mega (Tamura et al
185
2011) using maximum likelihood. The predicted model was applied in maximum
186
likelihood tree construction and support values were obtained using 1000 bootstrapping
187
events.
188
Ecological distribution of otsAB-harboring genera. We examined the correlation
189
between the presence/absence of trehalose synthesis otsAB genes in a genome and the
190
respective salt minimum, maximum and optimum concentrations. We used χ2
191
Contingency tables for correlation (Plackett 1983), since the dependent variable is
192
nominal (dichotomous). The independent variable was converted to nominal values as
193
follows. Values of salt minimum, maximum and optimum concentrations were ranked
194
and grouped into 6 categories: (very low (ranks 1-14), low (ranks 15-27), moderate
195
(ranks 28-40), moderate-high (ranks 41-53), high (ranks 54-66), and very high (ranks 67-
9
196
81)). With the two variables (dependent and independent) being nominal, χ2 Contingency
197
correlation was calculated, and the χ2 values obtained were compared to the tabulated χ2
198
value at α = 0.05. The probability of the result being due to chance (i.e. p-value) was
199
evaluated using the Chi-square calculator http://www.fourmilab.ch/rpkp/experiments
200
/analysis/chiCalc.html. Only in cases where the correlation was significant (p<0.05), the
201
obtained χ2 value was used to calculate Cramer's V statistics;
, where χ2 is the calculated χ2 value, n is the number of
202
203
Halobacteriales species with sequenced genomes (n=81), M is the number of rows (or
204
dependent variables, dichotomous), and N is the number of columns (or independent
205
variables), to measure the degree of association between the two variables.
206
To study the correlation between otsAB presence and the ecological distribution
207
of various genera, we reexamined the relative abundance of various Halobacteriales
208
genera in 13 different environments with salinities ranging from permanently hypersaline
209
(n=9), saline and fluctuating salinity habitats (where salinity is usually lower than 250 g.l-
210
1
211
diverse prokaryotic community) (n=3), and mostly non-saline with only occasional
212
development of temporal and spatial pockets (n=1, Zodletone spring). Relative
213
abundances of Halobacteriales genera with and without the OtsAB system in their
214
genome were compared between various groups of environments using Student t test.
215
Values were considered significantly different if the P-value associated with the t test
216
was <0.0038 (equivalent to an α value of 0.05 corrected for 13 pairwise comparisons
217
using the Bonferroni correction). The relative abundances values obtained were also used
218
in a principal-component analysis to evaluate the differences in genera composition
, exhibits fluctuation, and the Halobacteriales community is usually a fraction of a more
10
219
between the environments using the R statistical package (R Development Core Team
220
2011).
221
11
222
223
Results: Genes for Na+ extrusion, K+ uptake, and chloride homeostasis in
224
Haladaptatus paucihalophilus genome.
225
At least two Na+ extrusion mechanisms employing the proton electrochemical
226
gradient as the driving force for exporting Na+ exist in the genome: A Na+:H+ antiporter
227
of the NhaC family, and a multisubunit Na+:H+ antiporter similar to MnhABCDEFG. As
228
for potassium uptake, 2 clusters similar to the ATP-dependent low affinity K+ transporter
229
(Trk) exist in the genome, each consisting of the membrane component TrkH and the
230
NAD-binding component TrkA. A voltage-gated K+ channel of the Kef-type, most
231
probably functioning as a facilitated diffusion K+ uniporter driven by the membrane
232
potential, was also identified in the genome. Two voltage-gated Cl- channels of the EriC-
233
type exist in tandem in the genome upstream of a universal stress family protein. It is not
234
clear whether these would be employed in Cl- uptake or extrusion. However, two Na+-
235
dependent transporters of the neurotransmitter:Na+ symporter, NSS family, exist in the
236
genome in tandem. Some of these are known to co-transport Cl-, and could potentially act
237
as an energy-dependent inward chloride pump.
238
239
240
241
242
243
244
245
12
246
247
248
Cui H-L, Gao X, Sun F-F, Dong Y, Xu X-W, Zhou Y-G et al (2010). Halogranum
249
rubrum gen. nov., sp. nov., a halophilic archaeon isolated from a marine solar saltern. Int
250
J Syst Evol Microbiol 60: 1366-1371.
References
251
252
Desmarais D, Jablonski PE, Fedarko NS, Roberts MF (1997). 2-Sulfotrehalose, a novel
253
osmolyte in haloalkaliphilic archaea. J Bacteriol 179: 3146-3153.
254
255
Dulaney EL, Dulaney DD, Rickes EL (1968). Factors in yeast extract which relieve
256
growth inhibition of bacteria in defined medium of high osmolarity. Dev Ind Microbiol 9:
257
260-269.
258
259
Falicia Goh, Young Jae Jeon, Kevin Barrow, Brett A. Neilan, Burns BP (2011).
260
Osmoadaptive strategies of the archaeon Halococcus hamelinensis Isolated from a
261
hypersaline stromatolite environment. Astrobiology 11: 529-536.
262
263
Grieve CM, Grattan SR (1983). Rapid assay for determination of water soluble
264
quaternary ammonium compounds. Plant Soil 70: 303-307.
265
266
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H et al
267
(2007 ). Clustal W and Clustal X version 2.0. Bioinformatics 23 2947–2948.
268
13
269
Lundquist P, Mårtensson J, Sörbo B, Ohman S (1980). Turbidimetry of inorganic sulfate,
270
ester sulfate, and total sulfur in urine. Clin Chem 26: 1178-1181.
271
272
Markowitz VM, Chen I-MA, Palaniappan K, Chu K, Szeto E, Grechkin Y et al (2012).
273
IMG: the integrated microbial genomes database and comparative analysis system.
274
Nucleic Acid Res 40: D115-D122.
275
276
Minegishi H, Kamekura M, Itoh T, Echigo A, Usami R, Hashimoto T (2010). Further
277
refinement of the phylogeny of the Halobacteriaceae based on the full-length RNA
278
polymerase subunit B' (rpoB') gene. Int J Syst Evol Microbiol 60: 2398-2408.
279
280
Morgan JK, Luedtke BE, Thompson HA, Shaw EI (2010). Coxiella burnetii type IVB
281
secretion system region I genes are expressed early during the infection of host cells.
282
FEMS Microbiol Lett 311: 61-69.
283
284
Mori H, Lee J-H, Okuyama M, Nishimoto M, Ohguchi M, Kim D et al (2009). Catalytic
285
reaction mechanism based on alpha-secondary deuterium isotope effects in hydrolysis of
286
trehalose by European Honeybee trehalase. Biosci Biotechnol Biochem 73: 2466-2473.
287
288
Neves MJ, Terenzi HF, Leone FA, Jorge JA (1994). Quantification of trehalose in
289
biological samples with a conidial trehalase from the thermophilic fungus Humicola
290
grisea var. thermoidea. World Journal of Microbiology & Biotechnology 10: 17-19.
291
14
292
Ocon, Aurora, Hampp, Rudiger, Requena, Natalia (2007). Trehalose turnover during
293
abiotic stress in arbuscular mycorrhizal fungi. New Phytol 174: 879-891.
294
295
Plackett RL (1983). Karl Pearson and the Chi-Squared Test. Int Statist Rev 51: 59-72.
296
297
R Development Core Team (2011). R: A Language and Environment for Statistical
298
Computing. Reference Index., 2.13.2 edn. R Foundation for Statistical Computing:
299
Vienna, Austria.
300
301
Savage KN, Krumholz LR, Oren A, Elshahed MS (2007). Haladaptatus paucihalophilus
302
gen. nov., sp. nov., a halophilic archaeon isolated from a low-salt, sulfide-rich spring. Int
303
J Syst Evol Microbiol 57: 19-24.
304
305
Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D et al (2008).
306
Determination of structural carbohydrates and lignin in biomass. National Renewable
307
Energy Laboratory (NREL): Golden, CO.
308
309
Takagi M, Nakamura H, Sanui Y, Ueno K (1981). Spectrophotometric determination of
310
sodium by ion-pair extraction with crown ether complexes and monoanionic dyes. Analyt
311
Chim Acta 126: 185-190.
312
15
313
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011). MEGA5:
314
Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary
315
Distance, and Maximum Parsimony Methods. Mol Biol Evol 28: 2731-2739.
316
317
Uzawa H, Nishida Y, Sasaki K, Nagatsuka T, Hiramatsu H, Kobayashi K (2004).
318
Sulfatase-catalyzed assembly of regioselectively O-sulfonated p-nitrophenyl α-d-gluco-
319
and α-d-mannopyranosides. Carbohyd Res 339: 1597-1602.
320
321
Widdel F, Pfennig N (1981). Studies on dissimilatory sulfate-reducing bacteria that
322
decompose fatty acids. Arch Microbiol 129: 395-400.
16
323
324
325
326
Table S1. Microorganisms used in this study and their growth media showing the carbon source, salt concentration used in culturing
experiments, carbon sources in growth media, and yeast extract content in these media.
Strain
Growth medium
Culture
Salt concentration g.l-1
Carbon source(s)
Yeast extract
collection
(g.l-1)
Low Opt
High
Haloferax mediterranei
DSM 372
125
175
225
YE, CAA, Na citrate, Na glutamate 5
Halovivax ruber
DSM 514
125
175
225
YE, Peptone
1
Halogeometricum borinquense
DSM 372
125
225
275
YE, CAA, Na citrate, Na glutamate 5
Halostagnicola larsenii
DSM 1138
175
225
275
YE
5
Halococcus saccharolyticus
DSM 372
125
225
275
YE, CAA, Na citrate, Na glutamate 5
Halorhabdus utahensis
DSM 927
125
225
275
YE, glucose
1
Halorubrum lacusprofundi
DSM 372
125
175
275
YE, CAA, Na citrate, Na glutamate 5
Halosarcina pallida
JCM 645
75
175
225
YE, CAA
0.1
Natrialba magadii
DSM 371
125
175
275
YE, CAA, Na glutamate
5
Natrinema pellirubrum
DSM 372
125
175
275
YE, CAA, Na citrate, Na glutamate 5
Natronobacterium gregoryi
DSM 371
125
175
275
YE, CAA, Na glutamate
5
Natronococcus occultus
DSM 371
125
175
275
YE, CAA, Na glutamate
5
Halalkalicoccus jeotgali
DSM 954
125
175
275
YE, CAA
5
Halogranum rubrum
MR2A
125
225
275
YE, CAA, Na citrate, Na glutamate, 0.5
Na pyruvate, glucose, peptone
Haloterrigena turkmenica
DSM 372
125
175
275
YE, CAA, Na citrate, Na glutamate 5
Natronorubrum tibetense
DSM 371
125
175
275
YE, CAA, Na glutamate
5
Halobacterium salinarum
DSM 372
175
225
275
YE, CAA, Na citrate, Na glutamate 5
Haladaptatus paucihalophilus
DSM 1125
75
125, 175 225, 275
Na pyruvate
0
Eschericia coli K12
10
YE, tryptone
5
YE: yeast extract; CAA: casamino acid
M2RA: modified R2A medium as described in (Cui et al 2010), LB contains (g.l-1): yeast extract, 5; tryptone, 10; NaCl, 10.
17
327
Table S2. Primers used for RT-PCR.
Gene
Accession number qPCR primers
otsA1
EFW91797
F: 5’-GTGGGACGTGTTTCGAAGTT-3’
R: 5’-TAGCGAATCACTCCCGTTTC-3’
otsA2
EFW92049
F: 5’-AACTCACGGACGAGCAAGTT-3’
R: 5’-CCCGAGATGGTAATCCTGAA-3’
otsB
EFW92050
F: 5’-CAAGAACGAGTCGATGTGGA-3’
R: 5’-GAATCGAGAGTTGCGGAGAG-3’
treT
EFW90639
F: 5’-ACCGTCTGGATGGTGAACTC-3’
R: 5’-GTGCAGGAGGTTGTGAATCC-3’
GAPDH
EFW92006
F: 5’-TAACGGGTACGGGACTATCG-3’
R: 5’-ATAGCGGCGTAGAGTGGGTA-3’
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
18
356
357
Table S3. General genomic features as well as protein properties of Haladaptatus
paucihalophilus genome.
DNA, total number of bases
4,284,805
DNA coding number of bases
3,650,099 (85.2%)
DNA G+C number of bases
2,648,659 (61.8%)
DNA scaffolds
32
Genes total number
4,496
Protein coding genes
4,443
with function prediction
2,528
Average pI of all protein-coding genes
5.12
Percentage of acidic (D+E) residues
15.95
Percentage of basic (K+R) residues
8.79
Percentage of large hydrophobic residues (I+L+M+F)
18.98
RNA genes
53
rRNA genes
6
5S rRNA
2
16S rRNA
3
23S rRNA
1
tRNA genes
47
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
19
376Table S4. Comparison of average pI values of cytoplasmic versus membrane proteins in
377different halophilic archaeal and bacterial strains.
Microroganism
Average pI of proteins
Cytoplasmic
Membrane
Haladaptatus paucihalophilus
4.69
6.88
Haloarcula marismortui
4.1
6.89
Halobacterium salinarum
4.29
7.22
Halorubrum locusprofundii
4.23
6.56
Haloterigena turkmenica
4.42
5.53
Halopiger xanaduensis
4.44
5.36
Halostagnicola larsenii
4.44
5.44
Halovivax ruber
4.42
5.57
Halogeometricum borinquense
4.55
6.06
Halalkalicoccus jeotgali
4.57
6.19
Halomicrobium mukohataei
4.42
5.84
Haloferax sulfurifontis
4.49
6.11
Halorhabdus utahenesis
4.34
5.75
Haloquadratum walsbyi
4.56
6.04
Natrinema pellirubrum
4.51
5.63
Natronobacterium gregoryi
4.49
5.44
Natronococcus occultus
4.4
5.61
Natrialba magadii
4.44
5.38
Natronomonas pharonis
4.38
6.45
Salinibacter ruber
4.36
7.92
Halorhodospira halophila
5.12
8.56
20
378
Table S5. Intracellular levels of trehalose/2-sulfotrehalose, glycine betaine, and potassium in the strains tested.
Species
Salt conc
Osmolytes measuresa
(g.l-1)
Tre (µmol.mg STre (µmol SO4: GB (µmol.mg K+ (µmol.mg
protein-1)
µmol Tre)
protein-1)
protein-1)
Haloferax mediterranei
175
BDL
NA
8.02
16.26
Halovivax ruber
125
2.56
0
BDL
9.62
175
2.83
0
BDL
8.37
225
2.42
0
BDL
9.48
Halogeometricum borinquense
125
BDL
NA
BDL
7.67
225
BDL
NA
0.98
9.38
Halostagnicola larsenii
175
BDL
NA
BDL
4.19
225
BDL
NA
BDL
24.44
275
BDL
NA
BDL
29.7
Halococcus saccharolyticus
175
3.14
0
1.69
6.24
225
3.17
0
1.81
7.8
275
1.84
0
2.82
5.61
Halorhabdus utahensis
225
BDL
NA
0
9.07
275
BDL
NA
1.23
11.5
Halorubrum lacusprofundi
125
BDL
NA
0
6.5
175
BDL
NA
1.45
8.9
275
BDL
NA
2.74
22.9
Halosarcina pallida
75
3.07
0
0
3.92
225
3.02
0
1.73
5.6
Natrialba magadii
125
3.38
0.13
BDL
4.33
175
2.11
0.16
BDL
6.61
275
1.67
0.18
BDL
5.53
Natrinema pellirubrum
125
1.62
0.28
BDL
2.42
21
379
380
381
175
1.17
0.17
BDL
2.11
275
1.25
0.26
0.54
2.76
Natronobacterium gregoryi
125
5.88
0.28
BDL
4.36
175
2.43
0.27
BDL
6.62
275
1.66
0.13
BDL
5.12
Natronococcus occultus
125
3.31
0.74
0
4.69
175
1.70
0.31
2.85
3.75
275
1.22
0.27
3.63
3.83
Halalkalicoccus jeotgali
125
3.11
0.28
BDL
4.82
175
2.41
0.18
BDL
4.11
275
2.13
0.32
BDL
4.75
Halogranum rubrum
125
5.80
0
0.56
8.56
225
3.00
0
1.22
5.32
275
2.30
0
1.64
6.75
Haloterrigena turkmenica
125
2.32
0
0.23
3.17
175
2.19
0
1.47
4.67
275
1.28
0
2.05
3.86
Halobacterium salinarum
125
BDL
NA
1.58
6.1
175
BDL
NA
ND
8.88
225
BDL
NA
2.43
11.05
275
BDL
NA
2.65
12.5
Natronorubrum tibetense
125
3.62
0
BDL
5.24
175
2.09
0
0.82
4.54
275
1.49
0
0.9
4.37
A: osmolytes for which intracellular concentrations were measured in cell-free PCA extracts; trehalose (Tre), glycine betaine (GB),
and potassium are all shown in µmoles.mg protein-1. For 2-sulfotrehalose (STre), values shown are µmoles SO42-: µmoles trehalose.
BDL: below detection limit, NA: not applicable, BDL: Below detection limit, ND: not determined
22
382
383
23