1 Transcriptomic and Proteomic Insight into the Mechanism of Cyclooctasulfur- versus
2 Thiosulfate-Oxidation by the Chemolithoautotroph Sulfurimonas denitrificans
3
4 Florian Götz1,2,#, Petra Pjevac3,#, Stephanie Markert2,5, Jesse McNichol1,6, Dörte Becher7,
5 Thomas Schweder2,5, Marc Mussmann3,4, Stefan M. Sievert1,*
6
7 1Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
8 2Department of Pharmaceutical Biotechnology, Institute of Pharmacy, University of Greifswald, 9 D-17487 Greifswald, Germany
10 3Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, Research 11 Network ‘Chemistry Meets Microbiology’, University of Vienna, Althanstr- 14, 1090, Vienna, 12 Austria
13 4Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Celsiusstr. 1, 14 28359 Bremen, Germany
15 5Institute of Marine Biotechnology, Walther-Rathenau-Strasse 49, D-17489 Greifswald, 16 Germany.
17 6Present Address: Department of Biological Sciences, University of Southern California, Los 18 Angeles, CA 90089
19 7Institute of Microbiology, University of Greifswald, D-17487 Greifswald, Germany
20 #Contributed equally to this work
21 *corresponding author; email: [email protected]
22
23 Running title: S8 metabolism in Sulfurimonas denitrificans
1 24 Originality-Significance Statement: To the best of our knowledge, this is the first proteomic
25 and transcriptomic study of a chemolithoautotrophic campylobacterium, using Sulfurimonas
26 denitrificans as a model organism. To date, Sulfurimonas denitrificans is the only neutrophilic
27 microorganism with the demonstrated capability of cylcooctasulfur (S8) oxidation. However, the
28 mechanism by which this is accomplished is not known. Our results provide insight into the
29 active metabolism of S. denitrificans, revealing for the first time a differential regulation of the
30 sulfur-oxidation multienzyme complex (SOX) in response to the provided sulfur substrates.
31 Based on these findings, a model for the oxidation of S8 is proposed that might also apply to
32 other sulfur-oxidizing Campylobacteria that have been identified as key players in the oxidation
33 of zero-valence sulfur, a central intermediate in the sulfur cycle.
2 34 Abstract
35 Chemoautotrophic bacteria belonging to the genus Sulfurimonas (class Campylobacteria)
36 were previously identified as key players in the turnover of zero-valence sulfur, a central
37 intermediate in the marine sulfur cycle. S. denitrificans was further shown to be able to oxidize
38 cyclooctasulfur (S8). However, at present the mechanism of activation and metabolism of
39 cyclooctasulfur is not known. Here, we assessed the transcriptome and proteome of S.
40 denitrificans grown with either thiosulfate or S8 as the electron donor. While the overall
41 expression profiles under the two growth conditions were rather similar, distinct differences were
42 observed that could be attributed to the utilization of S8. This included a higher abundance of
43 expressed genes related to surface attachment in the presence of S8, and the differential
44 regulation of the sulfur-oxidation multienzyme complex (SOX), which in S. denitrificans is
45 encoded in two gene clusters: soxABXY1Z1 and soxCDY2Z2. While the proteins of both clusters
46 were present with thiosulfate, only proteins of the soxCDY2Z2 were detected at significant levels
47 with S8. Based on these findings a model for the oxidation of S8 is proposed. Our results have
48 implications for interpreting metatranscriptomic and -proteomic data and for the observed high
49 level of diversification of soxY2Z2 among sulfur-oxidizing Campylobacteria.
50
51
3 52 Introduction
53 Sulfur occurs in many oxidation states and is of central importance in biogeochemical
54 cycles (Sievert et al., 2007). As an essential component of biomass, sulfur is assimilated into
55 organic compounds and is also involved in energy yielding processes either as electron acceptor
56 or donor in chemolithautotrophic, photolithoautotrophic, and heterotrophic microorganisms
57 (Kletzin et al., 2004; Friedrich et al., 2005; Robertson and Kuenen, 2006; Sievert et al., 2007;
58 Finster, 2008; Dahl et al., 2008; Sievert and Vetriani, 2012). In various environments, elemental
59 sulfur accumulates in amounts visible to the naked eye, including marine sediments, microbial
60 mats, hydrothermal vents, glacial shields, oxygen minimum zones and volcanic soils (Ruby et
61 al., 1981; Woodruff and Shanks, 1988; Raiswell, 1992; Taylor and Wirsen, 1997; Alonso-
62 Azcárate et al., 2001; Engel et al., 2004; Zopfi et al., 2004; Lavik et al., 2009; Cosmidis and
63 Templeton, 2016; Lau et al., 2017). In these cases, sulfur occurs mostly in the form of zero-
0 64 valence sulfur (S ) with cyclooctasulfur (S8) as the most stable and common form (Steudel and
65 Eckert, 2003).
2- 0 66 During microbial sulfide (S ) oxidation, S in the form of S8, polymeric sulfur (Sµ) or
67 sulfur chains is often produced and deposited as an intermediate oxidation product (Friedrich et
68 al., 2001; Prange et al., 2002; Dahl and Prange, 2006; Sievert et al., 2007; 2008). In various S0-
69 rich natural and engineered environments, members of the genus Sulfurimonas and other sulfur-
70 oxidizing Campylobacteria, such as Sulfurovum and Sulfuricurvum, are highly abundant
71 (Macalady et al., 2008; Legatzki et al., 2011; Hubert et al., 2012; Meyer et al., 2013; Pjevac et
72 al., 2014; Gulmann et al., 2015; Zhang et al., 2015; Li et al., 2016; McNichol et al., 2016). It has
0 73 been proposed that especially Sulfurimonas and Sulfurovum species occupy the niche for S /S8
74 oxidation in marine sediments and at hydrothermal vents (Pjevac et al., 2014; Meier et al., 2017).
4 75 Considering the extremely low solubility and reactivity of S8 (Kamyshny, 2009; Franz et al.,
76 2007; Pjevac et al., 2014), neutrophilic S8-oxidizing microorganisms most likely need a specific
77 activation or solubilization mechanism to make S8 available for their energy metabolism, as has
78 been shown for acidophilic sulfur-oxidizing bacteria (Rohwerder and Sand, 2003; Mangold et
79 al., 2011; Chen et al., 2012). To date, Sulfurimonas denitrificans, the type strain of the genus
80 Sulfurimonas, is the only neutrophilic microorganism with the demonstrated capability of S8
81 oxidation (Pjevac et al., 2014).
82 S. denitrificans, a member of the globally distributed genus Sulfurimonas (Han and
83 Perner, 2015), was initially isolated from coastal marine sediments with thiosulfate and nitrate as
0 84 substrates, but subsequent studies showed that it can also use H2S, S , S8, and hydrogen (H2) as
85 electron donors (Timmer-Ten Hoor, 1975; Sievert et al., 2008; Pjevac et al., 2014). S.
86 denitrificans is thought to use the sulfur-oxidation multienzyme complex (SOX) for the
87 oxidation of reduced sulfur compounds (Friedrich et al., 2005, 2001; Sievert et al., 2008), which
88 was previously shown for the campylobacterium Sulfurovum sp. NBC37-1 (Yamamoto et al.,
89 2010). While genes of the SOX system in the model organism Paracoccus pantotrophus
90 (Friedrich et al., 2001; 2005) are found in one operon (soxABCDXYZ), S. denitrificans and other
91 Campylobacteria possess two operons of the sulfur oxidation system (SOX) (Sievert et al., 2008;
92 Yamamoto and Takai, 2011). One operon encodes the soxABX cluster and one of the two soxYZ
93 copies (Y1Z1), while the other operon encodes the soxCD genes and a second, highly divergent
94 soxYZ copy (Y2Z2) (Sievert et al., 2008; Meier et al., 2017). These two clusters were previously
95 hypothesized to be differentially regulated, for example in response to the availability of
96 different sulfur compounds (Sievert et al., 2008; Meier et al., 2017; Pjevac et al., 2018). Notably,
97 the differential expression of sox genes present in separate clusters has previously been reported
5 98 for the gammaproteobacterium Allochromatium vinosum (Hensen et al., 2006; Weissgerber et
99 al., 2013).
100 In the currently accepted model of the SOX multi-enzyme system, SoxYZ is the sulfur
101 carrier, and binds various reduced sulfur compounds covalently to a cysteine residue on SoxY,
102 which is coordinated by SoxAX (Quentmeier and Friedrich, 2001; Dahl and Prange, 2006;
2- 103 Grabarczyk and Berks, 2017). SoxB then hydrolyzes sulfate (SO4 ) to generate a cysteine-
104 persulfide left on SoxY. The sulfur dehydrogenase (Sox[CD]2) catalyzes the oxidation of the
105 outer sulfone sulfur of the cystein-persulfide on SoxY, which is subsequently hydrolyzed to
106 sulfate by SoxB (Friedrich et al., 2001, 2005). In a fully operational SOX pathway, sulfur is
107 oxidized to sulfate without a visible accumulation of sulfur intermediates in cells (Dahl and
0 108 Prange, 2006). In contrast, in a truncated SOX system lacking Sox(CD)2, S , Sµ or polysulfides
- 109 (Sn ) are formed as products of thiosulfate oxidation by the activity of SoxXYZAB (Friedrich et
110 al., 2005; Hensen et al., 2006). While work on acidophilic S0 oxidizers such as Acidithiobacillus
111 ferrooxidans or A. caldus has shown that the S8 ring is activated by reactive thiols and involves
112 outer membrane proteins (Rohwerder and Sand, 2003; Mangold et al., 2011; Chen et al., 2012),
113 the mechanism involved in the activation of insoluble and inert sulfur forms, like Sµ and S8, by
114 neutrophilic sulfur oxidizers such as S. denitrificans is at present unknown.
115 In order to investigate the mechanism of S8 oxidation in S. denitrificans, we analyzed and
116 compared its transcriptome and proteome under denitrifying conditions with either thiosulfate or
117 S8 as sole electron donors. Our results provide insight into the active metabolism of S.
118 denitrificans, revealing a differential regulation of the SOX system in response to the provided
119 sulfur substrate. Based on these findings, a model for the oxidation of cylcooctasulfur is
6 120 proposed that might also apply to other Campylobacteria that share the same genome
121 arrangement of the SOX system.
122
123 Material and Methods
124 Cultivation
125 Sulfurimonas denitrificans was grown in batch cultures at 22ºC in 2 L pyrex bottles
126 containing 1 L of artificial seawater medium (Timmer-Ten Hoor, 1975) purged with N2:CO2
127 (80:20, 1.1 atm) and sealed with butyl rubber stoppers. The bicarbonate concentration in the
128 medium was increased to 5 g L-1 to buffer the pH at approximately 7. Three replicate cultures
-1 -1 129 were supplemented with 5 g L S8 (S8-1, S8-2, S8-3), and two control cultures with 40 mmol L
130 thiosulfate (T-1, T-2) as electron donor. S8 for the experiments was freshly synthesized, as
131 described in Pjevac et al., (2014). Nitrate (20 mmol L-1) was added to all cultures as the electron
132 acceptor. In order to determine cell numbers, 4,6-Diamidino-2-phenylindole (DAPI) counts were
133 performed. Cells were harvested during exponential growth. For proteomics, 100 mL of culture
134 was harvested by centrifugation at 7,000 x g for 7 min at 20 °C in 50 mL centrifugation tubes.
135 For transcriptomics, 100 mL of culture was filtered onto 0.2 µm teflon filters and immediately
136 immersed in RNA later and stored at -20°C.
137 Protein isolation
138 For the isolation of proteins, cell pellets were resuspended in 1 mL 1x Tris-EDTA (TE)
139 buffer (pH 8; Sigma-Aldrich, St. Louis, MO, USA), containing cOmpleteTM Protease inhibitor
140 (Roche, Basel, Switzerland). To lyse cells, the suspensions were sonicated three times for 30 sec
141 at 5 x 10% amplitude using the Sonoplus HD2070 sonication probe (Bandelin, Berlin, Germany).
7 142 The obtained crude extracts were centrifuged for 10 min at 15,500 x g at 4°C. The raw protein
143 extracts (supernatants) were concentrated by means of Amicon tubes (Ultra-15 3 kDa cutoff
144 Merck Millipore, Kenilworth, NJ, USA), which were centrifuged for 30-60 min at 5,000 x g and
145 4°C as described in the manufacturer’s instructions. Protein concentrations were determined
146 using the Roti-Nanoquant protein assay (Carl Roth, Karlsruhe, Germany) as instructed by the
147 manufacturer. Subsequently, 4 x loading buffer (200 mmol L-1 Tris-HCl pH 6.8, 50 mmol L-1
148 EDTA pH 8.0, 40 % glycerol (v/v), 8 % SDS (w/v), 0.08 % bromophenol blue (v/v), 1 mmol L-1
149 dithiothreitol) was added to aliquots of ~25 µg protein, and samples were denatured for 10 min at
150 90°C. The denatured extracts and a Roti-Mark standard (Roti-Mark STANDARD, Carl Roth,
151 Karlsruhe, Germany) were transferred onto a polyacrylamide SDS mini gel (10% Mini-
152 PROTEAN, TGX Stain-free, BioRad, Hercules, CA, USA) and run approximately 20 min at 50
153 V and another 35 min at 150 V. The gel was fixed for 30 min in an ethanol: acetic acid: water
154 (40:10:50) solution. The gel was stained overnight in a Coomassie Brilliant Blue G250 (CBB)
155 staining solution (Carl Roth, Germany), followed by washing step in distilled water to remove
156 non-bound CBB before imaging of the protein bands.
157 Protein digestion
158 On a glass plate, cleaned with methanol and double distilled water (ddH2O), the entire
159 protein-containing gel lane was excised and divided into 10 equal-sized subsamples for each
160 sample. Each subsample was cut in small pieces and transferred into 2 mL Eppendorf cups. CBB
161 bound to the protein was removed by incubating the gel pieces multiple times in a wash solution
-1 162 containing 200 mmol L NH4HCO3 and 30% acetonitrile at 37°C for 30 min. De-stained gel
163 pieces were dried for 15 min in an Eppendorf Concentrator plus at 1,400 rpm, ambient
164 temperature and 20 mbar vacuum. For protein digestion, 2 µg mL-1 trypsin solution (Promega,
8 165 Madison, WI, USA) was added to dried gel pieces until covered (~50 µL) and incubated at room
166 temperature for 30 min. Additional trypsin was added to cover rehydrated gel pieces and
167 incubated at 37°C for 14-16 h. Thereafter, 50 µL ddH2O was added to the incubated gel pieces,
168 which were further sonicated for 15 min in an ultrasound bath (37 kHz, 600W \ FB15054
169 Fisherbrand) to elute the peptides. After sonication, the sample was centrifuged at 5,000 x g for
170 10 s. The peptide-containing supernatants were transferred into new tubes, and centrifuged at
171 250 x g under vacuum in an Eppendorf Concentrator to reduce the sample volume to 10 µL. The
172 10 µL peptide mixes were stored at -80 °C until measurement.
173 Mass spectrometry and proteome analysis
174 Peptide mixes were separated via chromatography on a reversed phase C18 column in a
175 nano-ACQUITY-UPLC (Waters Corporation, Milford, MA, USA) as described by Otto et al.
176 (2010). Mass spectrometry (MS) and MS/MS data were recorded with an online-coupled LTQ-
177 Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). MS data were
178 searched against a forward-decoy S. denitrificans protein database (Refseq Genome project:
179 NC_007575.1, NCBI [National Center for Biotechnology Information]), which also included
180 common laboratory contaminants, using Sequest (Thermo Fisher Scientific, San Jose, CA, USA;
181 v 27.11). The protein false discovery rate (FDR) was zero for all samples, with the exception of
182 one technical replicate of T-1, in which case the FDR was 0.2%. Identifications were filtered in
183 Scaffold (http://www.proteomesoftware.com) using the “Sequest” filter settings described by
184 Heinz et al., (2012). The total spectral count values of all proteins were exported for calculation
185 of the normalized spectral abundance factors (NSAF, Zybailov et al., 2006), which are given in
186 Supplementary Table 1. The average of the NSAF and the standard deviation were calculated for
187 the three biological replicates for S8 (S8 1-3). For thiosulfate, only two biological replicates were
9 188 available (T-1 and T-2). However, two technical replicates were run for sample T-1 to assess the
189 variability between runs. The NSAFs of the two technical replicates were averaged before being
190 averaged with the second biological replicate T-2 for statistical analyses. PSI-BLAST and
191 DELTA-BLAST (Boratyn et al., 2012) were used for domain analysis of hypothetical proteins.
192 We also used the tools BOCTOPUS (boctopus.bioinfo.se) and PRED-TMMB
193 (http://bioinformatics.biol.uoa.gr////PRED-TMBB) to search for beta-barrel topology. To detect
194 signal peptides, SignalP 4.0 (Petersen et al., 2011; http://www.cbs.dtu.dk/services/SignalP/) was
195 used. The mass spectrometry proteomics data are available through the ProteomeXchange
196 Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository
197 (Vizcaino et al., 2016) with the dataset identifier PXD009127 (Reviewer account details:
198 Username: [email protected] Password: sj7sSWSj).
199 RNA extraction, transcriptome sequencing and analysis
200 The membrane filters with the cells were cut in pieces, put in a microcentrifuge tube
201 containing extraction buffer provided with the mirVana miRNA isolation kit (Ambion, Carlsbad,
202 CA, USA), and subjected to bead-beating. Subsequently, total RNA was extracted using the
203 mirVana miRNA isolation kit, with subsequent turbo DNAse (Ambion) treatment and
204 purification with the Qiagen RNAeasy kit (Qiagen, Hilden, Germany) according to the
205 manufacturer’s instructions. Ribosomal RNA was not depleted. Total RNA extracts were sent to
206 the Max Planck Genome Centre (Cologne, Germany) for preparation of RNAseq-libraries and
207 subsequent sequencing of paired-end (2 x 150 bp) reads in the HiSeq2000 (Illumina).
208 Sequencing statistics are provided in Supplement Table 2. Sequence reads were quality trimmed
209 at a Phred score of 28 using the bbduk function of BBMap v35.82
210 (https://sourceforge.net/projects/bbmap/), and mapped to a reference file of all open reading
10 211 frames (ORFs) deposited in NCBI for the S. denitrificans genome (NCBI project NC_007575.1)
212 using the bbmap function of BBMap v35.82. Sequences were mapped as paired reads with a
213 minimal identity of 99%. For comparison between cultures (Supplement Table 1), read counts
214 were transformed to transcripts per million (TPM) values as described in Li et al. (2010). The
215 transcriptome sequence data has been deposited in the European Nucleotide Archive (ENA)
216 under the project accession number PRJEB25508.
217 Statistical analysis
218 To test for statistically significant differences between the two different growth
219 conditions, the NSAFs and TPMs of all biological replicates of each growth condition were used
220 as the input parameters to calculate P-values based on the Welch’s t-Test. Due to the small
221 number of biological replicates available for the thiosulfate treatment (n=2), the Welch’s rather
222 than the Student’s t-Test was selected as a more stringent manner to statistically evaluate
223 differences in gene transcription and protein expression between the two conditions. Transcripts
224 and proteins with a P-value smaller than 0.05 were considered to be statistically significantly
225 different between the two conditions.
226
227 Results
228 Growth characteristics
229 The growth rate of thiosulfate-grown cultures was 1.8-times higher compared to cultures
-1 -1 230 grown on cyclooctasulfur (2.7 day vs. 1.5 day ), corresponding to a doubling time of ~6 h and
231 ~11 h for thiosulfate- and S8-grown cultures, respectively (Supplement Figure S1). Thiosulfate-
232 grown cultures also had a significantly shorter lag phase, which might be related to the easier
11 233 accessibility to thiosulfate than to S8. One of the three S8-grown cultures had a significantly
234 extended lag phase, but once growth started, the growth rate was similar to the other two
235 cultures.
236 The transcriptome and proteome of thiosulfate- versus S8-grown cells are largely similar
237 Transcriptomic and proteomic data were obtained from either thiosulfate- or S8-grown
238 cultures sampled in early exponential phase. Overall, reads mapping to 97.7 % of the genes in
239 the genome (2,114 and 2,104 out of 2,164) were detected in the transcriptome of cultures grown
240 with thiosulfate or S8, respectively. In the proteome, 38.8% and 37% of proteins encoded in the
241 genome (822 and 784 out of 2,119 protein coding genes) were identified in thiosulfate- and S8-
242 grown cultures, respectively (Table 1). 122 and 84 proteins were detected exclusively in
243 thiosulfate- and S8-grown cultures, respectively, while 176 and 200 proteins were identified in
244 only one of the analyzed replicates of thiosulfate- and in S8-grown cultures, respectively (Table
245 1).
246 In general, a classification into clusters of orthologous genes (COG) showed similar
247 profiles of transcribed protein coding genes and identified comparable sets of proteins between
248 thiosulfate- and S8-grown cultures (Figure 1). Genes of the translational apparatus (including
249 tRNA ligases and ribosomal subunits) accounted for up to one third of the classifiable transcripts
250 and proteins under both conditions, followed by transcribed genes and proteins of the energy
251 metabolism (Figure 1). The most apparent difference among functional genes is the increased
252 transcription level of genes that encode proteins of the flagellar apparatus in S8-grown cultures
253 (Figure 1).
12 254 Since transcriptome sequencing was performed without rRNA depletion to minimize
255 sample processing biases, the majority of the transcriptomic reads (88.2-94.0%) mapped onto
256 rRNA-encoding genes from one of the four rRNA operons encoded by S. denitrificans
257 (Supplement Table 1). No differences were observed for ribosomal RNA in thiosulfate- and S8-
258 grown cultures. In addition, proteins of the small and large ribosomal subunits showed little
259 variance in their abundance, which is expected since all subunits and the ribosomal RNA form
260 functioning ribosomes. However, we found significant differences in the transcription of genes
261 encoding ribosomal small and large subunit proteins, which were transcribed at a significantly
262 higher level in thiosulfate-grown cultures. We also evaluated the expression of an alternative
263 indicator for growth - the translation elongation factor TU (EF-TU; WP_011371980.1) (Pedersen
264 et al., 1978), which also had a higher expression level in the transcriptome and higher levels of
265 abundance in the proteome in thiosulfate-grown cultures (Supplement Table 1).
266 Although the differences in transcriptomic and proteomic profiles between cells grown
267 with the two different electron donors were subtle (Supplement Figure S2), a small number of
268 proteins showed statistically significant differences in relative abundance (Supplement Table 1).
269 One of these proteins (WP_011373531.1) was present at significantly higher levels (2.9 x
270 increase) in S8-grown cultures, compared to thiosulfate-grown cultures. This hypothetical protein
271 was also differentially regulated in the transcriptome, but in the opposite manner - it was
272 transcribed at higher levels under the thiosulfate condition compared to the S8 condition. Based
273 on analyses using BOCTOPUS, PRED-TMMB and SignalP 4.0, WP_011373531.1 contains a
274 beta-barrel structure and a predicted signal peptide. It also contains a phenylalanine at the C-
275 terminal position, as well as hydrophobic amino acids at positions -3, -5, -7, and -9 relative to the
276 C-terminus, which have previously been reported to be important for the assembly into the outer
13 277 membrane (Struyvé et al., 1991; Tommassen, 2010). These indications suggest that it is an outer
278 membrane protein. Protein domain analysis (DELTA-BLAST and PSI-BLAST, NCBI) of this
279 hypothetical protein further showed that the first 177 amino acids of WP_011373531.1 have
280 similarity to the conserved domain of the outer membrane protein family OprD and the major
281 outer membrane protein of Campylobacter (Campylo_MOMP), suggesting an involvement in
282 transport and/or adhesion (Yoshihara et al., 1998; Zhang et al., 2000). However, based on the
283 low overall similarity, a functional classification cannot be inferred at present. A protein
284 BLAST-based comparison of alignment length and amino acid sequence identity revealed the
285 presence of homologs of this putative outer membrane protein (>70% alignment fraction, >30%
286 amino acid identity) in various campylobacterial species (e.g., Sulfurimonas, Sulfurovum,
287 Sulfuricurvum, Nitratiruptor) that are also implicated in elemental sulfur utilization (Campbell et
288 al., 2006; Sievert et al., 2008; Sievert and Vetriani, 2012).
289
290 Flagella-related proteins and transcripts are differentially abundant
291 Significant differences in gene transcription levels were observed for a large number of
292 genes involved in flagella biosynthesis and motility (Figure 2). Flagella-related genes were
293 generally upregulated in cultures grown with S8 as electron donor. In contrast, the differences in
294 the expression of proteins of the flagellar apparatus were not as pronounced between the growth
295 conditions, but the flagellar hook protein FlgE and flagellin were found in considerably higher
296 relative abundance in the proteome of S8-grown cultures (Supplement Table 1).
297
298 Genes and proteins of central biochemical pathways
14 299 Growing with nitrate as the electron acceptor with thiosulfate or cyclooctasulfur as the
300 sole electron donor, S. denitrificans gains energy through denitrification coupled to sulfur
301 oxidation. The generated energy mainly fuels carbon fixation through the reductive tricarboxylic
302 acid (rTCA) cycle. Accordingly, all transcripts and proteins of the rTCA cycle were identified. In
303 the genome, two copies of succinate dehydrogenase/fumarate reductase have been identified, one
304 being membrane-bound and one being cytoplasmic (Sievert et al., 2008). It has previously been
305 proposed that the membrane-bound form might play a direct role in the rTCA cycle by
306 transferring electrons derived from the oxidation of hydrogen or sulfur via the quinone pool
307 (Sievert et al., 2008). In line with this hypothesis, the putatively membrane-bound succinate
308 dehydrogenase/fumarate reductase (SDH; EC 1.3.5.1; WP_011372658.1-WP_011372660.1) was
309 found in the transcriptomes and proteomes under both growth conditions, while the cytoplasmic
310 succinate dehydrogenase/fumarate reductase (WP_011371673.1-WP_011371674.1) only
311 displayed a low transcription level and was not identified in the proteome (Supplement Table 1).
312 For the reduction of nitrate, we could confirm that all transcripts and all proteins involved
313 in the canonical dissimilatory denitrification pathway were present (Figure 3), including the
314 nitric oxide reductase cNOR, which had previously been questioned to be functional due to the
315 absence of norD and norQ in the genome (Sievert et al., 2008). In contrast, neither the transcripts
316 nor the proteins of the proposed alternative non-electrogenic quinone-oxidizing nitric oxide
317 reductase (gNOR, WP_011371739.1-WP_011371741.1; Sievert et al., 2008) could be detected
318 (Supplement Table 1). The comparatively low abundance of the nitrous oxide reductase (NorB,
319 NorC) in the proteome compared to the other enzymes of the denitrification pathway is most
320 likely due to the fact that NorB is an integral membrane protein and NorC is anchored in the
321 membrane. Membrane associated proteins are not extracted with the same efficiency as soluble
15 322 proteins by the used extraction procedure, resulting in their lower representation in the proteome.
323 The same bias applies for the membrane bound components of the nitrate reductase complex
324 (NapFGH).
325 With respect to sulfur compound metabolism, transcripts and proteins of the periplasmic
326 SOX (sulfur oxidation) multienzyme complex were detected, confirming the use of the SOX
327 system for sulfur oxidation in S. denitrificans, as has previously been shown for the
328 campylobacterium Sulfurovum sp. NCB37-1 (Yamamoto et al., 2010). The transcripts of both
329 sox operons, i.e., soxABXY1Z1 and soxCDY2Z2, showed a trend of increased transcription levels in
330 thiosulfate grown-cultures. However, the transcripts of soxABXY1Z1 were more strongly
331 upregulated with thiosulfate, as the transcription ratios for soxABXY1Z1 between S8 and
332 thiosulfate ranged from 0.3-0.6, while those of soxCDY2Z2 ranged from 0.65-0.8 (Figure 4). In
333 the proteomes of S8-grown cultures, SOX proteins of the soxABXY1Z1 operon were hardly
334 detected, while they were present in significantly higher abundance with thiosulfate (Figure 4).
335 In contrast, the sulfur dehydrogenase proteins, i.e., SoxCD, were significantly more abundant in
336 the proteome of S8- grown cultures, although still present with thiosulfate (Figure 4). Regardless
337 of the supplied electron donor, the SoxY and SoxZ proteins of the soxCDY2Z2 operon were not
338 only the two most abundant proteins of the SOX multienzyme system, but of both proteomes
339 under both growth conditions overall (Figure 4, Supplement Table 1), suggesting that they play
340 an important role under both conditions. In addition, a protein encoded by gene
341 WP_011373670.1 that is located next to soxCDY2Z2 is present in the transcriptome and proteome
342 under both growth conditions (Figure 4, Supplement Table 1). The 340 amino acids-long protein
343 has a signal peptide indicating a periplasmic location, and based on BLAST search it further
344 contains a metallo-hydrolase-like_MBL-fold with all the conserved residues of the active site
16 345 and three putative metal binding sites, suggesting it has a hydrolytic function. Transcript reads
346 and protein abundance of the sulfide:quinone oxidoreductase (SQR), a sulfide-oxidizng enzyme,
347 and the putative membrane-bound polysulfide reductase, a polysuldide-reducing complex, which
348 might be involved in the reduction of polysulfide, were low, and no significant differences
349 between growth conditions was observed (Figure 4).
350
351 Transcribed and translated genes involved in alternative energy metabolism
352 The S. denitrificans genome reflects the potential to use alternative energy sources to
353 generate energy for carbon fixation (Sievert et al., 2008). We were able to detect the
354 transcription of genes that are involved in the metabolism of compounds which were not
355 provided in the medium. The assessment of transcriptomic data revealed that genes of a cbb3-
356 type cytochrome c oxidase (fixNOQP cluster involved in aerobic respiration) as well as genes
357 encoding a membrane-bound [Ni,Fe]-uptake hydrogenase (hydABC cluster involved in hydrogen
358 oxidation) were transcribed in all cultures (Supplement Figure S3). In addition, FixO and HydB
359 were identified at low relative abundance in the proteomes under both cultivation conditions
360 (Supplement Table 1), whereas the proposed cytoplasmic hydrogenase putatively involved in
361 direct electron transfer as part of the rTCA cycle (WP_011373066.1; WP041672536.1; Sievert et
362 al., 2008) was not expressed under either cultivation condition (Supplement Table 1). Genes
363 encoding the membrane-bound formate dehydrogenase complex were transcribed at higher
364 relative levels in the S8-grown cultures (Supplement Figure S3), but no proteins of the complex
365 were identified (Supplement Table 1).
366
17 367 Discussion
368 In this study, we performed proteomic and transcriptomic analyses to investigate the
369 metabolism of Sulfurimonas denitrificans with either S8 or thiosulfate as the sole electron donor.
370 Transcriptomic and proteomic approaches complement each other to assess microbial activity
371 and metabolic regulation, as the half-life for mRNA is in the range of minutes (Selinger et al.,
372 2003), whereas that of proteins is in the range of hours (Maier et al., 2011). However, a protein
373 concentration of roughly 100 fmol per protein is necessary for successful detection (Silva et al.,
374 2006), which is much higher than the mRNA abundance required if a sufficient sequencing depth
375 is attained. Furthermore, isolation and detection of membrane-bound proteins remains a
376 challenge in proteomics. Thus, the combined application of proteomic and transcriptomic
377 profiling provides a comprehensive assessment of the microbial response to different
378 physiological conditions. In some instances, we did observe that there was not a strict correlation
379 between transcribed genes and detected proteins, which could be explained either by
380 posttranscriptional regulation (Picard, 2009; van Assche et al., 2015), the initiation of
381 transcription before proteins reached levels high enough for their detection, or the longer lifetime
382 of proteins compared to transcripts.
383 In the present study, the detection of 42% of the predicted proteins encoded in the
384 genome is comparable to was found for the gram-positive bacterium Bacillus subtilis, but lower
385 than for example the 70% reported for the thaumarchaeon “Candidatus Nitrosopelagicus brevis”
386 (Hahne et al., 2010, Santoro et al., 2015). The number of transcribed genes and identified
387 proteins was similar under both growth conditions. This similarity of the transcriptomic and
388 proteomic profiles was mirrored in the broad COG categories (Figure 1), and differences
389 between the growth conditions were restricted to individual mRNA and protein categories.
18 390 Ribosomal RNA levels as well as protein levels of ribosomal subunits did not vary
391 significantly between the two growth conditions, despite higher growth rates with thiosulfate.
392 Similar observations have been made previously and it was concluded that rRNA levels in
393 combination with the growth rates are not always a good indicator for microbial activity
394 (Blazewicz et al., 2013). However, in cultures grown with thiosulfate, transcription of ribosomal
395 subunit genes was significantly higher in relation to rRNA gene transcription levels, which,
396 together with elevated transcript and protein levels of the elongation factor TU, likely reflects the
397 higher growth rates and higher overall activity in cultures grown with thiosulfate. One of the
398 most abundant proteins of the proteome was identified as a hypothetical protein
399 (WP_011373531.1), which is likely to play an important role for Sulfurimonas species and other
400 Campylobacteria as apparent homologs are present in genomes of various Campylobacteria. In
401 S8-grown cultures, this protein showed an almost threefold increase in relative abundance.
402 Possibly, this protein could be involved in the activation of S8, similar to the outer membrane
403 protein identified in A. caldus (Ramírez et al., 2004; Mangold et al., 2011; Chen et al., 2012),
404 although no similarity was observed between these proteins. On the other hand, transcripts
405 showed an opposite trend with the level of WP_011373531.1 gene expression being higher under
406 the thiosulfate condition, making a specific role in S8 metabolism less certain. However, the
407 relative abundance of the transcripts is influenced by the absolute number of identified
408 transcripts and translation could further be prevented by posttranscriptional regulation, making
409 the proteomic data overall a better indicator for the involvement of the protein under different
410 growth conditions. Thus, although we are presently not able to assign a function to this protein,
411 our results indicate that it may play a fundamental role in the cells’ metabolism, including the
412 possibility of its involvement in S8 metabolism. To resolve the role of this protein in S.
19 413 denitrificans, and potentially other Campylobacteria, more work is needed. Such experiments,
414 however, were beyond the scope of this study, since they would rely on heterologous gene
415 expression assays or a genetic system for S. denitrificans, which is currently not available.
416
417 The expression of genes and proteins involved in energy metabolism
418 Chemoautotrophic bacteria gain energy through redox reactions. In our experiments, S.
419 denitrificans coupled the reduction of nitrate with the oxidation of thiosulfate or cyclooctasulfur
420 to gain energy for growth. All proteins of the canonical denitrification pathway were identified,
421 but no protein of the proposed alternative non-electrogenic gNOR system (Sievert et al., 2008).
422 To the best of our knowledge, we provide the first validation of a functional cNOR system that
423 lacks the components encoded by norD and norQ, which are absent from the genome of S.
424 denitrificans and other Campylobacteria (Sievert et al., 2008).
425 Besides the utilization of nitrate, S. denitrificans can utilize oxygen using a cbb3-type
426 cytochrome c oxidase (Sievert et al., 2008). All genes were transcribed under both conditions
427 (Supplementary Figure S3), which could be either an indicator for the presence of small amounts
428 of oxygen in our batch culture system, for example by leakage through the butyl stoppers, or for
429 constitutive transcription. In addition, genes for the membrane bound [Ni,Fe]-uptake
430 hydrogenase and genes for the formate dehydrogenase were transcribed (Supplement Figure S3),
431 although no hydrogen or formate was provided in the cultivation medium. Notably, even though
432 low level of transcripts for hydrogenase could be detected in the absence of hydrogen, the
433 transcripts for the hydrogenase are highly upregulated in the presence of hydrogen (unpublished
434 data).
20 435 The simultaneous transcription (at a low level) of genes for the utilization of alternative
436 substrates could be a mechanism to react quickly when either oxygen or hydrogen become
437 available in the environment. This high level of metabolic flexibility is a possible explanation for
438 the environmental success of Sulfurimonas species, which have been described as r-strategist
439 (Rogge et al., 2017) and primary colonizers (López-García et al., 2003).
440
441 Flagella likely facilitate attachment to S8
442 Cyclooctasulfur is a solid substrate, which likely requires the attachment of cells to make
443 it accessible (Mangold et al., 2011; Pjevac et al., 2014). Proteins of the flagellar apparatus have
444 been shown to play a critical role in attachment and biofilm formation, including in
445 Campylobacteria (e.g., O’Toole and Kolter, 1998; Pratt and Kolter, 1998; Otteman and
446 Lowenthal, 2002; Joshua at al., 2006; Haiko and Westerlund-Wikström, 2013). Previously,
447 Mangold and colleagues identified a flagellar basal-body rod protein (FlgC) to be up-regulated in
448 S0-grown vs. tetrathionate-grown cultures of A. caldus, facilitating attachment to the solid
449 substrate (Mangold et al., 2011). Interestingly, the only COG category that showed differences
450 between the two growth conditions encompassed genes of the flagellar apparatus. Specifically,
451 transcriptomic data showed a significantly higher expression of genes involved in flagella
452 biosynthesis and flagella motility in S8-grown cultures (Figure 2). Thus, we propose that the
453 increased transcription of flagella-related genes as well as the higher relative abundance of FlgE
454 (flagellar hook protein) and flagellin in S8-grown cultures is indicative of attachment and biofilm
455 formation cyclooctasulfur particles. On the other hand, genes related to chemotaxis showed a
456 trend of higher expression in thiosulfate-grown cultures, indicating that chemotaxis is more
457 important in sensing gradients of the soluble substrate thiosulfate. In this context, it is important
21 458 to note that while the original strain used in our experiments has been described as non-motile, S.
459 denitrificans has regained motility during cultivation in our laboratory (unpublished data). We
460 attribute this to the spontaneous excision of the transposon that was shown to interrupt a flagellin
461 biosynthesis operon, as its genome otherwise contains all genes required for a fully functional
462 flagellar apparatus (Sievert et al., 2008).
463
464
465 The mechanism of thiosulfate- and S8-oxidation
466 With the exception of the potential involvement of the hypothetical protein in the
467 activation and transport of S8 into the cell, we were not able to identify a possible mechanism for
468 the activation of cyclooctasulfur, as has for example been proposed for acidophilic sulfur-
469 oxidizing bacteria (Rohwerder and Sand, 2003; Mangold et al., 2011; Chen et al., 2012).
470 However, our data confirm the previously hypothesized differential regulation of the two SOX
471 gene clusters present in S. denitrificans in response to the available sulfur substrate (Sievert et
472 al., 2008; Meier et al., 2017; Pjevac et al., 2018), allowing us to propose a possible oxidation
473 pathway. In thiosulfate-grown cultures, the first gene cluster encoding soxABXY1Z1 was highly
474 transcribed and the corresponding proteins were found in significantly higher abundance
475 compared to growth with S8, confirming their involvement in thiosulfate oxidation as previously
476 proposed (Friedrich et al., 2001; 2005). The differential abundance of the SoxABXY1Z1-proteins
477 is in contrast to the phototrophic sulfur-oxidizing gammaproteobacterium A. vinosum, for which
478 no differences were observed in response to different sulfur compounds (Weissgerber et al.,
479 2014). Under both growth conditions, soxY2Z2 was highly transcribed and the corresponding
480 proteins were highly abundant in S. denitrificans, suggesting that it plays an important role in the
22 481 oxidation of both thiosulfate and S8 (Figure 4). In contrast, SoxCD proteins were significantly
482 more abundant in S8-grown cultures. Although the soxABXY1Z1-operon was present in the
483 transcriptome, it was nearly undetectable in the proteome, suggesting that it might be under post-
484 transcriptional control and not translated into functional proteins when S. denitrificans is grown
485 with S8 (Picard, 2009; van Assche et al., 2015) or translated at low levels preventing the
486 detection of the proteins by our methods. Thus, we propose that the oxidation of S8 is mediated
487 mainly by soxCDY2Z2, and we interpret the low abundance of transcripts and in particular of
488 proteins of the soxABXY1Z1 operon in S8-grown cultures as another reflection of baseline activity
489 and for metabolic flexibility, allowing S. denitrificans to quickly respond to the availability of
490 thiosulfate. This interpretation is in line with qPCR data that showed the presence of soxB
491 transcripts in low abundance in hydrogen-grown S. denitrificans cultures lacking reduced sulfur
492 compounds, and their increased abundance in the presence of thiosulfate, while soxD transcripts
493 remained more or less unchanged, pointing to its constitutive expression (unpublished data).
494
495 An alternative model for sulfur oxidation in S. denitrificans
496 Based on our observations on the differential abundance of the SOX proteins during
497 growth on thiosulfate versus S8 and previous results (Friedrich et al., 2001; 2005; Sauvé et al.,
498 2007; Grabarczyk and Berks, 2017), we propose a sulfur oxidation model for the oxidation of S8
499 by S. denitrificans that does not involve the SoxABXY1Z1 protein complex (Figure 5). At
500 present, we are not able to resolve how the sulfonate group bound to SoxY2Z2 is being
501 hydrolyzed to form sulfate. One option could be that SoxB, which might be present in low
502 abundance, could perform that function. However, another possibility might be that the putative
503 periplasmic metallo-hydrolase encoded by WP_011373670.1 could perform this function. This
23 504 scenario appears intriguing as this gene is located next to soxCDY2Z2 and homologs of this gene
505 are found in other sulfur-oxidizing Campylobacteria with conserved synteny. The protein’s
506 catalytic domain has similarities to SoxH of Parococcus pantotrophus. However, while soxH
507 expression is specifically induced by thiosulfate, it is not required for sulfur oxidation in P.
508 pantotrophus and its function is currently unknown (Rother et al., 2001), although it has been
509 described as a putative thiol hydrolase (Friedrich et al., 2001). Lastly, it might be possible that
510 SoxCDY2Z2 can catalyze the reaction by itself, although there is at present no evidence to
511 support this hypothesis. Future experiments need to be carried out to differentiate between these
512 options. Thiosulfate oxidation by S. denitrificans proceeds mainly as proposed in the current
513 sulfur oxidation model (Friedrich et al., 2001; 2005; Grabarczyk and Berks, 2017). First,
514 thiosulfate is activated via SoxXA and bound to a cysteine residue of SoxY in the SoxYZ1
515 complex, followed by the hydrolysis of the outer sulfonate group of the bound thiosulfate by
516 SoxB (Friedrich et al, 2001). However, we hypothesize that subsequently the sulfone group is
517 being transferred to SoxY2Z2, where it is oxidized and hydrolyzed to sulfate as described above,
518 in line with the high abundance of SoxCD and in particular SoxY2Z2 under both growth
519 conditions. We postulate SoxY2Z2 to interact specifically with SoxCD based on the operon
520 structure and the observed expression pattern. This way, we propose that SoxCDY2Z2 is always
521 present, whereas SoxABXY1Z1 is only needed in the presence of thiosulfate. Biochemical and
522 genetic studies will be required to validate this proposed model for the oxidation of S8 and
523 thiosulfate in S. denitrificans and other Campylobacteria with the same genetic arrangement of
524 the SOX system.
525 In addition to the described function in S. denitrificans, the soxY2Z2 genes of the
526 soxCDY2Z2 operon show a much higher level of diversification between different Sulfurimonas
24 527 and Sulfurovum species (up to 79% nucleotide sequence dissimilarity) than soxY1Z1 of the
528 soxABXY1Z1 operon (Meier et al., 2017). Moreover, the sequence similarity between the two
529 SoxYZ copies in S. denitrificans and other Campylobacteria is very low. The amino acid
530 sequence identity is 33% for the two SoxY proteins, and 28% (over a 61% alignment fraction)
531 for the two SoxZ proteins of S. denitrificans. In fact, no significant identity, except for the export
532 signal sequence, is detectable on the nucleotide level for either gene. This highly divergent
533 intragenomic duplication, and the intergenomic diversification of the SoxY2Z2 proteins, is
534 present in all currently available Sulfurimonas and Sulfurovum genomes (Meier et al., 2017).
535 Furthermore, a significantly higher expression level of SoxY2Z2 proteins when compared to
536 SoxY1Z1 proteins has been found in the metaproteomes of Campylobacteria-dominated biofilms
537 forming on black-smoker chimneys (Pjevac et al., 2018). The diversification of the SoxY2Z2
538 proteins could lead to different substrate preferences with respect to the chain length of the
539 bound polysulfide or to differences in overall substrate-binding affinities, and may therefore be
540 involved in niche partitioning between co-occurring Sulfurimonas and Sulfurovum species,
541 whereas the SoxY1Z1 proteins are specific for thiosulfate, explaining their lower diversity and
542 lower abundance in environmental samples, where thiosulfate is usually present in lower
543 concentrations than elemental sulfur or polysulfide (Zopfi et al., 2004). Potentially, the ratio of
544 the SoxCD to SoxAB proteins (SoxCD/SoxAB: S8:19; TS:0.9) and SoxY2Z2 to SoxY1Z1 proteins
545 (SoxY2Z2/ SoxY1Z1: S8:89; TS:9) , and to a lesser extent that of the transcripts (soxCD/soxAB:
546 S8:2.7; TS:1.5 and soxY2Z2/soxY1Z1: S8:3.3; TS:1.9), could be used as an indicator for the type of
547 sulfur compound being utilized by Sulfurimonas and possibly other sulfur-oxidizing
548 Campylobacteria in the environment.
549
25 550
551 Conclusions
552 We have applied transcriptomics and proteomics as two powerful approaches to elucidate
553 the biochemical pathways involved in the oxidation of two different sulfur compounds by the
554 campylobacterium S. denitrificans. We were able to show that the SOX system in S. denitrificans
555 is differentially regulated in response to the available sulfur substrate. Based on the available
556 data, we propose a model for the oxidation of cyclooctasulfur in S. denitrificans. We further
557 identified a hypothetical protein that may play a central role in the metabolism of reduced sulfur
558 compounds. In the future, genetic and biochemical studies are needed to elucidate its function,
559 including its potential involvement in the activation of S8, and to validate our proposed model for
560 sulfur oxidation in S. denitrificans, which is likely to also operate in other sulfur-oxidizing
561 Campylobacteria. The data presented here will aid in the interpretation of environmental
562 metatranscriptomic and –proteomic data and help to provide a biochemical basis for the observed
563 niche partitioning of different sulfur-oxidizing Campylobacteria.
564
565 Acknowledgements
566 We thank Dali Smolsky for helping with culturing and cell counts, Jörg Wulf for RNA
567 extraction, Jana Matulla for instructions on protein isolation, Sebastian Grund for mass
568 spectrometry measurements and Fabian Götz for helping with statistical analysis. We also would
569 like to thank two anonymous reviewers for their insightful and constructive comments that
570 improved the manuscript. Funding was provided by NSF OCE-1136727 and The Investment in
26 571 Science Fund at WHOI (SMS). Florian Götz was partly supported by a scholarship of the
572 Institute of Marine Biotechnology e.V. (IMaB).
573
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36 786 Table 1:
787 Overview of S. denitrificans protein identification counts for the two growth conditions
788 cyclooctasulfur (S8) and thiosulfate (S2O3). Exclusive identifications are proteins that were
789 detected in at least one replicate of one condition, but were absent in all replicates of the other
790 condition.
791
2- S8 grown cultures S2O3 grown cultures
Identified proteins total 784 822
Only in one replicate 200 176
Exclusive proteins 84 122
>1% NSAF 19 10
792
793
794
795
796
797
798
799
37 800 Figure 1: Distribution of COG categories in the S. denitrificans proteome (%NSAF =
801 Normalized Spectral Abundance Factor) and transcriptome (TPM = Transcripts Per Million) on
802 cyclooctasulfur (S8) or thiosulfate (S2O3).
803
804 Figure 2: The relative abundance of transcripts (TPM = Transcripts Per Million) and proteins
805 (%NSAF = Normalized Spectral Abundance Factor) related to flagellar biosynthesis and
806 motility. Light bars show the average values of S. denitrificans grown on cyclooctasulfur (S8)
807 and dark bars show the average of cultures grown on thiosulfate. Statistically-significant
808 differences are denoted with one (p<0.05) or two (p<0.01) asterisks. Genes indicated in bold
809 have also been identified in the proteomic dataset. Suden_0030: Flagellar hook capping protein =
810 WP_011371666.1; Suden_0031: Flagellar hook protein = WP_011371667.1; FlgE: Flagellar
811 hook = WP_011371668.1, Suden_0032; Suden_0172: Flagellin-like = WP_011371808.1;
812 Suden_0173: Flagellin-like = WP_011371809.1; Suden_0202: Flagellar hook-associated protein
813 2-like = WP_011371838.1; FliS: Flagellar protein = WP_041672386.1, Suden_0203; FliE:
814 Flagellar hook-basal body complex protein = WP_011371998.1, Suden_0363; FlgC: Flagellar
815 basal-body rod protein= WP_011371999.1, Suden_0364; FlgB: Flagellar basal-body rod protein
816 = WP_011372000.1, Suden_0365; FliF: Flagellar M-ring protein = WP_011372107.1,
817 Suden_0472; FliG: Flagellar motor switch protein = WP_011372108.1, Suden_0473;
818 Suden_0474: putative flagellar assembly protein = WP_011372109.1; Suden_0562: Flagellar P-
819 ring protein = WP_011372195.1; Suden_0733: Flagellar L-ring protein = WP_011372366.1;
820 FliL: Flagellar basal body-associated protein = WP_011372470.1, Suden_0840; Suden_1037:
821 putative flagellin = WP_011372667.1; FlgG: Flagellar basal-body rod = WP_011372733.1,
822 Suden_1103; Suden_1104: Flagellar basal body rod protein = WP_011372734.1.
38 823 Figure 3: Expression of S. denitrificans transcripts (upper panel; TPM = Transcripts Per
824 Million) and proteins (lower panel; %NSAF = Normalized Spectral Abundance Factor) involved
825 in denitrification. Light bars show the average values of S. denitrificans grown on
826 cyclooctasulfur (S8) and dark bars show the average of cultures grown on thiosulfate.
827 napAGHBFLD = Nitrate reductase (napA = WP_011373143.1, Suden_1514; napB =
828 WP_011373146.1, Suden_1517; napG = WP_041672542.1, Suden_1515; napH =
829 WP_041672543.1, Suden_1516; napF = WP_011373147.1, Suden_1518; napL =
830 WP_011373148.1, Suden_1519; napD = WP_011373150.1, Suden_1521); nirSF = nitrite
831 reductase (nirS = WP_011373599.1, Suden_1985; nirF = WP_011373602.1, Suden_1988);
832 norCB = nitric oxide reductase (norC = WP_011373597.1, Suden_1983; norB =
833 WP_011373598.1, Suden_1984); nosZ1,2 = nitrous oxide reductase (nosZ1 = WP_011372928.1,
834 Suden_1298; nosZ2 = WP_011373385.1, Suden_1770).
835
836 Figure 4: A) The relative abundance of expressed SOX (sulfur oxidation multienzyme complex)
837 and SQR (sulfide quinone reductase) proteins (%NSAF = Normalized Spectral Abundance
838 Factor) and transcripts (TPM = Transcripts Per Million). Light bars show the average values of
839 S. denitrificans grown on cyclooctasulfur (S8) and dark bars show the average of cultures grown
840 on thiosulfate. Statistically significant differences (p<0.05) are denoted with an asterisk. OMP
841 (outer membrane protein) = WP_011373531.1, Suden_1917; soxH-like = WP_011373670.1,
842 Suden_2056; SOX (sulfur oxidation multienzyme complex): soxZ2Y2DC: soxZ2 =
843 WP_011373671.1, Suden_2057; soxY2 = WP_011373672.1, Suden_2058; soxD =
844 WP_011373673.1, Suden_2059; soxC = WP_011373674.1, Suden_2060 ; soxXY1Z1AB: soxX =
845 WP_011371896.1, Suden_0260; soxY1 = WP_011371897.1, Suden_0261; soxZ1 =
39 846 WP_011371898.1, Suden_0262; soxA = WP_011371899.1, Suden_0263; soxB =
847 WP_011371900.1, Suden_0264) and SQR (sulfide quinone reductase = WP_011372252.1,
848 Suden_0619). B) The relative abundance of transcripts (upper panel; TPM = Transcripts Per
849 Million) and proteins (lower panel; %NSAF = Normalized Spectral Abundance Factor) of the
850 proposed polysulfide reductase (Suden_0498-Suden_0450: WP_011372132.1-
851 WP_011372134.1).
852
853 Figure 5: Model for the oxidation of elemental sulfur chains in S. denitrificans. The activation
2- 854 mechanism of cyclooctasulfur and the hydrolysis step resulting in the liberation of SO4 from the
855 outer sulfonate group (indicated with a “?”) are currently unknown. The ‘x7’ indicates that the
856 sulfur chain attached to SoxY2 might be oxidized completely (or in part) before accepting a new
857 sulfur chain. Sulfur is indicated as black dot, whereas oxygen is indicated as small white circles
858 on sulfur.
40 859 Supplementary Figure S1: Growth curves (dotted lines) of S. denitrificans cultures grown on
860 thiosulfate (upper panel) or cyclooctasulfur (lower panel), as determined via cell counts after
861 DAPI staining. Arrows indicate the time of sampling of the biological replicates.
862
863 Supplementary Figure S2: Distribution of the proteomic (left) and transcriptomic (right)
864 expression of S. denitrificans cultures grown on cyclooctasulfur (x-axis) vs. thiosulfate (y-axis).
865 Proteins and ORFs with statistically significant differences in expression are depicted in red.
866
867 Supplementary Figure S3: Gene expression (TPM = Transcripts Per Million) of the cbb3-type
868 cytochrome c oxidase (fixNOQP, fixN = WP_011371717.1, Suden_0081; fixO =
869 WP_011371718.1, Suden_0082; fixQ = WP_011371719.1, Suden_0083; fixP =
870 WP_011371720.1, Suden_0084), the membrane bound [Ni,Fe]-uptake hydrogenase (hydCBA,
871 hydC = WP_041672535.1, Suden_1434; hydB = WP_011373064.1, Suden_1435; hydA =
872 WP_011373065.1, Suden_1436) and the membrane bound formate dehydrogenase (fdhDCBAA,
873 fdhD = WP_011372449.1, Suden_0817; fdhC = WP_011372450.1, Suden_0818; fdhB =
874 WP_011372451.1, Suden_0819; fdhA = WP_011372452.1, Suden_0820; fdhA =
875 WP_041672204.1, Suden_0821; fdx = WP_011372455.1, Suden_0824). Light bars show the
876 average values of S. denitrificans grown on cyclooctasulfur (S8) and dark bars show the average
877 of cultures grown on thiosulfate.
878
879
41 880 Supplementary Table 1: Transcriptome and proteome of Sulfurimonas denitrificans
881 displayed with the locus tag, NCBI protein ID number, length of the gene in base pairs [bp], the
882 molecular weight of the protein in kilo Dalton [kDa], transcripts per million [TPM], percent
883 normalized spectral abundance factor [%NSAF] and the annotation. Both TPM and %NSAF are
884 given as an average of the biological replicates grown with cyclooctasulfur [S8] and thiosulfate
885 [S2O3]. In addition, the p-values for both the transcripts and proteins are provided, with p-values
886 <0.01 highlighted in light blue and p-values <0.05 highlighted in dark blue.
887
888 Supplementary Table 2: Transcriptomic data collected before and after quality control
889 (QC). S8-1-3 are the biological replicates of Sulfurimonas denitrificans grown with
890 cyclooctasulfur and T-1 and T-2 are the two biological replicates grown with thiosulfate.
Reads after Bases after Mapped
Library Reads total Bases total QC QC reads % mapped
S8-1 1.09E+07 1.64E+09 1.03E+07 1.09E+09 9.51E+06 93%
S8-2 7.85E+06 1.18E+09 7.09E+06 7.10E+08 6.57E+06 93%
S8-3 9.61E+06 1.44E+09 8.58E+06 7.72E+08 7.98E+06 93%
T-1 9.18E+06 1.38E+09 8.31E+06 8.38E+08 7.69E+06 92%
T-2 1.06E+07 1.58E+09 9.56E+06 9.67E+08 8.68E+06 91%
891
42
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