1 Supplementary information 2 Cultivation and characterization of a novel clade of deep-sea Chloroflexi: 3 providing a glimpse of the phylum Chloroflexi involved in sulfur cycling 1,2,3,4 1,2,3,4 1,2,4 1,2,3,4 1,2,4 1,2,4* 4 Rikuan Zheng , Ruining Cai , Rui Liu , Yeqi Shan , Ge Liu , Chaomin Sun 1 5 CAS Key Laboratory of Experimental Marine Biology & Center of Deep Sea 6 Research, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China 2 7 Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory 8 for Marine Science and Technology, Qingdao, China 3 9 College of Earth Science, University of Chinese Academy of Sciences, Beijing, 10 China 11 4Center of Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, China 12 13 * Corresponding author 14 Chaomin Sun Tel.: +86 532 82898857; fax: +86 532 82898857. 15 E-mail address: [email protected] 16 17 18 19 20 21 22 23 1 24 Supplementary methods 25 Proteomic analysis 26 Sample processing protocol 27 Strain ZRK33 was cultivated in the ORG medium supplemented without or with 200 28 mM Na2SO4 or 200 mM Na2S2O3 for 8 d at 28 °C and then the cells were collected. 29 These cells were sonicated three times on ice using a high intensity ultrasonic 30 processor in lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail). The remaining 31 debris was removed by centrifugation at 12,000 g at 4°C for 10 min. Finally, the 32 supernatant was collected and the protein concentration was determined with a BCA 33 kit (Solarbio, China) according to the manufacturer's instructions. For trypsin 34 digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 35 56 °C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in 36 darkness. The 100 mm TEAB was added to the diluted protein sample in a solution 37 with a urea concentration of less than 2 M. Finally, trypsin was added at a trypsin to 38 protein mass ratio of 1:50 for the first digestion overnight, with 1:100 trypsin and 39 protein. The mass was added for a second digestion for 4 h. Then the tryptic peptides 40 were dissolved in 0.1% formic acid (solvent A) and directly loaded into a home-made 41 reversed-phase analytical column (15-cm length, 75 μm inner diameter). The gradient 42 increased from 6% to 23% in solvent B (0.1% formic acid in 98% acetonitrile) over 43 26 min, from 23% to 35% in 8 min and increased to 80% in 3 min, then maintain 80% 2 44 for the last 3 min, and all at a constant flow rate of 400 nL/min on an EASY-nLC 45 1000 UPLC system. 46 The peptides were coupled to UPLC in Q ExactiveTM Plus (Thermo, USA) via 47 NSI source and tandem mass spectrometry (MS/MS). The applied electrospray 48 voltage was 2.0 kV. The full scan has an m/z scan range of 350 to 1,800, and at 49 70,000 resolution, intact peptides were detected in the Orbitrap. MS/MS was then 50 selected using the NCE set to 28 select peptides and fragments were detected in the 51 Orbitrap at a resolution of 17,500. A data-related process that alternates between one 52 MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. The automatic 53 gain control (AGC) was set to 5E4. The fixed first mass was set as 100 m/z. 54 Data processing protocol 55 (1) Database Search 56 The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8) 57 [1]. Tandem mass spectra were searched against some databases (such as 58 UniProt-GOA, InterPro, Kyoto Encyclopedia of Genes and Genomes (KEGG)) 59 concatenated with reverse decoy database. Trypsin/P was specified as cleavage 60 enzyme allowing up to 2 missing cleavages. The mass tolerance for precursor ions 61 was set as 20 ppm in First search and 5 ppm in Main search, and the mass tolerance 62 for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as fixed 63 modification and oxidation on Met was specified as variable modifications. FDR was 64 adjusted to < 1% and minimum score for peptides was set > 40. 3 65 (2) Enrichment of Gene Ontology analysis 66 Proteins were classified by GO annotation into three categories: biological process, 67 cellular compartment and molecular function. For each category, a two-tailed Fisher’s 68 exact test was employed to test the enrichment of the differentially expressed protein 69 against all identified proteins. The GO with a corrected P-value < 0.05 is considered 70 significant. 71 (3) Enrichment of pathway analysis 72 Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched 73 pathways by a two-tailed Fisher’s exact test to test the enrichment of the differentially 74 expressed protein against all identified proteins [2]. The pathway with a corrected 75 p-value < 0.05 was considered significant. These pathways were classified into 76 hierarchical categories according to the KEGG website. 77 (4) Enrichment of protein domain analysis 78 For each category proteins, InterPro (a resource that provides functional analysis of 79 protein sequences by classifying them into families and predicting the presence of 80 domains and important sites) database was researched and a two-tailed Fisher’s exact 81 test was employed to test the enrichment of the differentially expressed protein 82 against all identified proteins. Protein domains with a P-value < 0.05 were considered 83 significant. 84 (5) Enrichment-based Clustering 4 85 For further hierarchical clustering based on different protein functional classification 86 (such as: GO, Domain, Pathway, Complex). We first collated all the categories 87 obtained after enrichment along with their P values, and then filtered for those 88 categories which were at least enriched in one of the clusters with P value <0.05. This 89 filtered P value matrix was transformed by the function x = −log10 (P value). Finally 90 these x values were z-transformed for each functional category. These z scores were 91 then clustered by one-way hierarchical clustering (Euclidean distance, average linkage 92 clustering) in Genesis. Cluster membership was visualized by a heat map using the 93 “heatmap.2” function from the “gplots” R-package. 94 95 96 97 98 99 100 101 102 5 103 Supplementary results 104 Description of Sulfochloroflexaceae fam. nov. 105 Sulfochloroflexaceae (Sul.fo'ch.lo.ro.fle.xa.ce.ae. N.L. fem. n. Sulfochloroflexus type 106 genus of the family; suff. -aceae, ending to denote a family; N.L. fem. pl. n. 107 Sulfochloroflexaceae the family of the genus Sulfochloroflexus). 108 The description is the same as that for the genus Sulfochloroflexus. The type 109 genus is Sulfochloroflexus. 110 Description of Sulfochloroflexales ord. nov. 111 Sulfochloroflexales (Sul.fo'ch.lo.ro.fle.xa.les. N.L. fem. n. Sulfochloroflexus type 112 genus of the order; suff. -ales ending to denote an order; N.L. fem. pl. n. 113 Sulfochloroflexales order of the genus Sulfochloroflexus). 114 The description is the same as that for the genus Sulfochloroflexus. The type 115 genus is Sulfochloroflexus. 116 Description of Sulfochloroflexia classis nov. 117 Sulfochloroflexia (Sul.fo'ch.lo.ro.fle.xia. N.L. fem. n. Sulfochloroflexus type genus of 118 the class; N.L. fem. pl. n. Sulfochloroflexia, the class of the order 119 Sulfochloroflexales). 120 The class Sulfochloroflexia is defined on the basis of phylogenetic trees by 121 comparative 16S rRNA gene, genome, RpoB and EF-tu sequences analysis from a 122 wide variety of cultivated strains and environmental clones. The type order is 123 Sulfochloroflexales. 6 124 Supplementary figures 125 126 Supplementary Figure S1. Maximum likelihood phylogenetic tree of strain ZRK33 127 based on the genomes from all cultured Chloroflexi representatives using the 128 concatenated alignment of 37 single-copy genes. Actinoplanes derwentensis LA107 129 was used as the outgroup. Nodes with greater than 80% bootstrap support are 130 annotated with a black circle. Bar, 0.1 substitutions per nucleotide position. 131 132 7 133 134 Supplementary Figure S2. Maximum likelihood phylogenetic tree of RpoB from 135 genomes of strain ZRK33 and all cultured Chloroflexi representatives. Actinoplanes 136 derwentensis LA107 was used as the outgroup. Nodes with greater than 80% 137 bootstrap support are annotated with a black circle. Bar, 0.1 substitutions per 138 nucleotide position. 139 140 141 142 143 8 144 145 Supplementary Fig. S3. Maximum likelihood phylogenetic tree of elongation factor 146 Tu (EF-Tu) from genomes of strain ZRK33 and all cultured Chloroflexi 147 representatives. Actinoplanes derwentensis LA107 was used as the outgroup. Nodes 148 with greater than 80% bootstrap support are annotated with a black circle. Bar, 0.1 149 substitutions per nucleotide position. 150 151 152 153 154 155 9 156 157 Supplementary Fig. S4. Growth assays of strain ZRK33 cultured in the medium 158 supplemented with different sulfur-containing compounds. (A) Growth assays of 159 strain ZRK33 in the medium supplemented without or with 20 mM Na2SO4. (B) 160 Growth assays of strain ZRK33 in the medium supplemented without or with 20 mM 161 Na2S2O3. (C) Growth assays of strain ZRK33 in the medium supplemented without or 162 with 1 mM Na2SO3. (D) Growth assays of strain ZRK33 in the medium supplemented 163 without or with 1 mM Na2S. 164 165 166 167 168 169 170 171 172 10 173 174 Supplementary Fig. S5. Proteomic analysis of expressions of genes associated with 175 EMP glycolysis when strain ZRK33 was cultured in the medium supplemented with 176 200 mM sulfate or thiosulfate.