File S1 - Nickel-Resistance Determinants in Acidiphilium sp. PM Identified by Genome-Wide Functional Screening

Figure S1. Determination of heavy-metal cross-resistance of the four nickel-resistant clones. Serial dilutions of overnight-grown cultures were plated on LB-Ap plates containing 0.8 mM Cd, 1.25 mM Co, 4.5 mM Cu, or 1.5 mM Zn. Figure S2. Identification of the orfs involved in Ni resistance by subcloning. ORFs from the environmental DNA inserts of pSRNi5 (A), pSRNi6 (B) and pSRNi16 (C) were subcloned and tested for Ni resistance. Serial dilutions of overnight cultures were plated in LB-Ap plates containing 2.25 mM Ni. Assays were performed in triplicate using independent cultures. ORFs involved in Ni resistance are shown in grey. ORFs with predicted transmembrane helices are shaded with vertical bars. Asterisks indicate incomplete ORFs. pSRNi5_orf2: ATP-dependent protease hsIV; pSRNi5_orf3: ATP-dependent protease ATP-binding subunit HslU; pSRNi5_orf5: amidase; pSRNi6_orf1: 3-oxoacyl-(acyl-carrier-protein) reductase; pSRNi6_orf2: malonyl CoA-acyl carrier protein transacylase; pSRNi6_orf3: polysaccharide export protein; pSRNi6_orf4: non-specific protein-tyrosine kinase; pSRNi16_orf1: dihydroxy-acid dehydratase; pSRNi16_orf2: hypothetical protein; pSRNi16_orf3: RND efflux transporter. Figure S3. Determination of heavy-metal cross-resistance of protease HslVU (orf2-orf3). Serial dilutions of overnight-grown cultures were plated on LB-Ap plates containing 0.8 mM Cd, 1.25 mM Co, 4.5 mM Cu, or 1.5 mM Zn. Figure S4. Exploring possible horizontal gene transfer of operon hslVU in acidic environments. Phylogenetic trees of 14 acidic and 14 non-acidic species are shown as reconstructed from the 16S rRNA gene (A), and the concatenated amino acid sequences of HslV and HslU (B). Sequences belonging to Acidiphilium sp. PM are shown in bold text and those of acidic species are denoted by asterisks. Bootstrap values are indicated at the nodes. Scale bars correspond to 5% (A) or 10% (B) sequence divergence. The amino acid sequences for HslV and HslU used in the analysis are the following: Rhodospirillum rubrum F11, AEO50144 and AEO50145; Escherichia coli str. K-12 substr. DH10B, ACB04944 and ACB04943; Pseudomonas aeruginosa PAO1, AAG08438.1 and AAG08439; Bacillus subtilis subsp. subtilis str. 168, CAB13488 and CAB13489; Salinibacter ruber M8, CBH24765 and CBH24767; Ralstonia solanacearum GMI1000, CAD13571 and CAD13570; Borrelia burgdorferi N40, ADQ29063 and ADQ29174; Thermotoga maritima MSB8, AAD35606 and AAD35607; Planctomyces brasiliensis DSM 5305, ADY60041 and ADY60040; Acidithiobacillus ferrooxidans ATCC 23270, ACK80450 and ACK79019; Acidithiobacillus ferrooxidans ATCC 53993, ACH84568 and ACH84569; Acidithiobacillus ferrivorans SS3, AEM46768 and AEM46767; Acidithiobacillus caldus SM-1, AEK57130 and AEK57129; Acidithiobacillus caldus ATCC 51756, EET27426 and EET27425; Acidiphilium multivorum AIU301, BAJ80804 and BAJ80803; Acidiphilium sp. PM, EGO96503 and EGO96504; Acidiphilium cryptum JF-5, ABQ30620 and ABQ30619; Leptospirillum rubarum, EAY57767 and EAY57766; Leptospirillum sp. Group II '5-way CG', EDZ39580 and EDZ39579; Acidocella sp. MX-AZ02, EKM99984 and EKM99983; Acetobacter pasteurianus IFO 3283-01, BAH99172 and BAH99173; Gluconacetobacter diazotrophicus PAl 5, ACI52812 and ACI52813; Gluconobacter oxydans H24, AFW01689 and AFW01690; Roseomonas cervicalis ATCC 49957, EFH12145 and EFH12146; Azospirillum lipoferum 4B, CBS88212 and CBS88211; Thiomonas intermedia K12, ADG31586 and ADG31587; Sulfobacillus acidophilus DSM 10332, AEW05868 and AEW05867; Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446, ACV58411 and ACV58412; Leptothrix cholodnii SP-6, ACB36091 and ACB36092. (DOCX)