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UDP-glucose 4-epimerase. 233. HPG27_1429 riboflavin biosynthesis protein. 231 ... alanine racemase. 173. HPG27_518. 3-oxoacyl-(acyl carrier protein) ...
Electronic Supplementary Material (ESI) for Metallomics. This journal is © The Royal Society of Chemistry 2014

Metallomics

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ARTICLE   Supplemental Information

“Nickel-Responsive Regulation of Two Novel Helicobacter pylori NikR–Targeted Genes” M.D. Jones, I. Ademi, X. Yin, Y. Gong, and D.B. Zamble

Table S1. Primers for plasmid construction and qPCR. Promoter HPG27_1499 HPG27_866 Gene ureA HPG27_1499 HPG27_866 16S rRNA gyr

Primer name Plasmid construction p1499F p1499R p866F p866R qPCR qUreAF qUreAR q1499F q1499R q866F q866R q16SF q16SR qgyrF qgyrR Sequencing sKO866F sKO866R sKO866upF sKO866upR sKO866dwnF sKO866dwnR

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Sequence (5’-3’) CTGGGTCGACTCAGCGGAAACATAGT GATTTCGGCCGAAGCGAGCGATTAG CACTCGGCCGAACGCTTGTCATTCATTC CATCGTCGACTGCGTGCTTTGAAAGGG ACGCTGGAGAATTGGCTAAA CAGCTCTCGCTTCTTCCATAATA CTTGCATTTTGGCCAGTTTT GCTTAGCCCTGATCTTGTGG CGCGGCTTTAAATGTCTCGCCTTT ATCCACACCAAACCTCCAGGCATA CAGCGTCAGTAATGTTCCAG TAGAGTGTGGGAGAGGTAGG AGCGATAGAGAGGGCATTAGA CTCCATGGTGGTGAGTTTGT GCATTCTGCGTGCTTTGAAAGG GGGCTTATTACGCATGAAATCCTC GGCTATCAACACCAGCGATC CATAAGCGCATTGGGTGAGG GTGGTCGTGTGGAGAACTTTTC CCCTTGATTCGAACCGCTC

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Table S2. Putative HpNikR gene targets identified through ChIP-Seq. Gene Taga HPG27_998 HPG27_210 HPG27_663 HPG27_1285 HPG27_1419 HPG27_1036 HPG27_1429 HPG27_376 HPG27_1518 HPG27_513 HPG27_242 HPG27_452 HPG27_453 HPG27_1352 HPG27_768 HPG27_769 HPG27_160 HPG27_22 HPG27_903 HPG27_1164 HPG27_456 HPG27_457 HPG27_81 HPG27_1282 HPG27_27 HPG27_1286* HPG27_1287* HPG27_1091 HPG27_1499 HPG27_1500 HPG27_212 HPG27_1051 HPG27_1524 HPG27_631 HPG27_866 HPG27_1153 HPG27_507 HPG27_870 HPG27_1425 HPG27_1426 HPG27_1165 HPG27_288 HPG27_354 HPG27_361 HPG27_1229

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Putative Descriptionb 30S ribosomal protein S1 3-deoxy-manno-octulosonate cytidylyltransferase HopE (outer membrane protein) nicotinate-nucleotide adenyltransferase RplY (50S ribosomal protein L25) UDP-glucose 4-epimerase riboflavin biosynthesis protein UDP-3-O-[3-hydroxymyristoyl] N-acetylglucosamine deacetylase undecaprenylphosphate N-acetylglucosaminyl transferase 23S rRNA methyltransferase Type II DNA modification enzyme neuraminyllactose-binding hemagglutinin phospho-N-acetylmuramoyl-pentapeptide-transferase dimethyladenosine transferase hypothetical protein hypothetical protein peptidyl-prolylcis-transisomerase C HopD (outer membrane protein) prolipoprotein diacylglyceryl transferase ABC transporter, ATP-binding protein hypothetical protein sodium-and chloride-dependent transporter RpoD (RNA polymerase sigma factor) hypothetical protein hypothetical protein NikR (nickel responsive regulator) ExbB (biopolymer transport protein) 50S ribosomal protein L19 iron (III) ABC transporter, periplasmic iron-binding protein alkylhydroperoxide reductase hypothetical protein pyruvate flavodoxin oxidoreductase subunit delta hypothetical protein HorF (outer membrane protein) iron-regulated outer membrane protein ABC-transporter, ATP-binding domain CagA (cytotoxin-associated protein A) glyceraldehyde-3-phosphate dehydrogenase HopK-3 (outer membrane protein) hypothetical protein undecaprenyl pyrophosphate synthase putative N-carbamoyl-D-amino acid amidohydrolase hypothetical protein CheY (chemotaxis protein) hypothetical protein

Sequence Tagc 291 290 283 262 256 233 231 226 225 225 224 223 223 221 221 221 220 220 217 213 211 211 211 207 206 205 205 204 202 202 202 196 194 190 190 189 189 189 186 186 183 183 183 180 179

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Journal  Name   Gene Taga HPG27_573 HPG27_89 HPG27_1459* HPG27_257 HPG27_895 HPG27_134 HPG27_751 HPG27_1032 HPG27_1289* HPG27_876 HPG27_223 HPG27_890 HPG27_518 HPG27_988 HPG27_1414 HPG27_1415 HPG27_1120 HPG27_1158 HPG27_1398 HPG27_775 HPG27_1421 HPG27_795 HPG27_841 HPG27_1064 HPG27_1440 HPG27_736 HPG27_1435* HPG27_300 HPG27_418 HPG27_571 HPG27_531 HPG27_1522 HPG27_1523 HPG27_287 HPG27_1474 HPG27_65 HPG27_1155 HPG27_116 HPG27_1170 HPG27_1171 HPG27_1313 HPG27_145 HPG27_703 HPG27_325 HPG27_1199 HPG27_1391 HPG27_1025

ARTICLE   Putative Descriptionb ABC transporter. ATP-binding protein hypothetical protein FecA3 (putative nickel transport protein) guanosine pentaphosphate phosphohydrolase hypothetical protein cytochrome c oxidase (diheme subunit) Tig (trigger factor) ribonucleotide-diphosphate reductase subunit beta TonB (siderophore-mediated iron transport protein) HtpX (heat shock protein) NapA (neutrophil activating protein, bacterioferritin) alanine racemase 3-oxoacyl-(acyl carrier protein) synthase II GMP synthase phosphate permease conserved hypothetical nifU-like protein HopQ (outer membrane protein) F0F1 ATP synthase subunit C phosphopantetheine adenylyltransferase hypothetical protein hypothetical protein hypothetical protein FecE (iron (III) dicitrate ABC transporter) FlgK (flagellar hook-associated protein) hypothetical protein bifunctional aconitate hydratase 2/2-methylisocitrate dehydratase FrpB4 (putative nickel transport protein) arginyl-tRNA synthetase polyphosphate kinase ABC transporter, permease adenine phosphoribosyltransferase hypothetical protein FlgG (flagellar basal body rod protein) hypothetical protein putative type II methylase protein UreE (urease accessory protein) type II adenine specific DNA methyltransferase HorB (outer membrane protein) coproporphyrinogen III oxidase cytochrome c553 chemotaxis response regulator hypothetical protein hypothetical protein single-stranded-DNA specific exonuclease biotin synthesis protein branched-chain amino acid aminotransferase deoxycytidine triphosphate deaminase

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Sequence Tagc 179 178 177 177 177 176 176 174 174 174 173 173 172 172 171 171 170 170 169 169 168 168 168 167 166 165 164 164 164 164 163 161 161 161 159 158 157 156 155 155 154 154 154 152 150 150 149

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ARTICLE   Gene Taga Putative Descriptionb HPG27_1026 biotin carboxyl carrier protein HPG27_1066 type II DNA modification enzyme HPG27_1510 DNAse HPG27_41 HypE (hydrogenase expression/formation protein) HPG27_975 hypothetical protein HPG27_1214 NADH dehydrogenase subunit J HPG27_1497 FlgB (flagellar basal body rod protein) HPG27_1498 FtsW (cell division protein) HPG27_495 CagM (cag pathogenicity island protein) HPG27_190 lipopolysaccharide 1,2-glycosyltransferase HPG27_191 outer membrane protein a Known targets of direct HpNikR regulation are marked with an asterisk b As annotated in the NCBI Gene Database c As determined by SISSR

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Journal  Name   Sequence Tagc 149 149 148 148 148 147 147 147 137 26 26

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Figure S1. DNA-binding activity of apo-HpNikR. Apo-HpNikR (50 nM – 100 µM) was incubated with DNA containing the HPG27_1499 promoter sequence (a) or the HPG27_866 sequence (b) then resolved on 6% native polyacrylamide gels with 3 mM MgSO4. Binding was observed only at the maximum HpNikR concentrations used.      

 

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a)

HpNikR binding site

b) 5’ TCGACTCAGCGGAAACATAGTGGAGTATATTCTTAAAAAGTTAATGAAAAGATATTTCCTTTTAAATTTT AAGATTTTGTGAAAAAAAGTTTCATTTTCACTGCTTGTATTTCCTTG1AATAG2TG3TTATAATCG4CTTCAT AAATCATACG5AAAGGAATTGTCATGCTAATCGCTCGCTTCGGCCG 3’ Figure S2. DNA binding by HpNikR in excess nickel on the 1499 promoter. a) HpNikR (50 nM, 100 nM, 200 nM, 250 nM, 300 nM, 500 nM, 1 µM, 5 µM) was incubated with a 32P-labeled 184bp DNA probe in the presence of 35 µM NiCl2 for 30 minutes at room temperature prior to the addition of DNase I. The reactions were resolved on an 8% denaturing polyacrylamide gel and the area of protection is indicated. Lane G was loaded with a Maxam-Gilbert G reaction. The apparent DNA-binding affinity was determined to be 330 ± 40 nM with a Hill coefficient of 2.02 ± 0.04, calculated as an average of two independent experiments. The DNA strand was labeled on the 3’ end with α32P and Klenow fragment. b) DNA sequence of 1499 promoter (reverse complement of sequence in Figure 3b). The binding region is underlined and the guanines within in the footprint are bolded and noted on the footprint gel.  

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Figure S3. Expression of H. pylori genes upon transient nickel exposure. Quantitative PCR was performed for H. pylori G27 and its isogenic ΔnikR mutant grown to mid-log phase and exposed to 100 μM NiCl2 for 15 minutes. Fold expression is relative to 16S and gyr expression under the same conditions and normalized to WT without nickel. Statistically significant differences between expression levels are marked with diamonds (♦ = P < 0.05, ♦♦ = P < 0.01, Student’s t-test). The data represent the average of at least three independent biological and technical replicates, and the error bars indicate one standard deviation.  

 

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  Figure S4. Alignment of the HPG27_866/frpB2 promoters from H. pylori strains G27 and 26695. The HpNikR recognition consensus sequence proposed by Delany et al28 is circled by a dashed line and the consensus sequence proposed by Stoof et al29 is highlighted in blue. Both promoter sequences are the same distance upstream of the start codon.

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