Table S1. Bacterial strains (Related to Results and Experimental Procedures) Strain number Relevant genotype Source or reference 1045 AB1157 Graham Walker (Donnelly and Walker, 1989) 2458 3084 (MG1655) mazef (Christensen et al., 2003) 2562 AB1157 yihe-34::tn5 (kan) by transposon insertion 2666 MG1655 yihe-34::tn5 (kan) by P1 phage-mediated transduction of yihe-34::tn5 (kan) from 2562 to 3084. 2980 cysl95::tn10 Coli Genetic Stock Center: CGSC #7425 3009 cpxr::kan R Coli Genetic Stock Center: CGSC #10800 3084 MG1655 (wild type) (Christensen et al., 2003) 3086 3084 (MG1655) yihe by bacteriophage P1-mediated transduction from TL227 to 3084 3137 katg729::kan R Coli Genetic Stock Center: CGSC #10827 3336 3084 pacyc184-yihe 3337 3084 pacyc184 3338 3084 pacyc184-yihe S36A 3339 3084 pacyc184-yihe D217A 3340 3086 yihe) pacyc184 3341 3086 yihe) pacyc184-yihe 3342 3086 yihe) pacyc184-yihe S36A 3343 3086 yihe) pacyc184-yihe D217A 3344 2458 yihe by transduction from TL227 to 2458 3345 3344 mazef wt by transduction from 2980 to 3344 3357 3084, cpxr::kan R by transduction from 3009 to 3084 3371 3357 yihe by transduction from 3009 to 3396 3396 3086 Kan s by kanamycin marker excision (Baba et al., 2006) from 3086. 3401 Rosetta 2 pet28a-yihe 1
3402 BL21 (DE3) pet14b-yihe-s36a 3403 BL21 (DE3) pet14b-yihe-d217a 3404 BL21 (DE3) pet28a-maze 3405 BL21 (DE3) pet28a-mazef 3406 BL21 (DE3) pet14b-mazg 3407 3084 katg::kan R by transduction from 3137 to 3084 3418 3407 kan S by kanamycin marker excision (Baba et al., 2006) from 3407. 3419 3086 katg by transduction from 3086 to 3418 BL21 DE3 plyss Stratagene DH5 F - Φ80lacZΔM15 Δ(lacZYA-argF) U169 reca1 enda1 hsdr17 (rk, mk + ) phoa supe44 λ thi-1 gyra96 rela1 Invitrogen Rosetta 2 DE3 plyss EMD4Bioscience TL227 JW3831 ( yihe) Kan R (Baba et al., 2006) 2
Table S2. Minimal inhibitory concentration (Related to Results (Figures 1, 4, 6, and S1)) Antimicrobial MIC a KD3084 (wildtype) KD3086 ( yihe) Nalidixic acid 5 5 Ampicillin 35 35 Kanamycin 5 5 Mitomycin C 0.63 0.63 PD161144 0.32 0.32 Rifampicin 16 16 Tetracycline 2 2 Ciprofloxacin 0.01 0.01 Thiourea 250 mm 250 mm 2,2 -bipyridyl 300 M 300 M a Expressed in g/ml unless indicated otherwise. 3
Table S3. Plasmids and primers (Related to Results and Experimental Procedures) Plasmid/Primer Name Description Source or Reference Plasmids pacyc184 Low copy number plasmid vector used for complementation. Tet R, Cm R New England Biolabs (Ipswich, MA) pacyc184-yihe A native yihe cloned into pacyc184. Tet R pacyc184-yihe S36A A mutant yihe gene containing an S36A mutation cloned into pacyc184. Tet R pacyc184-yihe D217A A mutant yihe gene containing a D217A pet14b mutation cloned into pacyc184. Tet R IPTG-inducible expression vector having a 6x His Tag at 3 end. Amp R EMD4Biosciences (San Diego, CA) pet14b-mazg Expression vector for MazG purification. Amp R pet14b-yihe D217A YihE D217A expression vector for protein purification. Amp R pet14b-yihe S36A Expression vector for YihE S36A purification. Amp R pet28a pet28a- maze pet28a-mazef IPTG-inducible expression vector having a 6x His Tag at 3 end. Kan R. MazE expression vector for protein purification. Kan R MazEF expression vector for protein purification. Kan R EMD4Biosciences (San Diego, CA) pet28a-yihe Expression vector for YihE purification. Amp R Primers KatG_fwd 5 gga gaa cct ctt caa gta tga gtg gg KatG_rev 5 gga tcg ttg agg aaa cga cga g maze NdeI forward2 5 cta cat atg ata gcg gtt gag gaa agg gtt atg atc cac maze XhoI reverse 5 cgg gta ctc gag ggc tta cca tta cca gac tgt ctt 4
mazf XhoI reverse 5 gac cag ctc gag agt gag tta gta aca cta ccc aat c mazg NdeI forward 5 caa ttc ata tga atc aaa tcg acc gtt tgc acc gtt tgc t mazg XhoI reverse 5 gct cga gcg cgt aat tcc ctt aga gat caa pet14b-yihe1 5 cgg cat atg ggg atg aat aac agc gct pet14b-yihe2 5 tct ctc gag tta ata cat agg tgt taa ttg yihe_for_comp2 5 gca acg atc gca gaa act ggc gaa aga gtc agg yihe_rev_comp2 5 tac tct aga cga tta ata cat agg tgt taa ttg caa ag yihe_for_s36a yihe_rev_s36a yihe_for_d217a yihe_rev_d217a 5 ccg gtc tta ccc cgc tta acc gct atg aaa acc gtg tct atc aat 5 att gat aga cac ggt ttt cat agc ggt taa gcg ggg taa gac cgg 5 gcg atg gtc caa tgt ttg ttg cgc tgg atg atg cac gta atg gt 5 acc att acg tgc atc atc cag cgc aac aaa cat tgg acc atc gc 16S-RNA-fwd 5 agg cct tcg ggt tgt aaa gt 16S-RNA-rev 5 cgg gga ttt cac atc tga ct 5