PMC

Deletion of pspE in ΔmstA-sup decreases H₂S biosynthesis during exponential growth. a qRT-PCR of three thiosulfate sulfurtransferase genes. RNA was isolated from cells grown to OD₆₀₀ ~ 0.4. Only pspE expression was significantly increased in both ΔmstA-sup and ΔycjW. Values are means ± SD (n = 3). *p < 0.05 as determined by the Student's t test. b H₂S levels in ΔmstA-sup; ΔpspE were undetectable till OD₆₀₀ reached 0.8. Values are means ± SD (n = 3). *p < 0.05 as determined by the Student's t test. c ΔmstA-sup/ΔpspE had increased sensitivity to gentamicin compared with WT and ΔmstA-sup. Overexpression of pspE in ΔmstA increased tolerance to gentamicin compared with ΔmstA. Values are means ± SD (n = 3). d In cells lacking MstA which is responsible for H₂S production, S258N YcjW upregulates PspE as an alternate route of H₂S biosynthesis. Source data are provided as a Source Data file

Unique nonsynonymous SNP in ΔmstA-sup is mapped to the putative transcription factor ycjW. a PCR validation of whole-genome sequencing. Displayed are sequences from E. coli MG155, ΔmstA, and ΔmstA-sup of (−) strand. The SNP changes amino acid 258 from serine to asparagine. b ΔmstA/ΔycW strains with plasmid-expressed P_LL-ycjW or P_LL-ycjW S258N were measured for H₂S production. Only ΔmstA/ΔycW; P_LL-ycjW S258N had increased H₂S production with no significant differences with WT or ΔmstA-sup. At OD₆₀₀ 1.5, no significant differences exist between strains. c Cells exposed to 2 µg ml⁻¹ Gm. ΔmstA;ΔycW; P_LL-ycjW S258N had increased survival rate. ΔmstA/ΔycW; P_LL-ycjW was more sensitive to Gm compared with wild-type and ΔmstA-sup. d ΔmstA/ΔycW; P_LL-ycjW S258N improved tolerance to H₂O₂. Values are means ± SD (n = 3) for all experiments. *p < 0.05 as determined by the Student's t test. Source data are provided as a Source Data file

E. coli MG1655 lacking 3MSTA acquires phenotypic suppression and has increased H₂S levels and tolerance to Gm and H₂O₂. a ΔmstA-sup has increased survival rate compared with ΔmstA when treated with 2 µg  ml⁻¹ gentamicin in a time-kill assay. Values correspond to colony-forming units (c.f.u). b ΔmstA-sup also has increased tolerance after exposure to 5 mM H₂O₂ for 30 min. c H₂S production as measured with fluorescent probe, WSP5. Relative fluorescent units are normalized to OD₆₀₀ and minus the background fluorescent of PBS buffer + 100 µM L-cysteine and WSP5. H₂S reacts with lead acetate, leading to staining of strips (Sigma-Aldrich). Values are means ± SD (n = 3). *p < 0.05 as determined by the Student's t test. Source data are provided as a Source Data file

YcjW shows binding enrichment near ycjM and ycjU and regulates expression of operon ycjMNOPQRSTUV-ompG. a Represented on Integrative Genomics Viewer (IGV), are sorted, aligned sequences containing pileup data to reference genome NC_000913.3. MACS2 was used for peak calling. Enriched peaks are upstream of ycjM and ycjU. A 14 nucleootide sequence identified as the binding motif for YcjW. b YcjW protein was titrated to DNA:protein ratios of 1:0.5, 1:1, and 1:2. Unlabeled ycjM probe was added to the reaction in excess to compete for binding. Unbound (free) probe and YcjW-probe complexes are denoted as U and B, respectively. c YcjW and S258N YcjW proteins were titrated to DNA:protein ratios of 1:0.125, 1:0.25, 1:0.5, 1:1, and 1:2. with narP probe. Unbound (free) probe and YcjW-probe complexes are denoted as U and B, respectively. d qRT-PCR of a subset of genes in the ycjM-V and ompG operon. The absence of ycjW results in massive upregulation. mstA-sup and ycjW;P_LL—ycjW both showed moderate and significant increased expression, while mRNA levels are repressed in mstA. Values are means ± SD (n = 3). *p < 0.05 as determined by the Student's t test. e YcjW protein was pre-incubated with Kojibiose, trehalose, or sucrose before radiolabeld DNA probes were added to the mixture. Only kojibiose prevented complex formation at a concentration of 0.5 mM. In contrast, both sucrose and trehalose added in excess at 5 mM and 10 mM failed to disrupt binding. Unbound (free) probe and YcjW-probe complexes are denoted as U and B, respectively. Source data are provided as a Source Data file