Metabolic pathway for glyoxal (GO) and methylglyoxal (MG). GO can be reduced to glycolaldehyde and further to 1,2-ethandiol with the involvement of AKRs, such as YafB, YqhE, and YghZ. MG is converted to d-lactate, lactaldehyde, and acetol by glyoxalase, MG reductase, and AKRs, respectively. Glo III (hchA) catalyzes conversions of GO and MG to glycolic acid and d-lactate without cofactors. AldA, aldehyde dehydrogenase; FucO, l-1,2-propanediol oxidoreductase; Glo I, glyoxalase I; Glo II, glyoxalase II; Glo III, glyoxalase III; GldA, glycerol dehydrogenase; YqhD, aldehyde reductase; GSH, glutathione; SHG, S-2-hydroxyethyl glutathione; SLG, S-lactoylglutathione.

YafB expressions in the GO-resistant mutants. (A) The GO-resistant mutants show an expression of a 30-kDa protein, which was identified as YafB (*, 29.5 kDa, an aldo-keto reductase) by MALDI-TOF MS fingerprinting. Expression of YafB is highest in mutant 1. (B) Western blotting with specific antiserum for YafB; (C) results of a quantitative real-time PCR experiment showing yafB transcription in various mutants; (D) specific activities of YafB enzymes in the mutants. The data were standardized with the wild-type value as 1. Rough correlation between the data in panels C and D exists. Detailed procedures are described in Materials and Methods.

Phenotypes of GO-resistant mutants. (A) Results of spotting assays on LB plates containing various concentrations of GO. Mutant 1 exhibits normal growth on medium containing 7 mM GO, which is lethal to not only the MG1655 ΔgloA ΔyqhD strain but also the wild-type MG1655 strain. (B) Growth inhibition in various concentrations of GO. IC₅₀s (mM) are 3.23 (MG1655, wild type), 1.42 (ΔQG strain, parent), 8.55 (mutant 1), 4.41 (mutant 2), 4.99 (mutant 3), 6.39 (mutant 4), and 1.96 (mutant 5).

Genomic rearrangements in the GO-resistant mutants and their consequences. (A) Extents and locations of the deletions in the upstream region of yafB. Multibase deletions in the mutants are shown with dashed (1), dotted (2), and solid (3) lines. The intergenic sequences (lowercase letters) are distinguished from genes (uppercase letters). The location of the 4-bp deletion (GCAT) in the NsrR-binding site of mutant 5 is presented with the filled triangle (▲) below the dotted line. The putative NsrR- and Fnr-binding sites are underlined and in boldface. The transcription initiation site was determined previously (17), with its putative −10 consensus for RpoS (shading; EcoCyc, http://ecocyc.org/). Terminator and anti-antiterminator of aspU are shown with arrows with solid and dashed lines, respectively. (B) Schematic representation of multibase deletion mutations. Because of the differences in deletion sizes, PCR products, amplified with yafBup-F and yafBup-R primers, from the GO-resistant mutants are smaller than those of the wild type (328 bp). Putative NsrR- and Fnr-binding sites are presented with white and gray boxes, respectively. The anti-antiterminator and terminator of aspU are shown with dotted and solid lines (loops), respectively. (C) Locations of the seven rRNA operons (rrn) on the E. coli chromosome. Arrows indicate directions of transcriptions. The origin of chromosomal replication, oriC, is positioned at about 84.5 min. The distance between rrnD and oriC is much shorter than that between rrnH and oriC. Schematic representation of the recombination event in mutant 4. The sequences from rrnD are shown in gray. Locations of primers for RT-PCR are indicated with short arrows: rrl-F, primer 1 and 1′; rrf-F, primer 2 and 2′; aspU-F, primer 3; thrV-F, primer 3′; and yafB-R, primer 4. (D) Confirmation of read-through messages by RT-PCR. The amplified read-through transcripts are separated on a 1.2% agarose gel. In the cases of mutants 2 and 3, no transcripts were observed in lanes 3 due to a removal of the aspU gene. The read-through transcripts from the rrnH promoter in the wild type were detected by additional rounds of PCR as shown in the left panel. Similar transcripts were seen with mutant 5 (data not shown).

Glyoxal sensitivities of the strains with various levels of YafB expression. Strains tested were transformed with pBAD18 (vector control) or pBAD-YafB (+, arabinose-inducible yafB) and were spotted onto LB plates containing 0.2 mM arabinose. Various levels of glyoxal resistances were observed with the deletion and overproduction of the ΔyafB strain in the wild-type MG1655 background. Mutation 1 shows the highest GO resistance when transferred to the wild-type MG1655 strain.

Transcriptional repressions of yafB by NsrR and Fnr. (A) Construction of reporter phage λ for single-copy expression of lacZ. The plasmid-borne fusions were made first, and then they were crossed onto λRZ5 and inserted into the chromosome at the λ attachment site (attP) to generate strains carrying single-copy promoter fusions. The mutated version of the putative NsrR-binding site is indicated with an asterisk. Details are described in Materials and Methods. (B) The reporter phage containing nsrR-wt or nsrR-Δ were introduced into lacZ deletion strains; wild-type, ΔnsrR, Δfnr, or ΔnsrR Δfnr strains. (C) Transcription levels of yafB in Fnr and NsrR deletion mutants are elevated relative to that of the wild type. Derepression of yafB was higher in the double mutant (ΔnsrR Δfnr strain) than in single mutants. Exponentially growing cells in LB (OD₆₀₀ = 1.0) were treated for 15 min with 0, 1, and 3 mM GO. Total RNAs were used as templates for reverse transcription. The products were separated by 1.5% agarose gel electrophoresis, in which ompC was used as an internal reference. The RT-PCR data (top) were consistent with those of real-time qRT-PCR analysis (bottom).

Binding of NsrR and Fnr to the yafB promoter. Electrophoretic mobility shift assay was carried out with C-terminally His-tagged NsrR and Fnr. The probe DNA labeled with [γ-³²P]ATP was incubated with freshly prepared cell extracts from BL21(DE3) ΔnsrR Δfnr strains expressing NsrR, Fnr, or the empty vector pET21a. The yafB promoter region obtained by PCR from genomic DNA was used as a probe. The amount of cell extract is shown with the graded triangle on top. The reaction mixtures were incubated at 37°C for 30 min and run on polyacrylamide electrophoresis image gels for 16 h at 4°C. The NsrR-DNA complex and the free DNA probe are marked with an asterisk and an arrow, respectively.