Screening for Escherichia coli K-12 genes conferring glyoxal resistance or sensitivity by transposon insertions

• Published in: FEMS microbiology letters 2016-09, Vol.363 (18), p.fnw199
• Main Authors: Lee, Changhan, Kim, Jihong, Kwon, Minsuk, Lee, Kihyun, Min, Haeyoung, Kim, Seong Hun, Kim, Dongkyu, Lee, Nayoung, Kim, Jiyeun, Kim, Doyun, Ko, Changmin, Park, Chankyu
• Format: Article
• Language: English
• Subjects: Adenylate cyclase; Adenylyl Cyclases - genetics; Antioxidants; Antitoxins; Cellular stress response; Cyclic AMP; Cyclic AMP Receptor Protein - genetics; Deoxyribonucleic acid; DNA; DNA repair; DNA Transposable Elements; Drug Resistance, Bacterial - genetics; E coli; Escherichia coli K12 - drug effects; Escherichia coli K12 - genetics; Escherichia coli Proteins - genetics; Genes; Glyoxal - metabolism; Glyoxal - pharmacology; Medicin och hälsovetenskap; Microbiology; Mutagenesis, Insertional; Mutants; Mutation; Phenotypes; Polymerase chain reaction; RecA protein; Resistance factors; RNA, Transfer - genetics; Target recognition; Toxins; tRNA; tRNA Ala; Y chromosomes
• ISSN: 1574-6968; 0378-1097; 1574-6968
• DOI: 10.1093/femsle/fnw199

Glyoxal (GO) belongs to the reactive electrophilic species generated in vivo in all organisms. In order to identify targets of GO and their response mechanisms, we attempted to screen for GO-sensitive mutants by random insertions of TnphoA-132. The genes responsible for GO susceptibility were functionally classified as the following: (i) tRNA modification; trmE, gidA and truA, (ii) DNA repair; recA and recC, (iii) toxin–antitoxin; mqsA and (iv) redox metabolism; yqhD and caiC. In addition, an insertion in the crp gene, encoding the cAMP responsive transcription factor, exhibits a GO-resistant phenotype, which is consistent with the phenotype of adenylate cyclase (cya) mutant showing GO resistance. This suggests that global regulation involving cAMP is operated in a stress response to GO. To further characterize the CRP-regulated genes directly associated with GO resistance, we created double mutants deficient in both crp and one of the candidate genes including yqhD, gloA and sodB. The results indicate that these genes are negatively regulated by CRP as confirmed by real-time RT-PCR. We propose that tRNA as well as DNA are the targets of GO and that toxin/antitoxin, antioxidant and cAMP are involved in cellular response to GO. Genes screened for glyoxal susceptibility by transposon mutagenesis reveals cellular responses involving tRNA/DNA modification, toxin/antitoxin, antioxidant and cAMP regulation.