Tiling array study of MNNG treated Escherichia coli reveals a widespread transcriptional response

James A. Booth, Gard O. S. Thomassen, Alexander D. Rowe, Ragnhild Weel-Sneve, Karin Lagesen, Knut I. Kristiansen, Magnar Bjørås, Torbjørn Rognes, Jessica M. Lindvall
2013 Scientific Reports  
The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is known to trigger the adaptive response by inducing the ada-regulon -consisting of three DNA repair enzymes Ada, AlkB, AlkA and the enigmatic AidB. We have applied custom designed tiling arrays to study transcriptional changes in Escherichia coli following a MNNG challenge. Along with the expected upregulation of the adaptive response genes (ada, alkA and alkB), we identified a number of differentially expressed transcripts,
more » ... ed transcripts, both novel and annotated. This indicates a wider regulatory response than previously documented. There were 250 differentially-expressed and 2275 similarly-expressed unannotated transcripts. We found novel upregulation of several stress-induced transcripts, including the SOS inducible genes recN and tisAB, indicating a novel role for these genes in alkylation repair. Furthermore, the ada-regulon A and B boxes were found to be insufficient to explain the regulation of the adaptive response genes after MNNG exposure, suggesting that additional regulatory elements must be involved. A lkylating agents are present and generated both intra-and extracellularly. Such agents react with DNA and can subsequently give rise to mutations that can lead to cell death 1,2 . The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) reacts with DNA to produce different O-alkylated and N-alkylated DNA lesions 3 . After exposure of Escherichia coli (E. coli) to non-lethal doses of MNNG, transcription of the ada, alkB, alkA and aidB genes are induced under control of the ada-regulon. This stress response to alkylating agents is termed the adaptive response 2,4 . The Ada protein is a DNA methyltransferase that removes methyl groups from the premutagenic lesions O 6 -methylguanine and O 4 -methylthymine 2 . The AlkB protein repairs 1methyladenine and 3-methylcytosine in DNA by oxidative demethylation 5,6 . The DNA glycosylase AlkA catalyses the removal of several methylated base lesions 7 . Flavin adenine dinucleotide (FAD) containing AidB is involved in reducing the mutagenic effects of MNNG. It has been shown to potentially act at specific highly expressed genes including various DNA repair and constitutively expressed genes, but the mechanism of action is unknown 8, 9 . In addition to the role of Ada in the repair of alkylated DNA, the Ada protein also regulates the ada-regulon 10-12 . When the Ada protein covalently transfers a methyl group from a methylated phosphotriester in the DNA backbone to its own N-terminal Cys38, Ada is converted into a transcriptional activator of the ada, alkB, alkA and aidB genes 10 . Genome-tiling microarrays have enabled investigation of global expression patterns in organisms such as bacteria, mouse, human and yeast 13-18 , with or without a completely annotated genome. Investigations employing unbiased tiling of human chromosomes 21 and 22 have elegantly shown that large portions of the human genome are in fact transcribed 19 and are not, as previously thought, simply "junk" areas. Whether these transcripts represent novel mRNAs or non-coding RNAs (ncRNAs) 20 remains largely unclear. However, other more extensive datasets point towards the fact that these areas are of great importance 21-24 . In traditional gene-probing microarrays, all probes targeting the same gene are assumed to give independent measures of the same RNA expression 15, 25, 26 . When applying a tiling strategy to an entire genome, the analysis should not depend on the annotation, as this would restrict the analysis to annotated genes only, and would make the analysis impossible in the absence of an annotation. Therefore, one of the major challenges for tiling array OPEN
doi:10.1038/srep03053 pmid:24157950 fatcat:gwu5afsv5vd7xja6l5dvvteg5a