ABSTRACT
8-oxo-7,8-dihydroguanine, commonly referred to as 8-oxoG, is considered one of the most predominant oxidative lesions formed in DNA. Due to its ability to pair with adenines in its syn configuration, this lesion has a strong mutagenic potential in both eukaryotes and prokaryotes. Escherichia coli cells are endowed with the GO system, which protects them from the mutagenic properties of this lesion when formed both in cellular DNA and the nucleotide pool. MutY and MutM (Fpg) DNA glycosylases are crucial components of the GO system. A strong mutator phenotype of the Escherichia coli mutM mutY double mutant underscores the importance of 8-oxoG repair for genomic stability. Here, we report that in Caulobacter crescentus, a widely studied alpha-proteobacterium with a GC-rich genome, the combined lack of MutM and MutY glycosylases produces a more modest mutator phenotype when compared to E. coli. Genetic analysis indicates that other glycosylases and other repair pathways do not act synergistically with the GO system for spontaneous mutation prevention. We also show that there is not a statistically significant difference in the spontaneous levels 8-oxodGuo in E. coli and C. crescentus, suggesting that other yet to be identified differences in repair or replication probably account for the differential importance of the GO system between these two species. Environ. Mol. Mutagen. 61:246-255, 2020. © 2019 Wiley Periodicals, Inc.
Subject(s)
Bacterial Proteins/genetics , Caulobacter crescentus/genetics , DNA Glycosylases/genetics , DNA-Formamidopyrimidine Glycosylase/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Mutagenesis , Bacterial Proteins/metabolism , Caulobacter crescentus/metabolism , DNA Glycosylases/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA-Formamidopyrimidine Glycosylase/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Deletion , Gene Expression Regulation, Bacterial , Genomic Instability , Guanine/analogs & derivatives , Guanine/metabolismABSTRACT
In C. crescentus, iron metabolism is mainly controlled by the transcription factor Fur (ferric uptake regulator). Iron-bound Fur represses genes related to iron uptake and can directly activate the expression of genes for iron-containing proteins. In this work, we used total RNA sequencing (RNA-seq) of wild type C. crescentus growing in minimal medium under iron limitation and a fur mutant strain to expand the known Fur regulon, and to identify novel iron-regulated genes. The RNA-seq of cultures treated with the iron chelator 2-2-dypiridyl (DP) allowed identifying 256 upregulated genes and 236 downregulated genes, being 176 and 204 newly identified, respectively. Sixteen transcription factors and seven sRNAs were upregulated in iron limitation, suggesting that the response to low iron triggers a complex regulatory network. Notably, lexA along with most of its target genes were upregulated, suggesting that DP treatment caused DNA damage, and the SOS DNA repair response was activated in a RecA-dependent manner, as confirmed by RT-qPCR. Fluorescence microscopy assays using an oxidation-sensitive dye showed that wild type cells in iron limitation and the fur mutant were under endogenous oxidative stress, and a direct measurement of cellular H2O2 showed that cells in iron-limited media present a higher amount of endogenous H2O2. A mutagenesis assay using the rpoB gene as a reporter showed that iron limitation led to an increase in the mutagenesis rate. These results showed that iron deficiency causes C. crescentus cells to suffer oxidative stress and to activate the SOS response, indicating an increase in DNA damage.
