Transposition of Insertion Sequences was Triggered by Oxidative Stress in Radiation-Resistant Bacterium Deinococcus geothermalis

Abstract

During an oxidative stress-response assay on a putative Dps-like gene-disrupted Δdgeo_0257 mutant strain of radiation-resistant bacterium Deinococcus geothermalis, a non-pigmented colony was observed among the normal reddish color colonies. This non-pigmented mutant cell subsequently displayed higher sensitivity to H2O2. While carotenoid has a role in protecting as scavenger of reactive oxygen species the reddish wild-type strain from radiation and oxidative stresses, it is hypothesized that the carotenoid biosynthesis pathway has been disrupted in the mutant D. geothermalis cell. Here, we show that, in the non-pigmented mutant cell of interest, phytoene desaturase (Dgeo_0524, crtI), a key enzyme in carotenoid biosynthesis, was interrupted by transposition of an ISDge7 family member insertion sequence (IS) element. RNA-Seq analysis between wild-type and Δdgeo_0257 mutant strains revealed that the expression level of ISDge5 family transposases, but not ISDge7 family members, were substantially up-regulated in the Δdgeo_0257 mutant strain. We revealed that the non-pigmented strain resulted from the genomic integration of ISDge7 family member IS elements, which were also highly up-regulated, particularly following oxidative stress. The transposition path for both transposases is a replicative mode. When exposed to oxidative stress in the absence of the putative DNA binding protein Dgeo_0257, a reddish D. geothermalis strain became non-pigmented. This transformation was facilitated by transposition of an ISDge7 family IS element into a gene encoding a key enzyme of carotenoid biosynthesis. Further, we present evidence of additional active transposition by the ISDge5 family IS elements, a gene that was up-regulated during the stationary phase regardless of the presence of oxidative stress.

Active Transposition of Insertion Sequences by Oxidative Stress in Deinococcus geothermalis

“…Here, we detected two transposition events in wild-type (WT) strain and LysR family member gene disrupted strain (Δ dgeo _2840). Similar to our previous report ( Lee et al, 2019 ), phytoene desaturase ( dgeo _0524), a key enzyme of carotenoid biosynthesis, was disrupted by the integration of IS element, thereby detected a single phenotypically non-pigmented colony in each WT and Δ dgeo _2840 strain. Two separate types of IS element have been integrated into non-pigmented clones: IS Dge11 for WT and IS Dge6 for Δ dgeo _2840 strain.…”

“…We designed target-gene-encompassing primer sets with similar melting temperatures for the detection of transposition loci, and performed PCR of four carotenoid biosynthesis involved enzyme genes, following our previous study ( Lee et al, 2019 ), because the transposase-integrated sites were enlarged in the non-pigmented mutant, compared with WT and Δ dgeo_ 2840 mutant strains. From following PCR and agarose gel electrophoresis, the enlarged PCR products were selected, and then sequenced.…”

“…The strain D. geothermalis DSM 11300 T was obtained from the Korean Agricultural Culture Collection (KACC 12208). D. geothermalis was cultured on TGY medium containing 1% tryptone, 0.5% yeast extract, and 0.1% glucose at 48 • C. Escherichia coli DH5α was used as a competent cell for the transformation of recombinant DNA, and grown on Luria-Bertani medium at 37 • C. The dgeo_2840 gene locus disrupted mutant strain was constructed by integration of a kanamycinresistance cassette into a target gene through homologous recombination, following previous study (Lee et al, 2019). For the dgeo_2840 mutant strain, the homologous DNA sequence downstream of dgeo_2840, and also upstream region (roughly 1.0 kb), was amplified from genomic DNA using targetregion primers, and purified using a PCR purification kit (Bioneer, South Korea).…”

“…However, no individual transpositional action mechanism is clearly explained at all, because the key player transposase was controlled by multiple factors and phase points, such as transcriptional and translational control, and regulation at gene and protein network ( Nagy and Chandler, 2004 ; Siguier et al, 2017 ). We have found in previous studies that certain ISs types, IS Dge5 and IS Dge7 actively transposed to other sites on genomic DNA under a special DNA-binding protein Dgeo_0257 disrupted state through oxidative stress treatment ( Lee et al, 2019 ). IS Dge7 type integrated on a key enzyme of carotenoid biosynthesis result in colorless phenotype.…”

“…IS Dge7 type integrated on a key enzyme of carotenoid biosynthesis result in colorless phenotype. In addition, IS Dge5 type was transposed into two other sites in genome of this non-pigment mutant ( Lee et al, 2019 ). When carotenoid biosynthesis normally occurs, D. geothermalis strain is reddish in color.…”