Functional analysis of PprI in the DNA damage response mechanism of Deinococcus radiodurans

Analysis of the genome sequences of Deinococcus spp. discovered the radiation/desiccation response (RDR) motif, existing upstream of the radiation-inducible genes (RDR regulons) such as pprA and recA genes. The RDR motif serves as an operator sequence in the unique DNA repair response system. Another regulatory protein DdrO binds RDR motif. Following DNA damage, the metalloprotease activity of PprI cleaves DdrO, resulting in induction of the RDR regulon. However, the detailed functional site of PprI protein in the DNA damage response mechanism is poorly understood. In an effort to gain an insight into the role of the PprI in DNA damage response mechanism in D. radiodurans, firstly, we generated a pprI-deleted mutant strain and pprI expression plasmid. Consequently, we successfully generated complete deletion strains for the D. radiodurans pprI genes. This deletion strain SXPI carrying pRAD1 exhibited extreme sensitivity to gamma-rays compare to the wild type strain carrying pRAD1. In the strain SXPI carrying pEXpprI, the survival rate following treatment with gamma rays was restored to the similar level of that in the wild type. This result suggests that D. radiodurans PprI is a key protein for the repair of damage induced by gamma rays as shown in the previous study

Mutation analysis of the DNA damage response regulator protein PprI in the radioresistant bacterium Deinococcus radiodurans

Deinococcus radiodurans is a representative strain of radioresistant bacteria and has extremely high resistance to various DNA damaging agents such as gamma rays, ultraviolet rays, desiccation, free radical-generating substances, and DNA cross-linkers. The previous studies revealed that the expression of a unique DNA repair-related protein, PprA, was up-regulated by a regulatory protein, PprI, following DNA damage in D. radiodurans. The radiation/desiccation response (RDR) motif was found in the upstream regions of the radiation-inducible genes (RDR regulons) by comparative genome sequence analysis of Deinococcus. The RDR motif serves as an operator sequence in the unique DNA repair response system. The PprI consists of three domains, namely metalloprotease, DNA binding and GAF-like binding domains. Another regulatory protein DdrO binds the RDR motif and serves as a repressor. Following DNA damage, the metalloprotease activity of PprI cleaves DdrO, resulting in derepression of the RDR regulon. In this study, to delineate the functional site of PprI in regard to the DNA damage response mechanism, we constructed several pprI-expression plasmids carrying mutations which cause a single amino acid substitution. The pprI-deleted mutant strains carrying the mutated pprI genes (PIM_E119Q and PIM_E149Q) showed significant sensitivity to gamma rays as same as the pprI-deletion strain. Moreover, unlike the strain DAW carrying the wild-type pprI gene, the enhances of luciferase reporter activity in the luciferase reporter strains carrying the mutated pprI genes (DAM_E119Q and DAM_E149Q) were not observed following irradiation. These results suggest that E119Q and E149Q at metalloprotease domain of PprI play a critical role in the DNA damage response mechanism

Construction of Luciferase Reporter Strains for Functional Analysis of DNA Damage Response Regulators

Deinococcus radiodurans is a representative strain of radioresistant bacteria and has extremely high resistance to various DNA damage caused by gamma rays, ultraviolet rays, desiccation, free radical-generating substances, and DNA cross-linkers. The previous studies revealed that the expression of a unique DNA repair-related protein, PprA, was up-regulated by a DNA damage response regulator, PprI, following DNA damage in D. radiodurans. However, the detailed functional site of PprI protein is poorly understood. In an effort to gain an insight into the role of PprI in DNA damage response mechanism in D. radiodurans, we generated luciferase reporter strains that were regulated by pprI expression plasmids. Firstly, we constructed a luciferase reporter DNA cassette consisted of an engineered firefly luciferase gene (FL) and an Escherichia coli spectinomycin resistance gene. Next, this luciferase reporter DNA cassette was integrated into the genome of the wild-type and the pprI-deleted mutant strains, and the resulting FL reporter strains were designated DARP and SIRP, respectively. Consequently, in strain DARP, FL reporter activity was enhanced following 2 kGy gamma irradiation at 2 h post-irradiation time. On the other hand, FL reporter activity in the pprI-deletion strain SIRP was not observed following irradiation. Like strain DARP, the FL reporter activity in the strain SIRP carrying the pprI expression plasmid was enhanced following irradiation by functional complementation with the pprI expression plasmid. This result suggests that the D. radiodurans PprI is a key protein for DNA damage response regulator

Draft genome sequence of calcium-dependent Novosphingobium sp. strain TCA1, isolated from a hot spring containing a high concentration of calcium ions

Calcium-dependent Novosphingobium sp. strain TCA1 was newly isolated from a water sample of a hot spring containing a high concentration of calcium ions. Here, we report the draft genome sequence of this bacterium, which may be the basis for the research of calcium ion homeostasis. This bacterium was isolated from a water sample of Tsurumaki-onsen which is well known as a Japanese hot spring containing a high concentration of calcium ions. For sequencing, a library was constructed using the Nextera DNA library prep kit, with an insert length of approximately 300 bp; the library was then subjected to 100-bp paired-end sequencing on the Illumina HiSEQ2500 2x100-bp module. Consequently, the draft genome sequence of Novosphingobium sp. TCA1 was 6,173,804 bp with 64.3% GC content in total length and comprised of 136 large contigs (>500 bp), which was assembled by SPAdes v. 3.12.0. The draft genome sequence was predicted a total of 5,649 protein-coding genes and 53 tRNAs

Expression of translesion DNA polymerase genes from Deinococcus grandis in Escherichia coli

While Deinococcus radiodurans, the type species of the genus Deinococcus, does not possess any genes involved in translesion DNA synthesis, the draft genome sequencing of Deinococcus grandis KS0485 revealed that this bacterium possesses two lexA-imuB-dnaE2 gene clusters, which will be involved in translesion DNA synthesis. In this study, we expressed deinococcal dnaE2 and imuB genes in Escherichia coli to further delineate the function of these genes. The dnaE2 and imuB genes were cloned in pET-based vector and p15A-base vector, respectively, and introduced in E. coli BL21(DE3) cells. Mutant frequency was measured by colony formation assay based on rifampicin resistance. As a result, when both the dnaE2 and imuB genes were expressed in E. coli under IPTG induction, the mutant frequency was increased, confirming that the functionality of dnaE2 and imuB as mutagenesis genes. However, when dnaE2 or imuB gene was separately expressed in E. coli, no increase in mutant frequency was observed. These results suggest that both gene products are necessary to operate mutagenesis.第12回極限環境生物国際会議(Extremophiles2018)出