Roles Of The Two-component System-like DtrA/DtrB In Response To High Temperature And UV Stresses In Deinococcus Radiodurans

Deinococcus radiodurans, known as―the most adaptable bacteria‖in the world, showsextraordinary tolerance to DNA damages from diverse sources such as gamma-radiation, ultraviolet,desiccation and hydrogen peroxide, and can survive under high temperature, low temperature,osmotic conditions. It has been reported that two component systems （TCSs） played an importantrole in sensing, transmitting and responsing the stimuli. According to genomic analysis, Deinococcusradiodurans have large number of TCSs, containing20histidine kinases and26response regulators.However, only few TCSs of Deinococcus radiodurans have been identified and characterized. In thisstudy, we try to explore the function and regulatory mechnism of TCSs, then further explain theadaptive response to extreme stress in Deinococcus radiodurans.When analysising the genes expression in the null mutant of IrrE, a global regulator of extremeradiation resistance, we found DR_A0009and DR_A0010genes, encoding a putative two componentsystem, were upregulated significantly. DR_A0009encodes the histidine kinase and containsHisKA₃and HATPase_c domains; DR_A0010encodes the response regulator and containsRESPONSE_REGULATORY and HTH_LUXR₂domains. We used RT-PCT to verify that thesetwo genes were organized into an operon. The KEGG database predicted that DR_A0009/DR_A0010belongs to the NarL family and regulates the genes of desaturase expression. The predicted aminoacid sequences were above43%identical to the two component system DesK-DesR of Bacillussubtilis, and they had the same domains. DesK/DesR regulated the membrane phospholipiddesaturase when the temperature decreased. we found DR_A0009/DR_A0010were not affected bylow temperature, so they were renamed DtrA/DtrB.To study the biological functions of DtrA and DtrB, we constructed the ΔdtrA and ΔdtrB mutantsby inserting a Kan cassette in the middle of dtrA and dtrB sequences, individually. Under the optimaltemperature （30℃） and the low temperature（15℃）, the growth of the mutants did not show anysignificant difference from the wild type strain. Under other stress conditions, such as high osmotic（4M NaCl and3M sorbitol）, alkaline, acidity, desiccation stresses, there were also no significantdifferences between the mutant strains and wild type. However，the two mutants were sensitive tohigh temperature（42℃）, but enhanced UV radiation apparently.Using qRT-PCR to analysis the transcriptional dtrA and dtrB genes in Deinococcus radioduransafter heat shock （42℃） for3hours compared to the optimal conditions, the result showed that thetwo genes expression increased2.78fold and2.74fold respectively. QRT-PCR showed that most heatshock proteins （such as GroES, DnaK, Hsp20, Hsp1and Hsp2） in the two mutants weredownregulated significantly. The genes invovled in UV repair pathway（such as uvr-A, uvr-B, rad25and mutS） and other genes （such as yehZ, otsB, DR₁372, DR₁172, sodA and sodC） participated inosmotic, desiccation and oxidative regulation were upregulated apparently. This indicated dtrA/dtrBwere heat induced genes and they played an important role in heat stress and UV radiation. The unsaturated fatty acids played an important role in membrane fuidity and oxidative stressresistance. The persent of fatty acids were assayed and the result showed that the persent of fatty acidswas no significant difference between the wild type and the two mutants under the optimal growthconditions. But after heat shock （42℃） for three hours, the ratio of unsaturated fatty acids/saturatedfatty acid in the ΔdtrA and ΔdtrB mutants accounted for80%and82%of wild type content. QRT-PCRshowed that the mutation of dtrA and dtrB affected the genes involved in unsaturated fatty acidsynthesis, especially DR₀279and DR₀822. The result indicated that DtrA/DtrB regulated themetabolism of fatty acids.