J. Microbiol. Biotechnol. (2016), 26(12), 2106–2115 http://dx.doi.org/10.4014/jmb.1601.01017 Research Article Review jmb Identification and Functional Analysis of RelA/SpoT Homolog (RSH) Genes in Deinococcus radiodurans S Jinhui Wang, Ye Tian, Zhengfu Zhou, Liwen Zhang, Wei Zhang, Min Lin, and Ming Chen* Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R. China Received: January 11, 2016 Revised: July 20, 2016 To identify the global effects of (p)ppGpp in the gram-positive bacterium Deinococcus Accepted: August 10, 2016 radiodurans, which exhibits remarkable resistance to radiation and other stresses, RelA/SpoT homolog (RSHs) mutants were constructed by direct deletion mutagenesis. The results showed that RelA has both synthesis and hydrolysis domains of (p)ppGpp, whereas RelQ only First published online synthesizes (p)ppGpp in D. radiodurans. The growth assay for mutants and complementation August 24, 2016 analysis revealed that deletion of relA and relQ sensitized the cells to H2O2, heat shock, and *Corresponding author amino acid limitation. Comparative proteomic analysis revealed that the bifunctional RelA is Phone: +86-1082106106; involved in DNA repair, molecular chaperone functions, transcription, the tricarboxylic acid Fax: +86-1082106142; cycle, and metabolism, suggesting that relA maintains the cellular (p)ppGpp levels and plays a E-mail: [email protected] crucial role in oxidative resistance in D. radiodurans. The D. radiodurans relA and relQ genes are responsible for (p)ppGpp synthesis/hydrolysis and (p)ppGpp hydrolysis, respectively. (p)ppGpp integrates a general stress response with a targeted re-programming of gene S upplementary data for this regulation to allow bacteria to respond appropriately towards heat shock, oxidative stress, and paper are available on-line only at starvation. This is the first identification of RelA and RelQ involvement in response to http://jmb.or.kr. oxidative, heat shock, and starvation stresses in D. radiodurans, which further elucidates the pISSN 1017-7825, eISSN 1738-8872 remarkable resistance of this bacterium to stresses. Copyright© 2016 by The Korean Society for Microbiology Keywords: RelA, RelQ, (p)ppGpp, oxidative stress, Deinococcus radiodurans, RelA/SpoT homolog and Biotechnology Introduction synthetases (SAS), and small alarmone hydrolases (SAH). The long RSHs have (p)ppGpp synthetase, hydrolase, TGS, To adapt to environmental changes, bacteria utilize and ACT domains, whereas SASs and SAHs only contain guanosine tetraphosphate and guanosine pentaphosphate synthetase or hydrolase domain, respectively [3]. Gram- (collectively named (p)ppGpp) as signal molecules to negative bacteria such as Escherichia coli have two long regulate many physiological processes that are sensitive to RSHs, RelA and SpoT. RelA is considered to be a (p)ppGpp changing nutrient availability, such as growth [13, 47], synthetase. In response to amino acid starvation, RelA secondary metabolism [40], survival [18], persistence[29], senses uncharged tRNA stalled on the ribosome and then biofilm formation [11, 46], development [1], competence, synthesizes (p)ppGpp [50]. SpoT is a bifunctional enzyme and virulence [1, 15, 20, 38]. that is involved in both degradation and synthesis of Upon encountering a stress, (p)ppGpp is produced from (p)ppGpp and regulates (p)ppGpp levels in response to ATP and GTP (or GDP) and rapidly accumulates [45]. limitation of carbon sources, fatty acids, or iron [6, 43, 49, RelA/SpoT homolog (RSH) proteins, named for their 51]. Unlike E. coli, Vibrio cholerae has a novel RelA-SpoT- sequence similarity to the RelA and SpoT enzymes of independent (p)ppGpp synthetase gene [16]. Gram-positive E. coli, comprise a superfamily of enzymes that synthesize bacteria such as Bacillus subtilis have RelA, RelQ, and RelP. and/or hydrolyze the alarmone (p)ppGpp. The RSH The bifunctional enzyme RelA exists widely in gram- proteins can be divided into three major groups: long positive bacteria, whereas RelQ and RelP belong to SASs, RelA/SpoT homolog proteins (longRSH), small alarmone which only contain the synthesis domain [24, 26, 35, 36]. J. Microbiol. Biotechnol. RSH Genes in Deinococcus radiodurans 2107 (p)ppGpp-deficient mutants exhibit defects in growth D. radiodurans relA and relQ genes are responsible for regulation, decreases in viability under nutrient-limited (p)ppGpp synthesis/hydrolysis and (p)ppGpp hydrolysis, conditions, increases in susceptibility to oxidative stress, respectively. We show that relQ and relA responded to and defects in biofilm formation [46]. In Enterococcus several stresses, including oxidative stress, heat shock, and faecalis V583, the ΔrelA strain was highly sensitive to 45 mM amino acid limitation. Furthermore, comparative proteomic H2O2 and displayed reduced growth in a medium analysis showed that relA affected the expression of containing 1 M NaCl [52]. Mutants defective only in the multiple proteins involved in oxidation resistance. RSH synthase domain of Staphylococcus aureus did not show impaired bacterial growth under nutrient-rich Materials and Methods conditions; however, they were more sensitive to mupirocin, had lower viability when essential amino acids Bacterial Strains, Plasmids, and Growth Conditions were depleted from the medium, and were less virulent The bacterial strains and plasmids used in this study are listed than the wild-type [19]. in Table 1. The D. radiodurans strains were grown at 30°C in TGY Deinococcus radiodurans, a gram-positive bacterium, is broth (1% tryptone, 0.5% yeast extract, and 0.1% glucose) or on well known in its capacity to resist extreme ionizing TGY plates supplemented with agar (1.5%). Concentrations of 350 μg/ml spectinomycin or 8 μg/ml kanamycin were used for radiation, UV radiation, oxidative stress, and a variety of antibiotic selection of D. radiodurans mutant strains. The minimal DNA-damaging agents [10], allowing it to survive in medium was prepared as described by Venkateswaran et al. [48]: various extreme environments. In D. radiodurans, two fructose as the sole carbon source and L-histidine and L-cysteine as genes, DR_1838 (relA) and DR_1631 (relQ), are predicted to the amino acid supplements. The E. coli strains were grown at be associated with (p)ppGpp synthesis and hydrolysis. 37°C in LB broth (1% tryptone, 0.5% yeast extract, and 1% NaCl) However, the role of these genes in response to stresses or on LB plates supplemented with agar (1.5%) or antibiotic as remains unclear. In this study, we demonstrated that the required. All chemicals were of molecular biology grade and Table 1. Strains and plasmids used in this study. Strain/plasmid Genotype/construction Source/reference D. radiodurans R1 Wid-type (ATCC1399) Lab stock ΔrelA As R1 but relA::spe This study ΔrelQ As R1 but relQ::kan This study ΔrelArelQ As R1 but relA::spe DR_1631::kan This study DR Z3-relA As R1 but Z3-rleA This study DR Z3-rel NTD As R1 but Z3-rel NTD This study ΔrelA Z3-relA As ΔrelA but Z3-relA This study ΔrelA Z3-rel NTD As ΔrelA but Z3-relA NTD This study E. coli Trans10 Host for plasmid subclone TransGen CF1648 Wild-type E. coli Xiao et al. [51] CF1652 As CF1648 but relA::kana relA::kan Xiao et al. [51] CF1693 As CF1648 but relA::kana spoT::cm Xiao et al. [51] CF1648 Z3 As CF1648 with pRADZ3 This study CF1648 Z3-relA As CF1648 with Z3-relA This study CF1693 Z3 As CF1693 with pRADZ3 This study CR1693 Z3-relA As CF1693 with Z3-relA This study Plasmid pRADZ3 Shuttle plasmid between E. coli and D. radiodurans R1 Ap-R in E. coli Cm-R in D. radiodurans R1 Lab stock pBlueScript SK Cloning vector (Ap-R) Lab stock pRAZA pRADZ3 derivative carrying relA promoter This study pRAZQ pRADZ3 derivative carrying relQ promoter This study December 2016 ⎪ Vol. 26⎪ No. 12 2108 Wang et al. purchased from New England BioLabs, Takara Bio Inc., Promega, requirements of the ΔrelArelQ strain, the cells were grown in Sigma Chemicals Co., and General Electric Company. medium supplemented with only 17 amino acids (except His and Cys), and the growth curve was monitored. Construction of D. radiodurans relA and relQ Mutant Strains The relA and relQ mutants were constructed by direct deletion Conserved Domain Database Search of RelA and RelQ Proteins mutagenesis [53]. The primers that were used in this study are The protein sequences were submitted for a NCBI Conserved listed in Table S1. The homologous fragments 674 bp downstream Domain Database search (http://www.ncbi.nlm.nih.gov/Structure/ and 584 bp upstream of DR_1838 were amplified with the primers cdd/wrpsb.cgi) to identify various domains present in the relA-1F and relA-1R and with relA-2F and relA-2R, respectively, proteins. The default settings were used. The databases used were and the spectinomycin cassette was amplified with aadAF and CDSEARCH/cdd ver. 3.13. aadAR. The homologous fragments and spectinomycin cassette were cloned into the pBlueScript SK(+) vector by restriction H2O2 and Heat Shock enzyme digestion. The region containing homologous fragments TGY liquid cultures were inoculated at an OD600 value of 0.1 and the spectinomycin cassette was digested with KpnI and SacI, using overnight cultures. When the OD600 value reached 0.8, an and then transformed into the wild-type D. radiodurans. The relQ aliquot (1 ml) of the cultures was supplemented with a final mutants were generated by replacing the target gene with a concentration of 0, 20, 40, or 60 mM H2O2 for 0.5 h. The heat shock kanamycin cassette via a fusion PCR. The regions that were assay was performed by cultivating bacteria at OD600 =1.0, and located 339 bp upstream and 389 bp downstream of DR_1631 then shifting them to 48°C for 2, 3, or 4 h. were amplified with the primers relQ-1F and relQ-1R and with relQ-2F and relQ-2R, respectively, from the D. radiodurans RNA Preparation and qRT-PCR genome.