Conservation and diversity of radiation and oxidative stress resistance mechanisms in Deinococcus species

Abstract

Deinococcus bacteria are famous for their extreme resistance to ionising radiation and other DNA damage- and oxidative stress-generating agents. More than a hundred genes have been reported to contribute to resistance to radiation, desiccation and/or oxidative stress in Deinococcus radiodurans. These encode proteins involved in DNA repair, oxidative stress defence, regulation and proteins of yet unknown function or with an extracytoplasmic location. Here, we analysed the conservation of radiation resistance-associated proteins in other radiation-resistant Deinococcus species. Strikingly, homologues of dozens of these proteins are absent in one or more Deinococcus species. For example, only a few Deinococcus-specific proteins and radiation resistance-associated regulatory proteins are present in each Deinococcus, notably the metallopeptidase/repressor pair IrrE/DdrO that controls the radiation/desiccation response regulon. Inversely, some Deinococcus species possess proteins that D. radiodurans lacks, including DNA repair proteins consisting of novel domain combinations, translesion polymerases, additional metalloregulators, redox-sensitive regulator SoxR and manganese-containing catalase. Moreover, the comparisons improved the characterisation of several proteins regarding important conserved residues, cellular location and possible protein-protein interactions. This comprehensive analysis indicates not only conservation but also large diversity in the molecular mechanisms involved in radiation resistance even within the Deinococcus genus.

Figure 1.

Extreme radiation and oxidative stress resistance in Deinococcus involves multiple factors and well-regulated mechanisms.

Figure 2.

Schematic overview of ionising radiation and oxidative stress resistance-associated proteins in D. radiodurans. Many D. radiodurans gene deletion or disruption mutants with more than 10-fold increased sensitivity compared to the wild-type strain have been described (Table S1, Supporting Information), and the corresponding proteins are indicated in the figure. Red box, ionising radiation sensitive; green box, oxidative stress sensitive; blue box, ionising radiation and oxidative stress sensitive.

Figure 3.

Novel two-domain proteins. The canonical DNA repair proteins AlkA, PhrB, Ung, Nth and Dcm and carotenoid biosynthesis proteins CrtY and CruF are standalone proteins. Genes encoding fusions of two of these proteins were identified in several Deinococcus species. The total number of amino acid residues (aa) of the novel two-domain proteins is indicated at the right.

Figure 4.

Three groups of 3-methyladenine DNA glycosylase (MPG) proteins identified in 11 Deinococcus species. The phylogenetic analysis was carried out based on protein sequence alignment of 16 deinococcal MPG proteins (Table S2, Supporting Information) made with Clustal omega. GenBank accession numbers in parentheses follow the species name. The phylogenetic tree was developed using the neighbour-joining algorithm in MEGA 6.0. The scale indicates the number of amino acid substitutions per site, and the node numbers are bootstrap values based on 1000 replications.

Figure 5.

Phylogenetic relationship of glutaredoxin-like proteins identified in 11 Deinococcus species. The phylogenetic analysis was carried out based on protein sequence alignment of 35 deinococcal Grx/NrdH-like proteins (Table S3, Supporting Information) with some representative proteins taken from Uniprot: NrdH from E. coli (Uniprot Number P0AC65), M. tuberculosis (P95106), S. aureus (Q936D1) and B. cereus (Q819J1), and Grx from E. coli (P68688 and P0AC262) and P. aeruginosa (Q9HU55). Weblogo plots show sequences around the CxxC motif extracted from deinococcal proteins of each group. See further the legend to Figure 4.

Figure 6.

Phylogenetic relationship between two types of cold-shock proteins (Csp) identified in 11 Deinococcus species. The phylogenetic analysis was carried out based on protein sequence alignment of 19 deinococcal Csp homologues (Table S4, Supporting Information) made with Clustal omega. Two Csps of D. soli are identical to Csps of D. actinosclerus. See further the legend to Figure 4.

Figure 7.

Model for the radiation/desiccation response (RDR) mechanism in Deinococcus: induction of the RDR regulon after cleavage of a repressor by a separate and specific metalloprotease. Under standard conditions, DdrO probably binds as a dimer to the two half-sites of the palindromic motif RDRM, thereby inhibiting transcription of IrrE/DdrO-regulated genes. Exposure of Deinococcus to radiation or desiccation stimulates cleavage of DdrO by IrrE. Cleaved DdrO may not form dimers and no longer binds the RDRM, allowing rapid induction of RDR regulon genes (e.g. DNA repair genes and ddrO itself). Persistence of the signal leading to DdrO cleavage may induce, directly or indirectly, one or more pro-apoptotic genes.

Figure 8.

Phylogenetic relationship of LexA homologues identified in Deinococcus species. The phylogenetic analysis was carried out based on protein sequence alignment of 12 deinococcal LexA homologues (Table S6, Supporting Information) with some representative proteins taken from Uniprot: LexA from B. subtilis (Uniprot Number P31080) and E. coli (P0A7C2). See further the legend to Figure 4.

Figure 9.

IrrE/DdrO- and RecA/LexA-regulated expression of DNA repair genes within the same bacterium. The cinA-ligT-recA operon (ligT-cinA in D. proteolyticus) and genes for IrrE and DdrO are present in each Deinococcus species. Additional RecA (RecA-2) and the lexA-imuY-dnaE2 operon are found in D. deserti and D. peraridilitoris. Experimental data, obtained for D. deserti only, have shown that both recA genes are radiation-induced in an IrrE-dependent way, and that the presence of either recA-1 or recA-2 is sufficient for radiation resistance. Radiation exposure also induces expression of the lexA-imuY-dnaE2 operon leading to induced mutagenesis mediated by the translesion polymerases ImuY and DnaE2, and this induction requires recA-1 but not recA-2. The recA-2 product is thus functional for recombinational repair but not for induction of mutagenic lesion bypass. The lexA-imuY-dnaE2 operon is also radiation induced in the irrE mutant, indicating that basal level of RecA-1 is sufficient for this induction. The red symbols indicate transcriptional repression by DdrO or LexA, or repressor inactivation by IrrE- or RecA-mediated cleavage. HP, hypothetical protein.

Figure 10.

Three groups of FUR family proteins identified in 11 Deinococcus species. The phylogenetic analysis was carried out based on protein sequence alignment of 26 deinococcal FUR family proteins (Table S6, Supporting Information) with some representative proteins taken from Uniprot: Fur proteins from Vibrio cholerae (Uniprot Number P0C6C8), E. coli (P0A9A9), P. aeruginosa (Q03456), Campylobacter jejuni (P0C631) and H. pylori (O25671); Mur from Rhizobium leguminosarum (Q1MMB4); Zur from Streptomyces coelicolor (Q9L2H5), M. tuberculosis (P9WN85), B. subtilis (P54479), and E. coli (P0AC51); Nur from S. coelicolor (Q9K4F8); PerR from B. subtilis (P71986) and Streptococcus pyogenes (Q1JIU5); Irr from R. leguminosarum (I9X7E3) and Bradyrhizobium japonicum (O85719). GenBank accession numbers in parentheses follow the species name. The phylogenetic consensus tree was developed using the neighbour-joining algorithm in MEGA 6.0. The node numbers are bootstrap values based on 1000 replications.