Following the proteome dynamics of D. deserti after irradiation by a 2DE-gel approach highlighted the up-regulation of 21 proteins. Among these, ten proteins either have no homolog in D. radiodurans or were not found to be radiation-induced at the protein or RNA level in this organism (see Additional file 5: Table S2 and below), whereas eight others are known to be important players of the irradiation response in D. radiodurans: DdrB, SSB, PprA, RecA, GyrA/B, UvrD and DdrD. Concerning RecA, a notable difference is that two functionally different RecA proteins are up-regulated in D. deserti. The other sequenced Deinococcus species possess only one recA gene. Both RecAC and RecAP contribute to radiotolerance, but only RecAC is able to induce expression of DNA translesion polymerases in D. deserti
. The proteomic results obtained by Zhang et al.
 from the 2D-gel based analysis of D. radiodurans after irradiation did not reveal the presence of GyrB and RecA while transcriptomics revealed the up-regulation of their genes  (Additional file 5: Table S2). Lu et al.
 did see RecA induction, but apparently not GyrA, GyrB, DdrB, and DdrD accumulation. Here, we found a good correlation with the transcriptomics data obtained with D. radiodurans because GyrA, GyrB, RecA, DdrB and DdrD polypeptides are found accumulating in large quantities from 2 hours after irradiation. While this work was in progress, a new proteomic study with D. radiodurans was published, also showing up-regulation of GyrA, GyrB and DdrB, but not of DdrD (Additional file 5: Table S2). Using a 2DE-gel approach, Basu & Apte  identified ten and two different forms of induced SSB and DdrB, respectively, with each form in a different spot. These different forms are likely resulting from proteolytic processing of the C-terminal end of these proteins . In our study with D. deserti, we identified up-regulated SSB and DdrB in only one spot each. Interestingly, we found different spots for both GyrA and GyrB (see below). The use of different bacterial species and experimental conditions are likely explanations for the differences between our results and those previously published on D. radiodurans
Remarkably, we found the presence of several spots assigned to the same protein: four (spots sp_58, sp_59, sp_60, sp_61) and two (spots sp_101, sp_94) spots correspond to the GyrA and GyrB subunits, respectively. The different forms of Deide_12520 (GyrA) detected in the three HCA clusters are produced in higher amount after irradiation. The GyrA spots (spots sp_58, sp_59, sp_60, and sp_61) are only separated by small pI shifts. As reported previously by Zhu et al. , shifts in pI often correlate to protein modifications. These modifications include truncations or deletions, but also post-translational modifications of amino acid lateral chains such as phosphorylation or acetylation. They can also be due in a limited number of cases to alternative translation starts as revealed in an extensive survey of N-termini of proteins from D. deserti
, or N-terminal trimming by exo-peptidases. At the N-terminus and side chains of amino groups, post-translational modifications will lead generally to small (<0.2) isoelectric point shift. Here, we observed a sequence coverage of ~30% with the GyrA peptides in the four spots. We detected the [2-TGIHPVDITSEV-13] and the [790-VINIAERDSVISAFPIRR-807] peptides that are very close from the expected N-terminal and C-terminal extremities of the 811 amino acid-long GyrA. For this reason, a small truncation of the protein is unlikely, except maturation of the N-terminus (removal of the initial methionine) as evidenced by the most-N-terminal peptide. Regarding post-translational modification of GyrA, we could not detect any phosphorylation for serine, threonine or tyrosine residues, nor acetylation of the lysine side chains within the detected peptides. However, we found that GyrA N-terminus is acetylated. While a mixture of acetylated peptide and non-modified peptide is seen in spots sp_59, sp_60, and sp_61, only the acetylated form is detected in spot sp_58. For eukaryotic proteins acetylation is one of the most common covalent modifications with phosphorylation  while it is not frequent for prokaryotic proteins [45, 46] even if prokaryotic proteins are also extensively post-translationally modified . Amino-terminal acetylation occurs co-translationally with eukaryotic proteins  and post-translationally in the case of prokaryotic proteins . Acetylation may affect protein functions as their stability and DNA binding activity may be modified; the hyperacetylation of histones being the most illustrative example in this respect as it influences drastically transcription . Hwang et al.
 noted that N-terminal acetylation of proteins could act as a degradation signal in yeast. Interestingly, we found the Deide_20140 GCN5-related acetyltransferase being up-regulated in the earliest time-points after irradiation. Whether this enzyme is responsible for the acetylation observed on GyrA is an interesting hypothesis that deserves further investigation. However, at least under our experimental conditions, a potential Deide_20140-specific function is not essential for radiation resistance, since a Deide_20140 deletion mutant appeared to be as radiotolerant as the wild-type strain (data not shown). The D. deserti genome encodes various putative N-acetyltransferases, and some of these might have redundant activities.
We observed two up-regulated proteases: Deide_19590 (protease La, also called Lon protease) and Deide_02310 (related to dipeptidyl-aminopeptidase/acylaminoacyl-peptidase). Of these, Deide_02310 is not conserved in other sequenced Deinococcus species. The presence of these proteases in significant amounts is delayed compared to most DNA-repair related proteins. Their up-regulation, together with the presence of several other proteases (Additional file 2: Table S1) , could explain the significant trend observed in terms of number of spots detected in our 2DE-gels. A very active protein turn-over occurs because of protein damages and intense metabolic changes. Protein degradation after exposure to radiation was also reported for D. radiodurans
, which also encodes a high number of proteases. Other data indicated that Lon proteases play a key role in degradation of damaged proteins in D. radiodurans
. Proteolysis may also control the levels of radiation-induced proteins, as shown for E. coli where numerous SOS response proteins, including RecA and UvrA, are substrates for ClpPX, Lon and other proteases [53, 54]. Accumulated levels of some DNA repair proteins can be deleterious, and their activity must be restricted to regions of DNA damage. Translesion DNA polymerases are other examples of induced proteins that are rapidly degraded in vivo
. D. deserti encodes functional translesion DNA polymerases that are induced upon DNA damage , and their activity should be strictly controlled to prevent elevated levels of mutagenesis. Remarkably, the regulation of the dipeptidyl-aminopeptidase/acylaminoacyl-peptidase (Deide_02310) could explain the heterogeneity observed for the GyrA/B subunits.
Besides the already known proteins related to DNA-repair or metabolic changes described above, we detected the presence of several novel proteins that probably fulfill key roles in the radiation responses in D. deserti. Deide_20140 presents some far-related similarities with MshD (E value: 3.01e-09), the acetyl-transferase that catalyzes the final step of mycothiol biosynthesis in various members of the Actinomycetes. Mycothiol replaces glutathione in these species. Glutathione is a well-known antioxidant that helps protecting cells from reactive oxygen species such as free radicals and peroxides. Together with other antioxidants, such as Mn2+ complexes [5, 22], the Deide_20140 acetyl-transferase in D. deserti could contribute to tolerance to oxidative stress, which is generated by ionizing radiation-induced water radiolysis . Oxidative stress may also occur during dehydratation as described by Fredrickson and co-workers . Interestingly, we observed the up-regulation at the earliest stage after irradiation of the Deide_19260 protein (COG3947) that shows strong similarities with response regulators from two-component systems. COG3947 members usually comprise two structural domains: i) at their N-terminus, a CheY-like receiver with a phosphoacceptor site (Asp52 in Deide_19260) that is phosphorylated by histidine kinase homologs and ii) a DNA-binding transcriptional activator of the SARP family at their C-terminus. We tried to check the phosphorylation status of the CheY domain, but a peptide covering residue 52 was not detected in our experiments. Such aspartate-phosphorylation should be stable enough to be detected in our experimental conditions as previously found [57, 58].
It is worth to note that this putative two-component regulator is highly conserved in all radiotolerant Deinococcus species (70–84% identity) and in the thermophilic and radiotolerant Truepera radiovictrix (41% identity). However, the genetic context for Deide_19260 and its homologs is not conserved. None of the genes flanking Deide_19260 or its homologs encodes a histidine kinase in these species. The putative cognate histidine kinase for Deide_19260 is unknown. The Deide_19260 protein could be an important regulator for stress and/or DNA-damage response in D. deserti, besides the previously identified IrrE transcriptional activator. It will be of interest to study the targets of the SARP regulator. However, in contrast to the radiation-sensitive irrE mutant , deletion of Deide_19260 did not result in loss of radiation tolerance (data not shown). This could mean that Deide_19260 is not involved in radiation tolerance or that its function is redundant with another response regulator. The Deide_21840 up-regulated protein is related to PilT (COG2805), a nucleotide binding protein responsible for the retraction of type IV pili, likely by pili disassembly. This retraction provides the force required for travel of bacteria in low water environments. This protein is also required for DNA uptake in several bacteria . Three proposed roles for DNA uptake are genetic transformation, DNA repair, and to provide a source of nutrient . PilT may thus be an important element for survival at the population level of D. deserti upon adverse conditions. Another up-regulated protein, Deide_02842, presents far-related similarities with BglI restriction enzyme of Bacillus atrophaeus, previously known as Bacillus globigii
[61, 62]. We can predict that, like BglI, Deide_02842 is also a type-II site-specific ribonuclease, i.e. it cleaves specifically within or close to the recognition sequence in DNA. The Deide_02841 gene predicted to encode a DNA methylase is adjacent to Deide_02842. DNA methylation by this enzyme would protect D. deserti’s own DNA from cleavage by Deide_02842. Homologs of both genes are absent from other sequenced Deinococcus species. D. deserti may have acquired these genes by horizontal gene transfer, as suggested by their low GC percentage (46 and 50% versus an average of 63% for the total genome) and the nearby located transposase and integrase genes. Such restriction enzyme and methylase duo is known to be involved in the protection of bacterial cells by limiting incorporation of incoming foreign DNA, such as from bacteriophages, into the host genome. The Deide_13740 up-regulated protein (FtsY signal recognition particle GTPase) constitutes a universally conserved protein targeting pathway that ensures the co-translational delivery of substrates to the membrane-bound Sec translocon . Logically, the delivery of proteins synthesized in the cytosol to their correct cellular compartment is of utmost importance for the cell following gamma-irradiation. Deide_14090 shows strong sequence similarities with the ArgK kinase that phosphorylates periplasmic binding proteins involved in the LAO (lysine, arginine, ornithine)/AO transport systems .
To investigate if the proteins identified in this study may be up-regulated by a common mechanism, the upstream regions of the corresponding 22 genes were analyzed using MEME . A 17-bp palindromic motif was found upstream of 11 genes: gyrA, gyrB, ssb, pprA, ddrB, ddrD, uvrD, recA
P3 and Deide_02842 (Type II restriction enzyme). This motif corresponds to the radiation/desiccation response motif (RDRM) first identified after analysis of radiation-induced genes in D. radiodurans and D. geothermalis
. In a previous study we scanned the entire genome of D. deserti with this motif and a match with the RDRM was found in the upstream region of 25 genes, including 10 of the 11 genes mentioned above . Here, using another method (MEME), Deide_02842 is found as a new potential member of the RDRM regulon [4, 36]. It has been shown that radiation-induced transcription of at least some of these RDRM-containing genes is dependent on PprI (IrrE) [30, 31, 38]. Besides the RDRM upstream of 11 genes, no other motifs were found for the up-regulated proteins identified here, and transcription of the other 11 genes may be regulated in different manners. Alternatively, accumulation of the corresponding proteins may not be related to up-regulated transcription but rather to an increase of their translation and/or to protein-protein or protein-DNA interactions that increase their stability.
We have shown that DdrB and SSB are clearly accumulating in large quantities in the very earliest stages after irradiation, at least in our experimental conditions. These two single-stranded DNA binding proteins are probably of high importance to protect ssDNA that is formed after massive DNA damage and/or to initiate genome reconstitution by recruitment of other DNA repair proteins. That they are both up-regulated at the same early stage may indicate that they work concomitantly. Interestingly, Xu et al.  have recently shown that both proteins interact in vitro. Another recent study reported that DdrB is involved in DNA repair through a single-strand annealing (SSA) process that precedes the Extended Synthesis-Dependent Strand Annealing (ESDSA) . Finally, the specific post-translational modifications of GyrA detected from irradiated samples raises the importance of post-translational modifications of the Deinococcus proteome upon DNA damages. Whether the resulting heterogeneity impacts the cellular response is an open question.