Drying tests, using DNA-containing natural xeroprotectants were performed as previously described (Sauer and Galinski, 1998; Narváez-Reinaldo et al., 2010). Osmotic shock was applied by addition of 5 or 50% (wt/vol) polyethylene-glycol (PEG) 6000 to the media. In addition, xeroprotectant solutions were prepared synthetically by mixing commercial chemical products at the same molar ratios as their natural counterparts: fructose, glutamic acid, acetate, β-hydroxybutyrate, and lactate at a molar ratio of 16:4:1:0.8:1.4 for S4J2A2-S, and glucose, glutamine, glutamic acid, oxoglucuronic acid, and β-hydroxybutyrate at a molar ratio of 6.8:4:2:1.3:1 for S4J27-D.

Lipase from Burkholderia cepacia was purchased from Sigma-Aldrich (62309) and Src-SH3 was purified according to Cámara-Artigas et al. (2009) to 99% purity. For this purpose pET15b (a plasmid-encoded Src-SH3 domain) was expressed in Escherichia coli strain BL21. This domain, which contains an N-terminal 6His tag, was engineered with a thrombin cleavage site. Protein purity and concentration were determined as described previously in Cámara-Artigas et al. (2009). Plasmid pET15b containing the chicken Src-SH3 domain gene was a generous gift from Dr. E. Freire (Johns Hopkins University). Samples were prepared with a protein concentration of approximately 900 μM (2.0 mg/mL) by extensive dialysis against a large volume of 40 mM Hepes buffer at pH 7.0. Then 500 μL were mixed with an equal volume of water, trehalose 0.876 M (30% wt/vol), oligonucleotide 60 μM (1500 ng/μL) or glucose (equimolar with respect to trehalose 30%), each sample was then split in two aliquots; one was dried as described below and the other was kept as a positive control for DSC experiments. The protein concentration was then determined by reading absorbance at 280 nm. The molecular weight of Src-SH3 was 7000 Da and its extinction coefficient was 16,500 cm/M.

For the lipase assays, proteins were dried and rehydrated according to the method described by Narváez-Reinaldo et al. (2010). For DSC assays, the Src-SH3 protein fragment was dried as described for lipase but an additional 5-min incubation period at 100°C was included to remove the remaining water. Dry samples were kept dry overnight at 37°C in a desiccator. Then Hepes DSC buffer (20 mM, pH 7.0) was added to resuspend the samples, which were then used for the DSC measurements. Protein integrity after drying was measured as the UV-visible absorbance spectrum, and protein concentration was then calculated from absorbance at 280 nm, as described above.

For the removal of the DNA, DNAse I (2 units) from New England Biolabs (M3303S) and 1 μL MgCl₂ 25 mM were added to xeroprotectant samples containing DNA in the presence of the recommended buffer, and the mixtures were incubated at 37°C for 180 min. To stop the reaction 0.5 μL of EDTA (0.5 M) was added, followed by incubation at 75°C for 10 min.

To remove RNA, 73 μM RNAse free from DNAse and protease activity was added to 1.5 mg of each xeroprotectant preparation, and the mixtures were incubated at 37°C for 2 h (Thermo, EN0531). The efficiency of both treatments was validated by gel electrophoresis as described below.

Natural DNA from the different microorganisms was obtained reported elsewhere (Kado and Liu, 1981) based on phenol:chloroform:isoamyl alcohol extraction and precipitation with sodium acetate and alcohol. The quality of extraction was evaluated by gel electrophoresis in agarose gels (0.75% wt/vol) with Tris acetate-EDTA buffer. Nucleic acids were stained with ethidium bromide or GelRed™ and visualized with UV light. In addition, fish DNA was purchased from Sigma-Aldrich (74782). Synthetically produced 81-mer DNA was provided by Sigma at a 1-μmole scale, as desalted oligonucleotides purified by high-performance liquid chromatography.

Total RNA for Illumina sequencing was isolated from Microbacterium sp. 3J1 grown under osmotic shock by the addition of 5 or 50% (wt/vol) PEG 6000 to TSB (tryptic soy broth) growth media or control conditions using RNeasy Mini Kit (Qiagen, Germany). Following manufactured recommendations, polyethylene-glycol was not autoclave-sterilized, but absence of microbial contamination was analyzed by plating PEG drops in TSA-rich medium. In addition, serial dilutions of the different cultures were plated to confirm the absence of potential microbial contaminations in the cultures. DNA was removed by DNAses and rRNA was depleted by using specific probes for 16S and 23S rRNA. The quantity and quality of the total RNA were assessed using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA) and an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). The cDNA library was constructed and sequenced by the Life Sequencing Corporation (Valencia, Spain) following the TruSeq RNA Sample Preparation Kit. RNAseq libraries and were sequenced using a Illumina HiSeq^TM 2500 in combination with 50SR, rendering between 0.79 and 1.29 Gb information per sample.

To investigate whether the overproduced DNA was due to bacterial lysis, we used PMAxx dye (Biotium, Inc., Hayward, CA, USA). The technology is based on sample treatment with the photoactivatable, and cell membrane impermeant, nucleic acid intercalating PMAxx, a new and improved version of dye PMA^TM (propidium monoazide) followed by light exposure prior to extraction of DNA and amplification. Light activation of DNA-bound dye molecules results in irreversible DNA modification and subsequent inhibition of its amplification. To that end 4 μM of PMAxx were added to cultures of Microbacterium sp. 3J1 in late logarithmic phase (Abs_{660 nm} of 1.5) in TBS in the absence and presence of 50% (wt/vol) PEG. Following an incubation period of 10 min in the dark with occasional mixing, aliquot fractions were then illuminated with white light for 1 h to covalently modify the DNA in the medium sample from cells with compromised membranes. After photoinduced cross-linking, cells were pelleted at 8,000 g for 10 min prior to DNA isolation using DNeasy Blood & Tissue Kit (Qiagen).

At those times, serial dilutions of each culture were plated in TSA to estimate the cell number. The degree of cell lysis due to the addition of 50% PEG was determined by the amplification of the hypervariable V3 region of bacterial 16S rRNA gene using qPCR as described below with same amount of DNA as template.

For RT-PCR, total RNA was isolated using the TRIzol kit (Invitrogen) and then extracted using chloroform. Total RNA was cleaned up using RNeasy Mini Kit (Qiagen), and DNase was digested as recommended by the manufacturer. The RNA quantity was determined with a Nanodrop spectrophotometer (ND-1000; Thermo Fisher Scientific). Approximately 1 μg RNA was used for cDNA synthesis with SuperScript III First-Strand Synthesis System (Invitrogen). For priming, samples were incubated at 25°C for 10 min, followed by reverse transcription at 50°C for 50 min and inactivated at 85°C for 5 min. Once purified, cDNA was stored at –20°C till used. The RT-qPCR were performed in a LightCycler^® Nano Instrument (Roche Life Science) using FastStart SYBR Green Master (Roche Life Science). Quantitative-PCR were performed using 2 μg of the cDNA. Thermocycler program consisted in 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 62–64°C for 20 s, and 72°C for 15 s. After thermal cycles, the dissociation analysis (melting curve) was carried out to confirm specific amplification of PCR. All reactions were performed in triplicate with three biological replicates, including four different standard concentrations of cDNA to generate standard curves. Negative controls (deionized water instead of cDNA) were included in each run. The design and the specificity test of the oligonucleotides used in q-PCR assays were performed by using the NCBI-Primer BLAST tool (Primer-BLAST, RRID:SCR_003095). Primers 341F and 534R were used to amplify the hypervariable V3 region of the bacterial 16S rRNA gene. The list and sequence of oligonucleotides used in this study is depicted in Table 1.

The heat capacities of all samples were measured as a function of temperature using a high-precision differential scanning VP-DSC MicroCalorimeter (Microcal, Inc.). All DSC experiments were conducted at a scan rate of 1.5 K/min and a protein concentration of approximately 1.0 mg/mL. Thermal denaturation scans were recorded from 5 to 100°C. After normalization for protein concentration, the DSC thermograms were systematically corrected for the calorimeter time response as well as for instrumental baseline. Desiccated Src-SH3 samples were cooled inside the calorimeter and reheated to check the reversibility of the unfolding process under each experimental condition.

The results of each experiment correspond to at least three independent experiments. The statistical test used was to one-way ANOVA with Tukey’s test (Montgomery, 1999) in Figure 2, and Student’s t-test in Figures 4, 5, and 7 (Montgomery, 1999).

Naturally synthesized xeroprotectants produced and accumulated during water-stress by addition of 50% PEG and released after osmotic down-shock by the desiccation-tolerant bacterial strain Microbacterium sp. 3J1 were obtained as described in Section “Natural and Synthetic Xeroprotectants.” Then these naturally synthesized xeroprotectants were subjected to agarose gel electrophoresis to identify the possible presence of the biopolymer as described in Section “Nucleic Acid Purification and Electrophoresis” of Material and Methods. Several nucleic acid bands were found in agarose gels stained with ethidium bromide and GelRed™ (two intercalating agents in the nucleic acids), and the concentration of these nucleic acids increased with exposure time of the bacteria to desiccation. Maximum DNA production was reached in the late exponential phase (Figure 1A). Similar results were found when Arthrobacter siccitolerans 4J27 was used (data not shown). To analyze whether the DNA found after osmotic downshock was the result of bacterial lysis or whether it was newly synthesized in response to water stress, we added PMAxx to cultures of Microbacterium sp. 3J1, A. siccitolerans 4J27, and Pseudomonas putida KT2440 (the two first strains as desiccation tolerant strains and the latest as desiccation sensitive one) in presence of 50% PEG, as described in Section “PMAxx Labeling, RNA Extraction, cDNA Synthesis (RT-PCR), and Quantitative PCR (qPCR).” Then qPCR of the hypervariable V3 region of bacterial 16S rRNA gene was performed using 100 ng as a template to quantify the relative concentration of DNA under each condition. No statistical differences were found in the qPCR of the 16S rRNA gene of Microbacterium sp. 3J1 cultured with 50% PEG despite the addition of PMAxx showing that all DNA used as a template had an intracellular origin (Figure 1B). However, significant differences were found when P. putida was used, indicating that this microorganism is lysed upon addition of 50% PEG. Similar results to those presented by Microbacterium sp. 3J1 were found when A. siccitolerans 4J27 was used instead (data not shown). To determine whether the possible presence of DNA or RNA in the sample affected the protective effect of the mixture, naturally produced mixtures extracted from A. siccitolerans 4J27 (termed N4J27-S, Table 2) were treated as described in Section “RNAse and DNAse I Treatment” with RNAse and DNAse I (Santacruz-Calvo et al., 2013; Manzanera et al., 2014). Xeroprotectants, whether treated or not treated with the RNAse, showed a protective effect in lipase protein. Nonetheless, N4J27-S xeroprotectant treated with DNAse I showed a reduced protective effect on the protein. This result showed that the presence of DNAse I (8.5 μM) reduced the ability of a 10% (w/v) N4J27-S xeroprotectant to protect lipase from drying, whereas the addition of similar concentration of RNAse A (Thermo) did not affect the protective capacity of this xeroprotectant (Figure 2). As a means of ruling out a possible scavenger interaction of the protective molecules with an extra (added) protein, a sample containing bovine serum albumin (BSA) 8.5 μM as a mock protein was added to the same xeroprotectant. No significant effects were observed.

The addition of DNAse I to xeroprotectant N4J27-S (consisting of glucose, glutamine, glutamic acid, oxoglucuronic acid, and β-hydroxybutyrate at a proportion of 6.8:4:2:1.3:1), when tested with lipase, caused browning that was not observed when BSA was added instead of the nucleases. This result, and the fact that synthetic xeroprotectants triggered a darker browning effect than did the natural xeroprotectants, prompted us to test whether DNA reduced the browning caused by Maillard reactions. As the results of Maillard reactions, Amadori products (with brown color detectable at a wavelength of 410 nm) may be synthesized. Amadori products are intermediates in the production of an advance glycation end-product and one of the more damaging effect in desiccation sensitive organisms when exposed to dehydration. To detect if Amadori products were reduced by the presence of DNA, a mixture of glucose (0.27 M) and glutamine (0.34 M) (termed GlucGln here) was subjected to the drying protocol as described by Koehler et al. (1969) and by Hwang et al. (1993) in presence of different concentrations of DNA. The resulting Maillard reaction product was detectable by absorbance at 410 nm (Kwak and Lim, 2004). The addition of increasing concentrations (150; 1500; 15,000, and 23,000 ng/μL) of fish DNA led to decreases in absorbance (first evident at a concentration of 1500 ng/μL) that correlated with increasing DNA concentration (Figure 3).

To determine whether the addition of DNA to the synthetic xeroprotectant increased its protective action, we recorded this effect after adding different types of DNA. For this purpose, DNA extracted from the desiccation-sensitive bacteria P. putida KT2440 and from a eukaryotic organism (fish sperm) (800 ng/μL) was added to a synthetic mixture termed S4J27-S, designed to reproduce the effects of xeroprotectant N4J27-S and consisting of the commercial chemicals glucose, glutamine, glutamic acid, oxoglucuronic acid and β-hydroxybutyrate (6.8:4:2:1.3:1) (Narváez-Reinaldo et al., 2010). The protective effect against drying in lipase protein increased significantly, regardless of the source of the DNA molecule (Figure 4).

To rule out a potential effect of accompanying proteins or molecules co-purified during nucleic acid extraction, and to determine whether protection occurred with smaller DNA molecules, we tested the effect of synthetically produced DNA. Increasing concentrations of 81-mer oligonucleotides were added to 2.35 moles of lipase subjected to the desiccation protocol as described in Section “Drying Experiments.” The addition of 30 μM (750 ng/μL) of the oligonucleotide to the synthetic xeroprotectants S4J27-S and S4J2A2-S (the synthetic mixture designed to reproduce the xeroprotectant synthesized by the desiccation-tolerant microorganism Rhodococcus sp. strain 4J2A2 composed of fructose, glutamic acid, acetate, β-hydroxybutyrate and lactate at a ratio of 16:4:1:0.8:1.4) (Narváez-Reinaldo et al., 2010; Manzanera et al., 2015) led to a significant increase in protection of the lipase enzyme (Figure 5).

To test whether the protective effect also occurred in other types of proteins, we used differential scanning calorimetry (DSC) as described in Section “Differential Scanning Calorimetry” to test desiccation with the SRC homology 3 domain (Src-SH3) in the presence of oligonucleotides. Src-SH3 is a small, well-characterized protein domain first identified as a conserved sequence in the viral adaptor protein v-Crk and the non-catalytic parts of tyrosine kinases such as Src. Approximately 300 different SH3 domains are found in proteins encoded in the human genome, and they have been used as a model protein for DSC assays. Src-SH3 is characterized by fully reversible two-state thermal unfolding transitions at acidic pH values (Martinez et al., 1998; Bauer et al., 2005). The Src-SH3 protein also shows reversible two-state thermal unfolding transition at neutral pH values similar to the conditions under which lipase-drying experiments were performed. In the absence of the xeroprotectant, desiccation led to the loss of protein structure and its ability to renature, as indicated by a reduction of more than 70% in the area under the DSC transition peak (Figure 6). When desiccation occurred in the presence of trehalose (0.438 M) or oligonucleotides (30 μM), the unfolding transition was fully preserved albeit shifted to higher temperatures (ΔTm = 16°C), indicating a strong stabilizing effect of folded structures able to denature cooperatively. Whether these structures correspond to the fully folded native state, alternative conformations or aggregates remains to be determined by additional experiments beyond of the scope of this work. Reversibility of all transitions was high and similar to that of the wild-type SH3 in solution, indicating that these structures recover after thermal denaturation. The DNA xeroprotectant effect seems to be related to the desiccation procedure, since the effects of DNA addition on the DSC profile of the non-desiccated SH3 domain were negligible (i.e., much smaller than those induced by trehalose). Specifically, the addition of trehalose in assays with non-desiccated SH3 appeared to have a clear stabilizing effect on the SH3 native state in solution, as indicated by a ΔTm value of 3°C with respect to the reference sample. However, the addition of DNA in assays with non-desiccated SH3 had a negligible effect in solution, as indicated by a ΔTm of 1°C, which was within the range of expected experimental error.

To determine whether DNA was synthesized in response to water stress, Illumina HiSeq 2500 was used to sequence cDNA libraries from Microbacterium sp. 3J1 in response to mild and stringent changes in water activity by adding 5 and 50% (wt/vol) PEG, respectively, to growth media as described in Section “Transcriptional Quantification by RNAseq.” The relative quantification of mRNAs produced in response to water deficit showed induction of genes involved in the production of DNA polymerases, different types of DNA helicase, DNA gyrases, and different DNA topoisomerase homologs when slight changes in water activity were induced by adding 5% (wt/vol) PEG (Table 3). More stringent conditions achieved by increasing the PEG concentration from 5 to 50% to simulate drier environments led to an even higher upregulation of genes involved in the production of DNA polymerases, gyrases, and topoisomerases, i.e., 3.20-, 2.41-, and 3.70-fold, respectively, resulting in further increasing DNA production.

Apart from genes involved in DNA production under desiccating conditions, we observed the induction of genes involved in the uptake of exogenous DNA (e.g., genes coding for ComEC/Rec2 proteins) as described by Claverys et al. (2006). In addition, we found upregulation of the gene coding for the PF 06013 protein, which is related to a possible secretion system for DNA translocation in Gram-positive bacteria, and is also related by homology with the ESET-6 or YukA superfamily of proteins (Pallen, 2002). These proteins may comprise a type-IV secretion system used by many bacteria to deliver genetic material to the host cell (Christie, 2001). This secretion system must use ATPases and chaperones simultaneously to maintain the flux of molecules through their exchange channels (Christie, 2001). In this connection we observed the upregulation of ATPases and chaperone genes under desiccating conditions with RNAseq in Microbacterium sp. 3J1 (Table 3). Also, we found an increase in the transcription of genes coding for proteins involved in DNA recombination and repair, e.g., genes that code for TIGR02012, RmuC recombination factor, PF02559.8 and TIGR00416. Increased transcription was also seen for genes coding for integrase (PF02899.9), a primase (TIGR01613), proteins involved in DNA replication, repair and recombination (TIGR00611) or DNA metabolism, and proteins involved in chromosome recombination and structural maintenance (PF02463). In addition, we observed increased transcription of genes that code for proteins involved in chromosomal and plasmid partition (TIGR00180) (see Table 3).

Quantitative PCR (qPCR) was used as described in Section “PMAxx Labeling, RNA Extraction, cDNA Synthesis (RT-PCR), and Quantitative PCR (qPCR)” to characterize the rise in transcription levels of genes involved in the synthesis of new DNA and the uptake of exogenous DNA such as the genes that code for DNA polymerase, and the genes coding for ComEC/Rec2 proteins. Transcription levels of these genes were upregulated by 2 (Figure 7A) and 5 times (Figure 7B) respectively when cells were exposed to 50% PEG, compared to cells grown in TSB media supplemented with 5% PEG in Microbacterium sp. 3J1. However, no statistical difference was found in the expression of the gene coding for the DNA polymerase of the desiccation sensitive microorganism P. putida KT2440 (Figure 7C).