Desiccation-induced changes in cell viability.
The viability of Bradyrhizobium japonicum cells under hydrated and desiccating conditions was assessed by plate count analysis, following incubation periods of 4 and 72 h (Fig. ), and by direct microscopic analyses using BacLight after 4, 24 and 72 h of incubation (Fig. ). Cell viability, as visualized by direct microscopic observation, decreased most significantly after 24 h and thereafter remained relatively stable. Live/dead counts of cells incubated for 72 h showed a 49% survival ratio of desiccated cells relative to hydrated cells. In contrast, plate count analysis showed only 17% survival of desiccated cells after 72 h of incubation at 27% RH, suggesting the presence of a large number of viable-but-nonculturable (VBNC) cells.
FIG. 1. Viability of B. japonicum cells following incubation under desiccating (27% RH) and hydrated (100% RH) conditions. (A) Plate count data expressed as log10 CFU/ml for desiccated and hydrated cells at the 4- and 72-h incubation times. Error (more ...) Genome-wide transcriptional analyses of desiccated cells.
Microarray analyses were done to assess the global transcriptional responses of B. japonicum to desiccation stress. In order to specifically isolate transcriptional responses to desiccation, as opposed to genes involved in more general stress responses of starvation or transition to stationary phase, filtered B. japonicum cells incubated under severe desiccating conditions (27% RH) were compared to filtered cells concomitantly incubated under hydrated (control) conditions (100% RH). RNA from cells incubated under both conditions was extracted from cells following incubation for 4, 24, and 72 h.
Microarray analyses indicated that of the 8,480 B. japonicum
ORFs analyzed, 200 (636), 1,102 (1, 947), and 987 (2,206) genes were differentially expressed more than 2-fold (or 1.5-fold, as indicated in parentheses) in the 4-, 24-, and 72-h incubations, respectively (Fig. ). This represented approximately 2% (7%), 13% (23%), and 12% (26%) of the coding sequences analyzed. One hundred three (225) genes were universally up-regulated at all three incubation times, and an additional 16 (43
) and 262 (403) up-regulated genes were common to the 4/24-h and 24/72-h incubation times, respectively. Conversely, 28 (40), 182 (316), and 322 (516) genes were uniquely up-regulated in the three individual incubation periods. Commonly up-regulated expressed genes in the 24/72-h samples were referred to as late-induced genes, whereas those from the 4-h samples were termed early-induced genes. A substantially smaller number of common genes were down-regulated in samples from the three incubation times. Four (35) of these genes were universal to all three time points, and 30 (130) and 62 (195) genes were mutual to the 4/24- and 24/72-h incubation periods, respectively. In contrast, 11 (138), 443 (600), and 226 (762) genes were unique to the 4-, 24-, and 72-h incubation times, respectively.
FIG. 2. Venn diagrams showing statistically significant (α = 0.05) up- and down-regulated genes in the desiccated versus hydrated treatments from B. japonicum microarray analyses. Values shown represent more than 2- and 1.5-fold (in parentheses) (more ...)
Categories of genes that were significantly and differentially up- and down-regulated in response to desiccation, in the early and late periods, could be divided into 16 functional groups (Fig. ). The greatest number of induced and repressed genes encoded hypothetical proteins; thus, their specific physiological function could not be inferred. However, a large number of genes encoding transcriptional regulatory proteins and transposases and proteins involved in metabolism, transport, catabolism, and energy transfer were specifically induced and repressed in response to desiccation. In general, more late than early genes were induced following incubation under desiccating conditions (Fig. ). A complete list of genes significantly (α = 0.05) induced and repressed by incubation of cells under desiccated versus hydrated conditions is presented in Table S1 in the supplemental material.
FIG. 3. Functional categories of statistically significant (α = 0.05), differentially expressed genes. (A) Genes expressed only in the early (4-h) desiccation period. (B) Genes commonly expressed in the 24- and 72-h desiccation periods. Black (more ...) Functional roles of genes responding to desiccation stress.
To more precisely define desiccation-responsive processes, the microarray data sets were evaluated for genes meeting at least one of the following criteria: (i) there was simultaneous and differential expression of genes having similar physiological function, (ii) there was induction of multiple genes in a specific biochemical pathway, or (iii) induction of the same gene occurred in more than one time period and with an especially high level of expression (more than threefold). Ten categories of genes potentially involved in the tolerance of B. japonicum to desiccation were defined with these criteria (Table ). These included genes involved in sugar metabolism and transport, transcriptional regulation, oxidative stress and heat shock responses, modification and repair of nucleic acids, membrane modification, pyrroloquinoline synthesis, transport of cations, synthesis of pili and flagella, and formation of extracellular polysaccharides. Genes showing the greatest induction and meeting the above criteria encoded trehalose-6-phosphate synthetase (bll0322), a gluconolactone precursor protein (bll2817), an ABC transporter sugar binding protein (blr3200), nitrogen regulatory protein PII (blr0612), isocitrase lyase (blr2455), a cyclic AMP binding regulator (blr0536), a tryptophan-rich sensory protein (bll5190), σ54 (RpoN2) (blr0723), a σ54 modulation protein (blr0724), σ24 (RpoE), two-component response regulators (bll7795 and blr8144), an HspC2 (Hsp20) (blr4637), a probable DNA glycosylase protein (blr0857), an OpgC-like protein involved in the succinylation of osmoregulated periplasmic glucans (bll1464), putative alcohol dehydrogenases (bll0332 and bll0333), pyrroloquinoline quinone synthase protein A (bsr6735), pilus assembly proteins (bsl1442, bsl7141, bsl3118, bsl6587), flagellin (bll5845, and bll5846), a glycosyl transferase (bll2752), and a fasciclin I-like adhesin domain protein (bll5191).
Physiological processes and significant desiccation-induced genes in B. japonicum
Induction of genes involved in trehalose synthesis.
Desiccation stress has previously been shown to result in accumulation of the disaccharide trehalose in B. japonicum
). Consistent with this observation, we observed the induced transcription of genes encoding enzymes involved in two of the three B. japonicum
trehalose synthesis pathways in desiccated cells (Table ). Trehalose-6-phosphate synthase (bll0322) and trehalose 6-phosphate phosphatase (bll0323), participating in the OtsA/OtsB pathway in which glucose-6-phosphate and UDP glucose are transformed to trehalose, were significantly induced at all three incubation times assessed. Desiccation also resulted in the induction of trehalose synthase (blr6767) in the late period, which is responsible for transforming maltose to trehalose. A UTP-glucose-1-phosphate uridylyltransferase (blr1499) was induced 3.2- and 2.4-fold in the 24- and 72-h samples, respectively. This enzyme transforms UTP and α-d
-glucose 1-phosphate to produce UDP-glucose, a precursor for the OtsA/OtsB trehalose synthesis pathway. Interestingly, three other sugar metabolism-related ORFs (bll6764, bll6765, and bll6766), located upstream of the trehalose synthase gene (bll6767), were also significantly induced in both late desiccation samples. These genes encode a glycoside hydrolase, an amylomaltase-like 4-α-glucanotransferase, and an α-amylase (distantly related to trehalose synthase), respectively.
Real-time qRT-PCR was used to verify the induction of trehalose synthase genes observed in the microarray analyses. Results in Fig. show the standardized ratio of the induction of the three B. japonicum trehalose synthesis genes (otsA, treS, and treY) in the desiccated versus hydrated cells for the three incubation times. Consistent with the microarray analyses, the trehalose 6-P-synthase (otsA) and trehalose synthase (treS) genes were significantly induced in 24- and 72-h samples, with maximal otsA and treS expression at 24 and 72 h postdesiccation, respectively (Fig. ). In contrast to the microarray data, qRT-PCR also showed a significant induction of the treY gene, encoding MOTS (Fig. ). However, the absolute value of treY expression, under both desiccating and hydrating conditions, normalized to the housekeeping gene parA, was extremely small (below 0.0001); thus, the expression of TreY may play a lesser role in desiccation tolerance than do the other two trehalose synthase enzymes (Fig. ). The absolute normalized ratios of otsA/parA increased considerably in the late incubation period for both the hydrated and desiccated samples, reaching values exceeding 20-fold higher than those measured in the 4-h samples (Fig. ). This implies that although the induction of otsA was significantly higher in the desiccated samples, it was also substantially induced in the hydrated samples.
FIG. 4. qRT-PCR analysis of differentially expressed trehalose synthesis genes (otsA, treS, treY) in desiccated and hydrated B. japonicum cells normalized to the housekeeping gene parA. (A) Ratio of trehalose synthesis gene expression in desiccated versus hydrated (more ...) Biochemical analysis of trehalose synthase activity.
The activity of trehalose 6-P synthase (OtsA) and trehalose synthase (TreS) was measured in desiccated and hydrated cells from the 4- and 72-h incubation periods. The results, normalized for cell viability with the LIVE/DEAD assay, indicated that the specific activity of trehalose 6-P synthase for desiccated (27% RH), relative to hydrated (100% RH), cells was 79.5 ± 21.7 versus 52.7 ± 15.6 nmol/h/mg protein for the 4-h postincubation samples and 101.8 ± 43.3 versus 54.3 ± 9.2 nmol/h/mg protein for the 72-h postincubation samples. These values are in agreement with the microarray expression and qRT-PCR data, which showed significant induction of otsA and otsB when B. japonicum was incubated under desiccating conditions. In contrast, significant induction of TreS was not detected in desiccated cells relative to hydrated cells, where values of 44.3 ± 7.2 versus 41.1 ± 3.5 and 74.8 ± 4.3 versus 67.8 ± 0.5 nmol/h/mg protein were measured for the 4- and 72-h incubation times, respectively. It should be noted, however, that these values were normalized based on live/dead counts and not on viability plate counting; had the latter been done, the apparent activity of both enzymes would have significantly increased under conditions of desiccation.
NMR profiles of intercellular trehalose pools.
NMR analysis was done to determine if the induced transcription and activity of trehalose synthase genes, as seen in the transcriptional and biochemical assays, were consistent with an increase in the intracellular concentration of trehalose. Intracellular trehalose concentrations, based on the integrated value of the trehalose peak at 5.2 ppm, and the live/dead viability counts, were approximately 3.0 and 2.8 times greater in the desiccated samples, relative to the hydrated ones, following 24 and 72 h of incubation, respectively (Fig. ).
FIG. 5. Intracellular trehalose concentrations in desiccated and hydrated B. japonicum cells. Values are normalized based on cell viability determined by using LIVE/DEAD staining. Black bars, desiccated cells (27% RH); gray bars, hydrated cells (100% (more ...) Effect of rpoN mutations on desiccation tolerance.
The B. japonicum genome contains two σ54-encoding genes, rpoN1 and rpoN2. Microarray analyses of the desiccated versus hydrated samples revealed significant induction of rpoN2 under desiccating conditions (2.2-, 10.0-, and 13.9-fold induction at the 4-, 24-, and 72-h times, respectively), but there was no significant induction of rpoN1. Real-time qRT-PCR of cDNA extracted from cells incubated for 72 h using primers targeting both rpoN1 and rpoN2 showed a significant induction of rpoN2 (2.1-fold) in the desiccated versus hydrated cells, with no significant induction of rpoN1.
In order to assess the potential role of σ54
in the desiccation response, we compared the relative viability of rpoN1, rpoN2
, and rpoN1 rpoN2
) and wild-type B. japonicum
cells grown under desiccating and hydrating conditions (Fig. ). The viability of desiccated cells was significantly lower (P
< 0.001) in all three B. japonicum rpoN
mutants relative to the wild-type strain. In contrast, under hydrated conditions there was no significant difference (P
< 0.001) in cell viability between the wild-type and the rpoN1
mutant strains. However, under 100% RH conditions, the rpoN1 rpoN2
double mutant survived at a rate 8.5% lower than the mean survival rate of the rpoN1, rpoN2
, and wild-type strains (data not shown).
FIG. 6. Survival of B. japonicum rpoN mutant strains, relative to wild-type cells, following incubation under desiccating (27% RH) conditions. The significance of data was determined by comparison of means with ANOVA and Tukey-Kramer HSD. Columns followed (more ...) Influence of nonpermeating, solute-induced stress on the B. japonicum transcriptome.
Microarray analysis revealed that mild PEG-induced nonpermeating osmotic stress resulted in the differential expression of 184 and 43 up-regulated and 123 and 29 down-regulated genes, based on criteria of 1.5- and 2-fold induction, respectively. Of the 1.5-fold-induced genes, 30 were also induced in both early and later desiccated samples, and an additional 34 ORFs were upregulated only in the late (24- and 72-h) desiccated samples. Most notable were genes encoding trehalose 6-P-synthase (bll0322), an α-glucosidase (blr0901), potential EPS forming proteins (bll2362 and bll2752), transcriptional regulators (blr0536, bll7795, blr8144, and blr7797), a potential osmotic transport protein (blr7593), glutamine synthase II (blr4169), an organic hydroperoxide resistance protein (bll0735), and three pqq-associated alcohol dehydrogenases (bll0332, bll0333, and bll6220). Since many of the same proteins were produced under conditions of desiccation (Table ), similar mechanisms are likely involved in tolerance to reduced water potential imparted by both conditions of incubation. Interestingly, 11 of the genes specifically induced by mild PEG-mediated nonpermeating osmotic stress were localized to the B. japonicum symbiosis island and included the symbiosis-related genes nolB, nolT, nolU, nolV, nolW, rhcR, and rhcS. While relatively mild osmotic stress was imparted by the PEG concentration used in our studies, none of these genes were induced by desiccation stress. Reduced water activity also resulted in down-regulation of 16 flagellar genes and 8 heat shock proteins.