Characterization of B. megatarium FDU301
A strain of B. megatarium tolerant to arid condition (15% PEG200 (w/w), aw 0.985) was isolated from plaque area on the surface of a leaflet in an old book, and was named as FDU301. The sequence of its 16s rDNA gene was identical to that of B. megaterium NBRC15308 and B. megaterium QMB1551 (data not shown). The full genome of B. megaterium FDU301 has been sequenced and the data can be found in NCBI GenBank (accession numbers CP045267-CP045276).
As shown in Fig. 1a, FDU301 showed a typical "S" type growth curve in normal LB medium, with a short incubation period of 2 h, and reaching the plateau around 10 h. In the presence of 5% PEG200, the FDU301 grew faster and reached higher cell density than that in normal LB medium. As the concentration of PEG200 increased, the growth of bacteria slowed down and reached much lower cell density than that in normal LB medium. The bacteria hardly grew in the medium with 20% PEG200, indicating the limit of the strain to tolerate. Compared to B. megaterium NBRC15308, B. megaterium FDU301 grew much better in the arid medium (15% PEG200 (w/w)) (Fig. 1b).
Global overview of the RNA-Seq data
The transcriptome of B. megaterium FDU301 in the growth phase (4 h) under control (LB medium, L) and simulated arid condition (LB medium with 15% PEG200, P) was analyzed with RNA-seq. The RNA-seq data have been submitted to NCBI SRA (accession numbers PRJNA649685). After filtration, a total of 51,893,124 and 61,892,804 reads were obtained from L and P samples, respectively. For both samples more than 95% of the reads were mapped to the B. megaterium FDU301 reference genome (Table 1). In order to verify the transcriptomic results, ten differentially expressed genes (DEGs) were randomly selected and their transcriptional level were determined with real-time quantitative PCR (RT-qPCR). The results of RNA-seq and real-time quantitative PCR were generally consistent with each other, indicating that the transcriptomic results reflected the differences in gene expression under the arid and normal conditions (Additional file 1: Fig. S1).
As shown in Fig. 2a, the correlation between the three biological replicates of each sample (L and P) was high, indicating that the sequencing data was highly reproducible. Meanwhile the difference between treatment groups was obvious. Two groups were also well separated from the other in the result of principal component analysis (Fig. 2b). These showed that arid stress had a significant effect on the gene expression of FDU301.
The volcano map of DEGs was shown in Fig. 2c. Compared with the control group, the expression levels of 2941 genes were significantly different under the simulated arid conditions (FDR<0.05 & |log2FC|≥1), of which 1422 were up-regulated and 1519 were down-regulated (Additional file 2: Table S1).
Annotation analysis of DEGs
The 2941 DEGs (FDR<0.05 & |log2FC|≥1) were annotated with COG and KEGG database. According to COG annotation, DEGs were seen in most of the COG categories, which meant that the response of FDU301 to the arid stress was a complicated process (Additional file 3: Table S2). As shown in Fig. 3, the category with the highest proportion of up-regulated genes was inorganic ion transport and metabolism (P, about 41.56%). In terms of down-regulated genes, categories with more than 30% genes down-regulated included carbohydrate transport and metabolism (G); translation, ribosomal structure and biogenesis (J); energy production and conversion (C); lipid transport and metabolism (I); and amino acid transport and metabolism (E). These results suggested FDU301 had general suppressions in metabolism and protein production, and enhancement in the transport for inorganic ion, such as Fe, Zn, Ni, in face to the arid stress.
KEGG annotation showed that, the arid stress significantly up-regulated the transcription of genes associated with ABC transporters (Additional file 4: Table S3), and significantly down-regulated that of oxidative phosphorylation and glycolysis pathways (Additional file 5: Table S4).
Major changes in gene expression under arid stress
Selected genes with significant changes in transcription under simulated arid (15% PEG200 (w/w)) and normal conditions were further analyzed with RT-qPCR.
Oxidative stress-responsive genes. PerR is a key regulatory protein for oxidative stress response in Bacillus spp. (29). As shown in Fig. 4A, perR was up-regulated under arid condition. Several genes known to be regulated by perR were also upregulated significantly, including fur, dps, and katE (Fig. 4a). Fur encodes a major suppressor for the expression of many ferrous uptake operons, whereas dps and katE are related to avoiding DNA damage and removing ROS, respectively. These results were consistent with the ROS analysis of FDU301 cells grown in the medium with different concentration of PEG200 (Additional file 6: Fig. S2), suggesting that oxidative stress was one of the main challenges for the bacteria in the simulated arid condition.
Fe2+ transportation genes. Under oxidative stress, Fe2+ will react with H2O2 through fenton reaction to form hydroperoxide, which was highly active in destroying DNA. Up-regulation of many Fe2+ transportation-related genes were seen in B. megaterium FDU301 under arid condition. In fact, Fur is a suppressor for Fe2+ uptake, and Dps functions by binding Fe2+ and reduces the level of free Fe2+ in the cell. Meanwhile, the Fe2+ uptake gene feoB (30), was also found to be greatly upregulated in arid condition (Fig. 4a), suggesting a delicate balance of Fe2+.
Ectoine biosynthesis genes. The ectB and ectA were significant up-regulated by about 23.10-fold and 8.40-fold, respectively in arid condition (Fig. 4b). The two genes were involved in the biosynthesis of compatible solute ectoine (31). Meanwhile, genes related to the transportation and biosynthesis of another commonly used compatible solute, glycine-betaine, were not significantly changed, or even down-regulated (Table 2, Additional file 2: Table S1).
Spore formation genes. Under the arid condition, genes related to spore formation stage II (spoIIB, spoIIE, spoIIGA) were up-regulated by about 8.57 to 29.24-fold. SspD, the gene encoding small acid-soluble spore proteins (SASP), which is a major protective component of Bacillus spores, were also found to be highly expressed (Fig. 4c).
TipA gene. Under the simulated arid condition, tipA was one of the most dramatically up-regulated gene, in terms of the fold of change in the transcription level (Fig. 4d). TipA had not been noticed in previous studies on bacterial arid tolerance.
Respiratory and glycolysis genes. As shown in Fig. 4e, under the arid condition, genes related to oxidative phosphorylation (atpB, atpE, atpF, atpH, atpA, atpG, atpD, atpC) were down-regulated to various degrees. Meanwhile, some glycolysis-related genes (pgk, tpiA, frmA) were also down-regulated in arid condition (Fig. 4f). This might reflect the slow growing status of bacteria in arid condition.