Abstract :
[en] Microorganisms are often affected by various environmental factors.
These environmental conditions have an effect on their physiological and biochemical functions. Among these environmental factors, temperature plays an important role in the normal physiological behaviors of microorganisms. To adapt to different temperature environments, bacteria have evolved a variety of adaptive mechanisms to coordinate a range of gene expression and protein activity changes. In this study, we investigated the adaptation mechanisms of two Gram-positive bacteria at different temperatures. The main results of this thesis are as follows:
(1) Deinococcus radiodurans is a gram-positive, pink-pigmented, and high G+C bacterium. The heat response of D. radiodurans is considered to be a classical stress-induced regulatory system characterized by extensive transcriptional reprogramming. In this part, we investigated the key functional genes involved in heat stress that were expressed and accumulated in cells following heat treatment at 48°C for 2 hours (R48). Considering that protein degradation is a time-consuming process, we predicted that in order to maintain cellular homeostasis, the expression of the key functional proteins would be significantly decreased in cells that had partly recovered from heat stress (RH) relative to their expression in cells grown under optimal temperature (R30). Comparative transcriptomics identified fifteen genes that were significantly downregulated in RH relative to R30, seven of which were previously characterized as heat shock proteins. Among these candidates, three genes (dr_0127, dr_1083, and dr_1325) are more likely involved in response to heat stress as survival analysis of mutant strains lacking dr_0127, dr_1325, and dr_1083 showed a reduction in heat tolerance compared to the wild-type strain.
Based on our RNA-seq results and previous studies, we identified two novel heat-inducible ncRNAs in D. radiodurans, named DnrH and dsr11. Heat tolerance analysis showed that deleting DnrH significantly inhibited viability in response to high temperature conditions. Comparative phenotypic and qRT-PCR analyses of a DnrH mutant (∆DnrH) and wild-type (WT) suggested that DnrH is potentially involved in regulating the expression of the heat shock-related gene Hsp20. Microscale thermophoresis and genetic complementation showed that a 28-nucleotide (nt) sequence in the stem-loop structure of DnrH (143–170 nt) pairs with its counterpart in the coding region of Hsp20 mRNA (91–117 nt) via a 22 nt region. In vivo, mutation of the 22-nt region in the D. radiodurans genome led to a reduction in heat tolerance similar to that observed in the DnrH-mutant. Our
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results show that DnrH positively influences heat tolerance by increasing the transcription of Hsp20 mRNA, demonstrating, for the first time that a ncRNA may directly controls the expression of a heat stress-resistance gene. Similar to dnrH, we characterized another ncRNA, dsr11. Our result showed that the transcription level of dsr11 was upregulated 4.2-fold under heat stress by qRT-PCR analysis. Heat tolerance assays showed that deleting dsr11 significantly inhibited the viability under high temperature stress conditions. To assess the influence of dsr11 on the D. radioduans transcriptome, 157 genes were found differentially expressed in the knock-out mutant by RNA-Seq experiment. Combined RNA-Seq and bioinformatic analysis, we found that dr_0457 (biopolymer transport protein) was likely to be the direct targets of dsr11. Further microscale thermophoresis results demonstrated that dsr11 can directly bind to the mRNA of dr_0457. Our results indicated that dsr11 can enhance the tolerance to heat stress of D. radiodurans by binding to dr_0457 mRNA.
(2) Bacillus velezensis GA1 is a Gram-positive bacterium living in association with plant roots and which may provide some protective effects against phytopathogens (biocontrol). In this work, we evaluated the impact on GA1 of temperatures typical of soils and lower than the optimal one commonly used under laboratory conditions. Cold temperature negatively impacted the cell growth rate of GA1, reflecting a general reduction in the metabolic activity. In vitro cultures revealed that production of some metabolites involved in biocontrol changed markedly when the temperature was lowered. We observed that after several rounds of culture on RE liquid medium at 15 and 18°C the growth of GA1 became faster than before suggesting that the bacterium may somehow adapt to cold conditions. We also tested the behavior of GA1 at 18°C on tomato plants, and showed that biofilm formation on root was a bit slower than at 22°C which correlated with reduced populations as revealed by flow cytometry measurements.
In summary, this study deeply analyzed the adaptation pathways and mechanisms of two gram-positive bacteria in response to different temperatures. Our work will provide a theoretical basis for future applications in industry and agriculture.
