A cold shock protein from a thermophile bacterium promotes the high-temperature growth of bacteria and fungi through binding to diverse RNA species

Abstract

40High temperatures deleteriously affect cells by damaging cellular structures and changing the 41 behavior of diverse biomolecules, and extensive research about thermophilic microorganisms has 42 elucidated some of the mechanisms that can overcome these effects and allow thriving in 43 high-temperature ecological niches. We here used functional genomics methods to screen out a 44 cold-shock protein (CspL) from a high-productivity lactate producing thermophile strain 45 (Bacillus coagulans strain 2-6) grown at 37°C and 60°C. We subsequently made the highly 46 striking finding that transgenic expression of CspL conferred massive increases in high 47 temperature growth of other organisms including E. coli (2.4-fold biomass increase at 45°C) and 48 the eukaryote S. cerevisiae (a 2.7-fold biomass increase at 34°C). Pursuing these findings, we 49 used bio-layer interferometry assays to characterize the nucleotide-binding function of CspL in 50 vitro, and used proteomics and RNA-Seq to characterize the global effects of CspL on mRNA 51 transcript accumulation and used RIP-Seq to identify in vivo RNA targets of this 52 nucleotide-binding protein (e.g. rpoE, and rmf, etc.). Finally, we confirmed that a 53 nucleotide-binding-dead variant form of CspL does not have increased growth rates or biomass 54 accumulation effects at high temperatures. Our study thus establishes that CspL can function as a 55 global RNA chaperone. 56 57 Key words 58 Cold shock protein; CspL; global RNA chaperone; mRNA binding; heat shock response 59 60 104 105 Results 106Screening for high-temperature growth related genes from Bacillus coagulans 2-6 107 We previously isolated a Bacillus coagulans strain 2-6 (DSM 21869) from a milk processing 108 plant in Beijing by culturing samples from soil at 55 °C (Qin et al., 2009). This thermophile can 109 produce optically pure ʟ-lactic acid when cultured at 60 °C, but, despite having sequenced its 110 genome, we to date know relatively little about the mechanisms that drive the high-temperature 111 productivity of this strain (Su et al., 2014). We therefore observed the growth of B. coagulans 112 2-6 at 37 °C and 60 °C, then used RNA sequencing (RNA-Seq) and the iTRAQ proteomics 113 method to investigate how exposure to high temperature affects, respectively, its transcriptome 114 and proteome ( Figure 1A-figure supplement 1A). 115 At the mRNA level, there were 170 differentially accumulated mRNA transcripts (p < 0.05, ≥ 116 2-fold change) between the cells grown at 37 °C and 60 °C (106 mRNAs increased and 64 117 decreased for the 60 °C samples) ( Figure 1B, Supplementary file Tables 1 and 2). Prediction 118 using the MEME program indicated that there were no significantly different transcriptional start 119