Multiple Small RNAs Interact to Co-regulate Ethanol Tolerance in Zymomonas mobilis

Abstract

sRNAs represent a powerful class of regulators that influences multiple mRNA targets in response to environmental changes. However, very few direct sRNA-sRNA interactions have been deeply studied in any organism. Zymomonas mobilis is a bacterium with unique ethanol-producing metabolic pathways in which multiple small RNAs (sRNAs) have recently been identified, some of which show differential expression in ethanol stress. In this study, we show that two sRNAs (Zms4 and Zms6) are upregulated under ethanol stress and have significant impacts on ethanol tolerance and production in Z. mobilis. We conducted multi-omics analysis (combining transcriptomics and sRNAimmunoprecipitation) to map gene networks under the influence of their regulation. We confirmed that Zms4 and Zms6 bind multiple RNA targets and regulate their expressions, influencing many downstream pathways important to ethanol tolerance and production. In particular, Zms4 and Zms6 interact with each other as well as many other sRNAs, forming a novel sRNA-sRNA direct interaction network. This study thus uncovers a sRNA network that co-orchestrates multiple ethanol related pathways through a diverse set of mRNA targets and a large number of sRNAs. To our knowledge, this study represents one of the largest sRNA-sRNA direct interactions uncovered so far.

Genome-Scale Transcription-Translation Mapping Reveals Features of Zymomonas mobilis Transcription Units and Promoters

“…In particular, the existence of large numbers of intragenic and antisense TSSs suggests that Z. mobilis expresses noncoding RNAs from TUs with genomic densities similar to those of E. coli and B. subtilis . These results are consistent with findings that Z. mobilis uses sRNA-based regulation to manage ethanol-induced and other stress responses ( 12 – 14 ). More broadly, however, the function of pervasive noncoding transcription in bacteria remains uncertain ( 40 , 41 ); our results suggest that, in addition to regulatory sRNAs, pervasive noncoding transcription may have functions in Z. mobilis that remain to be discovered.…”

“…Of 15 experimentally validated Z. mobilis small RNAs ( Zm sRNAs) ( 12 ), we identified 27 TSSs within 25 bp of the predicted start site of 9 Zm sRNAs ( Data Set S1 ), including Zm s6 and Zm s4, which a recent report found to be upregulated by ethanol stress and to have a significant impact on ethanol tolerance and production in Z. mobilis ( 13 ). E. coli is thought to produce more than a thousand noncoding transcripts, some of which are functional as regulatory RNAs ( 37 , 39 ).…”

Molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression in Zymomonas mobilis

“…sphaeroides ( Berghoff et al, 2011 ). In addition, recent study in Z. mobilis identified two sRNAs, Zms4 and Zms6, that might affect a wide range of sRNAs through modulating hfq expression indirectly and specifically co-regulate some pathways important to ethanol stress, including sulfate assimilation and cysteine biosynthetic processes ( Han et al, 2020 ). All these results support the role of Hfq on sulfur metabolism in response to ethanol stress.…”

“…These findings suggested an important role of Hfq in dealing with ethanol stress in Z. mobilis . However, the full repertoire of Hfq-dependent gene regulation response to ethanol has not been elucidated, although omics efforts have been carried out to understand ethanol stress responses in Z. mobilis ( Zhang et al, 2010 ; Tan et al, 2016 ; Han et al, 2020 ). In this study, the role of hfq on ethanol tolerance in Z. mobilis was investigated by constructing mutant strains of hfq deletion or overexpression, and their molecular responses were characterized using genetic approaches and transcriptomic study.…”

PNPase and RhlB Interact and Reduce the Cellular Availability of Oxidized RNA in Deinococcus radiodurans

“…N-terminal His 6 tagged proteins were purified from the soluble lysate using Ni-NTA agarose (Qiagen) as described previously ( 58 ). EMSAs were performed to determine the binding affinity of the proteins to 8-oxoG-modified and unmodified RNA in vitro , as described previously ( 59 ). The oligonucleotides used in the affinity chromatography assays (same compositions except with no biotin on the 5′ end) were radiolabeled with 10 μCi of [γ- 32 P]ATP (Perkin-Elmer) by T4 polynucleotide kinase (New England BioLabs) and purified using an Oligo Clean & Concentrator kit (Zymo Research).…”