| The worldwide distribution of drought and saline environments restricts the efficiency of microbial application in the natural environment.Sphingomonas with abundant germplasm resources are widely used for plant growth promotion,biological control,and pollutant degradation.Sphingomonas has a very broad application prospect,as it can utilize both highand low-molecular-weight aromatic compounds and other pollutants.Besides,Sphingomonas can also help crops cope with drought and salt stress damage.In this study,we focused on the mechanism of S.melonis TY responding to osmotic stress and explored the gene regulation networks of S.melonis TY at transcriptional,post-transcriptional,and translational levels under hyperosmotic stress,and identified the main effects of hyperosmotic stress on S.melonis TY.In addition,the richness of novel s RNAs in S.melonis TY was explored using hyperosmotic stress as an induction condition.Two novel s RNAs including a hyperosmotic stress-induced s RNA SNC251 and a stably expressed s RNA SNC137 were in-depth investigated.SNC251 participates in the hyperosmotic stress response of S.melonis TY with the property of promoting the biofilm formation of S.melonis TY under hyperosmotic stress and activates the expression of Ton B-dependent receptor and envelope stress proteins,while SNC137 plays a role in maintaining the stability of the cellular metabolism process and promoting the growth of S.melonis TY under hyperosmotic stress.The main results of this research are as follows:The sensing systems and membrane protein expression were activated when S.melonis TY was under hyperosmotic stress,meanwhile,the components and structures of the membrane were adjusted adaptively according to the hyperosmtic stress intensity of external environments to maintain the membrane turgor.The extracellular hyperosmotic signals were then converted into intracellular signals by two-component signal transduction systems,and followed by the reset of global gene expression.During this procedure,the expression of other stress response regulators such as sigma-70 was also significantly upregulated,thus promoting the responding of downstream genes to hyperosmotic stress.In addition,the expression of genes in the transport system and genes associated with the biosynthesis of compatible solutes was also significantly upregulated,which promoted the accumulation of intracellular nutrients as well as compatible solutes,and also be favorable for cellular osmotic potential maintaining.The expression of genes related to oxidative stress response was triggered by hyperosmotic stress,and the activity of mechanosensitive ion channels and ion transporters were induced to maintain cellular homeostasis.The cellular concentration of c-di-GMP was significantly decreased under hyperosmotic stress conditions,thus affecting the expression of genes involved in flagella assembly and chemotaxis,which resulted in a significant decrease of motility and biofilm formation capability of S.melonis TY.A total of 290 non-coding RNAs were selected by in-depth analysis of the whole genome of S.melonis TY.A total of 90 s RNA transcripts were detected by RNA-seq,of which 83 s RNAs were expressed under osmotic stress,and s RNA SNC137 was found with the highest average expression level within all treatments.Network analysis of the expression of these 83 s RNAs under hyperosmotic stress revealed 7 different expression patterns.Weighted gene correlation network analysis(WGCNA)found that the expression of 18 s RNAs was significantly correlated with the hyperosmotic stress gradients,in which SNC251 had the highest correlation coefficient(r = 0.968)with hyperosmotic stress gradients.The location of SNC251 in the S.melonis TY genome was from 1,234,614 to 1,234,876 with a full length of 263 nt determined by RACE assay and verified with Northern blot analysis.The lac Z reporting system revealed that the transcription of SNC251 was initiated by its own promoter,rather than the product of the 3’ end of the upstream gene.Deletion of snc251 affected multiple cellular processes as well as the nicotine degradation capacity of S.melonis TY,while overexpression of SNC251 facilitated the biofilm formation by S.melonis TY under hyperosmotic stress.Two genes involved in the Ton B system were further verified to be activated by SNC251,which also indicated that SNC251 is a trans-acting s RNA.SNC137 was revealed located in the intergenic region of two genes with a full-length of231 nt based on 5’ and 3’ RACE experiments.SNC137 was transcripted by its own promoter and was verified as a trans-acting s RNA.Deletion of snc137 caused a wide range of effects on S.melonis TY,including ribosome pathways,organic nitrogen compound synthesis,protein metabolism,macromolecular compound metabolism,and cellular oxidative stress process.The expression of SNC137 was stable under various growth conditions and was not affected by hyperosmotic stress.However,the growth of S.melonis TY was significantly improved by overexpression of SNC137 under hyperosmotic stress.The target genes of SNC137 were screened in this research,thus its wide regulatory range was first revealed.Among these target genes,the gene BJP26＿RS13930,encoding extracellular polysaccharide synthesis,was screened under all three target gene screening strategies.Point mutation assays were introduced to validate the predicted core area of SNC137,and confirmed that SNC137 base-pairing with m RNA of the target gene BJP26＿RS13930 and BJP26＿RS10965 through a 54 nt conserved sequence at its 3’ end,which included 16 nt repeats at both ends.Finally,SNC137 was revealed as an Hfq in-dependent s RNA by Northern blot analysis and RIP-seq.In summary,this study revealed the responding mechanisms of S.melonis TY under hyperosmotic stress with multiple perspectives,and analyzed the influences on physiological activities of S.melonis TY under hyperosmotic stress.The post-transcriptional gene regulation networks of S.melonis TY under hyperosmotic stress were characterized and the acting mechanisms of s RNA was preliminarily explored.These results are of great significance to further reveal the stress response mechanisms of Sphingomonas and improve its environmental application efficiency. |
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