Identification of small RNAs involved in nitrogen fixation in Anabaena sp. PCC 7120 based on RNA-seq under steady state conditions

Ping He,Kangming Chen,Xiaodan Cai,Xuelin Fu

doi:10.1186/s13213-020-01557-w

Abstract

PurposeAnabaena sp. PCC7120 is a genetically tractable model organism for nitrogen fixation and photosynthesis research. The importance of small regulatory RNAs (sRNAs) as mediators of a number of cellular processes in bacteria has begun to be recognized. Bacterial sRNA binds to target genes through base pairing, and play a regulatory role. Many studies have shown that bacterial sRNA can regulate cell stress response, carbon and nitrogen fixation, and so on. However, little is known about sRNAs in Anabaena sp. PCC 7120 regarded to nitrogen fixation under later steady state.MethodsTo provide a comprehensive study of sRNAs in this model organism, the sRNA (< 200 nt) extracted from Anabaena sp. PCC 7120 under nitrogen step-down treatment of 12 days, together with the sRNA from the control, was analyzed using deep RNA sequencing. Possible target genes regulated by all identified putative sRNAs were predicted by IntaRNA and further analyzed for functional categorizations for biological pathways.ResultTotally, 14,132 transcripts were produced from the de novo assembly. Among them, transcripts that are located either in the intergenic region or antisense strand were kept, which resulted in 1219 sRNA candidates, for further analysis. RPKM-based differential expression analysis showed that 418 sRNAs were significantly differentially expressed between the samples from control (nitrogen addition, N+) and nitrogen depletion, (N−). Among them, 303 sRNAs were significantly upregulated, whereas 115 sRNAs were significantly downregulated. RT-PCR of 18 randomly chosen sRNAs showed a similar pattern as RNA-seq result, which confirmed the reliability of the RNA-seq data. In addition, the possible target genes regulated by unique sRNAs of Anabaena sp. PCC 7120 under nitrogen addition (N+) condition or that under nitrogen depletion (N−) condition were analyzed for functional categorization and biological pathways, which provided the evidences that sRNAs were indeed involved in many different metabolic pathways.ConclusionThe information from the present study provides a valuable reference for understanding the sRNA-mediated regulation of the nitrogen fixation in Anabaena PCC 7120 under steady state conditions.

Highlights

Cyanobacteria are a group of photoautotrophs originated nearly 3.5 billion years ago, during which they have evolved a diverse array of metabolic capabilities (Carr and Whitton 1973; Herrero and Flores 2008; Kaushik et al 2016; Srivastava et al 2017)
PCC 7120 showed different growth, heterocysts differentiated directly on AA/8(N−) medium (Fig. 1a, c) after 3 d culturing; only the vegetative cells grown on AA/8(N+) medium (Fig. 1b, d)
SRNA assembly and functional annotation To comprehensively identify small regulatory RNAs (sRNAs) involved in nitrogen fixation at a steady state, we compared the sRNAs profile (18–200 nt) of Anabaena sp

Summary

Introduction

Cyanobacteria are a group of photoautotrophs originated nearly 3.5 billion years ago, during which they have evolved a diverse array of metabolic capabilities (Carr and Whitton 1973; Herrero and Flores 2008; Kaushik et al 2016; Srivastava et al 2017) They have been studied as prokaryotic hosts for sustainable biofuel production due to their high photosynthetic efficiency, genetic manipulability, and diverse metabolic pathways (Dismukes et al 2008; Ducat et al 2011; Halfmann et al 2014b; Halfmann et al 2014a; Chen et al 2015; Burnat et al 2018; Singh et al 2019; Ishikawa et al 2019). Understanding of the underlying mechanisms of nitrogen fixation and diazotrophic growth in cyanobacteria will shed light on basic mechanisms of bacterial genetic regulation and physiology character. It can help the catalog of genetically modified cyanobacterial strains to increase and produce renewable chemicals and biofuels. The hetR gene is required for an early step of heterocyst differentiation, whenever under N− conditions or N+ conditions (Buikema and Haselkorn 1991)

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