Genome-wide transcription start site mapping of Bradyrhizobium japonicum grown free-living or in symbiosis - a rich resource to identify new transcripts, proteins and to study gene regulation.

Jelena Čuklina,Melanie Goebel,Julia Hahn,Mikhail S Gelfand,Nikolay Ljubimov,Konrad U Förstner,Elena Evguenieva-Hackenberg,Gabriella Pessi,Ulrich Omasits,Christian H Ahrens,Maxim Imakaev,Hans-Martin Fischer

doi:10.1186/s12864-016-2602-9

Abstract

BackgroundDifferential RNA-sequencing (dRNA-seq) is indispensable for determination of primary transcriptomes. However, using dRNA-seq data to map transcriptional start sites (TSSs) and promoters genome-wide is a bioinformatics challenge. We performed dRNA-seq of Bradyrhizobium japonicum USDA 110, the nitrogen-fixing symbiont of soybean, and developed algorithms to map TSSs and promoters.ResultsA specialized machine learning procedure for TSS recognition allowed us to map 15,923 TSSs: 14,360 in free-living bacteria, 4329 in symbiosis with soybean and 2766 in both conditions. Further, we provide proteomic evidence for 4090 proteins, among them 107 proteins corresponding to new genes and 178 proteins with N-termini different from the existing annotation (72 and 109 of them with TSS support, respectively). Guided by proteomics evidence, previously identified TSSs and TSSs experimentally validated here, we assign a score threshold to flag 14 % of the mapped TSSs as a class of lower confidence. However, this class of lower confidence contains valid TSSs of low-abundant transcripts. Moreover, we developed a de novo algorithm to identify promoter motifs upstream of mapped TSSs, which is publicly available, and found motifs mainly used in symbiosis (similar to RpoN-dependent promoters) or under both conditions (similar to RpoD-dependent promoters). Mapped TSSs and putative promoters, proteomic evidence and updated gene annotation were combined into an annotation file.ConclusionsThe genome-wide TSS and promoter maps along with the extended genome annotation of B. japonicum represent a valuable resource for future systems biology studies and for detailed analyses of individual non-coding transcripts and ORFs. Our data will also provide new insights into bacterial gene regulation during the agriculturally important symbiosis between rhizobia and legumes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2602-9) contains supplementary material, which is available to authorized users.

Highlights

Differential RNA-sequencing is indispensable for determination of primary transcriptomes
Seq with total RNA isolated from bacteria exponentially growing in oxic liquid cultures and from soybean root nodules
Following the dRNA-seq protocol, one half of the RNA samples was treated with terminal exoribonuclease (TEX), which degrades 5′-monophosphorylated transcripts

Summary

Introduction

Differential RNA-sequencing (dRNA-seq) is indispensable for determination of primary transcriptomes. We performed dRNA-seq of Bradyrhizobium japonicum USDA 110, the nitrogen-fixing symbiont of soybean, and developed algorithms to map TSSs and promoters. In the past five years differential RNA-sequencing (dRNA-seq) has become an essential technology for global analysis of gene expression allowing for the genomewide mapping of transcriptional start sites (TSSs) [1]. Such analyses of rhizobia in symbiosis with leguminous plants are still missing. Bacteroids convert molecular nitrogen into ammonium for the benefit of the plant in exchange for photosynthetically fixed carbon. A prominent, rhizobial model organism is Bradyrhizobium japonicum USDA 110, a symbiont of the soybean plant Glycine max [2, 3]

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