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

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.

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

“…Nevertheless, the actual production of proteins derived from intraRNAs has been little explored. Coding potential of intraRNAs have been explicitly suggested in internal open reading frames (intraORFs) and detection of peptides attributed to them has been observed in Caulobacter crescentus [15], Bradyrhizobium japonicum [16] and Shigella flexneri [17]. …”

Transcriptional Control of the Lateral-Flagellar Genes of Bradyrhizobium diazoefficiens

“…S3 in the supplemental material). In a recent study, a genome-wide transcription start site map for B. diazoefficiens USDA 110 grown in peptone/salts/yeast extract/arabinose medium was established (34), indicating different operon structures for the lateralflagellar region from those predicted by the bioinformatic analysis ( Fig. 2A).…”

“…2A). To resolve this contradiction, we designed primers to amplify, by reverse transcription-PCR (RT-PCR), eight intergenic regions that should differ in the resulting transcripts according to whether the distribution of polycistronic mRNAs from this genomic cluster under our conditions is as predicted by bioinformatics or as reported in the experimental tran- scription start site mapping (34). The regions chosen were as follows: region 1 from 3= flgJ L (bll6849) to 5= fliR L (bll6850; further encompassing the intergenic regions upstream and downstream from bll6849.5), region 2 from 3= flgE L (bll6858) to 5= bll6859, region 3 from 3= fliK L (bll6860) to 5= motC (bll6861), region 4 from 3= fliF L (bll6864) to 5= lafA2 (bll6865), region 5 from 3= lafA2 (bll6865) to 5= lafA1(bll6866), region 6 from 3= lafA1 (bll6866) to 5= fliP L (bll6867), region 7 from 3= flgB L (bll6876) to 5= flhB L (bll6877), and region 8 from 3= fliN L (bll6879) to 5= bll6880 ( Fig.…”

“…To further investigate the difference in the responses of lafA1 and lafA2 to FlbT L , we compared the nucleotide sequences and RNA predicted structures of the 5= UTRs of these transcripts. To this end, we used the sequence within the lafA2 5= UTR beginning at the experimentally identified transcription start site located 118 nucleotides upstream from the ATG initiation codon (34) and extending through the first 17 codons of the coding sequence (39) in comparison to a putative 5= UTR of lafA1 starting also 118 nucleotides upstream from the ATG and continuing through the first 17 codons of the coding sequence. Despite the high conservation among these sequences, we observed a gap near a sequence complementary to the ribosome binding site (RBS) in lafA2 (see Fig.…”

“…The operon prediction was run in different online servers: the ProOpDB online server (54), the DOOR 2 online server (55), and the MicrobesOnline server tools (56). Analysis of these results in comparison to the transcription start site map of B. diazoefficiens (34) was carried out using the Integrated Genome Browser (57). All the oligonucleotides were designed with Primer BLAST (58).…”