Abstract
Small open reading frames (sORFs) and genes for non-coding RNAs are poorly investigated components of most genomes. Our analysis of 1391 ORFs recently annotated in the soybean symbiont Bradyrhizobium japonicum USDA 110 revealed that 78% of them contain less than 80 codons. Twenty-one of these sORFs are conserved in or outside Alphaproteobacteria and most of them are similar to genes found in transposable elements, in line with their broad distribution. Stabilizing selection was demonstrated for sORFs with proteomic evidence and bll1319_ISGA which is conserved at the nucleotide level in 16 alphaproteobacterial species, 79 species from other taxa and 49 other Proteobacteria. Further we used Northern blot hybridization to validate ten small RNAs (BjsR1 to BjsR10) belonging to new RNA families. We found that BjsR1 and BjsR3 have homologs outside the genus Bradyrhizobium, and BjsR5, BjsR6, BjsR7, and BjsR10 have up to four imperfect copies in Bradyrhizobium genomes. BjsR8, BjsR9, and BjsR10 are present exclusively in nodules, while the other sRNAs are also expressed in liquid cultures. We also found that the level of BjsR4 decreases after exposure to tellurite and iron, and this down-regulation contributes to survival under high iron conditions. Analysis of additional small RNAs overlapping with 3’-UTRs revealed two new repetitive elements named Br-REP1 and Br-REP2. These REP elements may play roles in the genomic plasticity and gene regulation and could be useful for strain identification by PCR-fingerprinting. Furthermore, we studied two potential toxin genes in the symbiotic island and confirmed toxicity of the yhaV homolog bll1687 but not of the newly annotated higB homolog blr0229_ISGA in E. coli. Finally, we revealed transcription interference resulting in an antisense RNA complementary to blr1853, a gene induced in symbiosis. The presented results expand our knowledge on sORFs, non-coding RNAs and repetitive elements in B. japonicum and related bacteria.
Highlights
High-throughput transcriptomic and proteomic studies reveal many new genes in bacteria
Assuming that small genes are underrepresented in the original RefSeq annotation of the B. japonicum USDA 110 genome [11] and have been found among the recently annotated ones [15], we analyzed the length distribution of the new genes (Fig 1)
The predicted consensus secondary RNA structure of Br-REP2 is shown in Fig 9B and the corresponding alignment in S17 Fig. We found this repetitive element in the intergenic regions (IGRs) between blr1499 and bll1500 (Fig 9A)
Summary
High-throughput transcriptomic and proteomic studies reveal many new genes in bacteria. Bradyrhizobium japonicum USDA 110 (recently re-named to Bradyrhizobium diazoefficens USDA 110 [8]) is a slow-growing rhizobium belonging to the Alphaproteobacteria phylum. It lives in soil and can fix molecular nitrogen in symbiosis with the soybean plant acting as a natural fertilizer [9,10]. Differential RNA-seq (dRNA-seq) was used for mapping of transcription start sites (TSSs) onto the re-annotated genome of B. japonicum USDA 110 [15]. This re-annotation added 1391 new putative protein-coding genes (107 of them with proteomic evidence) to the 8317 previously annotated ones [11,15]. All newly annotated ORFs and most putative small RNAs (sRNAs) of B. japonicum have not been studied yet
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