Small Open Reading Frames, Non-Coding RNAs and Repetitive Elements in Bradyrhizobium japonicum USDA 110

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

DIGGER: exploring the functional role of alternative splicing in protein interactions.

Alternative splicing plays a major role in regulating the functional repertoire of the proteome. However, isoform-specific effects to protein-protein interactions (PPIs) are usually overlooked, making it impossible to judge the functional role of individual exons on a systems biology level. We overcome this barrier by integrating protein-protein interactions, domain-domain interactions and residue-level interactions information to lift exon expression analysis to a network level. Our user-friendly database DIGGER is available at https://exbio.wzw.tum.de/digger and allows users to seamlessly switch between isoform and exon-centric views of the interactome and to extract sub-networks of relevant isoforms, making it an essential resource for studying mechanistic consequences of alternative splicing

Nitrogen fixation and molecular oxygen: comparative genomic reconstruction of transcription regulation in Alphaproteobacteria

Biological nitrogen fixation plays a crucial role in the nitrogen cycle. An ability to fix atmospheric nitrogen, reducing it to ammonium, was described for multiple species of Bacteria and Archaea. Being a complex and sensitive process, nitrogen fixation requires a complicated regulatory system, also, on the level of transcription. The transcriptional regulatory network for nitrogen fixation was extensively studied in several representatives of the class Alphaproteobacteria. This regulatory network includes the activator of nitrogen fixation NifA, working in tandem with the alternative sigma-factor RpoN as well as oxygen-responsive regulatory systems, one-component regulators FnrN/FixK and two-component system FixLJ. Here we used a comparative genomics analysis for in silico study of the transcriptional regulatory network in 50 genomes of Alphaproteobacteria. We extended the known regulons and proposed the scenario for the evolution of the nitrogen fixation transcriptional network. The reconstructed network substantially expands the existing knowledge of transcriptional regulation in nitrogen-fixing microorganisms and can be used for genetic experiments, metabolic reconstruction, and evolutionary analysis

Transcription interference in blr1853.

<p><b>A)</b> Blr1853 locus and the structure of <i>lacZYA</i> reporter fusions used to analyze transcription interference in blr1853. Plasmid names are indicated. Blue straight line, DNA of the blr1853 locus; blue wave lines, asRNA AsR1-blr1853 (an abundant 65 nt form detected in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.g005" target="_blank">Fig 5C</a> and a long form previously detected by RT-PCR, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref015" target="_blank">15</a>]); orange line, <i>lacZYA</i> genes; thin black flexed arrows, active TSSs; thin gray flexed arrows, inactive TSSs; open boxes with promoter designations, promoters upstream of the TSSs; P_cyp, blr1853 promoter; P_int, internal promoter in the sense direction; P_as, internal promoter in the antisense direction. The genomic coordinates of the TSSs are given on top [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref015" target="_blank">15</a>]. Red stars indicate three mutations introduced in P_as (see B). The drawing is not to scale. <b>B)</b> Reporter fusions used to measure the activity of the wild type (wt) P_as and its mutated version P_as-mut3. Shown are parts of the cloned 63 nt sequence. The TSS of asRNA AsR1-blr1853 is indicated along with the –10 and –15 boxes of the P_as promoter. The mutated bases are in red. For other descriptions, see A). <b>C)</b> Beta-galactosidase activities of <i>B</i>. <i>japonicum</i> cells harboring the reporter constructs shown in A) and B). Measurements were performed with aerobic exponentially growing cultures. Shown are the results from three independent experiments with technical duplicates. Error bars indicate the standard deviation.</p

Non-annotated transcripts analyzed by Northern blot hybridization.

<p><b>A)</b> New sRNAs detected in liquid cultures and in nodules. <b>B)</b> New sRNAs detected only in nodules. <b>C)</b> mRNA-associated small transcripts with previously verified TSSs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref015" target="_blank">15</a>]. Total RNA from the exponential growth phase (E) and the stationary phase (S) of a liquid culture and from nodules (N) was used. For sRNAs detected in N only, control RNA from roots (R) was also included. After hybridization with sRNA-specific probes (indicated above the panels), the membranes were re-hybridized with probes specific for 5S rRNA from <i>B</i>. <i>japonicum</i> (5S <i>B</i>.<i>j</i>.) and, when root RNA was included, from <i>G</i>. <i>max</i> (5S <i>G</i>.<i>m</i>). In A) and B), the positions of the marker RNAs on the membranes (160 nt, 120 nt and 60 nt corresponding to 6S rRNA, 5S rRNA and a fragment detected by the 6S RNA-specific probe, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref029" target="_blank">29</a>]) are given on the right side (in nt). In C), the approximate lengths of the indicated bands are given (in nt), as calculated from the migration of the marker RNAs mentioned above. BjsR5a,b* and BjsR6a,b,c* – no discrimination between homologs in the Northern blot hybridization; BjsR8** – processing product according to dRNA-seq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.s012" target="_blank">S12 Fig</a>); E# – RNA isolated by TRIzol resulting in enrichment of sRNAs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.ref053" target="_blank">53</a>]; all other RNA samples were isolated by hot phenol.</p

Stabilizing selection for sORFs with proteomic evidence and bll1319_ISGA.

<p><b>A)</b> Distribution of the dN/dS ratio for homologs of 39 sORFs with proteomic evidence. <b>B)</b> Distribution of the dN/dS ratio for othologs of bll1319_ISGA. All pairs of homologs were considered to construct the histograms.</p

TBLASTX analysis of <i>B</i>. <i>japonicum</i> sORFs showing the distribution of homologs among bacteria.

<p><b>A)</b> Analysis of 39 sORFs with proteomic evidence. Indicated is their presence only in <i>B</i>. <i>japonicum</i> strains (<i>B</i>. <i>japonicum</i>), in the genus <i>Bradyrhizobium</i>, or in Alphaproteobacteria (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.s021" target="_blank">S4 Table</a>). Other – the sORF blr0566_ISGA present in Alphaproteobacteria and outside Alphaproteobacteria. <b>B)</b> Analysis of all 1080 sORFs (with and without proteomic evidence) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.s023" target="_blank">S6 Table</a>). Alphaproteobacteria_Rare – sORFs found in less than five Alphaproteobacteria other than <i>Bradyrhizobium</i> spp.; Alphaproteobacteria_Conserved – sORFs found in five or more Alphaproteobacteria other than <i>Bradyrhizobium</i> spp.; Other – sORFs found in organisms outside Alphaproteobacteria; No homologs, – sORFs found only in <i>B</i>. <i>japonicum</i> USDA 110. <b>C)</b> Analysis of 47 sORFs with homologs outside Alphaproteobacteria (belonging to the category “Other”). Sporadic – sORFs found in less than 20 Alphaproteobacteria and less than 20 organisms outside Alphaproteobacteria; More in Alphaproteobacteria – sORFs found in at least 20 Alphaproteobacteria and less than 20 organisms outside Alphaproteobacteria; More outside Alphaproteobacteria – sORFs found in less than 20 Alphaproteobacteria and at least 20 organisms outside Alphaproteobacteria; Conserved – sORFs found in at least 20 Alphaproteobacteria and at least 20 organisms outside Alphaproteobacteria (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165429#pone.0165429.s026" target="_blank">S9 Table</a>).</p

Characteristics of ten sRNAs analyzed by Northern blot hybridization.

<p>Characteristics of ten sRNAs analyzed by Northern blot hybridization.</p