# Riboregulation in Nitrogen-Fixing Endosymbiotic Bacteria
## 5 Antisense Transcription in _S. melidiot_  
Several studies on _S. melidiot_ anticipate a broad impact of _cis_-acting asRNA regulators on both the free living and host-associated lifestyles of this bacterium [26; 27; 97]. asRNAs are transcribed opposite to mRNA loci and may overlap either the 5'/3'-UTRs or the coding region of the message (Figure 1). The long and perfect complementarity between asRNAs and their targets favors duplex formation, which can influence transcription, translation and/or turnover rates of the mRNAs. Deep-sequencing of prokaryotic transcriptomes is continuously uncovering pervasive antisense transcription, which likely contributes to the posttranscriptional buffering of gene expression levels [98]. Such RNA-Seq surveys have revealed that _S. melidiot_ expresses more than 3000 asRNAs that are linked to \(\sim\)35% of the predicted protein-coding genes [26; 27]. Nonetheless, functional asRNAs in _S. melidiot_, like in other microorganisms, were firstly reported associated to extrachromosomal replicons [99].  
A systematic screening strategy to identify novel RNA regulators of N fixation revealed that both asRNAs and _trans_-sRNAs are overrepresented (41% and 24%, respectively) in _S. melidi_ pSymA symbiotic meaglasmid, with asRNAs particularly biased to _not_ and _nif_ genes. Further characterization of seven pSymA-borne asRNAs showed that most accumulate in response to different biological conditions, anticipating functional roles [97]. Among them, SMa_asRNA_244, antisense to the of _nodD2_ 3'-UTR, is slightly upregulated at high optical densities and upon luteolin addition (30%). It is worth noting that NodD2 is the only transcriptional regulator of _S. melidiot nod_ genes that is not activated by this plant flavonoid. Several asRNAs opposite to genes encoding the nitrogenase components NifE and NifK show also distinct expression profiles. SmelA031 and SMa_asRNA_277, also accumulate in early and late stationary phase of growth, respectively, while SMa_asRNA_279 responds to other stresses. Interestingly, the three transcripts are repressed in nodules and under low O\({}_{2}\) and N\({}_{2}\) concentrations, and plants inoculated with _S. melidi_ overexpressing SmelA031 are moderately affected in N fixation when compared to controls. Nitrogenase activity is extremely sensitive to O\({}_{2}\) and needs N\({}_{2}\) as substrate to function inside the nodules. Therefore, it is feasible that such post-transcriptional regulation modulates translation of this enzyme to avoid energy loss. These results first evidenced symbiotic-related asRNA regulation in rhizobia, but hundreds of rhizobial asRNAs await functional characterization. There are some works arguing that the majority of cellular asRNAs just arise from noisy transcription and are non-functional [100; 101]. However, there is an increasing number of rolesassigned to the widespread asRNAs, including both highly specific and global regulation such as DNA-repair and transcription interference [98]. Nevertheless, it is also advisable that, when searching for asRNAs with potential specific regulatory roles, requirements like upstream predicted promoters, high read coverage in reliable RNA-Seq profiling, and informative validated expression patterns are taken into account.