Regulation der Expression der opa-Gene von Neisseria gonorrhoeae durch Antisense-RNAs
Zusammenfassung der Projektergebnisse
In this project non-coding RNAs of N. gonorrhoeae were investigated. Previously, we had reported that opa gene loci are associated with antisense transcription. This observation led to the hypothesis, that asRNAs might influence opa transcript accumulation by binding preferentially to out of frame opa transcripts which are not covered by translating ribosomes thereby triggering enhanced degradation of these junk mRNAs. We have shown that out of frame opa mRNAs are indeed less abundant than their in frame counterparts, however, this effect could not be correlated with antisense transcription. Non-coding antisense transcripts derived from the opa loci were shown to be by far less abundant than their corresponding opa mRNA counterpart, arguing strongly against a regulatory mechanism based on asRNA-mRNA hybridization. We also planned to study the reciprocal impact of antisense transcripts derived from genes with opposite transcriptional orientation on mRNA accumulation using mRNAs of NGFG_02230 (opaK) and NGFG_00785 with extended complementarity in the 5’-regions as archetypical sense-antisense pair. However, we were unable to provide an extensive analysis, since construction of appropriate mutants failed in repeated attempts employing different strategies. Therefore, we concentrated on the characterization of a pair of trans-acting sRNAs, NgncR_162 and NgncR_163, showing 78% of sequence identity and being highly conserved in the genus Neisseria. The sibling sRNAs are highly abundant in logarithmic growth phase and are downregulated upon entry into stationary phase. Using pulse expression of the individual sRNAs in a sRNA double deletion mutant followed by RNAseq combined with in silico target prediction we defined the regulon of the sibling sRNAs, comprising 23 negatively and 14 positively regulated genes involved mainly in energy metabolism (citric acid cycle, methylcitrate cycle), biosynthesis of cofactors and prosthetic groups, transport and transcription regulation. Interestingly, the most highly regulated target mRNAs (NGFG_01721 and NGFG_00045) show inverse regulation and encode proteins supposed to be involved in amino acid transport. To our surprise our analysis suggested complete redundancy regarding the target spectrum of the individual sibling sRNAs. A subset of negatively regulated target genes had already been reported to be controlled by the sRNA homologs of N. meningitides, however positive regulation by the sibling sRNAs had not been reported before. We demonstrated that blocking of the RBS in the 5’-UTR of the target mRNA is a common mechanism of negative posttranscriptional regulation employed by the sibling sRNAs, however, the mechanisms of direct positive regulation, as already demonstrated for NGFG_00045 encoding a transport protein, remain to be analysed in further detail. Furthermore, we could demonstrate a role of another gonococcal sRNA, NgncR_237, in the control of type IV pilus biogenesis and homologous recombination.
Projektbezogene Publikationen (Auswahl)
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(2017) The sibling sRNAs NgncR_162 and NgncR_163 of Neisseria gonorrhoeae participate in the expression control of metabolic, transport and regulatory proteins. Microbiology 163:1720-1734
Bauer S, Helmreich J, Zachary M, Kaethner M, Heinrichs E, Rudel T, Beier D
