Transcription Termination within the Iron Transport-Biosynthesis Operon ofVibrio anguillarumRequires an Antisense RNA

Abstract

The iron transport-biosynthesis (ITB) operon in Vibrio anguillarum includes four genes for ferric siderophore transport, fatD, -C, -B, and -A, and two genes for siderophore biosynthesis, angR and angT. This cluster plays an important role in the virulence mechanisms of this bacterium. Despite being part of the same polycistronic mRNA, the relative levels of transcription for the fat portion and for the whole ITB message differ profoundly, the levels of the fat transcript being about 17-fold higher. Using S1 nuclease mapping, lacZ transcriptional fusions, and in vitro studies, we were able to show that the differential gene expression within the ITB operon is due to termination of transcription between the fatA and angR genes, although a few transcripts proceeded beyond the termination site to the end of this operon. This termination process requires a 427-nucleotide antisense RNA that spans the intergenic region and acts as a novel transcriptional terminator.Small RNAs, some of them antisense, have been found to regulate a wide variety of genes, either in a positive or negative fashion, although some RNAs can act both positively and negatively at different loci. Most work has been carried out in Escherichia coli, although with the explosion in the knowledge on bacterial genomes, small RNAs appear to be ubiquitous in bacteria (3,17,28,30,32). Functions that have been ascribed to some of these RNAs fall into one or more of the following processes: control of plasmid DNA replication (27), blocking of the ribosome binding site (2), freeing of the ribosome binding site (20), or targeted degradation of mRNA (21). In certain plasmids, such as pT181, harbored by some gram-positive bacteria, small RNAs have been associated with transcription attenuation by binding and folding the target RNA so that a Rho-independent terminator structure forms (7).We have previously identified two small RNAs in the fish pathogen Vibrio anguillarum: RNA␣, which acts on the iron transport gene expression, and RNA␤ (11,22,23). These two RNAs are transcribed as countertranscripts at different loci within the iron uptake-biosynthesis (ITB [iron transport-biosynthesis]) operon of the virulence plasmid pJM1 (15). This operon encodes the proteins FatD, -C, -B, and -A, which are involved in the transport of the ferric siderophore across the membranes, and AngR and -T, which are both active in the synthesis of the siderophore anguibactin and thus responsible for the ability of this bacterium to cause septicemia in salmonid fishes (26).Although transcribed as one operon, the level of fatDCBA mRNA is higher than the level of the full-length mRNA (fatDCBA angRT), thus causing differential expression of the genes encoding the ferric siderophore transport proteins as compared to the genes encoding the proteins involved in the siderophore biosynthesis. In this work, we report that the differential expression within this operon is mediated by RNA␤ and discuss its biological significance. MATERIALS AND METHODSBacterial strains and growth conditions. Vibrio...

Analysis of the Regulated Transcriptome of Neisseria meningitidis in Human Blood Using a Tiling Array

“…cis-acting antisense antagonist is translated, so translation of the ORF starts only when the mRNA concentration reaches a certain level (32). In the cases of asRNAs corresponding to genes that are part of an operon, asRNAs can be utilized by bacteria to set temporal gene expression thresholds for genes within an operon and to implement a quantitative adjustment of the mRNA amounts (31,43,58,65).…”

cis-Antisense RNA, Another Level of Gene Regulation in Bacteria

“…The binding of the asRNA to the growing polycistronic fatDCBA message leads to transcription termination at a potential hairpin close to the fatA stop codon. This also occurred in an in vitro transcription assay with the external addition of RNA␤, suggesting that other mechanisms, such as codegradation or transcriptional interference, could be excluded (89).…”

“…4A). Whereas RNA␣ represses fatA and fatB expressions under iron-rich conditions, RNA␤ causes the differential transcription of the full-length operon fatDCBA-angRT and a shortened fatDCBA message (89). The short form is about 17 times more abundant than the long form.…”

“…Previously, other mechanisms of asRNA function such as transcription termination (28,89) or transcript stabilization (66,90,97) were investigated only qualitatively. Because the transcription termination mechanism is not dependent on codegradation, this kind of regulation of the catalytic activity of the asRNA is possible if the asRNA dissociates from its target after action.…”

cis-Antisense RNA and Transcriptional Interference: Coupled Layers of Gene Regulation

“…Transcriptional attenuation is exemplified by interaction of RNAβ with the fatDCBA mRNA to induce transcriptional termination after the fatA gene in the fatDCBA-angRT operon in Vibrio anguillarum [62] (Figure 1b). This results in high levels of expression of fatDCBA mRNA, and consequently low levels of expression of angRT mRNA.…”