Date Published: September 12, 2013
Publisher: Public Library of Science
Author(s): Cornelius Schmidtke, Ulrike Abendroth, Juliane Brock, Javier Serrania, Anke Becker, Ulla Bonas, Matthew K. Waldor.
Small noncoding RNAs (sRNAs) are ubiquitous posttranscriptional regulators of gene expression. Using the model plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv), we investigated the highly expressed and conserved sRNA sX13 in detail. Deletion of sX13 impinged on Xcv virulence and the expression of genes encoding components and substrates of the Hrp type III secretion (T3S) system. qRT-PCR analyses revealed that sX13 promotes mRNA accumulation of HrpX, a key regulator of the T3S system, whereas the mRNA level of the master regulator HrpG was unaffected. Complementation studies suggest that sX13 acts upstream of HrpG. Microarray analyses identified 63 sX13-regulated genes, which are involved in signal transduction, motility, transcriptional and posttranscriptional regulation and virulence. Structure analyses of in vitro transcribed sX13 revealed a structure with three stable stems and three apical C-rich loops. A computational search for putative regulatory motifs revealed that sX13-repressed mRNAs predominantly harbor G-rich motifs in proximity of translation start sites. Mutation of sX13 loops differentially affected Xcv virulence and the mRNA abundance of putative targets. Using a GFP-based reporter system, we demonstrated that sX13-mediated repression of protein synthesis requires both the C-rich motifs in sX13 and G-rich motifs in potential target mRNAs. Although the RNA-binding protein Hfq was dispensable for sX13 activity, the hfq mRNA and Hfq::GFP abundance were negatively regulated by sX13. In addition, we found that G-rich motifs in sX13-repressed mRNAs can serve as translational enhancers and are located at the ribosome-binding site in 5% of all protein-coding Xcv genes. Our study revealed that sX13 represents a novel class of virulence regulators and provides insights into sRNA-mediated modulation of adaptive processes in the plant pathogen Xanthomonas.
The survival and prosperity of bacteria depends on their ability to adapt to a variety of environmental cues such as nutrient availability, osmolarity and temperature. Besides the adaptation to the environment by transcriptional regulation of gene expression bacteria express regulatory RNAs that modulate expression on the posttranscriptional level , . Small regulatory RNAs (sRNAs; ∼50–300 nt) have been intensively studied in the enterobacteria Escherichia coli and Salmonella spp. and, in most cases, regulate translation and/or stability of target mRNAs through short and imperfect base-pairing (10 to 25 nucleotides) , , , . The majority of characterized sRNAs inhibits translation of target mRNAs by pairing near or at the ribosome-binding site (RBS) , . In addition, sRNAs can promote target mRNA translation, e. g., the sRNAs ArcZ, DsrA and RprA activate translation of sigma factor RpoS , , . Regulation of multiple rather than single genes has emerged as a major feature of sRNAs affecting processes like iron homeostasis, carbon metabolism, stress responses and quorum sensing (QS) , , . In numerous cases, sRNAs are under transcriptional control of two-component systems (TCS), which themselves are often controlled by sRNAs . The activity and stability of most enterobacterial sRNAs requires the hexameric RNA-binding protein Hfq, which facilitates the formation of sRNA-mRNA duplexes and their subsequent degradation by the RNA degradosome , . Hfq is present in approximately 50% of all bacterial species and acts in concert with sRNAs to regulate stress responses and virulence of a number of animal- and human-pathogenic bacteria , .