A study of regulatory small RNAs in Vibrio salmonicida: construction of a knock-out mutant and a cDNA library

Abstract

The marine fish pathogen Vibrio salmonicida is the causative agent for vibriosis in Atlantic salmon (Salmo salar L.), Rainbow trout (Oncorhynchus mykiss) and Atlantic cod (Gadus morhua L.). V. salmonicidas virulence is regulated by Quorum sensing (QS) systems, which includes important regulatory small RNAs (sRNAs). sRNAs have the last years been identified in large numbers, and mostly in pathogenic bacteria strains. A cDNA library from small RNA species (120-340 nt) was constructed by size fractionation on a polyacrylamide gel. Due to time limitations, it was not possible to quality check the library properly. More colonies need to be screened in order to decide whether the cDNA library can be used in further studies or not. One sRNA proven to be very important in QS in several Vibrios is the quorum regulating RNA (Qrr). The expression of qrr has earlier been confirmed by Northern blotting. Furthermore, V. salmonicida has got only one predicted Qrr encoding gene, while other Vibrios have several. In order to gain more information about the role of Qrr in V. salmonicida, the construction of a qrr knock-out by deletion mutant was initiated. The knock-out construct was created, but the conjugation was not successful. The conjugation method for V. salmonicida is not yet established, so further optimization needs to be developed. A cDNA library of high quality can be subject to further experiments including ultra-high throughtput DNA sequencing followed by expression analysis like Northern blot. A qrr knock-out of V. salmonicida can be further investigated by methods like Microarray. Discovery of novel sRNAs can give us new insights in V. salmonicidas growth, virulence and stress adaptation and reveal possible targets for new antibiotics.