Novel Fur and Iron-Regulated Small Regulatory RNAs of Aggregatibacter actinomycetemcomitans

Objective: Four putative small regulatory RNA (sRNA) molecules, JA01-JA04, in the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) and their potential targets were identified using a bioinformatics approach. Previously, we found the expression of these sRNAs to be regulated by iron and the ferric uptake regulator protein (Fur) using RT-PCR, qPCR and the ferric uptake regulator titration assay. Here we further characterize the sRNA molecules, verify Fur binding, and identify potential targets. Methods: Total RNA from Aa HK1651R partially isolated by the Ribopure kit was enriched for sRNA using the mirVana kit. Approximate sRNA sizes were determined by Northern blots probed with biotinylated dsDNA corresponding to a portion of each sRNA. AaFur was overexpressed in the pET-30b expression vector, and cell lysates were probed with digoxigenin-labeled DNA corresponding to the promoters of each sRNA in an Electrophoretic Mobility Shift Assay (EMSA). Spotted Aa microarrays were probed with Cy-dye labeled HK1651R and the JA03 overexpression strain, and statistically analyzed. Results: The approximate size of JA01, JA02 and JA04 is 150,110, and 60 bp, respectively. EMSA verified that AaFur bound to the Fur box within each of the putative sRNA promoters. The gel shift was enhanced by the addition of E. coli anti-Fur antibody to cell lysate prior to incubation with labeled probe. Preliminary microarray analysis revealed that the genes for the queusosine biosynthesis protein (queA) and a DeoR family transcriptional regulator were significantly down-regulated. Conclusions: Northern blot confirmed the small size of the sRNAs JA01, JA02, and JA04. The size of JA03 could not be determined by Northern blot and will be determined by alternate methods. Each sRNA sequence is iron responsive and Fur regulated. Possible targets of JA03 have yet to be confirmed by qPCR.