Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are
frequently connected to the expression of virulence factors in diverse bacteria. Only a few
sRNAs have been described for Pasteurellaceae pathogens and no sRNA has yet been
described for Actinobacillus pleuropneumoniae, the causative agent of porcine
pleuropneumonia, responsible for considerable losses in the swine industry. To search for
sRNAs in A. pleuropneumoniae, we developed a pipeline for the computational analysis of
the bacterial genome by using four algorithms with different approaches, followed by
experimental validation. The coding strand and expression of 17 out of 23 RNA candidates
were confirmed by Northern blotting, RT-PCR and RNA sequencing. Among them, two are
likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied
GcvB and 6S sRNAs, and ten are putative novel trans-acting sRNAs, never before described
for any bacteria. The latter group has several potential mRNAs targets, many of which are
involved with virulence, stress resistance or metabolism, and connect the sRNAs in a
complex gene regulatory network. The sRNAs identified are well conserved among the
Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same
host. Our results show that the combination of newly developed computational programs can
be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of
gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus
providing clues for novel aspects of virulence that will be explored in further studies.