Efficient protein biosynthesis, the main rate-limiting factor during bacterial growth and replication, depends on the concentration of active ribosomes within the cell. Ribosomes may become stalled on an mRNA strand when a stop codon is not detected, and active rescue of the ribosome (a process called trans-translation) is required to release it. Almost all bacteria encode a small RNA (tmRNA), encoded by the ssrA gene, which mediates trans-translation. An alternate release factor protein, ArfA, has also been described in bacteria such as Escherichia coli, Salmonella enterica, and Yersinia pestis. Both the ssrA and arfA genes are present in the genome of Actinobacillus pleuropneumoniae, and the aim of this work was to characterize the respective contributions of these factors to stress response and virulence of this important swine pathogen.
We constructed DssrA and DarfA mutants in the serovar 8 strain MIDG2331, and compared their phenotypes with wild-type (WT) with regards to: in vitro growth; biofilm formation; resistance to antibiotics and stress conditions (oxidative, osmotic, temperature and ethanol); and virulence in the Galleria mellonella model of infection.
Our results indicated that the DssrA mutant was more sensitive to the antibiotics and stress conditions tested. Furthermore, the DssrA mutant showed reduced biofilm formation, and its virulence was highly attenuated in G. mellonella when compared with the WT and DarfA mutant. We were unable to generate a double mutant strain (DssrA/DarfA), suggesting that A. pleuropneumoniae has only these two ribosome rescue systems. The relative fitness of the DssrA and DarfA mutants under the conditions tested suggest that tmRNA is the main ribosome rescue factor in A. pleuropneumoniae, with the ArfA protein functioning as a less-efficient back-up system in the DssrA mutant.