SWATH based quantitative proteomics analysis reveals Hfq2 play an important role on pleiotropic physiological functions in Aeromonas hydrophila

The RNA-binding protein Hfq protein is a well-characterized post-transcriptional regulator and plays an important role in the regulation of various physiological functions. Most bacterial genomes have only one copy of hfq, but a few bacterial species carry another distinct copy of hfq (hfq2) on the chromosome. However, the physiological properties of Hfq2 remain elusive. Here, we successfully constructed an hfq2 knock-out strain of Aeromonas hydrophila ATCC 7966. Phenotype assays showed that hfq2 deletion significantly increased extracellular protease activity, chemotaxis and swarming motility; increased low temperature, acidic pH, and basic pH resistance; and increased sensitivity to H2O2 stress and high temperatures. A SWATH-based quantitative proteomics method was used to compare the differential expression of proteins between the Delta hfq2 mutant and the wild-type strain. Bioinformatic analysis showed that proteins associated with metabolic pathways were mostly upregulated, while those associated with ribosome subunits were mostly downregulated. Moreover, the deletion of hfq2 leads to the increased expression of several DNA- or RNA-binding regulators, including Hfq and the catabolite gene activator (Crp), and the decreased expression of OmpR. To our knowledge, this is the first study to demonstrate the effects of Hfq2 on physiological function at the protein level. Biological significance: Most of bacterial genome has only one hfq copy, while only few bacterial species have two distinct copies in chromosome, and there are few documents about the biological functions of Hfq2. The current phenotype assays showed that Hfq2 plays important roles on bacterial physiological functions such as chemotaxis, swarming motility, ECPase activity and response to various environmental stresses. To better understanding the biological behavior of this protein, a SWATH based quantitative proteomics method was used to compare the altered proteins between Delta hfq2 and wide type strain. Bioinformatics analysis showed that Delta hfq2 significantly affects central metabolic pathway and translation related proteins. Moreover, the deletion of hfq2 lead to the increased expression of post-transcriptional regulator Hfq and catabolite gene activator Crp, and the down regulation of two-component regulatory system regulator OmpR. Our results indicate that Hfq2 is not a pseudogene but plays important roles on the essential physiological functions in A. hydrophila. To our knowledge, this is the first report to demonstrate the molecular function of Hfq2 at proteomics level.