Effect of hfq2 gene in Aeromonas hydrophila on biofilm formation

The RNA-binding protein Hfq is an evolutionarily conserved sRNA chaperone protein that can regulate the RNA translation at the post-transcription level and is involved in the regulation of important physiological functions in bacteria. The Aeromonas hydrophila ATCC 7966 genome contains two copies of the hfq gene ( AHA_0924 and AHA_3797 ). AHA_0924 has been found to share pronounced homology with many popular bacterial species and its biological functions was well documented. However, only few bacterial species carry the second hfq gene copy and its role remains largely unknown. To better understand the physiological function of AHA_3797 ( hfq2 ), we first successfully knocked out hfq2 using homologous recombination technology and found that its biofilm formation capability was significantly reduced in this study ( P ∆hfq2 in biofilm status. Our results showed a total of 1538 proteins, including 131 proteins that were up-regulated and 140 proteins that were down-regulated. Selected altered proteins were validated by Western blot analysis, which indicated the proteomics results were reliable. The following Gene Ontology (GO) annotation enrichment analysis in molecular functional clustering showed that up-regulated proteins were mainly involved in receptor activity, transaminase activity, and metal ion binding function, while down-regulated proteins were mainly involved in oxidoreductase activity. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the most plentiful up-regulated proteins tended to be involved in the biosynthesis of antibiotics, while the down-regulated proteins mainly involved chemotaxis of bacteria. We also found ∆hfq2 caused much of the up-regulation of bacterial iron transport-related proteins, such as colicin I receptor and TonB-dependent copper receptor, and down-regulation of chemotaxis-associated proteins, such as CheY, in the biofilm. The deletion of hfq2 did not result in a significant change in the expression of homologous Hfq protein, suggesting that the roles of both Hfq in the biofilm formation process of A. hydrophila are different. To verify the role of iron homeostasis during hfq2 -mediated biofilm formation, we compared the biofilm formation between ∆hfq2 and the wide-type strain under iron limitation conditions. The results showed that the biofilm formation of the control strain decreased, whereas that of ∆hfq2 i ncreased significantly after the addition of 200 μmol/L ferrous iron chelating 2,2′-dipyridyl (DIP), suggesting that Hfq2 protein negatively regulates intracellular iron homeostasis and affects biofilm formation. In general, this is the first work to confirm that the deletion of the hfq2 gene of A. hydrophila affects the formation of biofilm, suggesting that this gene is not a pseudogene, but rather it plays an important regulatory role in the formation of bacterial biofilm. Establishing the mechanism by which Hfq2 protein regulates bacterial biofilm formation would be of considerable scientific significance and may be helpful in the prevention and control of pathogenic bacteria and the development of new antibacterial strategies in the future.