Quantitative Proteomics Analysis Reveals Hfq2 Play An Important Role On Pleiotropic Physiological Functions In Aeromonas Hydrophila

Hfq protein is an evolutionarily conserved sRNA chaperone protein,which is considered as a well-characterized post-transcriptional regulation global factor and plays an important role on the regulation of physiological functions in many kinds of bacteria.Most bacterial genomes have only one copy of hfq?hfq1?,but a few bacterial species carry another distinct copy of hfq?hfq2?on the chromosome.Meanwhile,the current molecular mechanisms of Hfq protein are largely based on Hfq1 protein.However,due to the absence of Hfq2 protein in model organisms such as Escherichia coli,the biological functions and its aggregation formation of this protein are lacking.Previous studies have reported that the physiological functions of different hfq copies of different bacteria are diverse,and the homology of Hfq2 is significantly different from Hfq1,which indicates that the function and regulation mechanism of Hfq2 protein may be different in various microorganisms.Thus,further research is necessary.We found that the genome-wide analysis of Aeromonas hydrophila ATCC 7966?NC₀08570.1?is different from most bacteria,its genome contains two distinct copies of the hfq gene,the first common hfq named AHA₀924,which has a high homology to most bacterial hfq and some biological functions of hfq have been well recorded;the other one we referred to hfq2 for short in this paper,which is AHA₃797 and it is an RNA chaperone of the Sm family with 121 residues?13.3 kDa?.Nevertheless,few reports on the biological functions of Aeromonas hydrophila Hfq2 protein until now.In order to study the biological function of Hfq2 in A.hydrophila,we first analyzed the structural domains of A.hydrophila Hfq1 and Hfq2proteins,both of them had the same two conserved regions as the model strain E.coli Hfq.It was found that Hfq2 has 50%homology with A.hydrophila Hfq1 and E.coli Hfq,whereas A.hydrophila Hfq1 has 87%homology with E.coli Hfq,indicating that Hfq2 protein may have some different biological functions from the common Hfq protein.We first successfully knocked out hfq2 using homologous recombination technology and determined its physiological phenotypes.The results showed that there were no effects on its growth when the hfq2gene is deleted,which indicate that the hfq2 gene was not necessary for the growth of A.hydrophila.However,the deletion of hfq2 significantly increased the activity of extracellular protease?ECPase?,chemotaxis and swarming motility;it has stronger tolerance under low temperature,acid pH and basic pH environment after deleted hfq2 gene,which can also increase sensitivity to H₂O₂ stress and high temperatures,and the ability to biofilm formation was significantly reduced;it also increased the sensitivity to polymyxin B and mycobacterium sulfate while slightly sensitive to neomycin sulfate;however,hemolysis ability was not affected,that indicated that Hfq2 plays an important role in the regulation of various basic physiological processes of A.hydrophila.To deeply understand the changes in intracellular protein expression after deletion of hfq2 gene and the regulation mechanism during biofilm formation,we further used SWATH based quantitative proteomics to compare proteins between?hfq2 and wild strains in planktonic status and biofilm status in this study.Firstly,LC MS/MS analysis showed that 862 proteins were identified in the planktonic status,compared with the wild-type strain including 234 proteins up-regulated and 72 proteins down-regulated,which accounts for 35.49%of the total identified proteins.Further bioinformatics analysis showed that proteins associated with metabolic pathways were up-regulated in the planktonic state,and ribosomal subunits are rich in abundance;in addition,deletion of hfq2 led to several DNA or RNA binding regulators?including Hfq and Crp?increasing,and a regulator of the two-component regulatory system OmpR was down-regulated,it suggests that Hfq2 plays an important role on maintaining the basic physiological function of A.hydrophila ATCC7966.Finally,the expression of some differential proteins was further verified by q-PCR and Western blotting,and the results were basically consistent with the quantitative proteomics data.We further compared the altered protein between wild-type and?hfq2 in biofilm status using proteomics method.A total of 1538 proteins were identified including 131 proteins were up-regulated and140 proteins were down-regulated,which account for 17.6%of the total identified proteins.Further bioinformatic analysis showed?hfq2 in the biofilm state led to the up-regulation of bacterial iron transport-related proteins,and chemotaxis-related proteins are greatly down-regulated.However,the absence of hfq2 did not cause significant changes in the expression of its homologous protein Hfq1,suggesting that the two Hfq homologous proteins had different roles in the biofilm formation process in A.hydrophila.To further verify the role of iron in the biofilm formation process mediated by Hfq2,we found that Hfq2 protein may affect the biofilm formation by the regulation of iron homeostasis using iron chelator addition assay.Finally,we further verified the expression of some differential proteins by q-PCR and Western blotting.To the best of our knowledge,this is the first demonstration of the effects of Hfq2 on the physiological functions of A.hydrophila at the protein level,which contributes to a new understanding of Hfq2.Studying Hfq2 may help prevent and control pathogenic bacteria,and provide a new control strategy for future development.