Bioinformatics of CusS in Escherichia coli in response to silver ion stress

Abstract:

[Objective] To decipher the regulatory mechanism of a sensor histidine kinase (CusS) in Escherichia coli K-12 in response to silver ion stress and provide scientific evidence for the prevention and treatment of this bacterium. [Methods] ProtParam, ProtScale, Protein-Sol, TMHMM, SignalP, LocTree3, NetNGlyc-1.0, NetPhosBac-3.0, SOPMA, I-TASSERF, STRING, and MEGA were employed to predict the physicochemical properties, hydrophilicity, solubility, transmembrane domain, signal peptides, subcellular localization, glycosylation sites, phosphorylation sites, secondary structure, tertiary structure, protein-protein interaction network of CusS, and the homology of CusS in Gram-negative bacilli, respectively. After that, ΔcusS was constructed by the Red homologous recombination system, and the growth of ΔcusS in different media was monitored. In addition, we evaluated the sensitivity of ΔcusS to silver and copper ions and common antibiotics based on the minimum inhibitory concentration (MIC). RT-qPCR was employed to determine the transcription levels of cusCFBA and cusR after cusS deletion. [Results] CusS was composed of 480 amino acid residues, with the relative molecular weight of 53 738.05, the atom number of 7 624, and the isoelectric point of 6.02. It was a hydrophilic and insoluble protein containing transmembrane domain, and no signal peptide, located in the intracellular membrane. CusS had 2 glycosylation sites, 24 serine phosphorylation sites, 14 threonine phosphorylation sites, and 3 tyrosine phosphorylation sites. In the secondary structure, α-helixes, β-sheets, β-turns, and random coils accounted for 55.42%, 11.67%, 3.75%, and 29.17%, respectively. The gene cusS was highly conserved in Escherichia and Shigella. The colony PCR and first-generation sequencing confirmed the successful construction of ΔcusS. The deletion of cusS had no influence on the growth or metabolism of the strain. However, cusS was the key gene for E. coli in response to the silver ion stress. [Conclusion] The deletion of cusS did not affect the growth but attenuated the protective response of E. coli to silver ion stress. Furthermore, the deletion of cusS significantly down-regulated the mRNA levels of the downstream genes cusCFBA and cusR. The bioinformatics analysis and phenotype characterization of CusS lays a foundation for unveiling the regulatory mechanism of CusS in E. coli in response to silver ion stress.