Identification of Key Genes during Ca2+-Induced Genetic Transformation in Escherichia coli by Combining Multi-Omics and Gene Knockout Techniques

Abstract

The molecular mechanism of the Ca²⁺-mediated formation of competent cells in Escherichia coli remains unclear. In this study, transcriptome and proteomics techniques were used to screen genes in response to Ca²⁺ treatment. A total of 333 differentially expressed genes (317 upregulated and 16 downregulated) and 145 differentially expressed proteins (54 upregulated and 91 downregulated) were obtained. These genes and proteins are mainly enriched in cell membrane components, transmembrane transport, and stress response-related functional terms. Fifteen genes with these functions, including yiaW, ygiZ, and osmB, are speculated to play a key role in the cellular response to Ca²⁺. Three single-gene deletion strains were constructed with the Red homologous recombination method to verify its function in genetic transformation. The transformation efficiencies of yiaW, ygiZ, and osmB deletion strains for different-size plasmids were significantly increased. None of the three gene deletion strains changed in size, which is one of the main elements of microscopic morphology, but they exhibited different membrane permeabilities and transformation efficiencies. This study demonstrates that Ca²⁺-mediated competence formation in E. coli is not a simple physicochemical process and may involve the regulation of genes in response to Ca²⁺. This study lays the foundation for further in-depth analyses of the molecular mechanism of Ca²⁺-mediated transformation. IMPORTANCE Using transcriptome and proteome techniques and association analysis, we identified several key genes involved in the formation of Ca²⁺-mediated E. coli DH5α competent cells. We used Red homologous recombination technology to construct three single-gene deletion strains and found that the transformation efficiencies of yiaW, ygiZ, and osmB deletion strains for different-size plasmids were significantly increased. These results proved that the genetic transformation process is not only a physicochemical process but also a reaction process involving multiple genes. These results suggest ways to improve the horizontal gene transfer mechanism of foodborne microorganisms and provide new ideas for ensuring the safety of food preservation and processing.

Keywords: Red homologous recombination; competent cell; proteome; transcriptome; transformation efficiency.