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.

INTRODUCTION

Horizontal gene transfer (HGT) is a common phenomenon in nature, especially among microorganisms (1), that is different from the conventional vertical gene transfer from parent to offspring and can cross interspecies isolation and transfer genetic information between genetically related or distant or near biological organisms. HGT enables recipient species to rapidly acquire new genes and new functions so that they can adapt to new environments or acquire new resources; this plays an important role in the evolution of both prokaryotes and eukaryotes and is also one of the hot spots of biological research in recent years. Transformation, phage-mediated transduction, and conjugation are three modes of HGT. Among them, transformation of genetic elements is a commonly used method in genetic manipulation. Genetic transformation has been proved to be related to the living environment, the physiological state, and the regulation of response to external information and donor information of microbial cells (2). Cells that can complete transformation are called competent cells, a special physiological state controlled by genes, and can absorb exogenous DNA from the surrounding environment and produce genetic changes (3). Bacteria such as Enterobacter (Hormaeche and Edwards 1960), Streptococcus, Bacillus, and Haemophilus spp. can all form competent cells. In order to cope with the external pressure, different kinds of bacteria have evolved their own independent regulation ways to control the establishment of the competent state, thus regulating the process of obtaining exogenous DNA (2, 3). At present, many laboratories around the world are committed to the study of artificial induction and the establishment of competence and genetic transformation mechanism under natural conditions, thereby hoping to provide a theoretical basis for disease prevention, research in pathogens, and the prevention and control of foodborne pathogens through the disclosure of genetic transformation mechanism (4).

Researchers have proposed transformation models of Gram-positive bacteria, represented by Bacillus subtilis, and Gram-negative bacteria, represented by Haemophilus influenzae and Neisseria gonorrhoeae (5, 6). These bacteria contain proteins that help DNA to cross the cell’s outer membrane, periplasmic space, and inner membrane for the natural transformation of exogenous DNA (7, 8).

However, the mechanism for how foodborne pathogenic bacteria respond to external conditions to complete HGT is still unclear, especially for the DNA transformation commonly used in genetic engineering. For example, how Escherichia coli responds to the induction of divalent metal ion Ca²⁺ to form the competent state and then complete the transformation of exogenous DNA is not clear (9). As a model organism, E. coli is not only an important recipient for gene cloning and expression (10). The unique way this bacterium establishes competence has led researchers to dispute its role and its status in gene transfer, and the emergence of many drug-resistant microorganisms makes this problem more important and prominent (11). Systematic study of the mechanism of competence formation and DNA transformation will help to explain the negative effects caused by genetic engineering, such as the spread of transgenic microorganisms and the variation of new pathogens, so that reasonable design strategies can be adopted in the construction of genetically modified organisms (GMOs) to minimize the possibility of DNA release and transfer (12, 13). Therefore, how to explore the key genes and their functions, the regulatory mechanisms among genes, and the interactions among proteins requires further study. With the rapid development of bioinformatics technology, several genes have been confirmed to be involved in the formation of the competent state and plasmid transformation within the genome (14, 15). In particular, the combined application of multi-omics technologies has enabled the functions of genes or proteins to be gradually resolved. The development of multi-omics technology will provide technical support to accurately reveal the natural transformation mechanism (16–18).

Here, we used transcriptomic and proteomic correlation analysis to screen key genes and proteins that are differentially expressed in the metabolic pathway of E. coli DH5α cells after Ca²⁺ mediation. On this basis, we constructed single-mutant strains to determine the changes in surface structure, physiology, membrane permeability, and transformation efficiency of naturally transformed cells under Ca²⁺-mediated transformation. We conducted an in-depth study of the molecular mechanism and morphological basis of Ca²⁺-mediated competence formation in E. coli cells. Our overall research scheme is shown in Fig. 1. The results lay a foundation for the study of the mechanism of E. coli competent formation and HGT and provide new ideas for ensuring the safety of food preservation and processing.

FIG 1.

Schematic representation of exploring the Ca²⁺-induced genetic transformation mechanism in E. coli by combining multi-omics and gene knockout techniques.

RESULTS

Transcriptome analysis of E. coli DH5α mediated by Ca²⁺.

As shown in Fig. S1 in the supplemental material, the bands of total RNA extracted from the 100 mM CaCl₂-treated group and the control group were clear and highlight with three bands, indicating that the concentration and integrity could meet the requirements of subsequent experiments. As shown in Tables S1 and S2, the quantity and quality of filtered bases and reads could meet the requirements of transcriptome analysis. When identifying differentially expressed genes (DEGs), a false discovery rate (FDR) of <0.05 and a |log₂ fold change (FC)| value of >1 were used as screening thresholds, as shown in Fig. 2A. The number of genes annotated in the reference genome is 4,140. A total of 3,951 known genes were detected in the Ca²⁺ treatment group, and 3,923 genes were detected in the control group. Among them were 333 DEGs, accounting for 8% of the detected genes. Among the DEGs, 317 genes were upregulated, accounting for 95% of the total DEGs, and 16 downregulated genes, accounting for 5% of the total DEGs.

FIG 2.

Analysis of E. coli DH5α transcriptome mediated by Ca²⁺. (A) Expression difference scatter diagram. Green dots denote downregulated genes (FDR < 0.05; the difference multiple is more than twice), red dots denote upregulated genes (FDR < 0.05, with more than twofold difference multiples), and black dots denote genes with no difference in expression. (B) Functional enrichment of significant differential genes GO. (C) Pathway enrichment map of DEGs. (D) Functional classification of DEGs.

GO functional annotation and KEGG pathway analysis of these DEGs were used to provide some clues about Ca²⁺-mediated genetic transformation. The biological functions related to DEGs were revealed by GO enrichment. For the three relatively independent ontologies in the GO database—namely, biological processes, cellular components, and molecular functions—three-level term enrichments and the corresponding significance analyses were carried out, respectively. The results showed that after 100 mM Ca²⁺ treatment, the significantly enriched top terms were as follows: for cellular components, these terms were periplasmic space, cell outer membrane, outer membrane, etc.; for molecular functions, the significantly enriched top terms were ATPase activity coupled to transmembrane movement of ions and phosphorylative mechanism, primary active transmembrane transporter activity, and PP-bond-hydrolysis-driven transmembrane transporter activity; and for biological processes, the significantly enriched top entries were mainly related to amino acid metabolism (Fig. 2B). Overall, the enrichment patterns of cellular components and molecular functions suggested that major cellular biological responses mediated by 100 mM Ca²⁺ were associated with transmembrane recognition and transport (Fig. 2C; see also Fig. S2 and Table S3). According to their functions, DEGs can be divided into cell membrane component-related genes, transcellular membrane transport-related genes, and cold-, heat-, and stress-mediated genes, etc. (Fig. 2D). Further attention was paid to the fold change of the related DEGs mentioned above, and genes related to transmembrane transport and stress were found to be highly expressed; in particular, osmB was annotated as a stress gene, and its expression was upregulated by 25 times, while ygiZ and yiaW were encoded as membrane protein genes, showing 13- and 7-fold upregulation, respectively.

To verify the reliability of the transcriptome data, we randomly selected 8 upregulated genes and 1 downregulated gene from the transcriptome-based DEGs and used quantitative reverse transcription-PCR (qRT-PCR) to verify their expression patterns (Fig. 3). According to the transcriptome and qRT-PCR results, the expression patterns of osmB, yiaW, yeeE, pspD, cspA, bhsA, and loiP were upregulated, and the expression patterns of yqiJ were downregulated in both assays, respectively. Only for genes ygiZ and bhsA did the log₂FC qRT-PCR results (0.05 and −0.09, respectively) contradict the transcriptome measurements (3.36 and 1.34, respectively). We guess that the amplification efficiency of primers may have a greater impact. Overall, based on these results, we consider the transcriptome data to be reliable.

FIG 3.

Verification of DEGs by qRT-PCR. Error bars represent the standard deviations (n = 3).

Proteomics analysis of E. coli DH5α mediated by Ca²⁺.

In order to ensure the direct comparability between the control group, the 100 mM CaCl₂ test group and the parallel samples within the group, the protein concentration had to be adjusted to be consistent. The SDS-PAGE showed that the protein bands in each lane were clear and morphologically intact (see Fig. S3). The quantitative results showed that the protein concentration of the control group was 5.67 ± 0.18 μg/μL and that of the 100 mM CaCl₂ treatment group was 5.54 ± 0.15 μg/μL; thus, the total protein concentrations of the two groups were similar. Therefore, the integrity and concentration of total protein both in the control group and in the 100 mM CaCl₂-treated group met the requirements and can be used for subsequent detection.

A total of 9,373 unique peptide sequences and 1,784 proteins were identified by proteomics. Compared to the control group, there were 145 differentially expressed proteins (DEPs) in the Ca²⁺ treatment group, including 54 upregulated proteins and 91 downregulated proteins. The DEPs were enriched in 16 secondary terms of three ontologies in the GO database (Fig. 4A). The ratio of the number of differential proteins to the number of all proteins under any GO term represented the enrichment degree of differential proteins for this term, and the results showed that 100 mM Ca²⁺ could mediate obvious changes in cell membrane composition and response to stimulus and transport activity in E. coli cells, which belong to the three first-level terms of cellular components, biological processes, and molecular functions, respectively. The KEGG enrichment results of DEP-coding genes in E. coli DH5α cells treated with 100 mM CaCl₂ are shown in Fig. 4B. A total of 65 DEPs were annotated by the KEGG pathway (see Table S4), and the annotation results indicated that the enrichment of the ABC transport pathway was the most significant, followed by the sulfur metabolism and two-component system. The results of the KEGG pathway annotation are shown in Table S4. DEPs can be functionally classified into proteins related to cell membrane, transmembrane transport, stress response, ribosomes, and various enzyme-catalyzed reactions (Fig. 4C). Comprehensive GO annotation, KEGG pathway annotation, and protein functional classification suggest that cell membrane components, transmembrane transport, and stress response-related proteins play important roles in Ca²⁺-mediated genetic transformation.

FIG 4.

Proteomic analysis of E. coli DH5α mediated by Ca²⁺. (A) Number ratio of DEPs to all proteins for each GO term. (B) Pathway enrichment map of DEPs. (C) Functional classification of DEPs. (D) Nine-quadrant diagram of the relationships between different expression levels of mRNA and protein.

A total of 1,523 genes and their corresponding proteins were involved in the correlation analysis of transcriptomes and proteomes (Fig. 4D). The correlation analysis map was divided into nine quadrants. The ordinate represents the log₂ value of the fold change in gene expression, and the abscissa represents the log₂ value of the fold change in protein expression. The 2-fold change in gene expression and the 1.5-fold change in protein expression were used as the thresholds for DEGs and proteins, respectively. The fifth quadrant indicates that the coexpressed mRNA and proteins are not differentially expressed, with a total of 1,020 genes accounting for 66.97% of all genes. The first, second, and fourth quadrants indicate that the abundance of protein expression is lower than that of mRNA, and it is regulated at the posttranscriptional or translation level. For example, miRNA regulates target genes to inhibit protein translation. Respectively, the first quadrant represents the upregulation of the transcription level and the downregulation of the protein level, with a total of 17 genes, which are mainly related to the synthesis and decomposition of amino acids, the efflux of metal ions, and some family member proteins. The second quadrant represents the upregulation of the transcriptional level, while the protein level changes, with a total of 43 genes, which are mainly related to the transcriptional regulatory factors of DNA, 50S ribosomal subunit protein L34, peroxidase, salt stress-mediated membrane proteins, induction of DNA binding response in the two-component system, regulation of nickel and cobalt efflux in the periplasm, and so on. The fourth quadrant represents no change in the transcriptional level, while the protein level is downregulated, with a total of 270 genes whose functions are mainly related to cellular metabolism, such as aspartate synthase A, d-gluconate dehydratase 1, galactose-1-phosphate uridine transferase, and so on. The sixth, eighth, and ninth quadrants indicate that the protein expression abundance is higher than that of mRNA and that the protein is regulated or accumulated at the posttranscriptional or translation level. The differential expression patterns of mRNAs in the third and seventh quadrants were consistent with the differential expression patterns of the corresponding proteins. Among them, the third quadrant contains 18 upregulated genes and proteins, and its function is mainly related to cell membrane transport. The seventh quadrant represents downregulated genes and proteins, one of which is functionally annotated as lipoprotein.

Overall, both transcriptomic and proteomic analyses consistently indicated that changes in the expression of genes and proteins related to cell membrane composition, transmembrane transport, and stress response were the major Ca²⁺-mediated changes in E. coli. 15 genes related to cell membrane components, transmembrane transport, or stress response are listed in Table 1, that are highly expressed and are mediated by Ca²⁺. However, whether these genes are really involved in the Ca²⁺-mediated formation of E. coli DH5α competent cells remains to be further investigated.

TABLE 1.

Genes that may be associated with response to Ca²⁺

Gene	Log2(FC)a	P	Gene function
osmB	4.61	8.65E–13	Osmotic and stress induced lipoproteins
ygiZ	3.36	3.43E–03	Inner membrane protein
yiaW	2.82	2.15E–03	DUF3302 family inner membrane protein
ycdU	1.65	6.69E–05	Putative inner membrane protein
yfdY	1.05	1.57E–04	DUF2545 family putative inner membrane protein
livK	1.69	1.11E–22	Leucine transporter subunit
metN	1.49	4.87E–54	dl-Methionine transporter subunit
argT	1.39	2.24E–29	Lysine/arginine/ornithine transporter subunit
cadB	1.09	9.12E–03	Putative lysine/cadaverine transporter
pstC	1.00	1.16E–13	Phosphate ABC transporter permease
loiP	2.73	1.21E–58	Phe-Phe periplasmic metalloprotease, OM lipoprotein; low salt inducible; Era-binding heat shock protein
yhbO	1.71	6.17E–19	Stress resistance protein
psiE	1.40	5.53E–55	Phosphate starvation-inducible protein
ves	1.24	1.14E–04	Cold- and stress-inducible protein
ygiW	1.05	9.20E–116	Hydrogen peroxide and cadmium resistance periplasmic protein; stress-induced OB-fold protein

^aFC, fold change for RNA-seq.

Construction and functional study of gene deletion strains.

The electrophoretic results are consistent with the theoretical gene targeting fragments (see Fig. S4A). The PCR amplification fragments of the five randomly selected transformants were all consistent with the theoretical size, indicating that the screened transformants were all positive clones in which the target gene had been successfully replaced (see Fig. S4B). As shown in Fig. S4C, the amplification lengths of the ygiZ and yiaW genes in the E. coli DH5α control group were 541 and 608 bp with identification primers, 1,958 and 2,118 bp after replacement with FRT-kan-FRT, and 373 and 453 bp after the elimination of kan fragments. The amplification length of osmB was 529 bp; after being replaced by FRT-kan-FRT, the amplification length was 1,771 bp, and the amplification length after eliminating the kan fragment was 288 bp. The electrophoresis results are consistent with the theoretical values, and the sequencing results are also consistent with the expected results, indicating that three target genes have been knocked out successfully.

The growth curves of the wild-type and mutant strains were almost the same, and the growth rates showed no significant changes (Fig. 5A). As shown in Fig. 5B, the transformation efficiencies of receptor cells of ΔyiaW, ΔygiZ, and ΔosmB mutants formed under 100 mM CaCl₂-mediated transformation of plasmids of different sizes were significantly greater than for wild-type E. coli DH5α (P < 0.001). The transformation efficiencies of plasmid pUC19 were 1.24, 1.68, and 1.12 times higher, respectively, than that for the wild type. The transformation efficiency of plasmid pET-32a increased by 1.58, 2.12, and 1.07 times, respectively. While the transformation efficiency of the strain to the plasmid gradually decreased with the increase of the plasmid, the transformation efficiency to plasmid p1304 increased by 1.13, 1.37, and 2.75 times, respectively. There were no positive transformants in the negative-control group treated with aseptic water of wild-type strain and mutant strain. Taken together, these results suggested that deletion of the genes yiaW, ygiZ, and osmB can increase the transformation efficiency of plasmids by receptor cells formed by E. coli DH5α mediated by 100 mM CaCl₂.

FIG 5.

(A) Growth curve of mutant strain. (B) Transformation efficiency of plasmid by mutant strain. Error bars represent the standard deviations (n = 3). ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant.

As shown in Fig. 6A, the microscopic morphology of wild-type E. coli DH5α bodies after treatment with 100 mM CaCl₂ had a dense and smooth surface with clear texture and intact morphology. Not all three mutant strains were significantly different in morphology and surface state from the control. Compared to wild-type strain E. coli DH5α (2.64, length/width) and except for the ΔygiZ mutant strain (4.32, length/width), the sizes (length/width) of the ΔygiW and ΔosmB strains were 2.54 and 2.30, respectively, which were not significantly different from that of the wild type. This showed that the change in transformation efficiency of the gene deletion strains was not only caused by the change in morphology. Flow cytometry analysis showed that, after 100 mM CaCl₂ treatment, the ΔyiaW, ΔygiZ, and ΔosmB mutant strains increased the positive cell ratio compared to wild-type E. coli DH5α by 7, 3, and 6.9%, respectively. This indicates that the genes ygiZ, yiaW, and osmB were deleted and that the intracellular membrane permeability was increased by 100 mM CaCl₂ treatment. However, there was no significant difference in intimal permeability between mutant strains and the wild-type strain (except the ΔosmB mutant) (Fig. 6B). As shown in Fig. 6C, except for the ΔyiaW mutant without a significant difference in electrical conductivity between the mutant strain and the wild type (P > 0.05), the electrical conductivities of the ΔygiZ and ΔosmB suspensions, which were 18.66 and 18.98 ms/cm, respectively, were higher than that of wild-type strain E. coli DH5α (18.54 ms/cm). As shown in Fig. 6D, there was a significant difference in intimal permeability between the ΔosmB strain and the wild type (P < 0.05). The results of the 1-N-phenyl-naphtylamine (NPN) assay on the permeability of the outer cell membrane of the strains showed that the fluorescence values of the ΔosmB mutant strain increased by 26.08% compared to the wild type (P < 0.01). The ΔygiZ and ΔyiaW strains were largely unchanged. This result indicated that deletion of the osmB gene simultaneously increased the permeability of the inner and outer membranes after treatment with 100 mM CaCl₂. In summary, the transformation efficiency and the outer membrane permeability of three mutant strains were increased by 100 mM CaCl₂ treatment. For one or more of the three strains, the morphology and surface state, the electrical conductivity, or the inner membrane permeability may play a role in improving the transformation efficiency.

FIG 6.

(A) Scanning electron microscope (×30,000). (a) Wild-type strain E. coli DH5α; (b, c, and d) ΔyiaW, ΔygiZ, and ΔosmB mutant strains, respectively. (B) Flow cytometry analysis chart. (C) Measurement of electrical conductivity of membrane cells. (D) Fluorescence determination of NPN in membrane cells. Error bars represent the standard deviations (n = 3). ***, P < 0.001; *, P < 0.05; ns, not significant.

DISCUSSION

Ca²⁺ mediates the formation of competent cells of E. coli.

Under normal conditions, the intracellular Ca²⁺ concentration is maintained at the micromolar level; high concentrations of Ca²⁺ can cause damage to the cell itself, and concentrations that are too high can lead to cell death. For eukaryotic cells, Ca²⁺ is an important second messenger in the cell and is involved in the mediation of many physiological processes, such as cell cycle, material transport, motility, gene expression, and metabolic activities (19–21). In the case of prokaryotes, Ca²⁺ is also of great significance to the cell. It has been shown that Ca²⁺ affects a variety of bacterial cell biochemical processes, such as steamed endospore formation, chemotaxis, material transport, and cytotoxicity. Bacteria can maintain intracellular calcium homeostasis and are able to produce Ca²⁺ responses instantaneously under conditions of nitrogen deficiency, environmental stress, and carbon metabolism (22–25). These and several other findings suggested that Ca²⁺ plays an important physiological regulatory role in bacterial cells (22). We found that 100 mM Ca²⁺ is much higher than the normal intracellular Ca²⁺ concentration and is a stress for the cell, which enters a competent state and has a strong capacity to take up exogenous DNA. Studies have shown that Ca²⁺-mediated formation of receptor cells in E. coli is a complex process accompanied by physical, chemical, and biometabolic changes (26). When the outer cell membrane contacts Ca²⁺, it excites a series of physicochemical changes, which are eventually translated into biological signals, causing an inward flow of Ca²⁺ that can subsequently stimulate a series of intracellular stress responses. Xie et al. modeled the metabolic heat output during the establishment of the competent state in E. coli by the calcium chloride method and concluded that the Ca²⁺-mediated establishment of the competent state in E. coli likely has its own intrinsic signal response regulatory mechanism and is not a simple physicochemical change process (27). It has also been suggested (i) that bacteria swell into a spherical shape in hypotonic calcium chloride solutions, (ii) that negatively charged DNA and lipopolysaccharide molecules on the cell membrane surface form covalent complexes with positively charged Ca²⁺ under electrostatic action, and (iii) that the flow of phospholipid molecules on the cell membrane surface slows down and pores form on the cell surface under the effect of a short period of thermal stimulation, facilitating cellular uptake of the DNA complexes (28). In this study, we investigated the changes in the transcriptional and translational levels of E. coli DH5α mediated by 100 mM Ca²⁺. The transcriptional results indicated that the Ca²⁺-mediated establishment of the competent state in E. coli was accompanied by changes in the expression patterns of 333 genes involved in cellular signaling, material transport, and metabolic processes. These changes may be related to the cellular efforts to control the inward flow of Ca²⁺ in the environment and the outward flow of Ca²⁺ in the cytoplasm under high Ca²⁺ stress. Although the results do not directly indicate how Ca²⁺ mediates the entry of cells into the competent state, the results provide further evidence that the process of Ca²⁺-mediated establishment of the competent state in E. coli involves the regulation of intracellular mechanisms rather than purely physicochemical changes.

Organisms achieve their gene expression processes mainly through gene transcription, protein synthesis, and the formation of metabolites. Therefore, there should theoretically be a high correlation between transcriptome and proteome data obtained from cells treated with the same conditions (29). In this study, the relationship between the 100 mM Ca²⁺-mediated differentially expressed mRNA and protein in E. coli DH5α showed that 503 transcripts were differentially expressed in both, indicating that most of the differential transcripts were differentially expressed in the proteomic data. The proteome differed to a greater extent relative to the transcriptome, and the transcriptome data differed to a lesser extent. Most of the differential proteins were consistent between the two, with a few differences. Analysis of log₂ scatter correlation density plots for differential proteins and differential transcripts showed that the proportion of both is mostly around 0, with more in the lower left and upper right corners (consistent transcriptional and protein up- and downregulation relationships) relative to the upper left and lower right corners (inconsistent transcriptional and protein up- and downregulation relationships), indicating a greater degree of consistency in differential expression trends between the two. Transcriptomic and proteomic association analysis uncovered a total of 15 genes that may be associated with the cellular response to Ca²⁺ under action (Table 1). These genes are involved in amino acid transport, lipoprotein synthesis, and transmembrane transport. However, these genes were not differentially expressed with ppdD, ybaV, yacI, pppA, hofB, hofC, and ompC that may respond to Ca²⁺ predicted by previous researchers, and it was therefore impossible to determine whether these genes are related to the E. coli response to Ca²⁺ from this study. Based on the transcriptome and proteome results presented above, we think that the formation of E. coli competent cells mediated by Ca²⁺ is not a simple physical and chemical process, but a biological process of a series of complex signal responses, and that there may be a molecular mechanism of Ca²⁺ response in E. coli DH5α cells.

Gene mining in the process of E. coli competent cell formation mediated by Ca²⁺.

Natural gene conversion is generally considered to be a process of stable uptake of exogenous DNA by host cells and is an important mechanism of gene level transfer, which is widely distributed in nature, but the transformation efficiency of this natural transformation phenomenon is very low (30, 31). The transformation efficiency of artificial competent DNA prepared by researchers with CaCl₂ can reach 5 × 10⁶ to 2 × 10⁷ CFU/μg DNA, which greatly improved the transformation efficiency (32). However, until now, the mechanism of how E. coli responds to the induction of the divalent metal ion Ca²⁺ to form a competent state and thus complete the transformation of exogenous DNA has been unclear. The whole genome of E. coli contains more than 4,000 open reading frames, approximately 10% of which encode outer membrane secretory proteins and 25% encode inner membrane proteins, which mainly function to maintain cell structure and material transport (33). Compared to Gram-positive bacteria such as B. subtilis and S. pneumoniae, the transformation mechanism of Gram-negative bacteria such as E. coli, H. influenzae, and N. gonorrhoeae is not in response to the quorum sensing of signal molecules secreted by cells in the medium but in response to external stimuli (pH change, temperature change, and divalent metal ions such as Ca²⁺, Ba²⁺, etc.), Although different species respond differently to specific stimuli, the transformation of Gram-negative bacteria is ultimately a physiological regulation process and a signal transduction gene level regulation process (26). It was found that GspD, HofQ, PpdD, YacI, and DprA proteins were not essential in plasmid transformation of E. coli. Aich et al. used bidirectional protein electrophoresis to study the differential proteins before and after 100 mM CaCl₂ treatment of E. coli and MALDI-MS analysis to sequence the OmpC and OmpA proteins (34). At the same time, the transformation efficiency of the pUC19 plasmid after overexpression of OmpC and OmpA proteins was analyzed by mutation and overexpression technique, and it was found that the transformation efficiency of the ompC gene deletion strain decreased by 40%. Moreover, it was found by using immunoprecipitation that heat shock protein GroEL could cooperate with OmpC protein overexpression to promote the efficient transformation of plasmid (35). Alshabib (36) used mutant construction, pulldown, gel blocking, and chromatin immunoprecipitation techniques to systematically study the transcriptional regulation mechanism of the key gene sxy during natural transformation of the Gram-negative bacteria Salmonella spp. and E. coli under starvation/nutrient deficiency. The regulatory factors (CRP and pilA) and transcriptional activation sites involved in sxy gene transcription were screened. The regulatory factors (CRP and pilA) gene was not only involved in the regulation of competent genes but also involved in the regulation of glucose metabolism, DNA replication, and small RNA and multiple transcription factors. Therefore, mining the key genes involved in the transformation process and analyzing their regulatory mechanisms still represent a key research direction for the future. The purpose of this study was to explore the mechanism of action of Ca²⁺ on E. coli DH5α at the molecular level. Based on the results of transcriptome and proteome analysis, 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.

In this study, transcriptome and proteome techniques were used to analyze the changes in transcription and protein level of E. coli DH5α competent cells mediated by 100 mM Ca²⁺. Bioinformatics correlation analysis was used to mine the correlation information between transcriptomes and proteomes to detect the genes and proteins related to Ca²⁺ mediation. Red homologous recombination was used to delete the three genes that were the most differentially expressed in order to investigate the effect of related genes on the Ca²⁺-mediated formation of E. coli DH5α competent cells. We found that Ca²⁺-mediated processes in E. coli DH5α competent cells are not simple physicochemical processes but rather a biological process with a complex series of signal responses. A molecular Ca²⁺ response mechanism may exist in E. coli DH5α cells. Through correlation analysis of transcriptome and proteome, cell membrane components, transmembrane transport, and response to stimulus were the major functional terms related to Ca²⁺-mediated responses in E. coli. Fifteen highly expressed genes, including yiaW, ygiZ, and osmB, related to these functional terms may play a key role in the process of cell response to Ca²⁺. The transformation efficiencies of yiaW, ygiZ, and osmB deletion strains for different-size plasmids were significantly increased. Not all gene deletion strains changed in size, but they all exhibited different membrane permeability and transformation efficiencies. Our study lays the foundation for an in-depth reveal of the molecular mechanism on Ca²⁺-mediated transformation.

MATERIALS AND METHODS

E. coli strains, plasmids, and reagents.

E. coli DH5α, plasmid pUC19 (Amp^r, 2,686 bp), plasmid pET-32a (Amp^r, 5,900 bp), and plasmid pCAMBIA1304 (p1304 Kan^r, 12,362 bp) all came from Yonggang Wang’s laboratory at Lanzhou University of Technology, Lanzhou, China. Plasmid pKD46 (Amp^r, 6,329 bp), plasmid pCP20 (Amp^r and Cm^r, 9,332 bp), and plasmid pKD4 (Amp^r and Kan^r, 3,267 bp) were purchased from Wuhan Miaoling Biotechnology Co., Ltd. The SanPrep column plasmid DNA extraction kit and the SanPrep column PCR product purification kit were purchased from Sangon Biotech (Shanghai) Co., Ltd.

Preparation of E. coli competent cells, CaCl₂ treatment, and transcriptome and proteome analysis.

E. coli DH5α competent cells were prepared by the CaCl₂ method (37). We inoculated 1 mL of Luria-Bertani (LB) liquid medium with E. coli DH5α and placed this in a shaking incubator at 37°C and 200 rpm, followed by incubation for 12 to 16 h. Next, we added 1 mL of overnight culture to 99 mL of fresh LB medium (1:100 dilution, no antibiotics), followed by shaking and incubation at 37°C and 200 rpm for 3 to 4 h or until the optical density at 600 nm (OD₆₀₀) reached 0.4. Then, we separated the culture into multiple Oakridge tubes and placed the samples on ice for 20 min. This was followed by centrifugation at 4°C at 4,000 rpm for 10 min. Next, we discarded the supernatant by tipping the tubes over a discard bin and aspirating away any remaining media. Next, we resuspended each pellet with 20 mL of ice-cold 0.1 M CaCl₂, followed by incubation on ice for 30 min. This was followed by centrifugation at 4°C at 4,000 rpm for 10 min. The supernatant was discarded, and the pellets were combined by resuspension in 5 mL of ice-cold 0.1 M CaCl₂ with 15% glycerol. The samples treated with 100 mM CaCl₂ solution were used as the test group (TEST), and the samples treated with aseptic water instead of CaCl₂ solution were used as the control group. There were three repeats for each group of samples (CK). All samples were transported to Guangzhou Chideo Biology Company on dry ice for transcriptome and proteome sequencing analysis. All samples were transported to Guangzhou Chideo Biology Company on dry ice for transcriptome and proteome sequencing analysis.

qRT-PCR verification.

The design of qRT-PCR primers is shown in Table S5. The total RNA of the treated group and the control group was extracted by BioTek high purity RNA extraction kit. The quality of RNA was detected by formaldehyde denaturing gel electrophoresis. The extracted RNA was quantified by using an ultramicroquantitative instrument to ensure that the amount of template added in the reverse transcription system was the same, and reverse transcription was carried out according to the instructions of TaKaRa reverse transcription kit (PrimeScript RT reagent kit with gDNA Eraser [Perfect Real Time]). qRT-PCR was performed according to the instructions of the TaKaRa real-time quantitative PCR kit (SYBR Premix Ex Taq II [Tli RNase H Plus]), using the 2^ΔΔCT algorithm.

Construction of mutant strains and confirmation of results.

On the basis of the above research, we used Red homologous recombination technique to knock out ygiZ, yiaW, and osmB genes in E. coli DH5α and then constructed three ΔyiaW, ΔygiZ, and ΔosmB mutants. The theoretical FRT-kan-FRT lengths of ygiZ, yiaW, and osmB gene targeting fragments were 1,766 bp, 1,846 bp, and 1,771 bp, respectively (38). We used plasmid pKD4 as the template for PCR amplification of targeting fragments, and the primers for targeting fragments are shown in Table S6. E. coli DH5α/pKD46 cells and E. coli DH5α/pKD46 competent cells were prepared according to the method of Jeon et al. (39). Finally, the three ΔyiaW, ΔygiZ, and ΔosmB mutants were constructed by homologous substitution of the target gene and stored in glycerol.

Determination of growth curve and transformation efficiency of mutant strain.

The growth curves of the activated wild-type strain, E. coli DH5α, and the three ΔyiaW, ΔygiZ, and ΔosmB mutants were determined. We added 1 mL of overnight cultures of these bacteria to 99 mL of fresh LB medium, followed by incubation with shaking at 37°C and 180 rpm. The OD₆₀₀ values of these bacterial solutions at 0, 2, 4, …, 24, 35, 40, 45, 50, 55, and 60 h were measured by using a UV-Vis spectrophotometer. Plasmids pUC19, pET-32a, and p1304 of the wild-type and mutant strains were extracted by using a SanPrep column plasmid DNA small extraction kit (C930KB3851). After successful transformation, the transformation efficiency (TE) of each plasmid was calculated according to the following equation:

$$\text{TE\hspace{0.17em}}(\text{CFU/}\mu \text{g)}=\frac{D\hspace{0.17em}\times \hspace{0.17em}C\hspace{0.17em}\times \hspace{0.17em}{T}_v}{\hspace{0.17em}{B}_v\hspace{0.17em}\times \hspace{0.17em}Q}$$

where D is the dilution degree, C is the plate colony number, T_v is the volume of conversion solution (μL), B_v is the volume of coating bacterial solution (μL), and Q is the plasmid quality number (ng).

Microscopic morphology observation of mutant strain.

According to the methods of Tong et al. (40) and Zhang et al. (41), competent wild-type and three mutant strain cells were observed by using a scanning electron microscope (JSM-6701F; Japan Electron Optics Company).

Study on the characteristics of cell membrane of mutant strain.

According to the methods of Zhang et al. (41) and Saravanan et al. (42), the cell membrane permeabilities of wild-type and mutant strains were determined by flow cytometry (FACSAria II; BD Company, USA). The wild type and the three mutant strains were used to prepare competent cells with 100 mM CaCl₂. The concentrations of these bacteria were adjusted to ~10⁸ CFU/mL. A final concentration of 50 μg/mL PI was added, followed by incubation at 37°C for 30 min protected from light, and then the samples were assayed by flow cytometry. These bacteria were treated with 0.1 M PBS to 10⁸ CFU/mL, and the conductivity was measured with a conductivity meter. We added a final concentration of a 10-μmol/L NPN solution to the treated suspension, followed by light shaking for 2 min at 37°C, and measured the fluorescence values (excitation wavelength, 350 nm; emission wavelength, 420 nm) with a fluorescence spectrophotometer (43).

Statistical analysis.

All data were analyzed by one-way analysis of variance (ANOVA) using SPSS statistical software (v13.0; SPSS, Inc., Chicago, IL). Significant differences were tested using one-way ANOVA, where a P value of <0.05 was considered significant. All graphs were plotted using OriginPro 2021 (learning version).

Data availability.

All data have been uploaded to the Sequence Read Archive database (SRP146061) of the National Center for Biotechnology Information (submission ID SRX4084295 and SRX4084294).