Development and mechanism exploration of a quantitative model for Escherichia coli transformation efficiency based on ultrasonic power

Abstract

Ultrasonic-mediated plasmid transformation is a promising microbial transformation strategy with broad application prospects that has attracted interest across various fields. Limited research exists on developing a quantitative model to understand the relationship between transformation efficiency and ultrasonic power. Within the ultrasonic range that did not damage plasmids, the maximum transformation efficiency reached at 4.84 × 10⁵ CFU/μg DNA. A kinetic model based on changes in membrane permeability was utilized to determine the membrane permeability at different power levels. The results indicated a linear correlation between ultrasonic power, transformation efficiency, and membrane permeability within a specific range. A quantitative relationship model was established based on ultrasonic power and transformation efficiency in E. coli. Electron microscopy revealed that E. coli cells subjected to ultrasonic treatment exhibited pore formation and cellular expansion. Furthermore, the integrity of the bacterial membrane was compromised as ultrasonic power increased. Nine genes associated with the functional terms of cell membrane components and transmembrane transport were identified in E. coli DH5α. According to qRT-PCR results, genes with these functions (including cusC, uidC, tolQ, tolA, ompC, yaiY) play crucial roles in ultrasound-mediated transformation of E. coli DH5α. This study suggested that ultrasound-mediated transformation in E. coli DH5α is not a simple physical–chemical process but rather involves the regulation of responsive membrane-related genes. This research establishes the groundwork for future comprehensive investigations into the molecular mechanism of ultrasound-mediated transformation and provides insights for the application of ultrasound technology in genetic engineering and related fields.

1. Introduction

The process of DNA transformation is a crucial technique in the field of molecular biology. Many bacteria are unable to naturally uptake exogenous DNA due to the negative charge carried by both the DNA and the cell membrane. Overcoming the electrostatic repulsion between DNA and the cell membrane, as well as the requirement for pores on the cell membrane for DNA entry, are key challenges in the natural uptake of DNA by cells [1]. Consequently, the entry of DNA into cells under natural conditions poses significant difficulties. The most commonly used methods for gene transformation include chemical transformation and electroporation, which are typically applied to facilitate the transformation of E. coli cells. Chemical transformation involves the utilization of microorganisms at specific growth stages, low-temperature operations, treatment with chemical reagents (CaCl₂/MgCl₂), and brief heat treatment, all of which require stringent environmental condition [2]. Additionally, conventional chemical methods are not practical for eukaryotic transformation. The technique of electroporation involves exposing cells to a high-voltage electric field environment, resulting in irreversible perforation of the cell membrane. This process ultimately leads to the leakage of cellular contents and potentially causing severe damage and even cell death [3].

The beforementioned methods require the repeated creation of competent cells before transformation and are challenging to complete large sample transformations in a single attempt. The fundamental principle of ultrasonic transformation method involves generating energy through low-frequency ultrasonic waves (20–100 kHz) to form gas cavities and microbubbles in the liquid, which encapsulate large biological molecules [4]. Ultrasonic transformation generates two primary forms of cavitation: stable cavitation and instantaneous cavitation. Stable cavitation formed physically balanced microbubbles within the liquid, and continuous acoustic cycling does not disrupt the physical equilibrium of these microbubbles [5]. During the formation process, the microbubbles can encapsulate nucleic acids, polysaccharides, and various other biological macromolecules in solution. In contrast, the microbubbles generated in the liquid due to instantaneous cavitation exist in a highly unstable physical state [6]. Numerous unstable microbubbles will continuously expand and eventually rupture under the sustained action of ultrasound. Simultaneously, the energy released from the microbubble rupture leads to the formation of numerous reversible nanochannels in the bacterial cell membrane [7]. Biomacromolecules can be incorporated into cells via the nanochannels present in cell membranes, thereby facilitating the processes of “uptake” or transformation. Ultrasound has emerged as a novel method for promoting gene transfection or transformation. Ultrasonic transformation has garnered significant attention in the field of medical biology due to its smaller biological damage and simplified operational methods. Furthermore, ultrasonic transformation allows for the simultaneous processing of numerous samples under consistent experimental conditions, offering distinct advantages for industrial applications [8].

Song et al. found that the application of a 40 kHz, 70 W ultrasound-mediated method can successfully transform certain Gram-negative bacteria. The transformation efficiency was reported to be 9 times higher than that of chemical transformation and 4 times higher than that of electroporation [9]. This preliminary evidence demonstrates the feasibility of ultrasound-mediated exogenous DNA transformation in prokaryotes. Song et al. has revealed that a specific range of ultrasonic frequencies can influence the efficiency of gene transformation. Low-frequency ultrasound-mediated transformation results in higher efficiency. This phenomenon is attributed to the increased transient cavitation generated by low-frequency ultrasound compared with high-frequency ultrasound, as well as the greater energy release from the rupture of individual microbubbles [10]. Zheng et al. investigated the influence of various factors on the efficiency of plasmid transfer into E. coli facilitated by low-frequency, low-intensity ultrasound. The ultrasound intensity was varied between 0.13 W/cm² and 0.51 W/cm², revealing that transformation efficiency improved with increasing ultrasound irradiation intensity. However, a decline in transformation efficiency was observed when the irradiation intensity reached 0.72 W/cm² [11].

Deeks et al. investigated the impact of six different plasmid sizes on the efficiency of bacterial transformation facilitated by shock waves and the integrity of DNA [4]. Lin et al. delivered pIKM2 to the thermophilic Gram-positive anaerobic bacterium X514 with a transformation efficiency of 66,102 transformants/µg DNA [12]. Wang et al.'s study reported that ultrasound can create nanochannels in cell membranes and walls, facilitating the successful introduction of exogenous DNA into host cells. Nonetheless, optimal expression of exogenous DNA in host cells was achieved only when ultrasound was applied under gentle conditions [13]. Wang and colleagues investigated the impact of ultrasonic power, ultrasonic treatment duration, microbial growth stage, washing buffer, and the presence of Mg²⁺/Ca²⁺ on the transformation efficiency. The maximum transformation efficiency of 3.24 × 10⁵ CFU/µg was achieved under the conditions of optimal ultrasonic power at 130 W, optimal ultrasonic treatment duration of 12 s, and in the presence of Mg²⁺. The changes in cell membrane permeability during microbial transformation mediated by ultrasound were observed through scanning electron microscopy. Pores were observed to form on the surface of bacteria, and these pores were found to be reversible. This study represents the first report of ultrasound-mediated microbial transformation observed through scanning electron microscopy [14].

The cell membrane is a selectively permeable biological membrane located at the outermost layer of cells, providing a relatively stable internal environment during the cell's life processes. The selective permeability of cells enables them to selectively retain, accept, or expel certain substances. Changes in the permeability of host cell membranes directly determine whether exogenous DNA can successfully and efficiently enter host cells [15]. Li et al. analyzed changes in membrane permeability under the influence of ultrasound using flow cytometry. They suggested that the outer membrane may be the primary target of ultrasound treatment. With increasing treatment duration, the inner membrane may become unstable [16].

Low-frequency ultrasonic treatment can cause microdamage to the surface of the cell membrane, leading to localized rupture of the cell wall. This change enhances the permeability of the cytoplasmic membrane, resulting in the leakage of intracellular material through the cell membrane [17]. Lu and colleagues conducted experiments on yeast cells using ultrasonic treatment under various conditions. They observed that the permeability of the cell membrane was most significantly altered when exposed to ultrasonic power of 500 W for 225 s. While the cells experienced some damage, it did not result in cell death. Eventually, the cells returned to their normal state after a certain period of time [18]. Tachibana et al. utilized an ultrasonic intensity of 48 W to irradiate HL-60 cell suspension and found that ultrasound affects the structure of the cell membrane's lipid bilayer, leading to increased membrane permeability. This enhancement allows cytarabine (Ara-C) to easily enter HL-60 cells (leukemia cancer cells), thereby enhancing cytotoxicity [19]. Lu et al. utilized the changes in the permeability of E. coli cell membranes under ultrasonic action to make reasonable assumptions. They established a kinetic model to study the changes in membrane permeability of E. coli under ultrasonic action by applying the theory of propagation and the universally accepted principle of mass conservation [18]. This model investigated the variations in target component concentrations over time and ultrasonic intensity, revealing the mechanism of membrane permeability changes under ultrasonic action. Rems demonstrated that ultrasonic waves can alter the permeability of cell membranes to varying degrees in biological cells. These changes in membrane permeability represent a complex process involving both the exocytosis of intracellular substances and the endocytosis of extracellular substances. Although the impact of ultrasound on cell permeability has been investigated, there has been no exploration of the quantitative relationship between transformation efficiency and ultrasound power [20].

This study utilized a membrane permeability kinetic model established based on the universally accepted mass transfer theory and the law of mass conservation [21]. Additionally, fluorescence staining experiments on E. coli were conducted to quantify membrane permeability under different ultrasonic power levels [22]. By elucidating the patterns of membrane permeability changes under ultrasonic influence, the study summarized the quantitative relationship between transformation efficiency and membrane permeability. The intricate kinetic process of cell membrane permeability and transformation efficiency was examined, offering insights into the mechanistic process through which cells were influenced by ultrasound in relation to transformation efficiency. A quantitative relationship model was established based on ultrasonic power and plasmid transformation efficiency in E. coli. Cell membrane permeability was assessed using laser confocal microscopy, changes in cell morphology and ultrastructure were examined using scanning electron microscopy, and modifications in intracellular chemical bonds of bacterial cells were investigated using FT-IR spectroscopy. This study investigated the structural and physiological changes of bacterial cells under various ultrasonic stresses. It also examined the expression changes of E. coli membrane-related genes under different ultrasonic stresses using qRT-PCR analysis, revealing the mechanism of ultrasonic transformation. This research provides a reference for the application of ultrasound technology in genetic engineering and related fields, offering a theoretical basis for further in-depth studies.

2. Material and methods

2.1. Plasmid and culture conditions

E. coli DH5α and plasmid pUC19 were provided by the Laboratory of Life Science and Engineering, Lanzhou University of Technology in Gansu, China. E. coli DH5α was used as the recipient strain for the plasmid, and this strain was cultured in Luria-Bertani (LB) medium at 37 °C. Plasmid pUC19 carries the ampicillin resistance (Amp^r) gene, enabling the selection of cells containing the pUC19 plasmid using medium containing ampicillin (100 μg/mL).

2.2. Measurement of the survival rate of E. coli DH5α cells

E. coli DH5α was cultured in LB liquid medium until it reached the logarithmic growth phase, with an OD₆₀₀ ranging between 0.5 and 0.6 [13]. The bacteria were immersed in a water bath at the center of an ultrasonic device (Dual-frequency ultrasonic cleaner operates at 40 kHz with adjustable power from 180 to 600 W) and exposed to ultrasonic waves at room temperature for a duration of 0 to 20 s. Cells treated under various ultrasonic conditions were diluted to 10⁻⁴ and 10⁻⁵ folds, plated separately, and then incubated at 37 °C for 16 h. Bacterial colony counts were determined using the plate counting method. The survival rate (%) was calculated as the number of viable cells in the sample after ultrasonic treatment divided by the number of viable cells in the sample.

2.3. Plasmid DNA integrity assessment

To achieve optimal transformation efficiency, plasmid DNA must maintain its supercoiled (SC) and open circular (OC) forms [17]. Shear stress generated by ultrasound can cause significant structural damage to plasmid DNA. Introducing structural changes through shear stress can ultimately convert supercoiled (SC) plasmid DNA into open circular (OC), linear, or fragmented DNA [23].

The impact of ultrasonic waves on the delivery of plasmid DNA was tested by subjecting the plasmids to ultrasonic treatment at varying power levels of 180 W, 240 W, 300 W, 360 W, 420 W, 480 W, 540 W, and 600 W for durations ranging from 0 to 20 s. Following ultrasonic exposure, the samples were loaded onto a 0.8 % agarose gel containing ethidium bromide for gel electrophoresis, which was conducted at 100 V for 30 min. Then, the plasmid integrity results were visualized under UV light. Subsequently, the electrophoresis results were analyzed using Image Lab software to determine the relative quantification of plasmids after ultrasonication.

2.4. Establishment of ultrasound-mediated plasmid transformation system

E. coli DH5α was cultured in LB liquid medium until it reached the logarithmic growth phase. The cell pellet was then collected, washed twice with physiological saline, and resuspended in a pre-chilled CaCl₂ solution (0.1 mol/L). Plasmid pUC19 (final concentration 1 ng/μL) was added to the cell suspension, followed by sonication (40 kHz, power ranging from 180 W to 600 W). Subsequently, LB liquid medium was added, and the mixture was incubated at 37 °C for 1 h. A volume of 100 μL of the mixture was spread on LB-Amp (100 μg/mL) agar plates and incubated overnight at 37 °C for transformation analysis. Three replicates were performed for each group, and the number of transformants per 1 μg of transforming DNA was calculated as the transformation efficiency. It was ensured that the plasmid concentration was consistent in each transformation system [24]. Illustration of the bacterial DNA transformation process was facilitated by ultrasound (Fig. 1A).

Fig. 1.

The ultrasound-induced DNA transfer process and the model of cell membrane permeability were illustrated. (A): The process of ultrasound-induced DNA transfer. (B): Cell membrane permeability model. E- concentration of intracellular fluorescein; F- concentration of extracellular fluorescein; k₁- rate constant of fluorescein loss caused by cell death (constant value); k₂- rate constant of fluorescein permeating from extracellular to intracellular space (constant value); k₃- rate constant of fluorescein permeating out of intracellular and then re-permeating into intracellular space (constant value), For the materials and ultrasound equipment used, k₁, k₂, k₃, and T are all constant values.

2.5. Variations in membrane permeability and the establishment of a kinetic model of E. coli DH5α under different ultrasonic conditions

2.5.1. Measurement of cell membrane permeability

The fluorescein diacetate (FDA) staining method was employed to characterize the impact of ultrasound on cell membrane permeability [25]. To prepare a storage solution of FDA with a concentration of 2 mg/mL in acetone, it was stored at 4 °C in the dark. E. coli DH5α cells in the logarithmic growth phase were cultured and then subjected to ultrasonication under various power conditions after being resuspended in pre-chilled CaCl₂ solution, as detailed in Section 2.4. Subsequently, the FDA solution was added to the cell suspension to achieve a final FDA concentration of 0.25 mg/mL. The mixture was then maintained at 20 °C for 5 min, followed by immediate observation using a fluorescence microscope and analysis using a fluorescence spectrophotometer with a maximum excitation wavelength of 269 nm and an emission wavelength of 517 nm [26].

2.5.2. Laser confocal microscopy observation

The strain treatment procedure was conducted as described in Section 2.5.1, with cells incubated in the dark at room temperature or 37 °C for 15–30 min. Subsequently, cells were washed three times with PBS buffer or distilled water for 5 min each to remove excess dye. The cells were then pipetted onto clean glass slides, dispersed evenly using a micropipette, covered with a coverslip, air-dried, and observed using a laser confocal microscope with an oil immersion lens (magnification × 100) for imaging and documentation.

2.5.3. Simulation of the kinetic model of E. coli cell membrane permeability

Based on the experimental conditions and mass transfer theory, the following assumptions are made: (1) The rate of loss of permeable substances due to cell death is a constant k₁; (2) N = kc, where permeable substances pass through the cell membrane at a constant rate. When substances permeate from the extracellular into the intracellular space, the mass transfer rate N₂ is directly proportional to the extracellular concentration F of the substance, with a coefficient of k₂. Considering on the opposite direction, when substances permeate from the intracellular to the extracellular space, the mass transfer rate N₃ is directly proportional to the intracellular concentration E of the substance, with a coefficient of k₃. The total amount of permeable substances inside and outside the cell is denoted as T, where F = T + E [18]. (3) Simultaneous permeation of substances occurs bidirectionally, i.e. the extracellular into the intracellular space and from the intracellular space to the extracellular, as illustrated in Fig. 1B [27].

According to the law of conservation of mass and the assumptions made, the following equation can be derived:

$$\frac{\mathit{dE}}{\mathit{dt}}=-k_1-k_{2}E+k_{3}F$$ (1)

The parameter k is independent of the variables t, E, and F, but it is related to ultrasonic intensity and frequency. The relationship between F, E, and T is expressed as F = T − E, where T represents the total amount of intracellular and extracellular permeating substances. Substituting this relationship into Eq. (1) yields the following result:

$$E(t)=\frac{k_{2}T-k_1}{k_2+k_3}·e^{-(k_2+k_3)t}+\frac{k_{3}T-k_1}{k_2+k_3},E(t)=y,b=-(k_2+k_3),a=\frac{k_{2}T-k_1}{k_2+k_3},c=\frac{k_{3}T-k_1}{k_2+k_3}$$ (2)

The relationship between the concentration of intracellular substances and time can be simulated as follows:

$$y={\mathit{ae}}^{\mathit{bt}}+c$$ (3)

By utilizing the fluorescence probe method, we observed changes in the fluorescence intensity of FDA over time in E. coli treated with specific ultrasonic power. Data analysis was conducted, and a kinetic model was simulated to interpret the experimental measurement results. This process derived a fitting equation for the concentration of intracellular permeable substances over time after ultrasonic treatment at different power levels.

2.5.4. Modeling the quantitative relationship among cell membrane permeability, ultrasonic power, and transformation efficiency

The penetration of substances into E. coli cell membranes occurred simultaneously with ultrasonic action. By taking the derivative of Eq. (3), Eq. (4) can serve as an instantaneous membrane permeability evaluation index. Integrating Eq. (4) provided a measure of membrane permeability over a specific time interval.

$$\frac{\mathit{dE}}{\mathit{dt}}=ab{e}^{\mathit{bt}}$$ (4)

$${\int }_0^t\frac{\mathit{dE}}{\mathit{dt}}dt={\int }_0^t\mathit{ab}{e}^{\mathit{bt}}{\left(\mathit{dt}=a{e}^{\mathit{bt}}\right|}_0^t$$ (5)

By quantifying the membrane permeability under different ultrasonic power levels based on Eq. (5) and combining it with transformation experiments of E. coli under ultrasonic treatment, a quantitative relationship model among transformation efficiency, membrane permeability, and ultrasonic power within a specific range of ultrasonic power was inferred and fitted. The R² value was calculated to observe the degree of agreement between the theoretical results curve and the experimentally fitted curve.

2.6. Analysis of Fourier transform infrared spectroscopy (FT-IR)

To explore the influence of different ultrasonic powers on the structural changes of bacteria and the peptidoglycan structure of the cell wall, E. coli was subjected to various ultrasonic treatments. Subsequently, the bacteria were rinsed and resuspended in sterile PBS buffer to obtain a bacterial suspension. The bacterial cell samples were then vacuum freeze-dried. The peptidoglycan from E. coli cell walls treated with various ultrasonic powers was extracted using the optimized TCA method [28], followed by freeze-drying and stored in a desiccator to eliminate all moisture. A small amount of the dried sample was taken, thoroughly ground with KBr powder at a ratio of 50–100 times the sample mass, and pressed into pellets using a pellet press. KBr pellets were prepared separately as blank controls. Fourier-transform infrared spectroscopy (Nexus670 FT-IR, Nicolet, USA) was used to scan and analyze the samples in the wavelength range of 4000–600 cm⁻¹. An average spectrum was obtained for each sample from three parallel replicates, followed by baseline correction processing [29].

2.7. Analysis of macromolecule content of E. coli DH5α cells before and after ultrasonic treatment

To investigate the impact of ultrasound on cellular contents, changes in cell structure and intracellular macromolecular substance content were measured respectively within a specific range of ultrasound power (180 W-420 W) before and after ultrasound treatment.

2.7.1. Analysis of protein content of E. coli DH5α cells before and after ultrasonic treatment

E. coli DH5α cells at logarithmic growth phase were subjected to different ultrasonication conditions. The suspension was centrifuged at 5000 rpm for 10 min. The supernatant was discarded, and the precipitate was collected, washed three times with distilled water, and suspended in distilled water. The absorbance values at 595 nm of each suspension were measured using the Bradford method [30]. The regression equation for the standard curve of protein content in Bovine Serum Albumin (BSA) solutions, ranging from 0 to 100 μg/mL was determined as y = 0.0053x + 0.006, R² = 0.9991, indicating its suitability for quantitative analysis of protein content.

2.7.2. Analysis of alkaline phosphatase content of E. coli DH5α cells before and after ultrasonic treatment

The samples were prepared as described in Section 2.4. After cultivation, the cells were washed with distilled water and suspended in distilled water. A volume of 1.0 mL of suspension was mixed with 2.0 mL of a 0.2 % disodium 4-nitrophenylphosphate (PNPP) solution. The mixture was then incubated at 37 °C for 15 min, resulting in a yellow-colored reaction mixture [31]. The reaction was terminated by adding 0.5 mL of 13 % K₂HPO₄, and the absorbance was measured at 420 nm. Each treatment group was conducted in triplicate.

2.7.3. Analysis of phospholipid content of E. coli DH5α cells before and after ultrasonic treatment

Likewise, the samples were prepared as described in Section 2.4. Thereafter, 0.5 mM 1,6- Diphenyl-1,3,5-Hexatriene (DPH) was added into the suspension. The sample was diluted 15 times with 100 mM CaCl₂, incubated in the dark at room temperature for 40 min, and the baseline fluorescence was measured using excitation and emission wavelengths of 350 nm and 440 nm, respectively. Each treatment group was performed in triplicate [32].

2.8. The effect of ultrasound on the structure of E. coli DH5α

2.8.1. Characterization of E. coli DH5α cells structure before and after ultrasonic treatment by scanning electron microscopy

E. coli DH5α bacterial suspension at the logarithmic growth phase was centrifuged at 3000 rpm for 5 min. The supernatant was discarded, and the cell pellet was washed twice with PBS buffer before being resuspended in PBS buffer. One portion of the suspension was subjected to ultrasonication at different power levels, while another portion served as a control. Subsequently, the suspensions were centrifuged, the cell pellets were collected, and fixed in 2.5 % glutaraldehyde for 2 h. The cell precipitates were resuspended in sterile water three times to wash the bacteria, followed by sequential washes with ethanol concentration gradients (30 %, 50 %, 70 %, 80 %, 90 %, 100 %) and tert-butanol, before being resuspended in a tert-butanol solution. Finally, the samples were vacuum-dried in a freeze dryer pre-cooled to −10 °C and observed using a scanning electron microscope (SEM) [29]. The scan analysis of the viewing area involved utilizing elemental analysis functionality was conducted to observe changes in elements content.

2.8.2. Atomic force microscope observation

To enhance the assessment of ultrasound's effect on bacterial cells, high-resolution imaging and mechanical measurements were performed through atomic force microscopy (AFM). E. coli cells were treated following the procedure outlined in Section 2.8.1. A 2.5 % glutaraldehyde solution was added to the cell sediment at room temperature, followed by fixation for 15 min. Subsequently, 20 μL of the bacterial suspension was dropped onto a natural mica substrate, left to air dry, and analyzed using tapping mode AFM to assess the morphological changes in E. coli DH5α cells before and after ultrasound treatment. The scanning area measured 10 μm × 10 μm, and AFM was utilized to capture morphology images, phase images, and 3D images for qualitative examination of cell surface characteristics, facilitating the direct visualization of surface microstructures. The images were processed using Gwyddion software without any additional modifications.

2.9. Real-time quantitative PCR validation

To identify membrane-related regulatory genes involved in the ultrasonic transformation process of E. coli, nine membrane-related genes were screened from E. coli membrane genes, and specific primers were retrieved using BLAST (https://blast.ncbi.nlm.nih.gov/Blast). Real-time quantitative primers are presented in Table S1. The experimental group underwent various high-power ultrasonic treatments after pre-cooling CaCl₂ suspension for cell precipitation, while the control group was treated with sterile water. Following the procedures outlined in Section 2.4, high-purity RNA was rapidly extracted from the treated and control strains using the Bio Teke RNA extraction kit. Total RNA was reverse transcribed into cDNA using the reverse transcription kit (Honor™ II 1st strand cDNA synthesis Super Mix for qPCR (gDNA digester plus)), ensuring equal template amounts in the reverse transcription system. PCR reactions were performed using the real-time quantitative PCR kit (Unique Aptamer™ qPCR SYBR® Green Master Mix (Low Rox Plus)) with the following program: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s. The relative expression levels of the target genes were determined using the 2^−ΔΔCt method, with 16S rRNA as the reference gene, and all tests were performed at least three times [33].

2.10. Statistical analysis

All experiments were conducted with three replicates, and the data were presented as the mean ± standard deviation. Experimental data were input into SPSS 20.0 statistical software for one-way analysis of variance and Tukey's test to determine significant differences. A significance level of P < 0.05 indicates the presence of significant differences, while P > 0.05 indicates no significant differences.

3. Results and discussion

3.1. Measurement of survival rate of E. coli

To evaluate the potential harmful effects of ultrasonic treatment on cells, E. coli cells were exposed to ultrasonic treatment from 0 to 20 s and subsequently plated on LB solid agar plates. E. coli survival rates under the influence of ultrasound were illustrated in Fig. 2. The findings indicated that within the specified ultrasonic treatment duration, with power ranging between 180 W to 480 W, the survival rate of E. coli cells exceeded 90 %, with no compromise to cell viability. However, at two higher intensity power levels (540 W, 600 W), ultrasonic treatment exhibited significant inhibitory effects on bacterial growth. This suggested that the cell membrane may undergo irreversible structural changes under high-intensity treatment, even leading to cell damage. Interestingly, in some instances, there was an increase in the number of viable cells detected, possibly due to the dispersion of cells that had briefly aggregated during ultrasonic treatment. These data implied that ultrasonic treatment within the range of 180 W to 480 W can effectively deliver plasmid DNA to E. coli cells without causing cellular damage [10].

Fig. 2.

Survival rate of E. coli under various ultrasonic conditions. Viable colonies were determined by plating cells on agar plates. The percent viability (survival rate) was calculated as described in Material and methods.

3.2. Analysis of plasmid damage under various ultrasonication conditions

To mitigate the stability issues of plasmids caused by ultrasonic shearing forces, the impact of ultrasound on the structural changes of plasmid DNA was initially investigated (Fig. S1). After enzymatic digestion of the pUC19 plasmid, gel electrophoresis revealed a structurally intact band of the appropriate size, displaying a single band (Fig. S1, Lane 2). In the absence of ultrasonication treatment, the plasmid DNA was present as a mixture of supercoiled (SC) and open circular (OC) forms in the agarose gel (Fig. S1, Lane 3). The Quantity One gel analysis software was utilized to evaluate the electrophoretic bands. By assessing the intensity of the band signals, we can determine the extent of reduction in supercoiled plasmid DNA across various ultrasonication groups compared with the supercoiled plasmids in the control group. This ratio is established through a relative comparison with the non-ultrasonicated group of supercoiled plasmids, which is theoretically considered to possess 100 % integrity. This method facilitates a relative quantitative assessment of DNA integrity (Table S2). When subjecting plasmid DNA to ultrasonication with ultrasonic power below 420 W, minimal damage was observed within the first 0–20 s, with plasmid relative quantification remaining above 80 %. However, at ultrasonication powers exceeding 480 W, some DNA fragments were observed (Fig. S1, Lanes 31–42), with both SC and OC forms still present in the agarose gel. As ultrasonication power and duration increased, DNA fragmentation also increased. When the ultrasonic power reached 600 W, a significant amount of fragmented plasmid DNA was present in the agarose gel (Lanes 41–43).

The aforementioned results were consistent with previous studies, indicating that ultrasound-induced shear stress may lead to structural degradation or partial degradation of plasmid DNA [34]. As previously mentioned, an ultrasound power range of 180 W to 420 W and an ultrasound processing time of 0 to 16 s were determined. This resulted in supercoiled plasmid relative quantification exceeding 88.61 %. The results showed that under the specified ultrasonic conditions, the majority of plasmid DNA remained in a supercoiled configuration, exhibiting no significant degradation or damage. which suggested that the integrity of plasmid DNA can be maintained with high efficiency, thereby ensuring the reliability and accuracy of subsequent experimental analyses.

3.3. Transformation of E. coli mediated by ultrasound

At an ultrasonic power of 300 W, the transformation efficiency of plasmid pUC19 in E. coli varied with different ultrasonic treatments. The highest transformation efficiency (approximately 3.3 × 10⁵ CFU/μg DNA) was observed at an ultrasonic processing duration of 12 s, after which the efficiency gradually decreases. The results of the T-test analysis indicated significant differences among the groups (P < 0.05). When the ultrasonic exposure time was less than 8 s, the changes in cell structure induced by ultrasonic treatment were not significant (Fig. 3A). Consequently, the modification in host cell membrane permeability does not create enough ion channels for the uptake of exogenous DNA, leading to low transformation efficiency. When ultrasonic treatment duration exceeded 12 s, the increased shear stress caused by ultrasound may lead to physical damage to cell structures, even though the cells remain viable [35].

Fig. 3.

Efficiency of plasmid transformation under different ultrasonic conditions. (A): The X-axis denotes the treatment duration at a rated power of 300 W. The Y-axis represents the transformation efficiency (p < 0.05). (B): The X-axis denotes ultrasonic power treated for 12 s. The Y-axis represents transformation efficiency (p < 0.05).

These results showed that when the ultrasound treatment duration was 12 s and the ultrasound intensity was between 180 W and 420 W, the transformation efficiency increased withthe rise in ultrasound irradiation intensity. The transformation efficiency reached a maximum of 4.84 × 10⁵ CFU/μg DNA at 420 W, as illustrated in Fig. 3B. In the ultrasonic power range without causing structural damage to bacteria and plasmids, the permeability of the host cell membrane increases. This mechanism guarantees the replication and expression of a substantial quantity of pUC19 plasmids within the cell.

3.4. Model establishment

3.4.1. Changes in cell membrane permeability and kinetic modeling simulation

The cell membrane is a selectively permeable biological membrane located at the outermost layer of cells. It provides a relatively stable internal environment during the cell life processes. The selective permeability of cells enables them to retain, accept, or expel specific substances [36]. The FDA can be absorbed by living cells, enzymatically deacetylated to remove the acetyl group, and thus produce fluorescence, a phenomenon not observed in dead cells. FDA accumulates only in cells with intact cell membranes. An increase in cell permeability would result in the leakage of fluorescence from the interior to the exterior of the cell, leading to a reduction in FDA fluorescence intensity. Therefore, the assessment of cell membrane permeability in living cells can be reflected by utilizing the fluorescent probe FDA, which serves as an indicator of permeability. The changes in host cell permeability directly determine whether exogenous DNA can successfully and efficiently into enter host cells.

As shown in Fig. 4A, the fluorescence staining of cells with FDA after ultrasonic treatment at various power levels was conducted. Analysis of the fluorescence measurements revealed a decreasing trend in the percentage of FDA fluorescence with prolonged ultrasonic exposure duration. Furthermore, it was observed that increased ultrasonic power levels resulted in more significant reductions in fluorescence intensity. It can be inferred that an increase in ultrasonic power and prolonged ultrasonic treatment duration enhances cell membrane permeability. The figure illustrated a significant decrease in fluorescence intensity within the initial 60 s of ultrasonic treatment. This decline was primarily attributed to the disruption of cell walls and membranes following ultrasonic exposure, which leads to a substantial increase in membrane permeability. Subsequently, between 60 and 90 s, the fluorescence intensity changes at a much slower rate, possibly due to the nearing saturation of membrane permeability.

Fig. 4.

(A): Experimental values and permeability model simulations of FDA under ultrasonication; (B): Microscopic images depicting E. coli stained with FDA following various ultrasonic treatment procedures. (I): No ultrasonic treatment; (II): The E. coli sample was treated with ultrasound at 180 W for 12 s; (III): The E. coli sample was treated with ultrasound at 300 W for 12 s; (IV): The E. coli sample was treated with ultrasound at 420 W for 12 s. The laser confocal microscope had a magnification × 40.

Based on the kinetic model described above (Eq. (3), experimental results were simulated. From Fig. 4A and Table 1, the correlation coefficients R² all exceeded 0.98, indicating a good curve fit. This suggested that the equation can reflect the actual process of E. coli cell membrane changes under ultrasound treatment, indicating that the process of membrane permeability change conforms to an exponential model (Fig. 4A). The increase in ultrasound power from 180 W to 420 W was confirmed to result in an increase in the rate constant of cell membrane transmembrane transport, indicating an enhancement in cell membrane permeability. A kinetic model was developed to describe the fitted equation of the intracellular concentration of permeable substances over time after ultrasound treatment, reflecting the changes in membrane permeability.

Table 1.

Membrane permeability at different power for ultrasound treatment duration of 12 s.

Ultrasonic power	Equation	R2	Membrane permeability
180 W	y = 27.78293e−0.01302t + 72.58552	R2 = 0.99248	4.01854
240 W	y = 57.02469e−0.00971t + 53.05089	R2 = 0.99476	6.27202
300 W	y = 47.39866e−0.01542t + 53.31495	R2 = 0.98448	8.00706
360 W	y = 48.138e−0.01769t + 52.79895	R2 = 0.97288	9.20696
420 W	y = 52.63744e−0.01888t + 48.3583	R2 = 0.98645	10.6711

Note: Membrane permeability formula:${\int }_0^{12}\frac{\mathit{dE}}{\mathit{dt}}dt={\int }_0^{12}\mathit{ab}{e}^{\mathit{bt}}dt=a{e}^{12b}-a$

3.4.2. Laser confocal microscopy observation

As illustrated in Fig. 4B, the cells exhibited green fluorescence following FDA staining, and the cell surface appeared orderly and smooth. The overall observation revealed a distinct outline of the cell membrane and provided a comprehensive depiction of the bacteria's morphology. The statement suggested that the fluorescent probe has been successfully introduced into E. coli, and that the FDA fluorescence staining technique is applicable for deployment in E. coli. The fluorescence intensity diminishes with the escalation of ultrasonic power, resulting in a reduction in FDA fluorescence intensity. These changes suggested cellular damage, disruption of the cell membrane integrity, leakage of fluorescence from the cytoplasm, and increased permeability, but the cells did not die.

3.4.3. Modeling for quantitative relationship among ultrasonic power, membrane permeability, and transformation efficiency

Eq. (4) can serve as an instantaneous evaluation index of membrane permeability when integrated over a 12 s ultrasound exposure period. Based on the parameters a and b of the equation as shown in Fig. 4A and Eq. (5), the membrane permeability of E. coli over 12 s can be calculated, as illustrated in Table 1.

An analysis was performed on membrane permeability under various ultrasonic power treatments to study the correlation between membrane permeability and changes in ultrasonic power. A suitable equation was derived from the experimental data, as illustrated in Fig. 5. Within a specific range, the relationship between transformation efficiency and ultrasonic power variations conforms to the equation: y = 0.01036x + 0.36733 (R² = 0.98446) (Fig. 5A). The equation that fits the membrane permeability related to ultrasonic power is expressed as: y = 0.02707x + 0.48483 (R² = 0.98543) (Fig. 5B). The fitting equation for the transformation efficiency and membrane permeability is represented as: y = 0.37201x + 0.6331 (R² = 0.94101) (Fig. 5C). Five different ultrasound power levels were selected to validate the fitting equation, and the experimental data exhibited a high degree of conformity to the equation, with correlation coefficients exceeding 0.92 (Fig. 5D). This indicates a linear correlation among ultrasonic power, transformation efficiency, and membrane permeability within the specified range. The quantitative relationship model between transformation efficiency and ultrasonic power is expressed as T = aP + c, where “a” and “c” are constants. “T” represents transformation efficiency, and “P” represents ultrasonic power. This model was considered reliable within a specific range of ultrasonic power.

Fig. 5.

Relationship curve among transformation efficiency, membrane permeability, and ultrasonic power. (A): Equation for fitting ultrasonic power and transformation efficiency. (B): Equation for fitting ultrasonic power and membrane permeability. (C): Equation for fitting transformation efficiency and membrane permeability. (D): Validation of the fitting equation for ultrasonic power and transformation efficiency.

3.5. FTIR analysis

The changes in the composition of E. coli cells and peptidoglycan induced by ultrasound were investigated using FTIR spectroscopy (Fig. 6). The FT-IR spectra characteristics of E. coli cells and peptidoglycan treated with ultrasound at different power levels varied and corresponded to the ultrasound power exerted [37]. Overall, with increasing ultrasound power intensity, there were no significant shifts in the infrared absorption peak positions of the treated groups, but changes were observed in the spectral intensities of dominant peaks. The vibrational absorption peaks of E. coli cells and peptidoglycan were presented in Table 2.

Fig. 6.

Analysis of E. coli cells and peptidoglycans under various ultrasonic treatments using FTIR spectroscopy (A&B). (A): FTIR spectra of E. coli DH5α. after different ultrasound treatments; (B): FTIR spectra of bacterial peptidoglycan from E. coli DH5α; (C&D&E): Variations in protein, phospholipid, and alkaline phosphatase content of E. coli DH5α under different ultrasonic conditions. The ultrasonic treatment duration was set at 12 s. (C): Protein content of E. coli DH5α cells before and after ultrasonic treatment; (D): Cellular phospholipid content of E. coli DH5α cells before and after ultrasonic treatment; (E): Alkaline phosphatase content of E. coli DH5α cells before and after ultrasonic treatment.

Table 2.

Vibrational absorption peaks of E. coli cells and peptidoglycans.

Wavelength	Bacterial vibrational absorption peaks	Peptidoglycan vibrational absorption peaks
3500–3200 cm−1	–NH, –OH	–OH
3000–2800 cm−1	C–H（–CH2, –CH3）	C–H（–CH2, –CH3）
1660–1620 cm−1	–CONH2 (C = O)	–CONH2 (C = O)
1560–1520 cm−1	N–H, C = N	N–H
1500–1000 cm−1	P = O, C–N, P–OH PO43−	C–H, C–O–C, O–H

In Fig. 6A, the spectral region of fatty acids within the cell membrane showed significant increases in absorption bands around 2921 cm⁻¹ and 2850 cm⁻¹ (corresponding to symmetric and antisymmetric stretching vibrations of CH₂ and CH₃ in lipids) [38]. These enhancements indicate a weakening of intermolecular hydrogen bonding interactions between CH₂ and CH₃ functional groups. This weakening was attributed to an increase in the concentration of saturated lipids in the membrane [39], resulting in enhanced rigidity and permeability of bacterial cell membranes. These findings hold significant implications for assessing membrane fluidity levels [40]. In the context of protein structure, variations in the range of 1700.00 cm⁻¹ to 1500.00 cm⁻¹ were examined. This region corresponds to the amide I band (1650.81 cm⁻¹) and amide II band (1550.65 cm⁻¹) characteristic absorption peaks of proteins [41], which represent the vibrational bands of the protein main chain and can indicate changes in protein structure. With increasing ultrasonic power, the peak intensity gradually increases, indicating a rise in protein concentration after ultrasonication. One possible reason for this increase could be the induction of the synthesis of several membrane proteins, which is consistent with the overexpression of certain membrane biosynthesis-related genes identified through quantitative analysis. Furthermore, variations in the region of 1200.00 cm⁻¹ to 1240.00 cm⁻¹ were examined in terms of cell membrane structure. This range corresponds to the phosphate groups located at the head functional groups of phospholipid molecules. These bands correspond to the chemical bonds that form phospholipids and fatty acids, which were predominantly present in bacterial cell membranes. They serve as crucial sites for hydrogen bonding between phospholipid molecules on the cell membrane. These changes generally reflect ultrasound-induced cell membrane damage or alterations in the chemical bonds of cell membrane components [42]. The characteristic absorption peak of β-1,4 glycosidic bonds on the cell wall is observed in the region of 915.00 cm⁻¹ to 920.00 cm⁻¹, which can indicate variations in the peptidoglycan structure on the cell wall [43].

In summary, the ultrasonic effect primarily manifests in the sensitive region of microbial analysis at 1000–2000 cm⁻¹. It involves changes in the protein amide II band on the cell wall, as well as the C-N, O–H, N–H, C = O, and P = O functional groups. Additionally, alterations were observed in the methyl, methylene, and dimethyl groups on the cell membrane, indicating that the cellular structure of the bacterial strain underwent slight damage following ultrasonic treatment. These observations are consistent with the findings from scanning electron microscopy.

The infrared spectrum of the peptidoglycan extract was illustrated in Fig. 6B, showing characteristic absorption peaks of peptidoglycan without significant interference peaks. It was confirmed that the extracted peptidoglycan composition and structure of E. coli DH5α cells in this experiment conform to the standard peptidoglycan component structure and composition. The absorption peak at 1063 cm⁻¹ corresponds to the stretching vibration peak of C-O-C, potentially indicating the linkage of N-Acetylglucosamine and N-Acetylmuramic Acid through β-(1,4) glycosidic bonds. The infrared spectrum reveals the presence of α-configuration absorption peaks around 840 cm⁻¹ and β-type peaks near 880 cm⁻¹. According to Fig. 6B, the sugar extracted from the E. coli cell wall in this experiment was β-peptidoglycan. In comparison to the control, the ultrasonicated group not only exhibits certain changes in displacement but also alterations in the intensities of absorption peaks such as O–H, C–H, C = O, N–H, and C-O-C in sugars [44]. During treatment with 180 W ultrasound, there is a significant increase in the intensity of characteristic absorption peaks, including those associated with O–H, C–H, C = O, N–H, and C-O-C functional groups. As the ultrasound power increases, the variation in peak intensity diminishes, and the peak widths contract considerably. This phenomenon may be attributed to the action of 180 W ultrasound, which facilitates the disruption of the cell wall, thereby releasing a greater quantity of peptidoglycan components while preserving the integrity of most chemical structures [39]. This results in a notable enhancement of the absorption peak intensities. However, with the increase of ultrasonic power, it may lead to the degradation of certain peptidoglycan components or the cleavage of functional groups, potentially resulting in a reduction of the absorption peak intensities.

3.6. Effect of ultrasound on the content of bioactive macromolecules in E. coli cells

The impact of various ultrasonic treatments on the proteins, phospholipids, and alkaline phosphatase of E. coli DH5α was illustrated in Fig. 6 (C, D, & E). In the absence of ultrasonic treatment, proteins, phospholipids, and alkaline phosphatase were barely detectable. Upon exposure to ultrasonic waves, these three macromolecules started leaking out and rapidly releasing into the extracellular environment. With increasing ultrasonic power intensity, the release of substances into the extracellular environment gradually increased, exhibiting significant differences among the different treatment groups (P < 0.05). When the cavitation effect generated by ultrasound acts on host cells, it causes damage to the cell wall and cell membrane. Changes in the permeability of the host cell membrane and the structure of the cell wall result in the leakage of intracellular macromolecules into the extracellular environment (He et al., 2021). Under abovementioned ultrasound conditions, the survival rate of E. coli is minimally affected. This could be attributed to the relatively low level of ultrasound treatment, which causes minimal damage to host cells. The damaged host cells are more easily self-repairable, thereby providing impetus and a foundation for the process of exogenous DNA entering the cells.

3.7. E. Coli cell structure observation by SEM and AFM

3.7.1. SEM analysis

Ultrasonication has been demonstrated to enhance membrane permeability by inducing transient pore formation on the cell membrane through cavitation [45]. The membrane acts as a barrier to the absorption of exogenous or extracellular DNA. The enhancement of membrane permeability through the creation of surface pores on cells can increase transformation efficiency. Based on scanning electron microscopy, the mechanism underlying ultrasound-mediated transformation of microorganisms with an ultrasonic homogenizer involved the cavitation phenomenon, with reversible pore formation accompanied by cell expansion [14].

As shown in Fig. 7A, the surface of E. coli DH5α cells appeared smooth, uniform in shape, and exhibited a complete rod-like morphology when not subjected to ultrasonic treatment. Whereas, under ultrasonic treatment, the cell wall structure of E. coli DH5α cells was disrupted, resulting in a wrinkled and uneven cell surface, altered cell membrane permeability, and the movement of substances such as water molecules towards the extracellular space. This led to a darker appearance of the cells under electron microscopy (Fig. 7B, 7C, 7D). Most cells still maintained their original shape, with only a few showing visible pore formation and blurred cell envelopes (Fig. 7B, 7C). With an enhancement in ultrasonic power, more pores were formed on the membrane, the number of ruptured cells significantly increased, and at 420 W power, clusters of cell fragments were observed (Fig. 7D). Under abovementioned ultrasonic conditions, the integrity of the cells was largely preserved. In this scenario, the formation of pores in each cell allowed plasmids to enter into the cell.

Fig. 7.

SEM images showing changes in surface structure of E. coli DH5α cells before and after ultrasonic treatment with different power levels. (A): Cultivated normal E. coli; (B): The ultrasonic treatment was set at 180 W for 12 s; (C): The ultrasonic treatment was set at 300 W for 12 s; (D): The ultrasonic treatment was set at 420 W for 12 s. The scanning electron microscope was configured at a setting of 3 µm, with a magnification × 20000; (E): The ultrasonic treatment was set at 180 W for 12 s. Analysis of elements through scanning of the viewing area. The three viewing areas underwent scanning and analysis utilizing elemental analysis functionality, which identified elements such as Ca, P, C, and O within the sampled area via surface scanning.

3.7.2. Elemental analysis

Elemental analysis was conducted to scan and analyze the viewing area, revealing the presence of elements such as Ca, P, C, and O in the sample area. Spectral analysis of the areas was performed, and the results were presented in Fig. 7E. EDS analysis primarily evaluated the elemental composition of the surface of the sample. Consequently, the observed increase in Ca²⁺ may predominantly occur at the surface or cell wall regions of bacterial cells, Analyses of Ca²⁺ concentrations across different scanning areas indicated a marked elevation in Ca²⁺ levels in specific areas of E. coli that were substantially influenced by ultrasonic treatment. In particular, the Ca²⁺ concentration in area 3 was found to be 1.6 times greater than that in area 1 [8].The application of ultrasonic treatment disrupts the cell wall and membrane through the cavitation effect, thereby exposing additional functional groups (such as carboxyl, hydroxyl, and amino groups) that can bind with Ca²⁺, resulting in an increased attachment of Ca²⁺. Furthermore, ultrasonication may induce cell lysis, leading to the release of intracellular materials that could form precipitates or complexes with Ca²⁺, thereby contributing to the elevat d calcium levels observed in the EDS analysis (Table S3) [46].

3.7.3. AFM analysis

In contrast to SEM, AFM offers a more detailed depiction of the three-dimensional structure of the sample. This is particularly evident in applications such as imaging biological membranes and high-resolution, in situ imaging of macromolecules on membrane surfaces [47]. In this experiment, AFM was employed to investigate the impact of ultrasonic treatment on E. coli DH5α cells, displaying changes in cell structure in three-dimensional space, as shown in Fig. 8.

Fig. 8.

AFM images. of E. coli DH5α upon different ultrasound treatments. (A1-A4): The AFM topography images of normal E. coli and E. coli treated with ultrasound at power levels of 180 W, 300 W, and 420 W, respectively; (B1-B4): The AFM phase images of the identical samples of normal E. coli and E. coli treated with ultrasound at power levels of 180 W, 300 W, and 420 W, respectively; (C1-C4): the AFM 3D images of normal E. coli and E. coli treated with ultrasound at power levels of 180 W, 300 W, and 420 W, respectively. The duration of the ultrasound treatment was set at 12 s, covering a scanning area of 10 μm × 10 μm.

Untreated E. coli DH5α cells appeared a rod-shaped morphology with a smooth surface, dense internal contents, uniform shape, and low refractive height when observed through AFM imaging (Fig. 8A1, 8B1, 8C1). At 300 W, the cell surface turned blurred (Fig. 8A3, 8B3), and while deformation and roughening of the E. coli cell surface were observed at 420 W (Fig. 8A4, 8B4). After ultrasonic treatment, significant changes in cell surface morphology were evident when examined with an AFM, revealing cells with decreased density and irregular shapes. The cell membrane of E. coli DH5α showed a blurred appearance, leading to cell wall damage, altered membrane permeability, and the release of essential molecules such as phospholipids, proteins, and alkaline phosphatase. This process resulted in the leakage of intracellular contents and cell swelling (Fig. 8) [48].

The Gwyddion software facilitates the measurement of length and width parameters of cells, allowing for precise quantification of bacterial size and evaluation of bacterial morphological alterations [49]. The dimensional findings were displayed in Table S4. The length of normal cells was 1.4–1.8 µm and the width was 0.35–0.37 µm. Under the influence of ultrasound, cell length varies between 2.5 and 4 µm, with a maximum recorded length of 4.224 µm, whereas cell width ranges from 0.41 to 0.82 µm. The dimensions of E. coli cells expand when exposed to ultrasonic treatment, resulting in a significant increase in volume.

3.8. qRT-PCR validation

To investigate the cell membrane-related regulatory genes involved in the ultrasonic transformation of E. coli, nine membrane-related genes encoding membrane proteins were identified from E. coli genes (Fig. 9A), as shown in Table S5. Among these, six genes were associated with the outer membrane, including genes encoding outer membrane pore protein L, outer membrane porin C (OmpC), and putative β-glucuronate outer membrane porin (UidC). Two genes were related to membrane structure, and one gene was associated with the inner membrane, such as the gene encoding the DUF2755 family inner membrane protein (YaiY). The chromosomal localization analysis of the nine membrane-related genes revealed that the tolA and tolQ genes were closely arranged together. These genes encode similar protein products, forming a gene cluster with a similar structure and performing analogous functions. The other genes were dispersed at various locations on the same chromosome (Fig. 9B). Furthermore, potential interactions between the protein-encoding genes were explored using the STRING database (https://string-db.org/), and a protein-encoding gene interaction network was constructed to illustrate these interactions (Fig. 9C). These interactions were primarily enriched in cellular membrane components and functional terminologies related to transmembrane transport [50].

Fig. 9.

Schematic depicting the investigation of the genetic transformation mechanism of E. coli triggered by ultrasound through membrane gene screening and qRT-PCR. (A): Image illustrating the process of membrane gene selection in E. coli; (B): Chromosomal localization analysis diagram; (C): Diagram depicting the network of interactions among nine membrane-related genes; (D): Validation of membrane gene expression using qRT-PCR; (E): Illustration of the genetic transformation mechanism of E. coli induced by ultrasound.

To speculate on the changes in membrane-related genes during ultrasonication, confirm whether their expression is influenced by ultrasonic power and to further understand the underlying mechanisms. Transcripts of nine genes in E. coli were analysed by qRT-PCR under different ultrasonic power conditions (Fig. 9D). The qRT-PCR results revealed that, apart from the downregulation of ompL and wza gene expression, the transcriptional expression of the remaining 7 genes in the ultrasonicated strains was higher compared with that in the control. The relative expression of the bglH gene was slightly higher than that in the control, but the difference was not significant. The expressions of cusC, uidC, and yaiY genes under ultrasonic stress were upregulated due to an increase in ultrasonic power, with the maximum expression multiples being 4.69, 5.47, and 1.97 times higher, respectively. The transcriptional expressions of tolQ, tolA, and ompC genes exhibited a pattern of initial upregulation followed by downregulation, peaking at 240 W. The maximum expression multiples were 3.32, 2.64, and 2.78 times higher, respectively.

The findings of this study indicated that gene transcription related to a series of key cellular processes is downregulated under ultrasound stress. Waz is a crucial outer membrane auxiliary lipoprotein essential for the biosynthesis of capsular polysaccharides [51]. The downregulation of genes associated with the outer membrane polysaccharide export protein (waz) and the putative outer membrane porin-encoding gene (ompL) may inhibit transmembrane transport function, thereby altering the permeability of the cell membrane. The slight upregulation of the gene encoding carbohydrate-specific outer membrane porin (bglH) suggests a minor increase in the ability to break down carbohydrates and generate energy, with minimal changes in ATP production. Furthermore, under the influence of ultrasound, the expression of most genes encoding outer membrane proteins increases. Examples include cusC, uidC, and yaiY. Among these, cusC is responsible for the efflux of cations and plays a pivotal role in forming pores in the cell, allowing the passive diffusion of cations on the outer membrane. The putative β-glucuronide outer membrane pore protein-encoding gene (uidC) and the DUF2755 family inner membrane protein-encoding gene (yaiY) fulfil the function in cell membrane channels. These findings deduced that genes and proteins related to cell membrane permeability and integrity are upregulated under ultrasound stress. Genes (tolA and tolQ) encoding transmembrane proteins, which are part of the TolA-TolQ-TolR complex, play crucial roles in the inward invagination of the outer membrane during cell division. The synergistic effecty of these genes finally contribute to stabilizing the integrity of the cell envelope and they are also involved in the uptake of filamentous phage DNA [52].

The ompC gene, which encodes outer membrane pore protein C, facilitates the formation of pores, allowing small molecules to passively diffuse across the outer membrane [53]. The observed trend of gene expression, showing an initial upregulation followed by downregulation, may be attributed to the formation of pores on the cell surface under the influence of ultrasound, which leads to the upregulation of gene expression. As ultrasound stress increases, cells may inhibit gene expression to prevent excessive cellular damage. These findings demonstated that these genes may be involved in the ultrasound-induced transformation of E. coli. The potential mechanism of ultrasound-enhanced cell membrane permeability proposed in this study was summarized in Fig. 9E. It was suggested that ultrasound-mediated transformation in E. coli is not merely a simple physical–chemical process but involves the regulation of responsive membrane-related genes.

4. Conclusion

In this study, a quantitative model was established based on the relationship between ultrasonic power and plasmid transformation efficiency by quantifying membrane permeability at various ultrasonic power levels. This study represents the first investigation into the quantitative relationship between ultrasonic power and transformation efficiency in Escherichia coli, focusing on bacterial cell membrane permeability. The results of electron microscopy observations indicate that when ultrasound interacts with host cells, cell pores are formed. FTIR spectroscopy studies show changes in cell membrane permeability and cell wall structure, resulting in the release of intracellular macromolecules into the extracellular environment. Nine cell membrane-related genes were selected for qRT-PCR analysis to study transcription in E. coli under various ultrasound power conditions. Genes such as cusC, uidC, tolQ, tolA, and ompC, which are associated with cell membrane permeability and integrity, were significantly upregulated under ultrasound stress. This further demonstrates that the process of ultrasound transformation in E. coli involves the regulation of cell membrane mechanisms, rather than purely physical and chemical changes. The study has laid the foundation for a comprehensive understanding of the molecular mechanisms involved in ultrasound-mediated transformation. This research provides momentum and a fundamental framework for the exogenous DNA transformation process, and is important for analyzing related studies mechanistically.

CRediT authorship contribution statement

Feifan Leng: Writing – review & editing, Methodology, Funding acquisition, Formal analysis. Yubo Wang: Writing – original draft, Software, Formal analysis, Data curation. Ning Zhu: Validation, Supervision, Resources, Investigation. Xiaopeng Guo: Validation, Supervision, Resources, Investigation. Wen Luo: Validation, Supervision, Resources, Investigation. Yonggang Wang: Writing – review & editing, Resources, Methodology, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: