Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Outer membrane protein N expressed in Gram-negative bacterial strain of Escherichia coli BL21 (DE3) Omp8 Rosetta strains under osmoregulation by salts, sugars, and pHs

Abstract

This study presented the expression of the outer membrane protein N in E. coli BL21 (DE3) Omp8 Rosetta under its growth condition and by osmoregulation. The effects of osmotic stress caused by salts, sugars, or pH values on the survival of the target Gram-negative bacterial strain of E. coli BL21 (DE3) Omp8 Rosetta and OmpN expression remain unknown. Here, tryptone yeast extract with varied salts and concentrations was initially used to generate an LB broth medium. To show how salts and concentration affect bacterial growth, the optical density at 600 nm was measured. The findings supported the hypothesis that salts and concentrations control bacterial growth. Moreover, a Western blotting study revealed that OmpN overexpression was present in all tested salts after stimulation with both glucose and fructose after being treated individually with anti-OmpN and anti-histidine tag polyclonal antibodies on transferred nitrocellulose membrane containing crude OmpN. Following the presence of the plasmid pET21b(+)/ompN-BOR into E. coli BL21 (DE3) Omp8 Rosetta, which was expressed in the recombinant OmpN protein (BOR), OmpN expression was demonstrated for all monovalent cations as well as MgCl2. All of the tested salts, except for BaCl2, NaH2PO4, and KH2PO4, showed overexpression of recombinant BOR after Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Using CH3COONa, both with and without IPTG induction, there was very little bacterial growth and no OmpN expression. With NaCl, a pH value of 7 was suitable for bacterial development, whereas KCl required a pH value of 8. According to this research, bacterial growth in addition to salts, sugars, and pH values influences how the OmpN protein is produced.

Citation: Chumjan W, Sangchalee A, Somwang C, Mookda N, Yaikeaw S, Somsakeesit L-o (2023) Outer membrane protein N expressed in Gram-negative bacterial strain of Escherichia coli BL21 (DE3) Omp8 Rosetta strains under osmoregulation by salts, sugars, and pHs. PLoS ONE 18(8): e0288096. https://doi.org/10.1371/journal.pone.0288096

Editor: Bashir Sajo Mienda, Federal University Dutse, NIGERIA

Received: March 22, 2023; Accepted: June 19, 2023; Published: August 3, 2023

Copyright: © 2023 Chumjan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This research project was supported by the Rajamangala University of Technology Isan (Contract no. ENG21/65) and Thailand Science Research and Innovation (TSRI) (Contract no. FF66-P1-024). WC was funded by the Rajamangala University of Technology Isan (Contract no. ENG21/65), Thailand Science Research and Innovation (TSRI) (Contract no. FF66-P1-024). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The author have declared that no competing interest exist.

Introduction

To measure the development of bacteria in their growth environment, salt is typically used to make a variety of tryptone-yeast media preparations, known as a Luria Bertani broth (LB broth) [1–7]. Studies using monovalent and divalent cations have been carried out both in vivo and in vitro in order to assess bacterial growth and determine the ion conductance of numerous outer membrane proteins [8–16]. Previous studies have revealed that the influence of the anion considerably outweighs that of the cation. The order of the effects of the anion was as follows: F>SO4>PO4>Cl>NO3>Br>I>CNS, whereas the order of the cation was typically Ca>Sr>Mg>Cs>Rb>K>Na>Li [17]. Another outcome was that sodium and potassium chloride did not inhibit the E. coli bacterium, while calcium and iron salts did. The best concentration of bact. coli growth was found at 0.20 M, regardless of the NaCl content (0.05, 0.10, 0.20, 0.30, or 0.40 M) or different pH values [18]. The results demonstrated the accelerating action of the salts on an increase in the speed of organism growth when compared to a medium containing 1 per cent peptone with either 0.2 M NaCl or 0.1 M Na2SO4 [19].

Typically, Escherichia coli models for bacterial growth and the generation of outer membrane proteins are derived from gram-negative bacterial strains. E. coli DH5α and E. coli BL21 (DE3) Omp8 Rosetta are the two major strains of E. coli used for cloning and the production of outer membrane proteins [20–25]. Each is a distinct bacterial strain. BL21 (DE3) Omp8 Rosetta is E. coli BE, whereas E. coli DH5α is E. coli K-12. In addition, all outer membrane proteins are expressed in E. coli BL21 (DE3) Omp8 Rosetta, with the exception of LamB, OmpF, OmpC, and OmpA because it lacks the genes required for producing those porins [26]. Outer membrane protein N, as well as OmpF, OmpC, and PhoE, were first found and identified in E. coli K-12. Except for PhoE, they are all cation selective [27]. Both the ion conductance and cation selectivity of OmpF were investigated using black lipid membranes. OmpF channels preferred the permeation of alkali metal ions over chloride, according to selectivity tests at low concentrations, which also indicated a preference for smaller cations. These findings imply that the permeating cation and charged residues lining the channel walls interact explicitly [10]. Chemically changing OmpF to Ca2+ selectivity led to a reduction in pore conductance, which was another conclusion. At Ca2+ mole fractions of <10−3, the conductance of glutathione-modified LECE decreased. Zero-current potential tests showed that the selectivity of Ca2+ versus monovalent cations was the highest in the presence of Li+ and the lowest in the presence of Cs+. Thus, it could be illustrated that the LECE pore was sufficiently sized to permit the passage of monovalent cations after Ca2+ had been bound [8].

One of the reasons for the production of outer membrane proteins is osmotic stress caused by salts, sugars, pH value, and temperature, which activates the gene involved in the expression of those proteins [28,29]. In Klebsiella pneumoniae, for example, high osmotic stress causes the KbvR to be upregulated, which increases the expression of OmpK36 [30]. Moreover, higher levels of osmotic stress caused by NaCl impact OmpC expression increases Escherichia coli’s kanamycin resistance [31,32]. A hyperosmotic adaptation at basic pH value was developed by E. coli wild type, which had both the ompF and ompC genes [33]. Herein, the authors chose and used the E. coli BL21 (DE3) Omp8 Rosetta strain as a model to investigate the effects of salts that obtain the monovalent and divalent cations in the medium as well as to monitor bacterial growth and OmpN expression levels. To create the tryptone-medium preparation, various salts were used. To monitor bacterial growth, the optical density was measured at 600 nm. Additionally, a Western blotting analysis of OmpN protein expression was carried out.

Materials and methods

Bacterial strains, sugars, chemicals, LB broth preparations, and instruments

The E. coli BL21 (DE3) Omp8 Rosetta bacterial strains were given by Dr Wipa Suginta, a professor at the Vidyasirimedhi Institute of Science and Technology (VISTEC) in Rayong, Thailand. A 1,000 ml solution of dH2O, 10 g of tryptone, 10 g of sodium chloride (NaCl), and 5 g of yeast extract were used to make a LB broth medium with a pH value of 6.5. Also, an alternative LB broth medium that had different salts was developed, and the pH values were presented (S1 and S2 Tables). All chemicals including tryptone, yeast extract, glucose, fructose, lithium chloride (LiCl), potassium chloride (KCl), rubidium chloride (RbCl), caesium chloride (CsCl), magnesium chloride (MgCl2), barium chloride (BaCl2), calcium chloride (CaCl2), ammonium chloride (NH4Cl), sodium acetate (CH3COONa), disodium phosphate (Na2HPO4), sodium phosphate (NaH2PO4) and potassium phosphate (KH2PO4) were purchased from Titan Biotech Ltd., India, Vivantis, Malaysia and Sigma-Aldrich, Denmark. At the Department of Chemistry, Faculty of Engineering, Rajamangala University of Technology Isan, Khon Kaen campus, the optical density at 600 nm was measured using a spectrophotometer (Cecil EC1011, 1,000 series).

Bacterial growth at varied concentrations and different salts

The bacterial cell culture was made by mixing 30 per cent glycerol stock (2–5 μl) of a bacterial strain of E. coli BL21 (DE3) Omp8 Rosetta into a 50 ml LB broth medium and incubated at 37°C with regular shaking for 18 hours at 200 rpm (overnight culture). Numerous studies have demonstrated how the presence of NaCl in the medium affects bacterial growth [5]. Additionally, in vitro investigations utilizing black lipid membrane analyses have shown that various salts also impact the ion conductance of the outer membrane proteins [10,14]. Thus, the author used various salts (such as LiCl, NaCl, KCl, RbCl, CsCl, MgCl2, BaCl2, CaCl2, NH4Cl, CH3COONa, Na2HPO4, NaH2PO4 and KH2PO4) to design and prepare the LB broth media at various concentrations (0, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, 1.5 and 2 M) and also measured the pH values of the medium. The incubated overnight culture was then pipetted and directly transferred to each of the test tubes containing the salts mentioned above, with a final optical density of 600 nm (OD600nm) equal to 0.1. All test tubes containing bacteria were shaken continuously at 37°C for 18 hours. Bacterial growth was then assessed using a spectrometer at OD600nm. Another starter culture left by assessment using a spectrometer was then directly added into 5 ml of LB broth medium containing various salts and concentrations until a final OD600nm equalled 0.1, and after which all test tubes were shaken continuously at 200 rpm (at 37°C) until the OD600nm reached 0.5–0.7. Subsequently, each tube was filled with 125 μl of 1 M glucose and fructose to final concentration of 0.025 M and followed by shaking at 200 rpm for 3 hours. OmpN expression was evaluated on a 12 per cent SDS-PAGE gel for 60 minutes at 120 volts. Bacterial cells containing OmpN protein were employed for Western blot detection.

In addition, pH value and temperature tests were carried out. Briefly, LB broth media containing 0.25 M NaCl and KCl were made separately at different pH values, ranging from pH 4 to 10, with citric acid or Tris-base adjustments. The E. coli BL21 (DE3) Omp8 Rosetta starter culture was used to prepare the competent cells, and the recombinant plasmid (pET21b(+)/ompN) was transformed and selected in LB broth agar medium containing ampicillin (100 μg/μl) and kanamycin (25 μg/μl). After incubation at 37°C for 18 hours, the colonies were picked up and then grown in LB broth media (~25 ml) containing ampicillin (100 μg/μl), kanamycin (25 μg/μl) and 1% glucose at any pH value (including pH 4, 5, 6, 7, 8, 9, and 10) and shaken at 200 rpm for 18 hours at 37°C. After that, the starter culture was then transferred to a fresh LB broth medium (4 ml) containing ampicillin (100 μg/μl) and kanamycin (25 μg/μl) and an OD600nm equal to 0.01. After shaking all the test tubes containing bacterial strains at 200 rpm for 4 hours at 37°C until the OD600nm reached up 0.6–0.7, 0.5 mM IPTG was added directly to each test tube and shaken continuously at 200 rpm and a temperature of 37°C for 3 hours. The recombinant OmpN protein expression was then separated on a 12 per cent SDS-PAGE gel at 120 volts for 60 minutes, followed by Western blotting detection.

For temperature effect on OmpN expression, E. coli BL21 (DE3) Omp8 Rosetta harboring recombinant plasmid (pET21b(+)/ompN) was grown in a LB broth medium containing ampicillin (100 μg/μl), kanamycin (25 μg/μl) and 1% glucose and shaken at 200 rpm for 18 hours at 37°C. The starter culture was then transferred to a fresh LB broth medium (4 ml) containing ampicillin (100 μg/μl), kanamycin (25 μg/μl) and an OD600nm equal to 0.01. All test tubes containing bacterial strains were shaken at 200 rpm and 37°C until the OD600nm reached 0.6–0.7, 0.5 mM IPTG was added to each tube separately and then shaken continuously at 200 rpm for 3 hours at 20, 25, 30, 35, and 37°C. The recombinant OmpN protein expression was then analysed on a 12 per cent SDS-PAGE gel at 120 volts for 60 minutes followed by Western blotting detection.

Confirmation of OmpN expression level by Western blotting analysis

The E. coli BL21 (DE3) Omp8 Rosetta strains containing the OmpN and recombinant OmpN protein were separated on a 12 per cent SDS-PAGE gel. Using a semi-dry gel blotting technique, the protein band from an SDS-PAGE gel was transferred to a nitrocellulose membrane (Amersham, Biosciences). The transferred membrane was then incubated overnight at 4°C in a blocking solution containing 1x PBS (pH 7.4) and 5% skim milk (non-fat). It was then incubated for 1 hour at room temperature the following day. The membrane was incubated with anti-OmpN and anti-histidine tag polyclonal antibody dilution (1:20,000) in 2 per cent skim milk, 1x PBS (pH 7.4). After incubation with antiserum, the membrane was washed five times in 1x PBS containing 0.1 per cent Tween 20 (PBS-T), then incubated for 1 hour at room temperature in a 1:20,000 dilution of goat anti-rabbit IgG (HRP) in 1x PBS containing 2 per cent skim milk, 1x PBS (pH 7.4). The membrane was then washed three times in PBS-T and twice in 1x PBS for a total of five minutes per wash. The chemiluminescence detection stage was carried out by incubating the membrane for 3 minutes at room temperature with a 1:1 ratio of chemiluminescence substrate. In a dark room, the membrane was wrapped in saran wrap and exposed to an X-ray film.

Statistical analysis

Each test was performed at least three times, and the mean and standard error of each test were revealed. The statistical evaluation was done using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). A statistical analysis that compared groups of tests for bacterial growth, such as E. coli BL21 (DE3) Omp8 Rosetta strains grown in tryptone-yeast medium prepared with various salts supplement versus no salts addition in tryptone-yeast medium as a control, was done using the unpaired t-test. ***P <0.001 was used to evaluate statistical significance.

Results

The effect of salts and concentrations on the growth of Gram-negative bacterial growth

Herein, we described the E. coli BL21 (DE3) Omp8 Rosetta bacterial strain, which typically expressed OmpN, our target outer membrane protein N, in the outer membrane. The first experiment showed bacterial growth at an optical density of 600 nm at varying salt concentrations. On LB broth media containing LiCl, NaCl, KCl, RbCl, and CsCl, respectively, the bacterial strains were cultivated (Fig 1A). With the exception of CsCl, all salts displayed strong growth at 0.25 M, the optimum concentration. Bacterial growth was inhibited at both the highest and lowest dosages. With no additional salt, no bacterial growth was observed. Additionally, MgCl2, CaCl2, and BaCl2 were added to the LB broth medium to screen the bacterial growth (Fig 1B). The growth was reduced at 0.25 M concentration, but showed strong results at 0.1 M and an optical density of 600 nm. Bacterial growth in LB broth media containing either CaCl2 or KCl was compared (S1 Fig). The bacterial strain started to grow in the presence of CaCl2 at 0.025 M to 0.1 M but was inhibited from 0.25 M to 2.0 M. When compared to KCl at 0.1 M, CaCl2 had a stronger impact on bacterial growth at 600 nm.

thumbnail
Download:
Fig 1. coli BL21 (DE3) Omp8 Rosetta strain bacterial growth at optical density 600 nm in LB broth with salts at different concentrations (0–2.0 M).

E. LiCl, NaCl, KCl, RbCl, CsCl (A), and MgCl2, CaCl2, and BaCl2 (B) are all ingredients in LB broth. It is important to note that all of the data came from three different experiments.

https://doi.org/10.1371/journal.pone.0288096.g001

We also tested the impact of ammonium ions from NH4Cl and CH3COONH4. NH4Cl at concentrations between 0.025 and 1.0 M was found to promote bacterial growth, while CH3COONH4 did not. In comparison to CH3COONa, NH4Cl increased bacterial growth considerably at 0.25 M (Fig 2A). We also showed how the phosphates KH2PO4, NaH2PO4, and Na2HPO4 affected bacterial growth at 600 nm (Fig 2B). Bacterial growth was achieved by these salts at concentrations between 0.025 M and 0.25 M. In comparison to the addition of NaH2PO4 and Na2HPO4 to LB broth media, KH2PO4 showed greater bacterial growth at 0.1 M. With the exception of CsCl, CaCl2, CH3COONH4, and NaH2PO4, all salts significantly influenced the growth of E. coli BL21 (DE3) Omp8 Rosetta when their effects on bacterial growth were tested at 0.25 M (Fig 3A). After these bacterial strains were grown in either CsCl or CH3COONH4, no bacterial growth was observed. All salts, especially CaCl2, demonstrated a significant effect on bacterial growth (Fig 3B). After the bacteria were cultivated for 18 hours at 37°C, the increased pH value of the medium was observed (Table 1). The findings indicated that the E. coli BL21 (DE3) Omp8 Rosetta strains produced ammonia (NH3) as a by-product of the metabolism of carbon and nitrogen sources for the bacteria to live and raise the pH value of the LB broth. High pH value was observed for KCl during its optimal growth concentration range between 0.025 M and 0.75 M because it accelerated this bacterial growth (Table 2).

thumbnail
Download:
Fig 2. coli BL21 (DE3) Omp8 Rosetta strain bacterial growth at optical density 600 nm in LB broth with salts at different concentrations (0–2.0 M).

E. CH3COONa, CH3COONH4, and NH4Cl (A), and Na2HPO4, NaH2PO4, and KH2PO4 (B). It is important to note that all of the data came from three different experiments.

https://doi.org/10.1371/journal.pone.0288096.g002

thumbnail
Download:
Fig 3. coli BL21 (DE3) Omp8 Rosetta strain bacterial growth at optical density 600 nm was observed in LB broth medium.

E. With all salts at 0.25 M (A) and at the optimum growth concentration (B). ***P<0.001 relative to control (at 0 M). It is important to note that all of the data came from three different experiments.

https://doi.org/10.1371/journal.pone.0288096.g003

thumbnail
Download:
Table 1. The pH values displayed before and after E. coli BL21 (DE3) Omp8 Rosetta addition and cultured in LB broth containing salts at the optimum growth concentration at 37°C, 200 rpm for 18 hours.

https://doi.org/10.1371/journal.pone.0288096.t001

thumbnail
Download:
Table 2. The pH values displayed before and after E. coli BL21 (DE3) Omp8 Rosetta addition and cultured in LB broth prepared with various concentration of KCl at 37°C, 200 rpm for 18 hours.

https://doi.org/10.1371/journal.pone.0288096.t002

OmpN protein expression with and without induction of mono-sugars on Gram-negative bacterial strain of E. coli BL21 (DE3) Omp8 Rosetta

A Western blot was used to discern the level of OmpN protein expression using an anti-OmpN polyclonal antibody based on the bacterial growth profiles. At four hours, the E. coli BL21 (DE3) Omp8 Rosetta starting culture was transferred into various salts with a growth concentration such as 0.25 M for LiCl, NaCl, KCl, RbCl, CH3COONa and NH4Cl; 0.1 M for MgCl2, BaCl2 and CaCl2; 0.075 M for Na2HPO4, NaH2PO4 and KH2PO4 (Fig 4A). In comparison to the positive control (LB broth medium containing 0.17 M NaCl), CH3COONa caused a reduction in bacterial growth. A Western blotting analysis using an anti-OmpN polyclonal antibody revealed low OmpN expression in the absence of sugar stimulation. When a bacterial strain was cultivated in LB broth media with either NaH2PO4 or Na2HPO4, a high expression level was observed (Fig 4B). The nitrocellulose membrane was stained with Ponceau S to allow for internal protein loading control (Fig 4C). The outcome revealed various levels of protein loading, confirming that OmpN protein expression level is dependent on bacterial growth. Furthermore, the OmpN protein expression was stimulated using glucose and fructose. The nitrocellulose membrane from transferred SDS-PAGE presented for glucose (Fig 5A and 5B) and fructose (Fig 5C and 5D) after being incubated with an anti-OmpN polyclonal antibody and stained with Ponceau S. OmpN expression was found to be significantly expressed for 3 hours after fructose and glucose stimulation.

thumbnail
Download:
Fig 4. OmpN expression in E. coli BL21 (DE3) Omp8 Rosetta strains without sugar stimulation.

This bacteria grew in LB broth medium with various salts at the optimum growth concentration (A). Crude OmpN expression was separated by 12 per cent SDS-PAGE and transferred to nitrocellulose membrane for Western blotting analysis by an anti-OmpN polyclonal antibodies (B). Ponceau S solution was used to stain the nitrocellulose membrane as an internal sample loading control (C).

https://doi.org/10.1371/journal.pone.0288096.g004

thumbnail
Download:
Fig 5. E. coli BL21 (DE3) Omp8 Rosetta strains of Gram-negative bacteria with sugar stimulation for OmpN expression.

Western blot using an anti-OmpN polyclonal antibodies and the nitrocellulose membrane stained with Ponceau S solution were demonstrated in the presence of glucose (A-B) and fructose (C-D).

https://doi.org/10.1371/journal.pone.0288096.g005

Additionally, it was shown that OmpN expression in the outer membrane of Gram-negative bacteria under osmotic stress was confirmed by recombinant BOR production. Initially, the recombinant pET21b(+)/ompN-BOR was used to transform into the E. coli BL21 (DE3) Omp8 Rosetta strain, which led to the induction of recombinant BOR expression by 0.5 mM IPTG. Before IPTG induction, bacterial growth was seen at all salts tested for four hours (Fig 6A). For CH3COONa, there was less bacterial growth. 12 Per cent SDS-PAGE (Fig 6B) revealed low crude OmpN extraction levels for CH3COONa in the absence of IPTG induction, which prevented anti-OmpN polyclonal antibodies from detecting OmpN. For BaCl2, less OmpN expression was seen. Furthermore, significant OmpN expression was seen in the LB broth medium containing LiCl, NaCl, KCl, RbCl, and MgCl2 (Fig 6C). Following the incubation of nitrocellulose membrane with anti-histidine tag polyclonal antibodies, no protein band was found. Recombinant BOR protein was only identified in positive control (Fig 6D) after the nitrocellulose membrane was stained with Ponceau S. Fig 6E shows an internal check of the protein loading.

thumbnail
Download:
Fig 6. The E. coli BL21 (DE3) Omp8 Rosetta strain’s recombinant BOR expression profile.

Without IPTG induction, the bacterial growth at optical density of 600 nm was present (A). Crude recombinant BOR protein was subsequently separated by 12 per cent SDS-PAGE and stained with Coomassie brilliant blue (B). Anti-OmpN (C) and anti-histidine tag polyclonal antibodies (D) were used to treat nitrocellulose membrane. For internal sample loading control, the nitrocellulose membrane was stained with Ponceau S solution (E).

https://doi.org/10.1371/journal.pone.0288096.g006

Crude recombinant BOR protein was extracted using 2x SDS-PAGE loading buffer and boiled at 100°C for ten minutes after being induced with 0.5 mM IPTG for three hours. The micro-centrifuged tube having a crude sample protein was then centrifuged again, and the protein was put onto two pages of 12 per cent SDS-PAGE. One was used to transfer into a nitrocellulose membrane and the other was coloured with Coomassie brilliant blue (Fig 7A). Except for BaCl2, CH3COONa, and KH2PO4, all salts tested included in each LB broth medium showed an OmpN protein band following incubation with anti-OmpN polyclonal antibodies (Fig 7B). The same nitrocellulose membrane was washed with mild stripping solution and used for another incubation with anti-histidine tag polyclonal antibodies (Fig 7C). After being exposed to both antibodies, the nitrocellulose membrane was stained with Ponceau S (Fig 7D).

thumbnail
Download:
Fig 7. Recombinant BOR protein expressed in the E. coli BL21 (DE3) Omp8 Rosetta strain by 0.5 mM IPTG induction for three hours.

Crude recombinant BOR was separated by 12 per cent SDS-PAGE and stained with Coomassie brilliant blue (A). The treatment of nitrocellulose membrane was carried out with anti-OmpN (B) and anti-histidine tag polyclonal antibodies (C). For an internal sample loading control, the nitrocellulose membrane was stained with Ponceau S solution (D).

https://doi.org/10.1371/journal.pone.0288096.g007

The pH effect of bacterial growth and recombinant OmpN protein expression on Gram-negative bacterial strain of E. coli BL21 (DE3) Omp8 Rosetta

The recombinant plasmid pET21b(+)/ompN-BOR, which caused recombinant BOR protein expression, was carried by the E. coli BL21 (DE3) Omp8 Rosetta bacterial strain, which was used to confirm recombinant BOR protein expression upon bacterial growth. It was decided to modify the pH value of the LB broth medium containing 0.25 M salts of either NaCl (Fig 8A) or KCl (Fig 8B) to 4, 5, 6, 7, 8, and 10. A single colony of the E. coli BL21 (DE3) Omp8 Rosetta bacterial strain, which carried the plasmid pET21b(+)/ompN-BOR, was picked up, grown for a further four hours, and then stimulated with 0.5 mM IPTG for three hours. For bacterial growth, pH values of 7 and pH 8 were shown to be the optimum pH ranges when 0.25 M NaCl and 0.25 M KCl were used, respectively. Crude recombinant BOR protein was extracted using a 2x loading buffer and boiled for 10 minutes at 100°C and then separated into two pages of 12 per cent SDS-PAGE after being centrifuged at 13,680 x g for 10 minutes. Coomassie brilliant blue was used to stain one page, and another was transferred into a nitrocellulose membrane. With anti-histidine tag polyclonal antibodies, both pages of the nitrocellulose membrane were incubated. The outcomes showed that, with 0.25 M NaCl, large levels of recombinant BOR protein expression were seen at pH values of 6 and 7. However, these levels were lower at pH values of 5 and 8, while pH values of 4 and pH 10 showed no protein band. A high level of recombinant BOR protein expression was seen at pH value 8 when 0.25 M KCl was present in LB broth medium, but pH values of 6, 7, and 9 showed reduced expression. There was no evidence of protein expression at pH values of 4 or 10.

thumbnail
Download:
Fig 8.

Recombinant BOR protein expressed in the E. coli BL21 (DE3) Omp8 Rosetta strain at various pH values. The bacterial strains harbouring the plasmid of pET21b(+)/ompN-BOR were grown in LB broth with 0.25 M NaCl (A) and 0.25 M KCl (B). Crude recombinant BOR protein was separated by 12 per cent SDS-PAGE and stained with Coomassie brilliant blue. Nitrocellulose membrane was treated with anti-histidine tag polyclonal antibodies.

https://doi.org/10.1371/journal.pone.0288096.g008

Discussion

OmpN was expressed from the target ompN gene, which was initially identified in the genome of E. coli K-12. OmpN was extracted and investigated in vitro using black lipid membranes (BLMs). The conductance of a completely opened channel was represented as 1.63 ±0.06 nS. Additionally, the pNa/pCl ratio of the OmpN channel was 4.8 ±0.8, indicating cation-selectivity [27]. Agreement with our 3D structure was achieved based on the corrected DNA sequencing of synthesized ompN to OmpN from E. coli K-12 using OmpK36 (7q3t.1.A), which shares 70.55% of its sequence [34]. It was revealed that the recombinant BOR in term of the vacuum electrostatics of its trimeric structure are presented in a 3D structure (Fig 9). Cation selectivity is the property of a channel that favours cations over anions because red has more negative than positive surface content (blue). Therefore, prior research led to the findings in this research, and we addressed bacterial proliferation at different salt concentrations in this article. The E. coli BL21 (DE3) Omp8 Rosetta bacterial strain was the target of our investigation since it usually expresses OmpN, a protein necessary for nutrient uptake in the outer membrane. In contrast to the lack of bacterial growth in LB broth medium without any salt supplement, our prior research showed that all salts, with the exception of CsCl, could sustain bacterial strain survival at 0.25 M. With the exception of CaCl2, these bacteria can survive at 0.25 M in MgCl2 and BaCl2. At 0.1 M, however, it grows well. Additionally, we noticed that the E. coli BL21 (DE3) Omp8 Rosetta strains, with the exception of CH3COONa and NH4Cl, could not grow at all on LB broth medium containing CH3COONH4. Greater evidence of bacterial growth was also detected in LB broth medium containing the other three distinct salts Na2HPO4, NaH2PO4, or KH2PO4. We demonstrated how changing the concentrations of phosphate from sodium and potassium affected the growth of the target bacterium. Like the chlorides of MgCl2, CaCl2, and BaCl2, high bacterial growth was noted at 0.1 M, but not at 0.25 M, as was the case for the chlorides of other salts from other monovalent metal ions. Many papers have clarified that all microbial growth requires a medium with salinity stress that is produced by salt [16,35–39]. Long ago, Sherman et al. showed that E. coli could grow at varied pH values and NaCl concentrations when peptone was present at 1%. The outcomes demonstrated that 0.2 M at low to high concentrations of H-ion had the best influence on bacterial growth [18]. Other observations on the bioluminescence of the three luminous bacterial species under their growth conditions of two-dimensional diffusion gradients of NaCl and H+ concentration. All of the Photobacterium leiognathi, Photobacterium phosphoreum, and Vibrio fischeri grew in the range of 0.9–3% NaCl, pH value less than 7, and temperature at 20°C, except for Vibrio fischeri at 15°C [40].

thumbnail
Download:
Fig 9. The trimeric structure of recombinant BOR protein was built using an OmpK36 template (7q3t.1.A).

Vacuum electrostatics using pymol produced the surface charges, both negative and positive.

https://doi.org/10.1371/journal.pone.0288096.g009

Bacterial proliferation led to OmpN expression. OmpN expression was detected by Western blot analysis using an anti-OmpN polyclonal antibody. It was shown to be lower without sugar induction and higher after the bacterial strain was stimulated with either 0.025 M glucose or fructose. Furthermore, OmpN expression was confirmed in the outer membrane of the E. coli BL21 (DE3) Omp8 Rosetta strain using recombinant OmpN expression induced by IPTG, known as BOR. After the pET21b(+)/ompN-BOR plasmid was used to transform into the E. coli BL21 (DE3) Omp8 Rosetta strain, the colony was picked up and allowed to grow. OmpN expression occurred without the need for IPTG induction, and anti-histidine polyclonal antibodies were unable to detect any recombinant BOR. When bacterial strains were cultivated in LB broth medium that included various salts in a particular order, specifically MgCl2>RbCl>KCl>NaCl>LiCl, high levels of OmpN expression were seen. When the bacterial strain was cultivated on LB broth medium containing CH3COONa, no protein band was visible. BaCl2, CH3COONa, and KH2PO4 in LB broth medium had no effect on OmpN expression after 3 hours of 0.5 mM IPTG induction. However, MgCl2 had a reduced effect. The expression of the recombinant BOR also showed a pattern of variation with pH value. In a LB broth medium containing 0.25 M NaCl or KCl, the pH value was changed to a range of 4 to 10. Low or absent OmpN expression was a result of decreased bacterial growth in response to lower and higher pH value.

Previous research revealed that salt stress causes numerous genes to become more active [41,42]. ompF and ompC genes are two examples of genes that are upregulated and express both OmpF and OmpC [43,44]. Both OmpF and OmpC, together with PhoE, were found in E. coli K-12 [20]. Upregulation occurred under osmolarity to produce both outer membrane proteins. High osmolarity was favoured for the production of OmpC, whereas OmpF was produced with low osmolarity [45]. Black lipid membranes were displayed to characterise the function. Both OmpF and OmpC were extracted and purified for use in in vitro experiments. Both proteins were tested and demonstrated a fully opened channel conductance for ion transport at various salt concentrations in the buffer. The ion conductance was reported to be small at low concentrations and larger at high concentrations, as is typically found for all porins [15,46]. In in vitro investigations, CsCl was found to have a substantial ion conductance of OmpF in a series of LiCl, NaCl, and CsCl that was contained in a buffer at 0.1 M, but LiCl had a very low ion conductance [8]. Similar to our findings in vitro investigations, monovalent cations showed a significant contribution to bacterial growth while divalent cations worked as a restriction to inhibit bacterial growth. The outer membrane protein N was extensively expressed for monovalent cations, while reduced expression was reported for divalent cations, and no OmpN protein expression was observed for sodium acetate as a result of bacterial development on the LB broth medium containing salts. Following stimulation of the bacterial strain with either 0.025 M glucose or fructose, OmpN expression was shown to be overexpressed. Similar to the recombinant BOR protein, overexpression was seen with the presence of 0.5 mM IPTG for 3 hours, but no protein band was seen for barium chloride, sodium acetate, or potassium phosphate.

Conclusion

The E. coli BL21 (DE3) Omp8 Rosetta strain is a gram-negative bacterial strain resistant to the drug kanamycin. This strain is typically used for producing outer membrane proteins. It lacks OmpF, OmpC, OmpA, and LamB, four of the main porins. According to earlier studies by the author, this bacterial strain produces the outer membrane protein N and VhChiP from the chip gene in Vibrio harveyi [24]. There is no evidence of the effect of osmotic stress on the other three factors such as salts, sugars, and pH value. The isolation of OmpN from E. coli K-12 and functional characterisation by black lipid membranes were only shown in an in vitro investigation by Prilipov et al [27]. The outcomes demonstrated the cation selectivity of its channel conductance and its preference for cation over anion. Also, the liposome swelling assay shows that the channel prefers monosugars over disugars. An earlier study enabled the author to test the notion that osmotic stress from salts, sugars, and any pH value affected OmpN expression in this bacterial strain.

The authors’ finding indicated that growth in varied salt supplements in an LB broth medium had an impact on the bacterial growth of the E. coli BL21 (DE3) Omp8 Rosetta strain. All salts tested, except for CaCl2, CH3COONH4, and NaH2PO4, had an impact on bacterial growth at 0.25 M, whereas CsCl inhibited bacterial growth at all doses. This demonstrated that bacterial growth is dependent on salt [5,17], much as Bacillus cereus was dependent on NaCl [7]. To induce OmpN expression and identify it using anti-OmpN polyclonal antibodies under bacterial growth conditions, glucose and fructose were used. After the addition of either glucose or fructose for three hours, high OmpN expression was observed. Low OmpN expression was observed in the absence of sugar stimulation. Additionally, OmpN expression on the outer membrane of the E. coli BL21 (DE3) Omp8 Rosetta strain was verified using recombinant BOR protein. All salts, with the exception of BaCl2, CH3COONa, and KH2PO4, showed high levels of OmpN expression after 0.5 mM IPTG induction for 3 hours. Another observation of recombinant BOR protein expression at different pH values was that it was not detected at pH values of 4 and 10, when LB broth medium contained both NaCl and KCl. For NaCl, high OmpN expression levels were seen at pH values of 6 and 7, while KCl showed high OmpN expression levels at pH value of 8. All results lead to the conclusion that osmoregulation by the salts, sugars, and pH values studied influences bacterial growth as well as OmpN expression levels. Salt, sugar, and pH value are necessary for OmpN and recombinant BOR protein expression. This information will be used by the author to do additional research on how ions affect the conductance of the OmpN channel [8–10,14,27,46]. Additionally, the interaction of the recombinant OmpN protein with sugar or antibiotics using black lipid membranes will be investigated [24].

Supporting information

S1 Fig. Bacterial E. coli BL21 (DE3) Omp8 Rosetta strain growth at optical density 600 nm in LB broth with KCl and CaCl2 at concentration of 0 to 2.0 M.

It is important to note that all of the data came from three different experiments.

https://doi.org/10.1371/journal.pone.0288096.s001

(TIF)

S1 File.

https://doi.org/10.1371/journal.pone.0288096.s002

(PPTX)

S1 Table. The pH values of LB broth medium containing monovalent and divalent cations was present.

https://doi.org/10.1371/journal.pone.0288096.s003

(DOC)

S2 Table. The NH4Cl, CH3COONa, CH3COONH4, Na2HPO4, NaH2PO4, and KH2PO4 components of LB broth medium were present, as well as the pH value.

https://doi.org/10.1371/journal.pone.0288096.s004

(DOCX)

Acknowledgments

The authors would also like to thank the Department of Chemistry, Faculty of Engineering at the Rajamangala University of Technology Isan, Khon Kean campus and the Biochemistry-Electro-chemistry Research Unit, School of Chemistry, Institute of Science at Suranaree University of Technology for partially supporting this research.

References

  1. 1. Raevuori M, Genigeorgis C. Effect of pH and sodium chloride on growth of Bacillus cereus in laboratory media and certain foods. Appl Microbiol. 1975;29: 68–73. pmid:234158
  2. 2. Tripathi NK, Shrivastva A, Biswal KC, Rao PL. Methods: Optimization of culture medium for production of recombinant dengue protein in Escherichia coli. Ind Biotechnol. 2009;5: 179–183. http://dx.doi.org/10.1089/ind.2009.3.179.
  3. 3. Greif G, Greifová M, Karovićová J. Effects of NaCl concentration and initial pH value on biogenic amine formation dynamics by Enterobacter spp. bacteria in model conditions. J Food Nutr Res. 2006;45: 21–29.
  4. 4. Marsden AE, Grudzinski K, Ondrey JM, DeLoney-Marino CR, Visick KL. Impact of salt and nutrient content on biofilm formation by Vibrio fischeri. PLoS ONE. 2017;12(1):e0169521. https://doi.org/10.1371/journal.pone.0169521.
  5. 5. Sutherland JP, Bayliss AJ, Braxton DS. Predictive modelling of growth of Escherichia coli O157: H7: the effects of temperature, pH and sodium chloride. Int J Food Microbiol. 1995;25: 29–49. https://doi.org/10.1016/0168-1605(94)00082-h.
  6. 6. McClure PJ, Kelly TM, Roberts TA. The effects of temperature, pH, sodium chloride and sodium nitrite on the growth of Listeria monocytogenes. Int J Food Microbiol. 1991;14: 77–91. https://doi.org/10.1016/0168-1605(91)90039-r.
  7. 7. Raevuori M, Genigeorgis C. Effect of pH and sodium chloride on growth of Bacillus cereus in laboratory media and certain foods. Appl Microbiol. 1975;29: 68–73. https://doi.org/10.1128/am.29.1.68-73.1975.
  8. 8. Miedema H, Vrouenraets M, Wierenga J, Gillespie D, Eisenberg B, Meijberg W, et al. Ca2+ selectivity of a chemically modified OmpF with reduced pore volume. Biophys J. 2006;91: 4392–4400. https://doi.org/10.1529%2Fbiophysj.106.087114.
  9. 9. García-Giménez E, López ML, Aguilella VM, Alcaraz A. Linearity, saturation and blocking in a large multiionic channel: divalent cation modulation of the OmpF porin conductance. Biochem Biophys Res Commun. 2011;404: 330–334. pmid:21134352
  10. 10. Danelon C, Suenaga A, Winterhalter M, Yamato I. Molecular origin of the cation selectivity in OmpF porin: single channel conductances vs. free energy calculation. Biophys Chem. 2003;104: 591–603. pmid:12914905
  11. 11. Kennell WL, Egli C, Hancock RE, Holt SC. Pore-forming ability of major outer membrane proteins from Wolinella recta ATCC 33238. Infect Immun. 1992;60: 380–384. pmid:1370429
  12. 12. Hindahl MS, Crockford GW, Hancock RE. Outer membrane protein NmpC of Escherichia coli: pore-forming properties in black lipid bilayers. J Bacteriol. 1984;159: 1053–1055. https://doi.org/10.1128%2Fjb.159.3.1053-1055.1984.
  13. 13. Brzostek K, Hrebenda J, Benz R, Boos W. The OmpC protein of Yersinia enterocolitica: purification and properties. Res Microbiol. 1989;140: 599–614. https://doi.org/10.1016/0923-2508(89)90192-7.
  14. 14. Whitfield C, Hancock RE, Costerton JW. Outer membrane protein K of Escherichia coli: purification and pore-forming properties in lipid bilayer membranes. J Bacteriol. 1983;156: 873–879. pmid:6313621
  15. 15. Alcaraz A, Queralt-Martín M, García-Giménez E, Aguilella VM. Increased salt concentration promotes competitive block of OmpF channel by protons. Biochim Biophys Acta Biomembr BBA-Biomembranes. 2012;1818: 2777–2782. pmid:22789813
  16. 16. Yang L, Zhan L, Han H, Gao H, Guo Z, Qin C, et al. The low-salt stimulon in Vibrio parahaemolyticus. Int J Food Microbiol. 2010;137: 49–54. https://doi.org/10.1016/j.ijfoodmicro.2009.11.006.
  17. 17. Holm GE, Sherman JM. Salt effects in bacterial growth I. Preliminary paper. J Bacteriol. 1921;6: 511–519. pmid:16558936
  18. 18. Sherman JM, Holm GE. Salt effects in bacterial growth II. The growth of bact. coli in relation to H-ion concentration. J Bacteriol. 1922;7: 465–470. https://doi.org/10.1128/jb.7.5.465-470.1922.
  19. 19. Sherman JM, Holm GE, Albus WR. Salt effects in bacterial growth III. Salt effects in relation to the lag period and velocity of growth. J Bacteriol. 1922;7: 583–588. pmid:16558978
  20. 20. Ruan L, He W, He J, Sun M, Yu Z. Cloning and expression of mel gene from Bacillus thuringiensis in Escherichia coli. Antonie Van Leeuwenhoek. 2005;87: 283–288. https://doi.org/10.1007/s10482-004-4775-5.
  21. 21. Guzev KV, Isaeva MP, Novikova OD, Solov’Eva TF, Rasskazov VA. Molecular characteristics of OmpF-like porins from pathogenic Yersinia. Biochemistry. 2005;70: 1104–1110. https://doi.org/10.1007/s10541-005-0231-z.
  22. 22. Dekker N, Merck K, Tommassen J, Verheij HM. In vitro folding of Escherichia coli outer‐membrane phospholipase A. Eur J Biochem. 1995;232: 214–219. https://doi.org/10.1111/j.1432-1033.1995.tb20801.x.
  23. 23. Hejair HM, Zhu Y, Ma J, Zhang Y, Pan Z, Zhang W, et al. Functional role of ompF and ompC porins in pathogenesis of avian pathogenic Escherichia coli. Microb Pathog. 2017;107: 29–37. https://doi.org/10.1016/j.micpath.2017.02.033.
  24. 24. Suginta W, Chumjan W, Mahendran KR, Janning P, Schulte A, Winterhalter M. Molecular uptake of chitooligosaccharides through chitoporin from the marine bacterium Vibrio harveyi. PLoS ONE. 2013;8(1):e55126. https://doi.org/10.1371/journal.pone.0055126.
  25. 25. Aunkham A, Schulte A, Winterhalter M, Suginta W. Porin involvement in cephalosporin and carbapenem resistance of Burkholderia pseudomallei. PLoS ONE. 2014;9(5):e95918. https://doi.org/10.1371/journal.pone.0095918.
  26. 26. Prilipov A, Phale PS, Van Gelder P, Rosenbusch JP, Koebnik R. Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli. FEMS Microbiol Lett. 1998;163: 65–72. https://doi.org/10.1111/j.1574-6968.1998.tb13027.x.
  27. 27. Prilipov A, Phale PS, Koebnik R, Widmer C, Rosenbusch JP. Identification and characterization of two quiescent porin genes, nmpC and ompN, in Escherichia coli BE. J Bacteriol. 1998;180: 3388–3392. https://doi.org/10.1128%2Fjb.180.13.3388-3392.1998.
  28. 28. Altendorf K, Booth IR, Gralla JAY, Greie JC, Rosenthal AZ, Wood JM. Osmotic stress. EcoSal Plus. 2009;3(2). https://doi.org/10.1128/ecosalplus.5.4.5.
  29. 29. Bremer E, Krämer R. Responses of microorganisms to osmotic stress. Annu Rev Microbiol. 2019;73: 313–334. pmid:31180805
  30. 30. Wang M, Tian Y, Xu L, Zhang F, Lu H, Li M, et al. High osmotic stress increases OmpK36 expression through the regulation of KbvR to decrease the antimicrobial resistance of Klebsiella pneumoniae. Microbiol Spectr. 2022;10:e00507–22. https://doi.org/10.1128%2Fspectrum.00507-22.
  31. 31. Angeles A, Chang T, Coombe L, Rogers T. Regulation of osmotic stress by OmpC but not OmpF increases kanamycin resistance in Escherichia coli. J Exp Microbiol Immunol. 2013;17: 73–77.
  32. 32. Lo J, Van Tol T, Yeung S, Zou K. EnvZ is not essential for the upregulation of OmpC following treatment with sublethal kanamycin in Escherichia coli. Journal of Experimental Microbiology and Immunology. 2014;18: 65–69.
  33. 33. Kaeriyama M, Machida K, Kitakaze A, Wang H, Lao Q, Fukamachi T, et al. OmpC and OmpF are required for growth under hyperosmotic stress above pH 8 in Escherichia coli. Lett Appl Microbiol. 2006;42: 195–201. https://doi.org/10.1111/j.1472-765x.2006.01845.x.
  34. 34. Dutzler R, Rummel G, Albertí S, Hernández-Allés S, Phale PS, Rosenbusch JP, et al. Crystal structure and functional characterization of OmpK36, the osmoporin of Klebsiella pneumonia, Structure. 1999;7: 425–434. https://doi.org/10.1016/s0969-2126(99)80055-0.
  35. 35. Qurashi AW, Sabri AN. Osmoadaptation and plant growth promotion by salt tolerant bacteria under salt stress. Afr J Microbiol Res. 2011;5: 3546–3554. http://dx.doi.org/10.5897/AJMR11.736.
  36. 36. Singleton PW, El Swaify SA, Bohlool BB. Effect of salinity on Rhizobium growth and survival. Appl Environ Microbiol. 1982;44: 884–890. pmid:16346113
  37. 37. Soussi M, Santamaria M, Ocana A, Lluch C. Effects of salinity on protein and lipopolysaccharide pattern in a salt‐tolerant strain of Mesorhizobium ciceri. J Appl Microbiol. 2001;90: 476–481. https://doi.org/10.1046/j.1365-2672.2001.01269.x.
  38. 38. Heinz J, Waajen AC, Airo A, Alibrandi A, Schirmack J, Schulze-Makuch D. Bacterial growth in chloride and perchlorate brines: halotolerances and salt stress responses of Planococcus halocryophilus. Astrobiology. 2019;19: 1377–1387. https://doi.org/10.1089/ast.2019.2069.
  39. 39. Zabihi R, Mowla D, Karimi G, Setoodeh P. Examination of the impacts of salinity and culture media compositions on Clostridium acetobutylicum NRRL B-591 growth and acetone-butanol-ethanol biosynthesis. J Environ Chem Eng. 2019;7: 102835. http://dx.doi.org/10.1016/j.jece.2018.102835.
  40. 40. Waters P, Lloyd D. Salt, pH and temperature dependencies of growth and bioluminescence of three species of luminous bacteria analysed on gradient plates. Microbiology. 1985;131: 2865–2869. https://doi.org/10.1099/00221287-131-11-2865.
  41. 41. Xu C, Ren H, Wang S, Peng X. Proteomic analysis of salt-sensitive outer membrane proteins of Vibrio parahaemolyticus. Res Microbiol. 2004;155: 835–842. https://doi.org/10.1016/j.resmic.2004.07.001.
  42. 42. Liu ZY, Li XP, Zhang TQ, Wang YY, Wang C, Gao CQ. Overexpression of ThMYB8 mediates salt stress tolerance by directly activating stress-responsive gene expression. Plant Sci. 2021;302: 110668. pmid:33288032
  43. 43. Schnaitman CA, McDonald GA. Regulation of outer membrane protein synthesis in Escherichia coli K-12: deletion of ompC affects expression of the OmpF protein. J Bacteriol. 1984;159: 555–563. https://doi.org/10.1128/jb.159.2.555-563.1984.
  44. 44. Forst S, Comeau D, Norioka S, Inouye M. Localization and membrane topology of EnvZ, a protein involved in osmoregulation of OmpF and OmpC in Escherichia coli. J Biol Chem. 1987;262: 16433–16438. https://doi.org/10.1016/S0021-9258(18)49274-5.
  45. 45. Pratt LA, Hsing W, Gibson KE, Silhavy TJ. From acids to osmZ: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli. Mol Microbiol. 1996;20: 911–917. https://doi.org/10.1111/j.1365-2958.1996.tb02532.x.
  46. 46. Kojima S, Nikaido H. High salt concentrations increase permeability through OmpC channels of Escherichia coli. J Biol Chem. 2014;289: 26464–26473. https://doi.org/10.1074/jbc.m114.585869.