Effects of NaCl Concentrations on Growth Patterns, Phenotypes Associated With Virulence, and Energy Metabolism in Escherichia coli BW25113

Abstract

According to the sit-and-wait hypothesis, long-term environmental survival is positively correlated with increased bacterial pathogenicity because high durability reduces the dependence of transmission on host mobility. Many indirectly transmitted bacterial pathogens, such as Mycobacterium tuberculosis and Burkhoderia pseudomallei, have high durability in the external environment and are highly virulent. It is possible that abiotic stresses may activate certain pathways or the expressions of certain genes, which might contribute to bacterial durability and virulence, synergistically. Therefore, exploring how bacterial phenotypes change in response to environmental stresses is important for understanding their potentials in host infections. In this study, we investigated the effects of different concentrations of salt (sodium chloride, NaCl), on survival ability, phenotypes associated with virulence, and energy metabolism of the lab strain Escherichia coli BW25113. In particular, we investigated how NaCl concentrations influenced growth patterns, biofilm formation, oxidative stress resistance, and motile ability. In terms of energy metabolism that is central to bacterial survival, glucose consumption, glycogen accumulation, and trehalose content were measured in order to understand their roles in dealing with the fluctuation of osmolarity. According to the results, trehalose is preferred than glycogen at high NaCl concentration. In order to dissect the molecular mechanisms of NaCl effects on trehalose metabolism, we further checked how the impairment of trehalose synthesis pathway (otsBA operon) via single-gene mutants influenced E. coli durability and virulence under salt stress. After that, we compared the transcriptomes of E. coli cultured at different NaCl concentrations, through which differentially expressed genes (DEGs) and differential pathways with statistical significance were identified, which provided molecular insights into E. coli responses to NaCl concentrations. In sum, this study explored the in vitro effects of NaCl concentrations on E. coli from a variety of aspects and aimed to facilitate our understanding of bacterial physiological changes under salt stress, which might help clarify the linkages between bacterial durability and virulence outside hosts under environmental stresses.

Keywords: Escherichia coli; bacterial survival; biofilm formation; glycogen; transcriptome; trehalose.

FIGURE 1

Schematic illustration of the potential interactions between abiotic stress and bacterial pathogens. A group of indirectly transmitted bacterial pathogens shed from human hosts were spread to environment via a variety of routes, where they encounter many abiotic stresses for a certain duration. During the period, it is possible that bacterial pathogenic phenotypes could be altered from low virulence to high virulence due to the influences of abiotic stresses. These bacteria may cause severe consequences after being transmitted to new hosts.

FIGURE 2

Effects of glucose and NaCl concentrations on the growth rates of Escherichia coli BW25113. (A) E. coli growth curves in 1 × M9 minimal medium with three different glucose concentrations (0.2, 0.4, and 0.8%) measured via logarithmic value of CFU with base 10. (B) E. coli growth curves in LB broth supplemented with four different NaCl concentrations (0, 1, 3.5, and 5%) measured via logarithmic value of CFU with base 10. Three independent replicates were performed for the number of viable cells, the average values and standard error means were used to draw the curves.

FIGURE 3

Effects of NaCl on E. coli biofilm formation, oxidative stress resistance, and motile ability. (A) Effects of different NaCl concentrations on E. coli biofilm formation. (B) Influences of different NaCl concentrations on E. coli oxidative stress resistance. (C) Effects of different NaCl concentrations on E. coli motile abilities. Tukey’s Honestly Significant Difference (HSD) test was performed, which compared the means of every group to the means of every other group simultaneously. Means denoted by a different letter indicated significant differences among groups (P-value <0.05).

FIGURE 4

Influences of NaCl concentrations (0, 1, 3.5, and 5%) on E. coli glucose consumption rate, glycogen accumulation, and trehalose content at three time points, that is, 16, 20, and 24 h. (A–C) Glucose consumption rate. (D–F) Glycogen accumulation capacity. (G–I) trehalose content. Tukey’s Honestly Significant Difference (HSD) test was performed. Means denoted by a different letter indicated significant differences among groups (P-value <0.05).

FIGURE 5

Effects of NaCl on growth rate, biofilm formation, oxidative resistance, and motile ability in E. coli ΔotsA and ΔotsB. (A,B) Effects of different NaCl concentrations on the growth rates of E. coli mutants. (C,D) Effects of different NaCl concentrations on E. coli mutant biofilm formation. (E,F) Influences of different NaCl concentrations on the oxidative stress resistance of E. coli mutants. (G,H) Effects of different NaCl concentrations on E. coli mutant motile abilities. Tukey’s Honestly Significant Difference (HSD) test was performed, which compared the means of every group to the means of every other group simultaneously. Means denoted by a different letter indicated significant differences among groups (P-value <0.05).

FIGURE 6

Influences of NaCl concentrations (0, 1, 3.5, and 5%) on E. coli ΔotsA and ΔotsB in terms of glucose consumption rate and glycogen accumulation at 20 h. (A,B) Glucose consumption rates. (C,D) Glycogen accumulation capacity. Tukey’s Honestly Significant Difference (HSD) test was performed. Means denoted by a different letter indicated significant differences among groups (P-value <0.05).

FIGURE 7

Transcriptomic study of E. coli BW25113 under four NaCl concentrations of 0, 1, 3.5, and 5%. Since the phenotypes of E. coli under 0% and 1% NaCl are more consistent, while the phenotypes of E. coli under 3.5% and 5% NaCl are more similar, we combine 0% and 1% NaCl samples together as Group 1 and 3.5% and 5% NaCl samples together as Group 2 for the analysis. (A) Volcano plot for DEGs. (B) Heatmap plot for DEGs. (C) PCA analysis for group clusters. (D) Hub nodes among up-regulated genes. (E) Hub nodes among down-regulated genes. (F) Bar plot of GO term enrichment analysis. (G) Bubble plot of GO term enrichment analysis. (H) KEGG pathway enrichment for up-regulated genes. (I) KEGG pathway enrichment for down-regulated genes.