Identification and functional analysis of novel protein-encoding sequences related to stress-resistance

Abstract

Currently, industrial bioproducts are less competitive than chemically produced goods due to the shortcomings of conventional microbial hosts. Thus, is essential developing robust bacteria for improved cell tolerance to process-specific parameters. In this context, metagenomic approaches from extreme environments can provide useful biological parts to improve bacterial robustness. Here, in order to build genetic constructs that increase bacterial resistance to diverse stress conditions, we recovered novel protein-encoding sequences related to stress-resistance from metagenomic databases using an in silico approach based on Hidden-Markov-Model profiles. For this purpose, we used metagenomic shotgun sequencing data from microbial communities of extreme environments to identify genes encoding chaperones and other proteins that confer resistance to stress conditions. We identified and characterized 10 novel protein-encoding sequences related to the DNA-binding protein HU, the ATP-dependent protease ClpP, and the chaperone protein DnaJ. By expressing these genes in Escherichia coli under several stress conditions (including high temperature, acidity, oxidative and osmotic stress, and UV radiation), we identified five genes conferring resistance to at least two stress conditions when expressed in E. coli. Moreover, one of the identified HU coding-genes which was retrieved from an acidic soil metagenome increased E. coli tolerance to four different stress conditions, implying its suitability for the construction of a synthetic circuit directed to expand broad bacterial resistance.

Keywords: bacterial robustness; industrial biotechnology; metagenomics; stress resistance; synthetic biology.

Figure 1

Schematic representation of the approach used in this study and the frequency of stress-related proteins in public metagenomic databases. (A) Schematic representation of in silico and experimental approaches used to identify and validate stress-related genes using HMM profiles, metagenomic mining, and experimental characterization. (B) Representation of gene frequency in different metagenomes selected from public databases. Frequency calculation was made by normalizing the number of found hits by the size of the metagenomic library (Gb). *Metagenomes from the Atacama Desert and Dourados are not presented in the (B) panel, since these metagenomic data did not include all classes of the proteins plotted.

Figure 2

Functional characterization of E. coli DH10B clones carrying hup, clpP, and dnaJ genes. (A) Growth curve of E. coli DH10B clones in liquid M9GlycAA medium during 8 h; optical density at 600 nm was measured every 30 min (a 2.5× factor should be applied due to the optical path length). To test the effects of stress factors, E. coli DH10B clones were grown to exponential phase at 30°C, then (B) treated at 55°C for 30 min, (C) exposed to acid medium (pH = 3.0) for 60 min or (D) to 12 mM hydrogen peroxide for 60 min. Serial dilutions were made and CFU counts were used to determine the survival percentage. E. coli DH10B carrying empty pUC19 plasmid was used as a negative control. (E) Effect of UV radiation resistance in E. coli clones. Overnight cultures of E. coli clones were irradiated with a germicidal lamp (254 nm UV) during 0, 5, 10, 15, 20, and 25 s. (F) Effect of salinity on growth rates of E. coli DH10B clones. Growth rates were determined from the exponential curve fitting function as described in Section 2. E. coli DH10B was used as a negative control. Student’s t-test was used for comparison of the values obtained in the experiments and significance determination (p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001). All graphs represent the average from at least three independent experiments. Standard deviation from experiments is represented as shaded regions in (A) and vertical bars in (B–F).

Figure 3

Clustering of E. coli clones considering functional characterization in the five stress conditions. Hierarchical cluster of E. coli DH10B clones carrying the pUC19 plasmid with genes clpP, hup, and dnaJ. Scale bar denotes the Z-score fold change. Strong color represents an increased response level to stress factors.