Enhancement of acid tolerance of Escherichia coli by introduction of molecule chaperone CbpA from extremophile

Abstract

Molecular chaperone CbpA from extreme acidophile Acidithiobacillus caldus was applied to improve acid tolerance of Escherichia coli via CRISPR/Cas9. Cell growth and viability of plasmid complementary strain indicated the importance of cbpA^Ac for bacteria acid tolerance. With in situ gene replacement by CRISPR/Cas9 system, colony formation unit (CFU) of genome recombinant strain BL21-ΔcbpA/AccbpA showed 7.7 times higher cell viability than deficient strain BL21-ΔcbpA and 2.3 times higher than wild type. Cell morphology observation using Field Emission Scanning Electron Microscopy (FESEM) revealed cell breakage of BL21-ΔcbpA and significant recovery of BL21-ΔcbpA/AccbpA. The intracellular ATP level of all strains gradually decreased along with the increased stress time. Particularly, the value of recombinant strain was 56.0% lower than that of deficient strain after 5 h, indicating that the recombinant strain consumed a lot of energy to resist acid stress. The arginine concentration in BL21-ΔcbpA/AccbpA was double that of BL21-ΔcbpA, while the aspartate and glutamate contents were 14.8% and 6.2% higher, respectively, compared to that of wild type. Moreover, RNA-Seq analysis examined 93 genes down-regulated in BL21-ΔcbpA compared to wild type strain, while 123 genes were up-regulated in BL21-ΔcbpA/AccbpA compared to BL21-ΔcbpA, with an emphasis on energy metabolism, transport, and cell components. Finally, the working model in response to acid stress of cbpA from A. caldus was developed. This study constructed a recombinant strain resistant to acid stress and also provided a reference for enhancing microorganisms' robustness to various conditions.

Keywords: Acid tolerance; Acidithiobacillus caldus; CRISPR/Cas9 genome editing; Extreme acidophile; Molecule chaperone; Transcriptomics.