Constitutive expression of the sRNA GadY decreases acetate production and improves E. coli growth.

Alejandro Negrete,Joseph Shiloach

doi:10.1186/s12934-015-0334-1

Abstract

BackgroundEscherichia coli responds to acid stress by applying various physiological, metabolic, and proton-consuming mechanisms depending on the growth media composition, cell density, growth phase, pH, and aerobic or anaerobic growth conditions. It was reported that at mild acidic conditions (pH 5.8), the Hfq-associated sRNA GadY is activated. It was also reported that the two decarboxylase systems—the lysine decarboxylase system (LDS) and the glutamate decarboxylase system (GDS)—are activated to maintain intracellular balance of protons. The purpose of this study was to establish the role of GadY in high density growth of E. coli and to evaluate the possibility of using this small RNA to create an acid-resistant strain suitable for industrial applications.ResultsParental E. coli K-12 and constitutively expressing GadY strains were grown to high cell densities in a bioreactor at pH 7.0 and pH 6.0. At pH 7.0, both strains grew to similar cell densities of 43 OD, but the constitutively expressing GadY strain produced around 6 g/L acetate compared with 10 g/L by the parental strain. At pH 6.0, the parental strain grew to an OD of 20 and produced 10 g/L of acetate while the GadY strain grew to an average OD of 31 and produced 4 g/L acetate. After analyzing 17 genes associated with acid stress, it was found that at pH 7.0 LDS was expressed in the early exponential phase and GDS was expressed in the late exponential phase in both strains. However, at pH.6.0, GDS was expressed in the late exponential phase only in the parental strain and not in the constitutively expressing GadY strain, while there was no difference in the LDS expression pattern; it was expressed in the early exponential phase in both strains. This indicates that GadY affects GDS expression at low pH since the GDS was not detected in the GadY strain at pH 6.0.ConclusionsThe constitutive expression of GadY improves E. coli growth at pH 6.0 by deactivating the expression of the GDS in the late exponential growth phase. The expression of GadY also decreases acetate production regardless of pH, which decreases the inhibitory effect of this acid on bacterial growth.

Highlights

Escherichia coli responds to acid stress by applying various physiological, metabolic, and protonconsuming mechanisms depending on the growth media composition, cell density, growth phase, pH, and aerobic or anaerobic growth conditions
Two proton-consuming acid resistance systems active at mild acidic conditions have been described in E. coli: (1) the lysine decarboxylase system (LDS) that is activated in the early exponential growth phase and consists of the enzyme CadA and the lysine/cadaverine antiporter CadB [5,6,7], and (2) the glutamate decarboxylase system (GDS) that is induced in the late exponential phase and is comprised of the isozymes GadA, GadB, and the glutamate/γ-aminobutyric acid (GABA) antiporter GadC [8, 9]
The genes evaluated by RT-qPCR were: gadA, gadB, and gadC from the GDS; cadA, cadB, and cadC genes from the LDS; and the adiA from the arginine decarboxylase system (ADS)

Summary

Introduction

Escherichia coli responds to acid stress by applying various physiological, metabolic, and protonconsuming mechanisms depending on the growth media composition, cell density, growth phase, pH, and aerobic or anaerobic growth conditions. Growing E. coli to high cell densities is the preferred method for large scale production of recombinant proteins [1] During this process, the bacteria are being exposed to various stress parameters that can possibly affect their growth and production capability. It was established that exposing E. coli to acid stress triggers physiological and metabolic changes and activates proton consuming systems [4]; each consists of two cytoplasmic decarboxylases that catalyze a proton-dependent decarboxylation reaction of an amino acid, and a Negrete and Shiloach Microb Cell Fact (2015) 14:148 membrane antiporter that exchanges external substrate for internal product [5]. Two additional proton consuming systems have been described: (1) the arginine decarboxylase system (ADS) that includes the AdiA enzyme and the arginine/agmatine antiporter AdiC which is activated in extreme acid environments under anaerobic conditions [5], and, (2) the ornitine decarboxylase system (ODS) which include the SpeF enzyme and the ornithine/putrescine antiporter PotE that is not well understood [5, 10]

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