Variants of the Bacillus subtilis LysR-Type Regulator GltC With Altered Activator and Repressor Function.

Miriam Dormeyer,Till Ischebeck,Ralf Heermann,Björn Richts,Fabian M Commichau,Sabine Lentes

doi:10.3389/fmicb.2019.02321

Miriam Dormeyer, Till Ischebeck + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2019.02321

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Abstract

The Gram-positive soil bacterium Bacillus subtilis relies on the glutamine synthetase and the glutamate synthase for glutamate biosynthesis from ammonium and 2-oxoglutarate. During growth with the carbon source glucose, the LysR-type transcriptional regulator GltC activates the expression of the gltAB glutamate synthase genes. With excess of intracellular glutamate, the gltAB genes are not transcribed because the glutamate-degrading glutamate dehydrogenases (GDHs) inhibit GltC. Previous in vitro studies revealed that 2-oxoglutarate and glutamate stimulate the activator and repressor function, respectively, of GltC. Here, we have isolated GltC variants with enhanced activator or repressor function. The majority of the GltC variants with enhanced activator function differentially responded to the GDHs and to glutamate. The GltC variants with enhanced repressor function were still capable of activating the PgltA promoter in the absence of a GDH. Using PgltA promoter variants (PgltA∗) that are active independent of GltC, we show that the wild type GltC and the GltC variants with enhanced repressor function inactivate PgltA∗ promoters in the presence of the native GDHs. These findings suggest that GltC may also act as a repressor of the gltAB genes in vivo. We discuss a model combining previous models that were derived from in vivo and in vitro experiments.

Highlights

Glutamate is the most abundant cellular metabolite that serves as an amino group donor in many anabolic reactions (Gunka and Commichau, 2012; Park et al, 2016)
The mutagenized plasmids were introduced into the indicator strain BP852 (PgltA-lacZ gltC− rocG− gudB1), which contains a translational PgltA-lacZ fusion to monitor the activity of GltC and synthesizes the active glutamate dehydrogenases (GDHs) GudB1
We expected that the indicator strain synthesizing GltC variants with enhanced activator and repressor function would form dark blue and white colonies, respectively

Summary

Introduction

Glutamate is the most abundant cellular metabolite that serves as an amino group donor in many anabolic reactions (Gunka and Commichau, 2012; Park et al, 2016). B. subtilis can take up glutamate from the environment via the high-affinity and low-affinity glutamate transporters GltT and GltP, respectively (Tolner et al, 1995; Zaprasis et al, 2015). It has Genetic Dissection of the Transcriptional Regulator GltC been shown that the substrate specificity of GltT is relaxed because the transporter can mediate the uptake of aspartate as well as of the herbicides glyphosate and glufosinate (Zhao et al, 2018; Wicke et al, 2019)

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