Dataset Information

A glutamate-dependent acid resistance gene in Escherichia coli.

ABSTRACT: Stationary-phase cultures of Escherichia coli can survive several hours or exposure to extreme acid (pH 2 to 3), a level well below the pH range for growth (pH 4.5 to 9). To identify the genes needed for survival in extreme acid, a microliter screening procedure was devised. Colonies from a Tn10 transposon pool in E. coli MC4100 were inoculated into buffered Luria broth, pH 7.0, in microtiter wells, grown overnight, and then diluted in Luria broth, pH 2.5, at 37 degrees C for 2 h. From 3,000 isolates screened, 3 Tet(r) strains were identified as extremely acid sensitive (<0.1% survival at pH 2.5 for 2 h). Flanking sequences of the Tn10 inserts were amplified by inverse PCR. The sequences encoded a hydrophobic partial peptide of 88 residues. A random-primer-generated probe hybridized to Kohara clones 279 and 280 at 32 min (33.7 min on the revised genomic map EcoMap7) near gadB (encoding glutamate decarboxylase). The gene was designated xasA for extreme acid sensitive. xasA::Tn10 strains grown at pH 7 to 8 showed 100-fold-less survival in acid than the parent strain. Growth in mild acid (pH 5 to 6) restored acid resistance; anaerobiosis was not required, as it is for acid resistance in rpoS strains. xasA::Tn10 eliminated enhancement of acid resistance by glutamic acid. xasA was found to be a homolog of gadC recently sequenced in Shigella flexneri, in which it appears to encode a permease for the decarboxylated product of GadB. These results suggest that GadC (XasA) participates in a glutamate decarboxylase alkalinization cycle to protect E. coli from cytoplasmic acidification. The role of the glutamate cycle is particularly important for cultures grown at neutral pH before exposure to extreme acid.

SUBMITTER: Hersh BM

PROVIDER: S-EPMC232665 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

A glutamate-dependent acid resistance gene in Escherichia coli.

Hersh B M BM Farooq F T FT Barstad D N DN Blankenhorn D L DL Slonczewski J L JL

Journal of bacteriology 19960701 13

Stationary-phase cultures of Escherichia coli can survive several hours or exposure to extreme acid (pH 2 to 3), a level well below the pH range for growth (pH 4.5 to 9). To identify the genes needed for survival in extreme acid, a microliter screening procedure was devised. Colonies from a Tn10 transposon pool in E. coli MC4100 were inoculated into buffered Luria broth, pH 7.0, in microtiter wells, grown overnight, and then diluted in Luria broth, pH 2.5, at 37 degrees C for 2 h. From 3,000 iso ...[more]

PMID: 8682809

Similar Datasets

Project description:Lactic acid bacteria often encounter a variety of multiple stresses in their natural and industrial fermentation environments. The glutamate decarboxylase (GAD) system is one of the most important acid resistance systems in lactic acid bacteria. In this study, we demonstrated that GlnR, a nitrogen regulator in Gram-positive bacteria, directly modulated γ-aminobutyric acid (GABA) conversion from glutamate and was involved in glutamate-dependent acid resistance in Lactobacillus brevis The glnR deletion strain (ΔglnR mutant) achieved a titer of 284.7 g/liter GABA, which is 9.8-fold higher than that of the wild-type strain. The cell survival of the glnR deletion strain was significantly higher than that of the wild-type strain under the condition of acid challenge and was positively correlated with initial glutamate concentration and GABA production. Quantitative reverse transcription-PCR assays demonstrated that GlnR inhibited the transcription of the glutamate decarboxylase-encoding gene (gadB), glutamate/GABA antiporter-encoding gene (gadC), glutamine synthetase-encoding gene (glnA), and specific transcriptional regulator-encoding gene (gadR) involved in gadCB operon regulation. Moreover, GABA production and glutamate-dependent acid resistance were absolutely abolished in the gadR glnR deletion strain. Electrophoretic mobility shift and DNase I footprinting assays revealed that GlnR directly bound to the 5'-untranslated regions of the gadR gene and gadCB operon, thus inhibiting their transcription. These results revealed a novel regulatory mechanism of GlnR on glutamate-dependent acid resistance in LactobacillusIMPORTANCE Free-living lactic acid bacteria often encounter acid stresses because of their organic acid-producing features. Several acid resistance mechanisms, such as the glutamate decarboxylase system, F1Fo-ATPase proton pump, and alkali production, are usually employed to relieve growth inhibition caused by acids. The glutamate decarboxylase system is vital for GAD-containing lactic acid bacteria to protect cells from DNA damage, enzyme inactivation, and product yield loss in acidic habitats. In this study, we found that a MerR-type regulator, GlnR, was involved in glutamate-dependent acid resistance by directly regulating the transcription of the gadR gene and gadCB operon, resulting in an inhibition of GABA conversion from glutamate in L. brevis This study represents a novel mechanism for GlnR's regulation of glutamate-dependent acid resistance and also provides a simple and novel strategy to engineer Lactobacillus strains to elevate their acid resistance as well as GABA conversion from glutamate.

Dataset Information

A glutamate-dependent acid resistance gene in Escherichia coli.

Publications

A glutamate-dependent acid resistance gene in Escherichia coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets