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J. Bacteriol., Jul 1996, 3978-3981, Vol 178, No. 13
BM Hersh, FT Farooq, DN Barstad, DL Blankenhorn and JL Slonczewski
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.
Copyright © 1996, American Society for Microbiology
A glutamate-dependent acid resistance gene in Escherichia coli
Department of Biology, Kenyon College, Gambier, Ohio 43022, USA.
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