Stationary-phase cultures of Escherichia coli can survive several hours of 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 microtiter 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؇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.

The Caenorhabditis elegans Bcl-2-like protein CED-9 prevents programmed cell death by antagonizing the Apaf-1-like cell-death activator CED-4. Endogenous CED-9 and CED-4 proteins localized to mitochondria in wild-type embryos, in which most cells survive. By contrast, in embryos in which cells had been induced to die, CED-4 assumed a perinuclear localization. CED-4 translocation induced by the cell-death activator EGL-1 was blocked by a gain-of-function mutation in ced-9 but was not dependent on ced-3 function, suggesting that CED-4 translocation precedes caspase activation and the execution phase of programmed cell death. Thus, a change in the subcellular localization of CED-4 may drive programmed cell death.

The misregulation of programmed cell death, or apoptosis, contributes to the pathogenesis of many diseases. We used Nomarski microscopy to screen for mutants containing refractile cell corpses in a C. elegans strain in which all programmed cell death is blocked and such corpses are absent. We isolated a mutant strain that accumulates refractile bodies resembling irregular cell corpses. We rescued this mutant phenotype with the C. elegans mucolipidosis type IV (ML-IV) homolog, the recently identified cup-5 (coelomocyte-uptake defective) gene. ML-IV is a human autosomal recessive lysosomal storage disease characterized by psychomotor retardation and ophthalmological abnormalities. Our null mutations in cup-5 cause maternaleffect lethality. In addition, cup-5 mutants contain excess lysosomes in many and possibly all cell types and contain lamellar structures similar to those observed in ML-IV cell lines. The human ML-IV gene is capable of rescuing both the maternal-effect lethality and the lysosome-accumulation abnormality of cup-5 mutants. cup-5 mutants seem to contain excess apoptotic cells as detected by staining with terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. We suggest that the increased apoptosis seen in cup-5 mutants is a secondary consequence of the lysosomal defect, and that abnormalities in apoptosis may be associated with human lysosomal storage disorders. P rogrammed cell death or apoptosis regulates cell number during metazoan development (1). The misregulation of programmed cell death, resulting in either too many or too few dying cells, contributes to the pathogenesis of many human diseases (2). For example, animal models of human retinitis pigmentosa indicate that retinal degeneration occurs by apoptotic death, and that blocking this death can prevent retinal degeneration and restore vision (3-5). Mutations that disrupt Fas-mediated apoptosis in the immune systems of mice and humans lead to lymphoproliferative disorders (6, 7). The inhibition of apoptosis caused by the misexpression of the proto-oncogene Bcl-2 is a primary cause of human follicular lymphoma (8), a cancer of the immune system.Studies of the nematode Caenorhabditis elegans have played an important role in defining the components that regulate and execute programmed cell death (9). To seek new genes that can affect programmed cell death, we screened for suppressors of a gain-of-function (gf) allele of the C. elegans Bcl-2-like gene ced-9. The ced-9 gene normally protects cells that should survive from undergoing programmed cell death (10), and the ced-9(gf) allele n1950 causes all programmed cell death to be blocked (11). In this paper, we describe one ced-9(gf) suppressor, n3194, which proved to be a mutation in the C. elegans counterpart of the human mucolipidosis type IV (ML-IV) gene (12, 13), cup-5 (coelomocyteuptake defective; ref. 14). Materials and MethodsGenetics and Strains. Strains were cultured as described (15) LGIV: ced-3(n717), unc-31(e928), ced-5(n1812). We used three-factor mapping to localiz...