Escherichia coli has an ability, rare among the Enterobacteriaceae, to survive extreme acid stress under various host (e.g., human stomach) and nonhost (e.g., apple cider) conditions. Previous microarray studies have exposed a cluster of 12 genes at 79 centisomes collectively called an acid fitness island (AFI). Four AFI genes, gadA, gadX, gadW, and gadE, were already known to be involved in an acid resistance system that consumes an intracellular proton through the decarboxylation of glutamic acid. However, roles for the other eight AFI gene products were either unknown or subject to conflicting findings. Two new aspects of acid resistance are described that require participation of five of the remaining eight AFI genes. YhiF (a putative regulatory protein), lipoprotein Slp, and the periplasmic chaperone HdeA protected E. coli from organic acid metabolites produced during fermentation once the external pH was reduced to pH 2.5. HdeA appears to handle protein damage caused when protonated organic acids diffuse into the cell and dissociate, thereby decreasing internal pH. In contrast, YhiF-and Slp-dependent systems appear to counter the effects of the organic acids themselves, specifically succinate, lactate, and formate, but not acetate. A second phenomenon was defined by two other AFI genes, yhiD and hdeD, encoding putative membrane proteins. These proteins participate in an acid resistance mechanism exhibited only at high cell densities (>10 8 CFU per ml). Densitydependent acid resistance does not require any demonstrable secreted factor and may involve cell contactdependent activation. These findings further define the complex physiology of E. coli acid resistance.
SummaryEscherichia coli survives pH 2 environments through an acid resistance (AR) system regulated by the transcriptional activator GadE. Numerous proteins control gadE at an upstream, conserved, 798 bp intergenic region. We show this region produces three transcripts starting at -124 (T1), -324/-317 (T2) and -566 (T3) bp from the gadE start codon. Transcriptional lacZ fusions to gadE promoter regions revealed P1 and P3 were active while P2 alone was not. However, pairing P3 with P2 activated P2 and increased expression 20-fold above P3 alone. The fusions were transferred to Salmonella, which lacks this AR system, and plasmid-borne E. coli-specific regulators EvgA, YdeO, GadE and GadX were introduced. Data revealed that YdeO and GadX activate P3, P2 and P3P2, while GadE autoactivates P1 and represses P3 and P3P2. The developing model indicates that different signals activate YdeO, GadX, or an MnmE-dependent regulator, which stimulate gadE transcription from the P3 and P2 promoters. Once made, GadE activates P1 and represses P3 and P2. The P1 region also enables efficient downstream transcription and translation of the P3 or P2 transcripts. Evidence indicates the entire 750 bp sensory integration locus is necessary for a versatile response.
Escherichia coli can survive pH 2 acid stress by using several acid resistance systems. The most efficient of these employs glutamate decarboxylase (GadA/GadB) to consume protons, and an antiporter (GadC) to exchange the intracellular decarboxylation product for external glutamic acid. Expression of the essential transcriptional activator of this system, GadE, is controlled by several regulators in a hierarchical fashion. In this study, two additional activators have been identified. The AraC-family regulators GadX and GadW, previously found to activate gadA/BC in vitro, are now shown in vivo to directly activate gadE expression, which, in turn, activates the gadA/BC genes. In vivo results using E. coli and Salmonella enterica show that these regulators actually have little direct effect on gadA and gadBC promoters. The numerous gadE induction pathways converge on a 798 bp control region situated upstream of the gadE promoter region. Deletions of this control region exposed the region between "798 and "360 nt (relative to the translational start) to be required for maximum gadE-lacZ expression in Luria-Bertani (LB) medium and to be the primary focus of GadX and GadW control. The GadE protein itself, which binds to three GAD box sequences present between "233 and "42 nt, helped activate GadE expression in LB, but only when the "798 to "360 region was absent. These regulatory regions and proteins appear to integrate a variety of physiological signals that forecast a need for GadE-dependent gene expression and acid resistance.
A transgenic approach in mice allows the functional interrogation of an odorant receptor locus in vivo and reveals characteristics of its monogenic and monoallelic expression.
The metabolism of copper by cultured fibroblasts from brindled male (Mobr/Y) and normal male ( + / Y ) mice was studied using radioactive copper ("Cu). Brindled fibroblasts accumulated more 64Cu than normal fibroblasts. The accumulation of "Cu by both brindled and normal fibroblasts was enhanced if the cells were preincubated in a coppersupplemented medium or if fetal calf serum was omitted from the medium. Brindled fibroblasts also retained more 64Cu on continued incubation. The greater accumulation might, therefore, represent an effect of reduced copper efflux. The demonstrated expression of the inherited defect in copper metabolism of brindled mice in cultured fibroblasts emphasizes the value of this model for further study of copper metabolism and its genetic control.
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