The gastric acid barrier presents a major obstacle to enteric pathogens and, as a result, is considered a key has strategy for preventing gastrointestinal infections. Yet some organisms, such as E. coli O157:H7, require extremely low oral infectious doses, suggesting the presence of extraordinary counter-defenses against acid stress. Our laboratory has discovered three highly effective and inducible acid resistance (AR) systems that protect pathogenic and commensal strains of Escherichia coli during passage through host gastric acid barriers. AR system 1 is glucose repressed, requires the alternative sigma factor 's and cAMP receptor protein for expression and will protect cells at pH 2.5 in minimal media. AR systems 2 and 3 depend upon amino acid decarboxylase/antiporter systems that utilize extra-cellular glutamate (AR system 2) and arginine (AR system 3) to protect cells during extreme acid challenges. However, the molecular basis of acid resistance provided by these three systems remains a mystery. The factors involved in system 1 AR are unknown and although some components of systems 2 and 3 have been identified, additional, unrecognized factors are clearly required. This application represents a comprehensive research plan employing biochemical and genetic approaches designed to probe the mechanisms and control of these critical acid survival systems. Focus is placed on systems 1 and 2 since systems 2 and 3 appear mechanistically similar. Specific aims are designed to examine the impact of AR systems 1 and 2 on pH homeostasis, characterize system-specific proton and counterion movements, probe the function of a critically important antiporter, identify requisite accessory proteins and define the complex transcriptional and post-transcriptional regulation of both systems. The molecular response of microbes to environmental stress is an exciting area of modern biology but often the functions of specific members are unknown and their roles in helping the cell survive the inducing stress obscure. The inducible acid resistance systems described provide a rare opportunity to learn how a microorganism senses a given stress (acid) and responds to that stress in a focused, purposeful manner to escape death. In a broader sense, knowledge gained from this study will also provide insights into fundamental questions concerning proton circulations, protein structure-function relationships and gene expression as each relates to acid stress