During staphylococcal growth in glucose-supplemented medium, the pH of a culture starting near neutrality typically decreases by about 2 units due to the fermentation of glucose. Many species can comfortably tolerate the resulting mildly acidic conditions (pH, ϳ5.5) by mounting a cellular response, which serves to defend the intracellular pH and, in principle, to modify gene expression for optimal performance in a mildly acidic infection site. In this report, we show that changes in staphylococcal gene expression formerly thought to represent a glucose effect are largely the result of declining pH. We examine the cellular response to mild acid by microarray analysis and define the affected gene set as the mild acid stimulon. Many of the genes encoding extracellular virulence factors are affected, as are genes involved in regulation of virulence factor gene expression, transport of sugars and peptides, intermediary metabolism, and pH homeostasis. Key results are verified by gene fusion and Northern blot hybridization analyses. The results point to, but do not define, possible regulatory pathways by which the organism senses and responds to a pH stimulus.Facultative pathogens such as staphylococci produce a wide variety of accessory proteins. Many of these are involved in, or required for, infectivity and are collectively referred to as the virulon. It is widely believed that the genes encoding these proteins are regulated according to the exigencies of the local sites in which the organism is able to establish an infection. The evidence in support of this very logical view is, at best, rather sketchy, largely for two reasons. Firstly, it has, until very recently, been unapproachably difficult to determine the putative locale-specific gene expression patterns. Secondly, it has been no easy matter to identify the specific environmental factors that would determine these patterns. As a preamble to the ultimate objective of defining in vivo environmental factors that influence the expression of accessory genes involved in local pathogenesis, we have begun to reexamine the effect of certain nutrients and other chemicals, including glucose, acidic pH, salt, salicylic acid, and subinhibitory concentrations of antibiotics, on the expression of virulence genes during growth in vitro. These substances have widespread effects on the transcription of virulence genes and other accessory genes, which are presumably initiated through signaling elements and mediated through the complex regulatory network that governs the expression of all accessory genes (34). Our entrée into the transduction of environmental signals was provided by the observation that all of these substances affected transcription of a key signaling locus, sae, which is involved in the regulation of the expression of many exoproteins (36). In that study, which was initiated by a determination of the effect of glucose, it was shown that sae has a complex transcriptional pattern that undergoes a very striking switch during in vitro growth at neutral pH, in which...