BACTERIAL GENETICS BY FLOW-CYTOMETRY - RAPID ISOLATION OF SALMONELLA-TYPHIMURIUM ACID-INDUCIBLE PROMOTERS BY DIFFERENTIAL FLUORESCENCE INDUCTION

The ability of Salmonella typhimurium to survive and replicate within murine macrophages is dependent on a low phagosomal pH. This requireme nt for an acidic vacuole suggests that low pH is an important environm ental stimulus for the transcription of genes necessary for intracellu lar survival. To study the behaviour of acid-inducible genes in respon se to the phagosomal environment, we have applied a novel enrichment s trategy, termed (d) under bar ifferential (f) under bar luorescence in duction (DFI), to screen an S. typhimurium library for promoters that are upregulated at pH 4.5. DFI utilizes a fluorescence-enhanced green fluorescent protein (GFP) and a fluorescence-activated cell sorter (FA CS) to perform genetic selection. In the presence of an inducing stimu lus, such as low pH, a FACS is used to sort highly fluorescent bacteri al clones bearing random promoters fused to the mutant GFP protein (GF Pmut). This population is then amplified at neutral pH and the least f luorescent population is sorted. Sequential sorts for fluorescent and nonfluorescent bacteria in the presence or absence of inducing conditi ons rapidly enriches for promoter fusions that are regulated by the in ducing stimulus. We have identified eight acid-inducible promoters and quantified their expression in response to pH 4.5 and to the phagosom e milieu. These acid-inducible promoters exhibited extensive homology to promoter regions of genes encoding for cell-surface-maintenance enz ymes, stress proteins, and generalized efflux pumps. Only a subset of these promoters was induced in macrophages with kinetics and levels of expression that do not necessarily correlate with in vitro pH-shock i nduction. This suggests that while low pH is a relevant inducer of int racellular gene expression, additional stimuli in the macrophage can m odulate the expression of acid-inducible genes.