The twin-arginine translocase (TAT) in some bacterial pathogens, including Pseudomonas aeruginosa, Burkholderia pseudomallei, and Mycobacterium tuberculosis, contributes to pathogenesis by translocating extracellular virulence determinants across the inner membrane into the periplasm, thereby allowing access to the Xcp (type II) secretory system for further export in Gramnegative organisms, or directly to the outside surface of the cell, as in M. tuberculosis. TAT-mediated secretion appreciably contributes to virulence in both animal and plant models of bacterial infection. Consequently, TAT function is an attractive target for small-molecular-weight compounds that alone or in conjunction with extant antimicrobial agents could become novel therapeutics. The TAT-transported hemolytic phospholipase C (PlcH) of P. aeruginosa and its multiple orthologs produced by the above pathogens can be detected by an accurate and reproducible colorimetric assay using a synthetic substrate that detects phospholipase C activity. Such an assay could be an effective indicator of TAT function. Using carefully constructed recombinant strains to precisely control the expression of PlcH, we developed a high-throughput screening (HTS) assay to evaluate, in duplicate, >80,000 small-molecular-weight compounds as possible TAT inhibitors. Based on additional TAT-related functional assays, purified PlcH protein inhibition experiments, and repeat experiments of the initial screening assay, 39 compounds were selected from the 122 initial hits. Finally, to evaluate candidate inhibitors for TAT specificity, we developed a TAT titration assay that determines whether inhibition of TAT-mediated secretion can be overcome by increasing the levels of TAT expression. The compounds N-phenyl maleimide and Bay 11-7082 appear to directly affect TAT function based on this approach.T he twin-arginine translocase (TAT) secretion system was first discovered as a Sec-independent mechanism by which proteins involved in photosynthesis are transported into the thylakoids of plant chloroplasts (10). Orthologs of plant TAT proteins were then identified in Escherichia coli and subsequently in other bacteria and archaea (58). However, no orthologs or functional analogs of TAT proteins have thus far been identified in animal cells.The TAT system in plants translocates proteins into the thylakoid compartment of chloroplasts, whereas in bacteria and archaea, it can export proteins out of the cytoplasm and across the cytoplasmic membrane. In Gram-negative organisms (e.g., E. coli, Pseudomonas aeruginosa), proteins secreted by the TAT system are most frequently terminally localized in the periplasm, but some (e.g., phospholipases C) can be further secreted by the Xcp (i.e., type II) system, thereby becoming extracellular (41,65,66). In Gram-positive bacteria (e.g., Bacillus spp., Mycobacterium spp., and Streptomycetes spp.) (26, 27, 36), TAT substrates typically remain cell associated upon secretion through the cytoplasmic membrane, but at least three proteins (i.e., agara...