Virulence factor regulator (Vfr) enhances Pseudomonas aeruginosa pathogenicity through its role as a global transcriptional regulator. The crystal structure of Vfr shows that it is a winged-helix DNA-binding protein like its homologue cyclic AMP receptor protein (CRP). In addition to an expected primary cyclic AMP-binding site, a second ligand-binding site is nestled between the N-terminal domain and the C-terminal helix-turn-helix domain. Unlike CRP, Vfr is a symmetric dimer in the absence of DNA. Removal of seven disordered N-terminal residues of Vfr prevents the growth of P. aeruginosa.Pseudomonas aeruginosa is a ubiquitous, Gram-negative opportunistic pathogen that is a leading source of morbidity and mortality for individuals with compromised immune systems or cystic fibrosis. P. aeruginosa switches from an environmental organism to a pathogen through the regulation and elaboration of multiple virulence factors. Virulence factor regulator (Vfr) is a central player in a transcriptional cascade that controls this transition.Vfr is a 24-kDa protein belonging to the winged-helix family of transcription regulators (40). Vfr positively regulates quorum sensing by promoting the transcription of lasR, upregulates transcription of the type III secretion system, represses flagellar gene transcription, and positively regulates twitching motility (1, 2, 9, 41). Vfr binds cyclic adenosine 3Ј5Ј monophosphate (cyclic AMP [cAMP]) in vitro with a dissociation constant of 1.6 M (39). Deletion of both cytoplasmic P. aeruginosa adenyl cyclases results in a transcription profile similar to a vfr deletion (41). Furthermore, like the strain carrying the vfr deletion, the double adenyl cyclase mutant has reduced virulence compared to the wild-type strain in a mouse pneumonia model (38). Thus, cAMP is a biologically relevant ligand for Vfr in vivo.The amino acid sequence of Vfr is 67% identical to that of cAMP receptor protein (CRP) from Escherichia coli (40), a long-standing model for the study of transcription regulation and a structurally well-characterized protein (22). When overexpressed, Vfr complements the ␤-galactosidase and tryptophanase-deficient phenotypes of a crp deletion in E. coli (40). Conversely, CRP expressed in a vfr deletion strain of P. aeruginosa promotes sub-wild-type levels of lasR transcription. However, CRP is unable to rescue the protease-or exotoxin A-deficient phenotypes of the vfr mutant (40). Recent work suggests that this discrepancy might be due at least in part to cAMPindependent regulation at the lasR promoter (15).Mounting evidence supports a model for CRP function in which a dimer of CRP binds a cAMP ligand in the N-terminal domain of each monomer to achieve the protein conformation needed for high-affinity specific DNA binding and DNA bending that precedes transcriptional activation. CRP demonstrates low affinity for specific DNA sequences, with a K d of ϳ10 Ϫ4 M, in the absence of cAMP (16). Its specificity and affinity for its promoter sites increase to 10 Ϫ7 to 10 Ϫ8 M in cAMP concentrations...

Calculating the cost of an emergency medical services (EMS) system using a standardized method is important for determining the value of EMS. This article describes the development of a methodology for calculating the cost of an EMS system to its community. This includes a tool for calculating the cost of EMS (the "cost workbook") and detailed directions for determining cost (the "cost guide"). The 12-step process that was developed is consistent with current theories of health economics, applicable to prehospital care, flexible enough to be used in varying sizes and types of EMS systems, and comprehensive enough to provide meaningful conclusions. It was developed by an expert panel (the EMS Cost Analysis Project [EMSCAP] investigator team) in an iterative process that included pilot testing the process in three diverse communities. The iterative process allowed ongoing modification of the toolkit during the development phase, based upon direct, practical, ongoing interaction with the EMS systems that were using the toolkit. The resulting methodology estimates EMS system costs within a user-defined community, allowing either the number of patients treated or the estimated number of lives saved by EMS to be assessed in light of the cost of those efforts. Much controversy exists about the cost of EMS and whether the resources spent for this purpose are justified. However, the existence of a validated toolkit that provides a standardized process will allow meaningful assessments and comparisons to be made and will supply objective information to inform EMS and community officials who are tasked with determining the utilization of scarce societal resources.