RESUMEN
Vibrio cholerae causes the human disease cholera by producing a potent toxin. The V. cholerae virulence pathway involves an unusual transcription step: the bitopic inner-membrane proteins TcpP and ToxR activate toxT transcription. As ToxT is the primary direct transcription activator in V. cholerae pathogenicity, its regulation by membrane-localized activators is key in the disease process. However, the molecular mechanisms by which membrane-localized activators engage the transcription process have yet to be uncovered in live cells. Here we report the use of super-resolution microscopy, single-molecule tracking, and gene knockouts to examine the dynamics of individual TcpP proteins in live V. cholerae cells with < 40 nm spatial resolution on a 50 ms timescale. Single-molecule trajectory analysis reveals that TcpP diffusion is heterogeneous and can be described by three populations of TcpP motion: one fast, one slow, and one immobile. By comparing TcpP diffusion in wild-type V. cholerae to that in mutant strains lacking either toxR or the toxT promoter, we determine that TcpP mobility is greater in the presence of its interaction partners than in their absence. Our findings support a mechanism in which ToxR recruits TcpP to the toxT promoter for transcription activation.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/ultraestructura , Proteínas de Unión al ADN/genética , Técnicas de Inactivación de Genes , Microscopía , Mutación , Activación Transcripcional , Vibrio cholerae/patogenicidadRESUMEN
Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane.
Asunto(s)
Bacterias/ultraestructura , Rastreo Celular/métodos , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , NanotecnologíaRESUMEN
An evidence-based public health (EBPH) course was developed in 1997 by the Prevention Research Center at Saint Louis University School of Public Health to train the public health workforce to enhance dissemination of EBPH in their public health practice. An on-line evaluation of the course was conducted among participants who attended the course from 2001 to 2004 to determine the impact the course had on the implementation of EBPH within their Respective public health agencies (n = 107). The majority of these individuals were program directors, managers, or coordinators working in state health departments. Results from the evaluation Revealed that 90 percent of participants indicated that the course helped them make more informed decisions in the workplace. Respondents identified improvement in their ability to communicate with their coworkers and Read Reports. When asked to identify potential barriers, participants specified that time constraints were the biggest impediment to using EBPH skills in the workplace. These data suggest the importance of professional training opportunities in EBPH for public health practitioners. Future endeavors should focus on overcoming the barriers to the dissemination of EBPH.