In vivo UV laser footprinting of the Pseudomonas putidasigma 54Pu promoter reveals that integration host factor couples transcriptional activity to growth phase.
The occupation of the final sigma(54)-dependent Pu promoter of Pseudomonas putida by the integration host factor (IHF) under different growth conditions has been monitored in its native state and stoichiometry (i.e. monocopy) with UV laser footprinting technology. We present evidence that an abrupt change in intracellular IHF concentrations occurs when P. putida cells enter stationary phase. This change results in enhanced binding of the factor to the promoter and in the ensuing bending of the target DNA. Since Pu activity depends rigorously on DNA bending, promoter occupation is in turn translated into a much higher transcriptional output when cells leave exponential growth. Inspection of the residual activity of Pu in an IHF(-) strain reveals that IHF predominantly locks the capacity of the promoter to specific growth stages and also that additional physiological signals are entered in the system through final sigma(54)-RNA polymerase. The results substantiate the notion that final sigma(54) promoters process metabolic co-regulation signals through factor-induced changes in the architecture of the cognate DNA region. Further, they validate UV laser technology as a suitable tool to visualize nondisruptive alterations of DNA shape in vivo.
Valls
Marc
M
Department of Microbial Biotechnology, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, Campus de Cantoblanco, 28049 Madrid, Spain.
Buckle
Malcolm
M
de Lorenzo
Victor
V
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D034982:Integration Host Factors; D007834:Lasers; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics* / Q000254:growth & development; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000235:genetics*; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D012808:Sigma Factor; C440503:integration host factor, Pseudomonas; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54