• Evaluation ofopd as a reporter gene in Streptomyces lividans

      Pierson, Vicki Lynn (1995)
      The transition from primary to secondary metabolism in Streptomyces is environmentally influenced, but it is unclear how environmental factors achieve this effect. In other genera, certain nonessential gene products such as virulence factors, pigments and bacteriocins can be regulated by multiple environmental factors, many of which can also alter DNA superhelicity. It was postulated that the primary to secondary metabolic transition in Streptomyces may be regulated by genes with promoters sensitive to transient changes in superhelicity. These promoters could be isolated in their native loci via an integrative vector bearing a reporter gene to monitor in situ expression. An {dollar}att\sp-{dollar} temperate promoter-probe phage utilizing the xylE gene of Pseudomonas putida used for this purpose failed to express xylE for unknown reasons. A promoter-probe suicide vector developed for interspecific conjugation gave a frequency of integration approximately equal to the rate of spontaneous mutation to resistance. As the work progressed, the need for a dependable reporter gene in Streptomyces became apparent. A gene for organophosphate degradation (o p d), parathion hydrolase, was evaluated for its utility as a reporter gene. It can be expressed from a variety of native and heterologous promoters, either inducible or constitutive, at various stages of growth. These promoters express o p d under the same conditions as they express their native products, and activity can be screened by single colony (microtiter plate) assay, quantitative by spectrophotometric assay, and detected even at low levels by immunoblotting. As a reporter gene, opd offers several advantages. It has no known analog in Streptomyces, so background interference is negligible, as is unwanted interaction with chromosonal sequences. It is metabolically benign, even at high levels of expression, and requires no cofactors for activity. Since the protein is quite stable, it may be particularly useful in detecting transiently expressed promoters. This protein is naturally secreted by Streptomyces and may be particularly useful in studying this process, although it is not limited to this application since adding intra- and extracellular fractions gives an accurate measurement of total gene expression.