Characterization of a Negative-Acting Sequence Downstream of the Phenazine Biosynthetic Promoter That Reduces Gene Expression in Pseudomonas Chloroaphis

Abstract

The bacterium Pseudomonas chlororaphis strain 30-84, isolated from the roots of wheat, biosynthesizes heterocyclic, nitrogen containing secondary metabolites termed phenazines. Phenazines have many practical implementations, such as the inhibition of pathogenic microorganisms and the formation of biofilms. However, in order to maximize the utility of phenazines, the complex regulatory mechanisms that control the phenazine biosynthetic pathway need to be better understood. This paper focuses on the characterization of a specific 185 base pair (bp) sequence located within the phenazine promoter region the presence of which attenuates phenazine gene expression. This region was identified by analysis of phenazine promoter subclones fused to a reporter gene. There are two likely mechanisms By which this region could reduce gene expression, one involves a DNA regulatory mechanism and a second involves a messenger RNA (mRNA) mechanism. Both of these possible mechanisms will be discussed and explored in this paper Various experiments were performed with the hope of characterizing this regulatory sequence. Some results helped to characterize the pathway, including every phenazine promoter subclone was subject to the mechanism of quorum sensing. The most interesting results suggest that there is a small segment of DNA in the 185 bp attenuator region that is capable of forming a stem-loop hairpin structure, according to mRNA MFOLD data. This data suggests that the attenuator subclone region may act via formation or relief of this stem-loop structure. If this is true, it is a big step towards further understanding of the phenazine promoter region. Through my work in the Pierson laboratory, we have learned more about the minimal size and regions required for an active phenazine promoter.