Mechanism of heme regulation of a P. aeruginosa non-coding RNA

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DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infection in compromised individuals, including neutropenic cancer patients and individuals with cystic fibrosis (CF). To establish successful infection, P. aeruginosa requires iron and employs several strategies for its acquisition, including the uptake of heme. Although required for survival, surplus iron or heme can lead to oxidative stress; thus, the ferric uptake regulator (Fur) protein regulates the uptake of these nutrients. In iron-replete environments, Fur blocks expression of genes required for iron and heme uptake, as well as two nearly identical genes encoding the PrrF1 and PrrF2 small regulatory RNAs (Wilderman et al., 2004). We previously showed that the PrrF RNAs negatively affect the expression of at least 50 genes, the products of which function in key metabolic pathways, and one of which profoundly affects production of quorum sensing factors (Oglesby et al., 2008). Consequently, the PrrF RNAs are capable of exerting wide-ranging effects on P. aeruginosa physiology and virulence. Heme is an abundant source of iron in the human body, and its acquisition by P. aeruginosa is hypothesized to play a significant role in infection. Because of the potentially toxic effects of heme, it is expected that a heme regulatory system coordinates expression of genes for heme uptake, degradation, and biosynthesis. With the substantial implications that heme has for virulence, identifying the mechanism(s) by which this compound acts as a signaling molecule will yield novel targets for therapeutic purposes. The PrrF RNAs in P. aeruginosa are encoded in tandem by two virtually identical genes, prrF1 and prrF2 (Fig. 1), while all other pseudomonads encode for the PrrF RNAs at distal loci. I and others in Dr. Vasil's laboratory showed that the prrF locus of P. aeruginosa encodes an additional, 325-nucleotide (nt) RNA, designated PrrH, which is repressed by heme as well as iron (Oglesby-Sherrouse & Vasil, 2010, Ochsner et al., 2000). Transcription of PrrH initiates at the 5' end of prrF1, proceeds through the prrF1-prrF2 intergenic sequence (95 nt), and terminates at the 3' end of prrF2 (Fig. 1). Thus, expression of prrH is dependent on read-through transcription at the prrF1 Rho-independent, or intrinsic, terminator. My data indicate PrrH regulates genes involved in heme biosynthesis (Oglesby- Sherrouse & Vasil, 2010) and virulence (Table 1), suggesting this tandem prrF organization imparts unique heme regulatory activities to P. aeruginosa. I hypothesize that a heme- regulated antiterminator (PHAT - Fig. 1) recognizes RNA sequence preceding or DNA sequence following the prrF1 terminator and relieves the hairpin structure of the nascent RNA, allowing stabilization of the transcription elongation complex (TEC) and continued transcription of the PrrH RNA. This proposal will determine the mechanism of prrH expression and heme regulation by I) determining the sequence requirements for expression and heme regulation of prrH and putative PrrH targets and II) identifying genes and proteins involved in expression and regulation of prrH.