D-112. Characterization of Staphylococcus aureus Nitric Oxide (NO)-Related Genes and their Potential Role in the Cid/Lrg Cell Death Regulon

K. C. Rice, J. L. Endres, K. W. Bayles;
Univ. of Nebraska Med. Ctr., Omaha, NE.

The S. aureus cidA and lrgA genes affect cell lysis under a variety of conditions during planktonic growth. It is hypothesized that these genes encode holins and antiholins, respectively, and serve as molecular control elements of cell lysis. Recently we have shown that cidA-mediated lysis plays an important role during biofilm development and that released genomic DNA is an important structural component of S. aureus biofilm. Cell death and lysis events are also important for later stages of biofilm development (maturation and detachment) in other organisms. A recent Pseudomonas aeruginosa study demonstrated that cell death and detachment from mature biofilm was enhanced in the presence of NO. The S. aureus scdA gene encodes a putative NO-sensing protein, and is located immediately upstream to the lytSR-lrgAB genes. Additionally, a novel NO reductase gene (nor) is located immediately downstream of lrgAB in the clinical isolate UAMS-1. Based on these observations, we hypothesized that NO may be involved in regulating cell death via the Cid/Lrg system. Northern blot analysis of UAMS-1 grown under low O2 conditions revealed that expression of scdA, nor, lrgAB and cidABC transcripts were all increased relative to an aerobic culture. Furthermore, lrgAB expression was reduced in an isogenic scdA mutant grown under low O2 conditions, and scdA expression itself appeared to be negatively auto-regulated. Static biofilms of UAMS-1 and the scdA mutant were grown in the presence of NO donor or NO donor + NO scavenger. Confocal microscopy of these biofilms showed that NO treatment altered the morphology of the scdA mutant biofilm but had no effect on UAMS-1, while the presence of NO scavenger resulted in flat, uniform biofilms in both strains. When UAMS-1 and isogenic nor mutant biofilms were grown in the presence of NO donor, the nor mutant displayed a 3-fold decrease in biofilm adherence relative to UAMS-1. Collectively, these results indicate that ScdA and Nor are involved in NO sensing and/or metabolism when S. aureus is grown under low O2 conditions or as biofilm, and may be involved in regulating Cid/Lrg mediated cell death and lysis.