B-247. The Role of Programmed Cell Death in the Development of Staphylococcal Biofilm

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

Our laboratory has demonstrated that the Staphylococcus aureus cid and lrg operons play important roles in the control of bacterial cell death and lysis. Recently, this system was shown to be important in biofilm development by demonstrating that a cidA mutant, which is defective in murein hydrolase activity and autolysis, displayed reduced biofilm adherence due to the decreased levels of extracellular DNA (eDNA) released into the biofilm matrix. In addition to providing evidence for the function of the cid/lrg system, these results provided evidence for the biological role of bacterial programmed cell death (PCD). To further examine the role of this system in biofilm development, the impact of various cid/lrg mutations on biofilm formation was assessed in an in vitro flow cell biofilm model. Both cidA and lrgAB mutations had dramatic effects on biofilm morphology and adherence. The cidA mutant biofilm lacked distinct tower structures that are produced by the parental strain, UAMS-1, and was visibly less adherent. In contrast, the lrgAB mutant biofilm was more adherent but still failed to form tower structures. As previously observed, the cidA mutant biofilm contained decreased eDNA levels while the lrgAB mutant biofilm contained increased eDNA levels. Biofilm produced by the previously described cidR regulatory mutant also lacked tower structures but produced biofilm containing patches of live and dead cells, suggesting that CidR controls the spatial pattern of cell death during biofilm development. Finally, a role for staphylococcal nuclease in biofilm formation was demonstrated, since a nuc mutant produced tower structures but exhibited increased levels of eDNA. Overall, these results indicate that cid- and lrg-mediated cell death and lysis play an important role in the release of eDNA that contributes to the biofilm matrix, as well as in the formation of biofilm structure. These data also suggest that cid-/lrg-mediated cell death and lysis are highly regulated processes, likely responding to specific metabolic signals during biofilm development.