The Functions of the CID and lRG Operons in S. Aureus Programmed Cell Death

Xinyan Zhang, University of Nebraska Medical Center


Staphylococcus aureus cid/lrg operons regulate the formation of S.aureus biofilm formation and programmed cell death based on previous in vivo work done in Dr. Bayles's lab. cid operon, which encodes CidA/CidB/CidC proteins, has been shown to be an effector in leading to the lysis and death of the S.aureus; While lrg operon, encoding LrgA and LrgB proteins, is an inhibitor of the lysis and death. Recent studies suggest that CidA behaves like holin proteins from bacterial phage, by increasing the murein hydrolysis activity under aerobic culturing conditions. LrgA, together with LrgB, appears to inhibit this function. Another important studies on S.aureus under carbon-overflow condition indicates that CidC plays a critical function in the autolysis of the bacteria by producing acetic acid, and finally lead to the death of the bacteria.

Based on these findings, we further characterize these proteins in this project with in vitro high concentration of purified proteins. Our result indicates that CidA/LrgA causes the leakage of smaller fluorescent dyes, but not large proteins, from the artificial membrane. And CidA showed a more rapid leakage compare to LrgA at the same concentration. This result may indicate that the function of CidA is penetrating the bacterial membrane and may induce the formation of nanometer level pores.

CidC is also overexpressed and purified. The assay has conclusively proved that CidC is a pyruvate oxidoreductase and binds to the biological membrane, It passes the electron to menaquinone in vivo and produces acetate instead of hydrogen peroxide. The activity of CidC is strongly pH dependent and replies on the cofactor thiamine pyrophsophate and Mg2+ ion.

In my last project, the effect of GC-rich sequence on the stability of thrombin-binding aptamer G-quadruplex was investigated. Two complementary GC-rich single strands are attached to the aptamer separately or both at the same time. The results showed that one single strand has a deleterious effect on the formation of the G-quadruplex but the other single strand has little effect on it, when both attached, they form a duplex on top of the G-quadruplex. The study has great indication on the mechanism of how the flanking sequence affects the stability of the thrombin-binding aptamer as well as the in vivo telomeric G-quadruplex.