CHARACTERIZATION OF NOVEL CYCLIC-DI GMP EFFECTORS AFFECTING B.BACTERIOVORUS` METABOLISM AND CELL CYCLE

The second messenger cyclic di-GMP (c-di-GMP) is a lifestyle regulator in many bacteria. c-di-GMP transduces signals by binding to effector proteins affecting, catalysis, protein interactions or gene expression. We used Bbellovibrio bacteriovorus as a model predatory bacterium in order to characterize the role of c-di GMP signaling in the control over cell cycle progress and replication. The unique characteristic of this bacterium is its obligate requirement for other gram-negative cells, which it invades and uses as a substrate. Another unique feature of B.bacteriovorus is its biphasic life cycle which includes a free-swimming attack phase (AP) and a periplasmic growth phase (GP). The transition between the AP to the GP appears to be linked to the RNA degradosome. c-di-GMP has been previously shown to significantly affect the life cycle of the B. bacteriovorus. Further, many putative, novel c-ci-GMP binding proteins were identified in a pull down experiment (Rotem et al. 2015). Polynucleotide phosphorylase (PNPase)A, a key component of the RNA degradosome was identified as as potential c-di-GMP binder in B.bacteriovorus. Selective and high affinity c-di-GMP binding (K_d=4.8µM)were demonstrated by microscale thermophoresis using a purified protein. Additionally, we show that the expression of central metabolism enzymes like an acetyl-CoA acyltransferase (degradative thiolase) and an NADP-dependent malate dehydrogenase which were also detected in the c-di-GMP pull down is affected by a putative c-di-GMP riboswitch, coined merRNA. The genes were expressed in GP in the WT strain but not in the GP in the merRNA deletion mutant.