C-di-GMP acts as a cell cycle oscillator to iihkdrive chromosome replication Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Christian Lori aus Malans (GR), Schweiz Basel, 2016 Originaldokument gespeichert auf dem Dokumentenserver der Universität ddfdfdfdBasel edoc.unibas.ch 1 Genehmigt von der Philosphisch-Naturwissenschaftlichen Fakultät auf Antrag von: Prof. Dr. Urs Jenal Prof. Dr. Christoph Dehio Basel, den 22. März 2016 Prof. Dr. Jörg Schibler Dekan Christian Lori: C-di-GMP acts as a cell cycle oscillator to drive chromosome replication, PhD Thesis, 2016 2 Abstract _____________________________________________________________________ Cyclic di-GMP (c-di-GMP) is an omnipresent bacterial second messenger molecule which has been recognized as a central regulator of lifestyle transitions. Generally, high levels of c-di-GMP promote a biofilm associated, surface attached lifestyle, while low levels of c-di-GMP favor a single cell, motile lifestyle. A wide range of c-di-GMP effector proteins are known which control various cellular functions. It has long been assumed that c-di-GMP is involved in the regulation of cell cycle progression. In this work the role of c- di-GMP on the G1-S transition is described in the aquatic bacterium Caulobacter crescentus. C. crescentus is an ideal model organism since G1-S transition is developmentally linked to the swarmer to stalked cell transition and therefore easily observable. Moreover, c-di-GMP influences several processes at the swarmer to stalked cell transition. Thus, disturbing the c-di-GMP-dependent processes causes specific phenotypes. In the first part of this work, the effect of c-di-GMP on core components of the C. crescentus cell cycle control machinery is assessed. It is described that the essential histidine kinase CckA (Cell cycle kinase A) is regulated by c-di-GMP. Binding of CckA to c-di-GMP activates the phosphatase activity of CckA and leads to dephosphorylation of the transcription factor CtrA (Central transcriptional activator A) which ultimately initiates chromosome replication. Furthermore it is shown that c-di-GMP is required in the predivisional cell to establish a CckA-dependent CtrA phosphorylation gradient. The second part describes the mechanism by which c-di-GMP activates CckA phosphatase activity. It was possible to isolate several mutations in CckA which specifically target certain activities of CckA and thereby give an insight into the intramolecular signaling mechanisms. Additionally, a recently solved crystal structure of CckA in complex with c-di-GMP will increase our understanding of the activation of phosphatse activity. The third part of this work deals with the regulation of several histidine kinases by a single domain response regulator. The single domain response Regulator MrrA (Multifunctional response regulator A) is shown to be a central part of the C. crescentus stress response pathway. MrrA is phorphorylated by two cognate histidine kinases and additionally acts as a repressor of one of the kinases. The downstream target of MrrA is the histidine kinase LovK which is the main activator of the general stress response. It is demonstrated that phosphorylated MrrA is an allosteric activator of LovK. 3 Taken together this work increases the understanding of how c-di-GMP regulates cell cycle progression and additionally gives insight into the modes of regulation of histidine kinases. 4 Contents _____________________________________________________________________ Introduction ..................................................................................................... 6 C-di-Nucleotide signaling ........................................................................... 6 Histidine kinases and two-component systems .................................... 29 Caulobacter crescentus cell cycle.................................................................... 32 CckA controls CtrA phosphorylation .................................................... 34 The general stress response ..................................................................... 36 Aim of the thesis ........................................................................................... 38 Results ............................................................................................................ 39 Cyclic di-GMP acts as a cell cycle oscillator to drive chromosome replication ................................................................................................... 39 Second messenger enforced bi-functionality of a central cell cycle switch .......................................................................................................... 82 Multifunctional single domain response regulator mediates SigT- dependent stress response in caulobacter crescentus ................................114 Additional results ....................................................................................169 Discussion & Outlook ...............................................................................183 Acknowledgments.......................................................................................187 References ....................................................................................................188 Curriculum vitae ..........................................................................................215 5 Introduction _____________________________________________________________________ C-di-Nucleotide signaling The following section on c-di-nucleotide signaling is written to be published as a review in Nature Reviews Microbiology. Text is written by Alberto Reinders (cyclases, phosphodiesterases, biofilm and motility) and myself (development, virulence, immunity, methods and “other c-di-nucleotides”). Abstract C-di-Nucleotides (cdN) are versatile signaling molecules used by bacterial and eukaryotic cells as second messengers. The best-studied example is bis-(3′-5′)- cyclic dimeric GMP (c-di-GMP). Known since the late 1980`s it is now regarded a widespread bacterial second messenger. Recent discoveries, aided by the development of new techniques, shed light on the various processes controlled by c-di-GMP. C-di-GMP effectors display a wide range of targets, ranging from core cell cycle events to biofilm formation, motility and virulence. Here we review the latest discoveries focusing on effector proteins and the output functions controlled by c-di-GMP. We also briefly review the recently emerging second messengers c-di-AMP as well as c-GMP-AMP (cGAMP). 6 DGCs & PDEs A planktonic lifestyle is incompatible to a sedentary lifestyle and requires profound reprogramming of cell physiology [1–5]. To trigger the transition and establish the lifestyle the cellular c-di-GMP concentrations have to be precisely set and readily adjusted if the environment requires adaptation. This requisite demands a highly fine-tuned network that can sense a plethora of stimuli to ultimately establish the appropriate c-di-GMP regime. This is achieved through the antagonistic enzyme families, which comprise two of the largest known enzyme families in the bacterial kingdom [6], namely diguanylate cyclases (DGC), which condense two GTP into c-di-GMP [7] and c-di-GMP-specific phosphodiesterases (PDE), which degrade it [8,9]. Diguanylate cyclases are characterized by their consensus GGDEF motif, while c-di-GMP-specific phosphodiesterases either contain an EAL or HD-GYP-motif [10,11]. These proteins either exist as stand-alone proteins or fused to one another to function as so-called “composite proteins”. While composite proteins comprise a large fraction of c-di-GMP-related enzymes and are an avid research target, their function and especially the regulatory mechanisms regulating either of the enzymatic activity still remain elusive. A recurring theme is that most enzymatic domains come along with N-terminally associated accessory domains that in most cases are believed to serve as input domains regulating the enzymatic output domain. The recent advances in structure and mechanism of c-di-GMP synthesizing and degrading enzymes are centered on the regulatory features of these remarkable enzymes. For nearly a decade, PleD from C. crescentus served as a cornerstone in understanding the catalytic and regulatory mechanisms of diguanylate cyclases [12], stating that induced dimerization of the GGDEF-domain drives condensation of c-di-GMP [12]. Moreover, PleD is a precedent in respect to the inherent regulation of catalysis. Most diguanylate cyclases share an allosteric product inhibition site (I-site), most likely to refrain a bacterial cell from excessive GTP consumption or accumulation of unphysiologically high c-di- GMP concentrations [13]. This feature however is not conserved throughout all active diguanylate cyclases. E.g., structural and biochemical insights into DgcZ (formerly YdeH [14]) from E. coli revealed that this particular cyclase does contain an inhibitory I-site, which nevertheless only shows its effect at unphysiologically high at c-di-GMP concentrations (ca. 40 µM) [14]. DgcZ is a constitutive dimer and its enzymatic activity is inhibited through subfemtomolar binding of zinc to the N-terminally 7 associated chemoreceptor zinc binding (CZB) domain. Mechanistically it was suggested that Zn2+-binding to the CZB arranges the GGDEF-domains of DgcZ such that their mobility is impeded, thereby hindering correct positioning of the substrates