An analysis of two-component regulatory systems in Myxococcus xanthus

Proteins of two-component regulatory systems (TCS) have essential functions in the sensing of external and self-generated signals in bacteria as well as in the generation of appropriate output responses. Accordingly, in Myxococcus xanthus TCS are important for fruiting body formation and sporulation...

Ausführliche Beschreibung

Gespeichert in:
1. Verfasser: Shi, Xingqi
Beteiligte: Sogaard-Andersen, Lotte (Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2008
Biologie
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Zusammenfassung:Proteins of two-component regulatory systems (TCS) have essential functions in the sensing of external and self-generated signals in bacteria as well as in the generation of appropriate output responses. Accordingly, in Myxococcus xanthus TCS are important for fruiting body formation and sporulation as well as normal motility. In this study, I analyzed the M. xanthus genome for the presence and genetic organization of genes encoding TCS. 272 genes that encode TCS proteins were identified including 21 genes in eight loci, which encode TCS proteins that are part of chemotaxis-like systems. Sebsequent analyses focused on 251 TCS proteins (non chemotaxis-like) consisting of 118 histidine protein kinases (HPKs), 119 response regulators (RRs) and 14 HPK-like genes. 71% of the TCS genes are organized in unusual manners as orphan genes or in complex gene clusters whereas the remaining 29% display the standard paired gene organization. Bioinformatics analyses suggest that TCS proteins encoded by orphan genes and complex gene clusters are functionally distinct from TCS proteins encoded by paired genes. Experimentally, microarray data and quantitative real-time PCR suggest that orphan TCS genes are overrepresented among TCS genes that display altered transcription during fruiting body formation. The genetic analysis of 25 orphan HPKs, which are transcriptionally up-regulated during development, led to the identification of two HPKs that are likely essential for viability and seven HPKs including four novel HPKs that have important function in fruiting body formation or spore germination. As an attempt to identify functional partners of orphan TCS proteins in M. xanthus, I focused on the RR FruA, which has a key role in the C-signal transduction pathway. To identify the FruA kinase, two candidate approaches were used. The first candidate approach is based on the hypothesis that a FruA kinase gene shares characteristics with the fruA gene, i.e. it is orphan, developmentally up-regulated at the transcriptional level and a null mutant is deficient in development. Yeast two-hybrid analysis was used to investigate potential interactions between FruA and developmentally regulated orphan HPKs. Three best FruA kinase candidates (SdeK, Hpk8 and Hpk12) and four potentially redundant candidates (Hpk9, Hpk11, Hpk13 and Hpk29) were identified. In vivo analyses of the three best FruA kinase candidates support a model in which SdeK is the main FruA kinase, Hpk12 is a minor FruA kinase and Hpk8 is a phosphatase of FruA~P. Furthermore, SdeK may have other downstream targets in addition to FruA and there may be other HPKs that phosphorylate or cross talk to FruA. To obtain direct evidence for an interaction between FruA and the FruA kinase candidates in vitro, the relevant proteins have been purified. To date, the Hpk8 and Hpk12 proteins have been shown to autophosphorylate in vitro. Intriguingly, Hpk8 does not appear to be phosphorylated on the conserved His residue but is likely phosphorylated on a Tyr residue. Preliminary phosphotransfer assay suggests that Hpk8 engages in phosphotransfer to or phosphorylation of FruA. A possible interaction in vitro between SdeK and Hpk12 with FruA still remains to be shown. Hpk37 belongs to the group of orphan HPKs that are transcriptionally up-regulated during development and essential for development. However, the yeast two-hybrid analyses to determine a possible direct interaction with FruA were inconclusive. In vivo analyses demonstrated that Hpk37 is likely involved in the production or response to (p)ppGpp or the A-signal suggesting that Hpk37 is not a FruA kinase. Domain analyses of Hpk37 and analyses of the genetic organization of the hpk37 locus suggest that regulation of Hpk37 activity could involve a unique methylation/demethylation mechanism similar to that resulting in adaptation in chemosensory pathways. In a second candidate approach to identify a FruA kinase, candidates were predicted using an in silico method (White et al., 2007). In vivo analyses of mutants carrying mutations in the genes encoding the six best candidates strongly suggest that these HPK are not FruA kinases.