Regulation by cyclic di-GMP in Myxococcus xanthus

The nucleotide-based second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes. Myxococcus xanthus undergoes major lifestyle changes in response to nutrient availability with the forma...

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1. Verfasser: Skotnicka, Dorota
Beteiligte: Søgaard-Andersen, Lotte (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2016
Biologie
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Zusammenfassung:The nucleotide-based second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes. Myxococcus xanthus undergoes major lifestyle changes in response to nutrient availability with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP in M. xanthus. We show that this bacterium synthesizes c-di-GMP. Manipulation of the cellular c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase in vegetative cells caused defects in type IV pili (T4P)-dependent motility whereas gliding motility was unaffected. An increased level of c-di-GMP caused reduced transcription of the pilA gene that encodes the major pilin of T4P, reduced assembly of T4P and altered cell agglutination whereas a decreased level of c-di-GMP caused altered cell agglutination. The systematic inactivation of the 24 genes in M. xanthus encoding proteins containing GGDEF, EAL or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation or binding, identified three genes encoding proteins important for T4P-dependent motility. These three proteins named DmxA, TmoK and SgmT all contain a GGDEF domain. Purified DmxA had diguanylate cyclase activity whereas the TmoK and SgmT (both hybrid histidine protein kinases) did not have diguanylate cyclase activity. During starvation, the c-di-GMP level in M. xanthus increases significantly. Manipulation of this level revealed that a low c-di-GMP level negatively affects the developmental program while an increased level does not interfere with development. Moreover, among the 24 genes encoding proteins containing GGDEF, EAL or HD-GYP domains, we identified two which are specifically involved in development: pmxA and dmxB. pmxA codes for an enzymatically active phosphodiesterase with an HD-GYP domain. dmxB codes for a developmentally induced, enzymatically active diguanylate cyclase. DmxB is essential for the increased c-di-GMP level and regulates exopolysaccharide accumulation during starvation. Our results show that c-di-GMP acts as an important signaling molecule during M. xanthus development, and suggest a model in which a minimal threshold level of c-di-GMP is essential for the successful progression and completion of the developmental program. Additionally, candidates for c-di-GMP effectors in M. xanthus were identified using a capture compound mass spectrometry approach. Some of the candidates were confirmed to bind c-di-GMP in vitro and deletion mutants for genes encoding those proteins were characterized in terms of T4P-dependent motility and development.
Beschreibung:134 pages.
DOI:https://doi.org/10.17192/z2016.0101