Three PilZ Domain Proteins, PlpA, PixA, and PixB, Have Distinct Functions in Regulation of Motility and Development in Myxococcus xanthus

Abstract

In bacteria, the nucleotide-based second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pili-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ-domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system, and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ-domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pili-dependent and gliding motility in a Frz-dependent manner as well as development. The acetyltransferase domain is required and sufficient for function during growth while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility independently of the Frz system likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA and PixB act in independent pathways and have distinct functions in regulation of motility. Importance c-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts in a manner that depends on the Frz chemosensory system to regulate motility via the acetyltransferase domain while the intact protein and c-di-GMP binding are essential for PixB to support development. By contrast, PixA acts acts in Frz-independent mannerto regulate motility. Together with previous observations, we conclude that PilZ-domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus.

The CRP-like transcriptional regulator MrpC curbs c-di-GMP and 3’, 3’ cGAMP nucleotide levels during development in Myxococcus xanthus

“…1B, Table S1B). PlpA is a PilZ-domain protein that regulates motility during growth but is not important for development and was reported not to bind c-di-GMP in vitro (36,61). Control experiments using RT-qPCR on the same RNA as for RNA-seq for selected genes (dmxA, dmxB, pkn1) reproduced the RNA-seq data (Fig.…”

“…1AB, Table S1B). Pkn1 is a Ser/Thr protein kinase with a C-terminal PilZ domain and is specifically important for development (36,59); it is not known whether the PilZ domain binds c-di-GMP. These observations are in agreement with previous findings that dmxB and pkn1 transcription is up-regulated during development (40,59).…”

“…Upon nutrient depletion, M. xanthus initiates a developmental program that culminates in the formation of multicellular, spore-filled fruiting bodies while cells that remain outside fruiting bodies differentiate to either so-called peripheral rods or undergo cell lysis (33)(34)(35). Nucleotide-based second messengers have important roles during both stages of the lifecycle: During growth, c-di-GMP is important for type IV pili-dependent motility and for regulation of motility (36,37). During development, the starvationinduced activation of the stringent response with synthesis of the second messenger (p)ppGpp is required and sufficient to initiate development (38,39).…”

“…The enhancer binding protein Nla24 also binds c-di-GMP and is important for motility during growth as well as development (40,55,56). Systematic inactivation of all 24 genes encoding PilZ-domain proteins identified PixA and PixB as c-di-GMP receptors that regulate motility (36). While PixA is important only during growth, PixB is crucial during growth and development (36).…”

“…Systematic inactivation of all 24 genes encoding PilZ-domain proteins identified PixA and PixB as c-di-GMP receptors that regulate motility (36). While PixA is important only during growth, PixB is crucial during growth and development (36). Finally, the ribbon-helix-helix proteins CdbA and CdbB bind c-di-GMP (57).…”