Three PilZ domain proteins, PlpA, PixA and PixB, have distinct functions in regulation of motility and development in Myxococcus xanthus

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 the 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 receptors. We confirm that PlpA, a stand-alone PilZ-domain protein, is specifically important for motility and that Pkn1, which is composed of a Ser/Thr 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 upstream of the Frz chemosensory system 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 downstream of the Frz chemosensory system by setting up the polarity of the two motility systems. Our results support a model whereby the three proteins PlpA, PixA and PixB act in parallel pathways and have distinct functions to regulation of motility.

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Three PilZ domain proteins, PlpA, PixA and PixB, have distinct functions in regulation of motility and development in Myxococcus xanthus

Journal of Bacteriology ◽

10.1128/jb.00126-21 ◽

2021 ◽

Author(s):

Sofya Kuzmich ◽

Dorota Skotnicka ◽

Dobromir Szadkowski ◽

Philipp Klos ◽

María Pérez‐Burgos ◽

...

Keyword(s):

Bacterial Species ◽

Myxococcus Xanthus ◽

Type Iv Pili ◽

Gliding Motility ◽

Intact Protein ◽

Dependent Manner ◽

Chemosensory System ◽

Type Iv ◽

Acetyltransferase Domain

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.

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Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation in Myxococcus xanthus

Journal of Bacteriology ◽

10.1128/jb.00403-10 ◽

2010 ◽

Vol 192 (17) ◽

pp. 4267-4274 ◽

Cited By ~ 19

Author(s):

Wesley P. Black ◽

Florian D. Schubot ◽

Zhuo Li ◽

Zhaomin Yang

Keyword(s):

Double Mutant ◽

Kinase Activity ◽

Myxococcus Xanthus ◽

Type Iv Pili ◽

Gliding Motility ◽

Chemosensory Proteins ◽

Downstream Target ◽

Signaling Complex ◽

Type Iv

ABSTRACT Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as a difD difE double mutant phenocopied the difE single mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifE∼P), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifD∼P). Here we report in vitro phosphorylation studies with all the Dif chemosensory proteins that were expressed and purified from Escherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifD∼P has an unusually long half-life for dephosphorylation in vitro, DifG efficiently dephosphorylated DifD∼P as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifE∼P.

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Three-dimensional observations of an aperiodic oscillatory gliding motility behaviour in Myxococcus xanthus using confocal interference reflection microscopy

10.1101/722231 ◽

2019 ◽

Author(s):

Liam M. Rooney ◽

Lisa S. Kölln ◽

Ross Scrimgeour ◽

William B. Amos ◽

Paul A. Hoskisson ◽

...

Keyword(s):

Myxococcus Xanthus ◽

Three Dimensional ◽

Basal Membrane ◽

Gliding Motility ◽

The Novel ◽

Force Transmission ◽

Topological Information ◽

Type Iv ◽

Microscopy Techniques

The Delta-proteobacterium, Myxococcus xanthus, has been used as a model for bacterial motility and to provide insights of bacterial swarming behaviours. Fluorescence microscopy techniques have shown that various mechanisms are involved in gliding motility, but these have almost entirely been limited to 2D studies and there is currently no understanding of gliding motility in a 3D context. We present here the first use of confocal interference reflection microscopy (IRM) to study gliding bacteria, and we reveal aperiodic oscillatory behaviour with changes in the position of the basal membrane relative to the coverglass on the order of 90 nm in vitro. Firstly, we use a model plano-convex lens specimen to show how topological information can be obtained from the wavelength-dependent interference pattern in IRM. We then use IRM to observe gliding M. xanthus and show that cells undergo previously unobserved changes in their height as they glide. We compare the wild-type with mutants of reduced motility, which also exhibit the same changes in adhesion profile during gliding. We find that the general gliding behaviour is independent of the proton motive force-generating complex, AglRQS, and suggest that the novel behaviour we present here may be a result of recoil and force transmission along the length of the cell body following firing of the Type IV pili.

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Analysis of type IV pilus and its associated motility in Myxococcus xanthus using an antibody reactive with native pilin and pili

Microbiology ◽

10.1099/mic.0.27614-0 ◽

2005 ◽

Vol 151 (2) ◽

pp. 353-360 ◽

Cited By ~ 23

Author(s):

Yinuo Li ◽

Renate Lux ◽

Andrew E. Pelling ◽

James K. Gimzewski ◽

Wenyuan Shi

Keyword(s):

Amino Acids ◽

Current Model ◽

Bacterial Species ◽

Myxococcus Xanthus ◽

Type Iv Pili ◽

Gliding Motility ◽

Soluble Form ◽

Truncated Form ◽

Type Iv ◽

Oligomerization Domain

Myxococcus xanthus possesses a social gliding motility that requires type IV pili (TFP). According to the current model, M. xanthus pili attach to an external substrate and retract, pulling the cell body forward along their long axis. By analogy with the situation in other bacteria employing TFP-dependent motility, M. xanthus pili have been assumed to be composed of pilin (PilA) subunits, but this has not previously been confirmed. The first 28 amino acids of the M. xanthus PilA protein share extensive homology with the N-terminal oligomerization domain of pilins in other bacterial species. To facilitate purification, the authors engineered a truncated form of M. xanthus PilA lacking the first 28 amino acids and purified this protein in soluble form. Polyclonal antibody generated against this protein was reactive with native pilin and pili. Using this antibody, it was confirmed that TFP of M. xanthus are indeed composed of PilA, and that TFP are located unipolarly and required for social gliding motility via retraction. Using tethering as well as motility assays, details of pili function in M. xanthus social motility were further examined.

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Myxococcus xanthus dif Genes Are Required for Biogenesis of Cell Surface Fibrils Essential for Social Gliding Motility

Journal of Bacteriology ◽

10.1128/jb.182.20.5793-5798.2000 ◽

2000 ◽

Vol 182 (20) ◽

pp. 5793-5798 ◽

Cited By ~ 100

Author(s):

Zhaomin Yang ◽

Xiaoyuan Ma ◽

Leming Tong ◽

Heidi B. Kaplan ◽

Lawrence J. Shimkets ◽

...

Keyword(s):

Cell Surface ◽

Myxococcus Xanthus ◽

Bacterial Chemotaxis ◽

Type Iv Pili ◽

Gliding Motility ◽

Genetic Studies ◽

Developmental Defects ◽

Bacterial Surface ◽

Type Iv ◽

Cell Surface Structures

ABSTRACT Myxococcus xanthus social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the dif genes. In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to M. xanthus S motility. We have demonstrated here that M. xanthus dif genes are required for the biogenesis of fibrils but not for that of type IV pili. Furthermore, the developmental defects of dif mutants can be partially rescued by the addition of isolated fibril materials. Along with the chemotaxis genes of various swarming bacteria and the pilGHIJ genes of the twitching bacteriumPseudomonas aeruginosa, the M. xanthus dif genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion. Genetic studies indicate that the dif genes are linked to theM. xanthus dsp region, a locus known to be crucial forM. xanthus fibril biogenesis and S gliding.

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The Orphan Response Regulator DigR Is Required for Synthesis of Extracellular Matrix Fibrils in Myxococcus xanthus

Journal of Bacteriology ◽

10.1128/jb.00189-06 ◽

2006 ◽

Vol 188 (12) ◽

pp. 4384-4394 ◽

Cited By ~ 24

Author(s):

Martin Overgaard ◽

Sigrun Wegener-Feldbrügge ◽

Lotte Søgaard-Andersen

Keyword(s):

Extracellular Matrix ◽

Response Regulator ◽

Cell Envelope ◽

Transcriptional Profiling ◽

Myxococcus Xanthus ◽

Gliding Motility ◽

Differentially Expressed ◽

Type Iv Pilus ◽

Receiver Domain ◽

Type Iv

ABSTRACT In Myxococcus xanthus, two-component systems have crucial roles in regulating motility behavior and development. Here we describe an orphan response regulator, consisting of an N-terminal receiver domain and a C-terminal DNA binding domain, which is required for A and type IV pilus-dependent gliding motility. Genetic evidence suggests that phosphorylation of the conserved, phosphorylatable aspartate residue in the receiver domain is required for DigR activity. Consistent with the defect in type IV pilus-dependent motility, a digR mutant is slightly reduced in type IV pilus biosynthesis, and the composition of the extracellular matrix fibrils is abnormal, with an increased content of polysaccharides and decreased accumulation of the FibA metalloprotease. By using genome-wide transcriptional profiling, 118 genes were identified that are directly or indirectly regulated by DigR. These 118 genes include only 2, agmQ and cheY4, previously implicated in A and type IV pilus-dependent motility, respectively. In silico analyses showed that 36% of the differentially expressed genes are likely to encode exported proteins. Moreover, four genes encoding homologs of extracytoplasmic function (ECF) sigma factors, which typically control aspects of cell envelope homeostasis, are differentially expressed in a digR mutant. We suggest that the DigR response regulator has an important function in cell envelope homeostasis and that the motility defects in a digR mutant are instigated by the abnormal cell envelope and abnormal expression of agmQ and cheY4.

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Genetic and functional evidence that Type IV pili are required for social gliding motility in Myxococcus xanthus

Molecular Microbiology ◽

10.1111/j.1365-2958.1995.mmi_18030547.x ◽

1995 ◽

Vol 18 (3) ◽

pp. 547-558 ◽

Cited By ~ 203

Author(s):

Samuel S. Wu ◽

Dale Kaiser

Keyword(s):

Myxococcus Xanthus ◽

Type Iv Pili ◽

Gliding Motility ◽

Type Iv

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AglZ Is a Filament-Forming Coiled-Coil Protein Required for Adventurous Gliding Motility of Myxococcus xanthus

Journal of Bacteriology ◽

10.1128/jb.186.18.6168-6178.2004 ◽

2004 ◽

Vol 186 (18) ◽

pp. 6168-6178 ◽

Cited By ~ 70

Author(s):

Ruifeng Yang ◽

Sarah Bartle ◽

Rebecca Otto ◽

Angela Stassinopoulos ◽

Matthew Rogers ◽

...

Keyword(s):

Response Regulator ◽

Myxococcus Xanthus ◽

Coiled Coil ◽

Gliding Motility ◽

Isolated Cells ◽

Genetic Studies ◽

C Terminus ◽

Heptad Repeats ◽

Two Hybrid

ABSTRACT The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro.

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PilB and PilT Are ATPases Acting Antagonistically in Type IV Pilus Function in Myxococcus xanthus

Journal of Bacteriology ◽

10.1128/jb.01793-07 ◽

2008 ◽

Vol 190 (7) ◽

pp. 2411-2421 ◽

Cited By ~ 92

Author(s):

Vladimir Jakovljevic ◽

Simone Leonardy ◽

Michael Hoppert ◽

Lotte Søgaard-Andersen

Keyword(s):

Atpase Activity ◽

Myxococcus Xanthus ◽

Type Iv Pili ◽

Atp Binding ◽

Dynamic Surface ◽

Conserved Residues ◽

Type Iv ◽

Atp Binding And Hydrolysis

ABSTRACT Type IV pili (T4P) are dynamic surface structures that undergo cycles of extension and retraction. T4P dynamics center on the PilB and PilT proteins, which are members of the secretion ATPase superfamily of proteins. Here, we show that PilB and PilT of the T4P system in Myxococcus xanthus have ATPase activity in vitro. Using a structure-guided approach, we systematically mutagenized PilB and PilT to resolve whether both ATP binding and hydrolysis are important for PilB and PilT function in vivo. PilB as well as PilT ATPase activity was abolished in vitro by replacement of conserved residues in the Walker A and Walker B boxes that are involved in ATP binding and hydrolysis, respectively. PilB proteins containing mutant Walker A or Walker B boxes were nonfunctional in vivo and unable to support T4P extension. PilT proteins containing mutant Walker A or Walker B boxes were also nonfunctional in vivo and unable to support T4P retraction. These data provide genetic evidence that both ATP binding and hydrolysis by PilB are essential for T4P extension and that both ATP binding and hydrolysis by PilT are essential for T4P retraction. Thus, PilB and PilT are ATPases that act at distinct steps in the T4P extension/retraction cycle in vivo.

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