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

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.

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

2021 ◽  
Author(s):  
Sofya Kuzmich ◽  
Dorota Skotnicka ◽  
Dobromir Szadkowski ◽  
Philipp Klos ◽  
Maria Perez-Burgos ◽  
...  
Keyword(s):  
Response Regulator ◽  
Myxococcus Xanthus ◽  
Type Iv Pili ◽  
Gliding Motility ◽  
Chemosensory System ◽  
Developmental Program ◽  
Type Iv ◽  
Genes Encoding ◽  
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 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.

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

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 353-360 ◽  
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.

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

2010 ◽  
Vol 192 (17) ◽  
pp. 4267-4274 ◽  
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.

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

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.

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

2000 ◽  
Vol 182 (20) ◽  
pp. 5793-5798 ◽  
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.

scholarly journals PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili

2019 ◽  
Author(s):  
Jennifer L. Chlebek ◽  
Hannah Q. Hughes ◽  
Aleksandra S. Ratkiewicz ◽  
Rasman Rayyan ◽  
Joseph Che-Yen Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Dependent Manner ◽  
Acinetobacter Baylyi ◽  
Bacterial Surface ◽  
Type Iv ◽  
Bona Fide ◽  
Almost All

AbstractBacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic appendages extend and retract from the cell surface. In many type IVa pilus systems, extension occurs through the action of an extension ATPase, often called PilB, while optimal retraction requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog of PilT called PilU. However, the function of this protein has remained unclear becausepilUmutants exhibit inconsistent phenotypes among type IV pilus systems and because it is relatively understudied compared to PilT. Here, we study the type IVa competence pilus ofVibrio choleraeas a model system to define the role of PilU. We show that the ATPase activity of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, however, contribute to pilus retraction in ΔpilTstrains. Thus, these data suggest that PilU is abona fideretraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a ΔpilUmutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additionalin vitroandin vivodata show that PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase is conserved inAcinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems.Author SummaryAlmost all bacterial species use thin surface appendages called pili to interact with their environments. These structures are critical for the virulence of many pathogens and represent one major way that bacteria share DNA with one another, which contributes to the spread of antibiotic resistance. To carry out their function, pili dynamically extend and retract from the bacterial surface. Here, we show that retraction of pili in some systems is determined by the combined activity of two motor ATPase proteins.

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

2008 ◽  
Vol 190 (7) ◽  
pp. 2411-2421 ◽  
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.

scholarly journals The Myxococcus xanthus pilQ(sglA) Gene Encodes a Secretin Homolog Required for Type IV Pilus Biogenesis, Social Motility, and Development

1999 ◽  
Vol 181 (1) ◽  
pp. 24-33 ◽  
Author(s):  
Daniel Wall ◽  
Paul E. Kolenbrander ◽  
Dale Kaiser
Keyword(s):  
Outer Membrane ◽  
Spontaneous Mutation ◽  
Myxococcus Xanthus ◽  
Type Iv Pili ◽  
Gliding Motility ◽  
Missense Mutations ◽  
Fruiting Body Formation ◽  
Subsequent Work ◽  
Type Iv ◽  
Pilus Biogenesis

ABSTRACT The Myxococcus xanthus sglA1 spontaneous mutation was originally isolated because it allowed dispersed cell growth in liquid yet retained the ability to form fruiting bodies. Consequently, most of today’s laboratory strains either contain the sglA1mutation or were derived from strains that carry it. Subsequent work showed that sglA was a gene for social gliding motility, a process which is mediated by type IV pili. Here sglA is shown to map to the major pil cluster and to encode a 901-amino-acid open reading frame (ORF) that is homologous to the secretin superfamily of proteins. Secretins form a channel in the outer membrane for the transport of macromolecules. The closest homologs found were PilQ proteins from Pseudomonas aeruginosa andNeisseria gonorrhoeae, which are required for type IV pili biogenesis and twitching motility. To signify these molecular and functional similarities, we have changed the name of sglAto pilQ. The hypomorphic pilQ1(sglA1) allele was sequenced and found to contain two missense mutations at residues 741 (G→S) and 762 (N→G). In addition, 19 independent social (S)-motility mutations are shown to map to the pilQ locus. In-frame deletions of pilQand its downstream gene, orfL, were constructed.pilQ is shown to be essential for pilus biogenesis, S-motility, rippling, and fruiting body formation, whileorfL is dispensable for these processes. ThepilQ1 allele, but not the ΔpilQ allele, was found to render cells hypersensitive to vancomycin, suggesting that PilQ1 alters the permeability properties of the outer membrane. Many differences between pilQ1 and pilQ +strains have been noted in the literature. We discuss some of these observations and how they may be rationalized in the context of our molecular and functional findings.

scholarly journals Exploitation of conserved eukaryotic host cell farnesylation machinery by an F-box effector of Legionella pneumophila

2010 ◽  
Vol 207 (8) ◽  
pp. 1713-1726 ◽  
Author(s):  
Christopher T.D. Price ◽  
Tasneem Al-Quadan ◽  
Marina Santic ◽  
Snake C. Jones ◽  
Yousef Abu Kwaik
Keyword(s):  
Legionella Pneumophila ◽  
Biological Function ◽  
Host Cells ◽  
Dependent Manner ◽  
Protein Farnesyltransferase ◽  
Type Iv ◽  
Eukaryotic Proteins ◽  
Bacterial Effectors

Farnesylation involves covalent linkage of eukaryotic proteins to a lipid moiety to anchor them into membranes, which is essential for the biological function of Ras and other proteins. A large cadre of bacterial effectors is injected into host cells by intravacuolar pathogens through elaborate type III–VII translocation machineries, and many of these effectors are incorporated into the pathogen-containing vacuolar membrane by unknown mechanisms. The Dot/Icm type IV secretion system of Legionella pneumophila injects into host cells the F-box effector Ankyrin B (AnkB), which functions as platforms for the docking of polyubiquitinated proteins to the Legionella-containing vacuole (LCV) to enable intravacuolar proliferation in macrophages and amoeba. We show that farnesylation of AnkB is indispensable for its anchoring to the cytosolic face of the LCV membrane, for its biological function within macrophages and Dictyostelium discoideum, and for intrapulmonary proliferation in mice. Remarkably, the protein farnesyltransferase, RCE-1 (Ras-converting enzyme-1), and isoprenyl cysteine carboxyl methyltransferase host farnesylation enzymes are recruited to the LCV in a Dot/Icm-dependent manner and are essential for the biological function of AnkB. In conclusion, this study shows novel localized recruitment of the host farnesylation machinery and its anchoring of an F-box effector to the LCV membrane, and this is essential for biological function in vitro and in vivo.

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