scholarly journals Conditional requirement of SglT for type IV pili function and S-motility in Myxococcus xanthus

Microbiology ◽  
2020 ◽  
Vol 166 (4) ◽  
pp. 349-358
Author(s):  
Vera Troselj ◽  
Darshankumar T. Pathak ◽  
Daniel Wall
Keyword(s):  
Myxococcus Xanthus ◽  
Critical Role ◽  
Point Mutations ◽  
Solid Surfaces ◽  
Temperature Sensitive ◽  
Fruiting Body Formation ◽  
Temperature Growth ◽  
Content Type ◽  
New Class ◽  
Type Iv

Myxobacteria exhibit complex social behaviors such as predation, outer membrane exchange and fruiting body formation. These behaviors depend on coordinated movements of cells on solid surfaces that involve social (S) motility. S-motility is powered by extension-retraction cycles of type 4 pili (Tfp) and exopolysaccharides (EPS) that provide a matrix for group cellular movement. Here, we characterized a new class of S-motility mutants in Myxococcus xanthus . These mutants have a distinctive phenotype: they lack S-motility even though they produce pili and EPS and the phenotype is temperature-sensitive. The point mutations were mapped to a single locus, MXAN_3284, named sglT. Similar to pilT mutants, sglT mutants are hyperpiliated and, strikingly, the temperature-sensitive phenotype is caused by null mutations. Our results indicate that SglT plays a critical role in Tfp function associated with pilus retraction and that the block in pili retraction is caused by a Tfp assembly defect in the absence of SglT at high-temperature growth.

scholarly journals Characterization of the Exopolysaccharide Biosynthesis Pathway in Myxococcus xanthus

2020 ◽  
Vol 202 (19) ◽  
Author(s):  
María Pérez-Burgos ◽  
Inmaculada García-Romero ◽  
Jana Jung ◽  
Eugenia Schander ◽  
Miguel A. Valvano ◽  
...  
Keyword(s):  
Repeat Unit ◽  
Myxococcus Xanthus ◽  
Type Iv Pilus ◽  
Fruiting Body Formation ◽  
Content Type ◽  
Body Formation ◽  
Polysaccharide Biosynthesis ◽  
Type Iv ◽  
Biosynthetic Machinery ◽  
Dependent Pathway

ABSTRACT Myxococcus xanthus arranges into two morphologically distinct biofilms depending on its nutritional status, i.e., coordinately spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. A secreted polysaccharide, referred to as exopolysaccharide (EPS), is a structural component of both biofilms and is also important for type IV pilus-dependent motility and fruiting body formation. Here, we characterize the biosynthetic machinery responsible for EPS biosynthesis using bioinformatics, genetics, heterologous expression, and biochemical experiments. We show that this machinery constitutes a Wzx/Wzy-dependent pathway dedicated to EPS biosynthesis. Our data support that EpsZ (MXAN_7415) is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for the initiation of the repeat unit synthesis. Heterologous expression experiments support that EpsZ has galactose-1-P transferase activity. Moreover, MXAN_7416, renamed WzxEPS, and MXAN_7442, renamed WzyEPS, are the Wzx flippase and Wzy polymerase responsible for translocation and polymerization of the EPS repeat unit, respectively. In this pathway, EpsV (MXAN_7421) also is the polysaccharide copolymerase and EpsY (MXAN_7417) the outer membrane polysaccharide export (OPX) protein. Mutants with single in-frame deletions in the five corresponding genes had defects in type IV pilus-dependent motility and a conditional defect in fruiting body formation. Furthermore, all five mutants were deficient in type IV pilus formation, and genetic analyses suggest that EPS and/or the EPS biosynthetic machinery stimulates type IV pilus extension. Additionally, we identify a polysaccharide biosynthesis gene cluster, which together with an orphan gene encoding an OPX protein make up a complete Wzx/Wzy-dependent pathway for synthesis of an unknown polysaccharide. IMPORTANCE The secreted polysaccharide referred to as exopolysaccharide (EPS) has important functions in the social life cycle of M. xanthus; however, little is known about how EPS is synthesized. Here, we characterized the EPS biosynthetic machinery and showed that it makes up a Wzx/Wzy-dependent pathway for polysaccharide biosynthesis. Mutants lacking a component of this pathway had reduced type IV pilus-dependent motility and a conditional defect in development. These analyses also suggest that EPS and/or the EPS biosynthetic machinery is important for type IV pilus formation.

scholarly journals Gliding Motility Revisited: How Do the Myxobacteria Move without Flagella?

2010 ◽  
Vol 74 (2) ◽  
pp. 229-249 ◽  
Author(s):  
Emilia M. F. Mauriello ◽  
Tâm Mignot ◽  
Zhaomin Yang ◽  
David R. Zusman
Keyword(s):  
Biofilm Formation ◽  
Protein Localization ◽  
Myxococcus Xanthus ◽  
Gliding Motility ◽  
Solid Surfaces ◽  
Biological Functions ◽  
Fruiting Body Formation ◽  
Body Formation ◽  
Periplasmic Flagella ◽  
Motile Bacterium

SUMMARY In bacteria, motility is important for a wide variety of biological functions such as virulence, fruiting body formation, and biofilm formation. While most bacteria move by using specialized appendages, usually external or periplasmic flagella, some bacteria use other mechanisms for their movements that are less well characterized. These mechanisms do not always exhibit obvious motility structures. Myxococcus xanthus is a motile bacterium that does not produce flagella but glides slowly over solid surfaces. How M. xanthus moves has remained a puzzle that has challenged microbiologists for over 50 years. Fortunately, recent advances in the analysis of motility mutants, bioinformatics, and protein localization have revealed likely mechanisms for the two M. xanthus motility systems. These results are summarized in this review.

scholarly journals Characterization of Zoospore Type IV Pili inActinoplanes missouriensis

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Tomohiro Kimura ◽  
Takeaki Tezuka ◽  
Daisuke Nakane ◽  
Takayuki Nishizaka ◽  
Shin-Ichi Aizawa ◽  
...  
Keyword(s):  
Vegetative Growth ◽  
Physiological State ◽  
Type Iv Pili ◽  
Solid Surfaces ◽  
Cellular Biology ◽  
Content Type ◽  
Type Iv ◽  
Transmission Electron ◽  
Rare Actinomycetes ◽  
Transcriptional Units

ABSTRACTThe rare actinomyceteActinoplanes missouriensisproduces terminal sporangia containing a few hundred flagellated spores. After release from the sporangia, the spores swim rapidly in aquatic environments as zoospores. The zoospores stop swimming and begin to germinate in niches for vegetative growth. Here, we report the characterization and functional analysis of zoospore type IV pili inA. missouriensis. The pilus gene (pil) cluster, consisting of three apparently σFliA-dependent transcriptional units, is activated during sporangium formation similarly to the flagellar gene cluster, indicating that the zoospore has not only flagella but also pili. With a new method in which zoospores were fixed with glutaraldehyde to prevent pilus retraction, zoospore pili were observed relatively easily using transmission electron microscopy, showing 6 ± 3 pili per zoospore (n = 37 piliated zoospores) and a length of 0.62 ± 0.35 μm (n = 206), via observation offliC-deleted, nonflagellated zoospores. No pili were observed in the zoospores of a prepilin-encodingpilAdeletion (ΔpilA) mutant. In addition, the deletion ofpilT, which encodes an ATPase predicted to be involved in pilus retraction, substantially reduced the frequency of pilus retraction. Several adhesion experiments using wild-type and ΔpilAzoospores indicated that the zoospore pili are required for the sufficient adhesion of zoospores to hydrophobic solid surfaces. Many zoospore-forming rare actinomycetes conserve thepilcluster, which indicates that the zoospore pili yield an evolutionary benefit in the adhesion of zoospores to hydrophobic materials as footholds for germination in their mycelial growth.IMPORTANCEBacterial zoospores are interesting cells in that their physiological state changes dynamically: they are dormant in sporangia, show temporary mobility after awakening, and finally stop swimming to germinate in niches for vegetative growth. However, the cellular biology of a zoospore remains largely unknown. This study describes unprecedented zoospore type IV pili in the rare actinomyceteActinoplanes missouriensis. Similar to the case for the usual bacterial type IV pili, zoospore pili appeared to be retractable. Our findings that the zoospore pili have a functional role in the adhesion of zoospores to hydrophobic solid surfaces and that the zoospores use both pili and flagella properly according to their different purposes provide an important insight into the cellular biology of the zoospore.

scholarly journals Neutral and Phospholipids of the Myxococcus xanthus Lipodome during Fruiting Body Formation and Germination

2015 ◽  
Vol 81 (19) ◽  
pp. 6538-6547 ◽  
Author(s):  
Tilman Ahrendt ◽  
Hendrik Wolff ◽  
Helge B. Bode
Keyword(s):  
Life Cycle ◽  
Lipid Profile ◽  
De Novo ◽  
Membrane Lipids ◽  
Myxococcus Xanthus ◽  
Model Organism ◽  
Ether Lipid ◽  
Complex Life Cycle ◽  
Fruiting Body Formation ◽  
Content Type

ABSTRACTMyxobacteria are well-known for their complex life cycle, including the formation of spore-filled fruiting bodies. The model organismMyxococcus xanthusexhibits a highly complex composition of neutral and phospholipids, including triacylglycerols (TAGs), diacylglycerols (DAGs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), cardiolipins (CLs), and sphingolipids, including ceramides (Cers) and ceramide phosphoinositols (Cer-PIs). In addition, ether lipids have been shown to be involved in development and signaling. In this work, we describe the lipid profile ofM. xanthusduring its entire life cycle, including spore germination. PEs, representing one of the major components of the bacterial membrane, decreased by about 85% during development from vegetative rods to round myxospores, while TAGs first accumulated up to 2-fold before they declined 48 h after the induction of sporulation. Presumably, membrane lipids are incorporated into TAG-containing lipid bodies, serving as an intermediary energy source for myxospore formation. The ceramides Cer(d-19:0/iso-17:0) and Cer(d-19:0/16:0) accumulated 6-fold and 3-fold, respectively, after 24 h of development, identifying them to be novel putative biomarkers forM. xanthussporulation. The most abundant ether lipid, 1-iso-15:0-alkyl-2,3-di-iso-15:0-acyl glycerol (TG1), exhibited a lipid profile different from that of all TAGs during sporulation, reinforcing its signaling character. The absence of all these lipid profile changes in mutants during development supports the importance of lipids in myxobacterial development. During germination of myxospores, only thede novobiosynthesis of new cell membrane fatty acids was observed. The unexpected accumulation of TAGs also during germination might indicate a function of TAGs as intermediary storage lipids during this part of the life cycle as well.

scholarly journals Cell Surface Attachment Structures Contribute to Biofilm Formation and Xylem Colonization by Erwinia amylovora

2011 ◽  
Vol 77 (19) ◽  
pp. 7031-7039 ◽  
Author(s):  
Jessica M. Koczan ◽  
Bryan R. Lenneman ◽  
Molly J. McGrath ◽  
George W. Sundin
Keyword(s):  
Biofilm Formation ◽  
Time Course ◽  
Erwinia Amylovora ◽  
Critical Role ◽  
Gene Clusters ◽  
Type I ◽  
Content Type ◽  
Type Iv ◽  
Attachment Structures

ABSTRACTBiofilm formation plays a critical role in the pathogenesis ofErwinia amylovoraand the systemic invasion of plant hosts. The functional role of the exopolysaccharides amylovoran and levan in pathogenesis and biofilm formation has been evaluated. However, the role of biofilm formation, independent of exopolysaccharide production, in pathogenesis and movement within plants has not been studied previously. Evaluation of the role of attachment inE. amylovorabiofilm formation and virulence was examined through the analysis of deletion mutants lacking genes encoding structures postulated to function in attachment to surfaces or in cellular aggregation. The genes and gene clusters studied were selected based onin silicoanalyses. Microscopic analyses and quantitative assays demonstrated that attachment structures such as fimbriae and pili are involved in the attachment ofE. amylovorato surfaces and are necessary for the production of mature biofilms. A time course assay indicated that type I fimbriae function earlier in attachment, while type IV pilus structures appear to function later in attachment. Our results indicate that multiple attachment structures are needed for mature biofilm formation and full virulence and that biofilm formation facilitates entry and is necessary for the buildup of large populations ofE. amylovoracells in xylem tissue.

scholarly journals Characterization of the exopolysaccharide biosynthesis pathway in Myxococcus xanthus

2020 ◽  
Author(s):  
María Pérez-Burgos ◽  
Inmaculada García-Romero ◽  
Jana Jung ◽  
Eugenia Schander ◽  
Miguel A. Valvano ◽  
...  
Keyword(s):  
Fruiting Body ◽  
Repeat Unit ◽  
Myxococcus Xanthus ◽  
Type Iv Pili ◽  
Fruiting Body Formation ◽  
Body Formation ◽  
Polysaccharide Biosynthesis ◽  
Type Iv ◽  
Biosynthetic Machinery ◽  
Dependent Pathway

AbstractMyxococcus xanthus arranges into two morphologically distinct biofilms depending on its nutritional status, i.e. coordinately spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. A secreted polysaccharide referred to as exopolysaccharide (EPS) is a structural component of both biofilms and is also important for type IV pili-dependent motility and fruiting body formation. Here, we characterize the biosynthetic machinery responsible for EPS biosynthesis using bioinformatics, genetics, heterologous expression, and biochemical experiments. We show that this machinery constitutes a Wzx/Wzy-dependent pathway dedicated to EPS biosynthesis. Our data support that EpsZ (MXAN_7415) is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for initiation of the repeat unit synthesis. Heterologous expression experiments support that EpsZ has galactose-1-P transferase activity. Moreover, MXAN_7416, renamed WzxEPS, and MXAN_7442, renamed WzyEPS, are the Wzx flippase and Wzy polymerase responsible for translocation and polymerization of the EPS repeat unit, respectively. Also, in this pathway, EpsV (MXAN_7421) is the polysaccharide co-polymerase and EpsY (MXAN_7417) the outer membrane polysaccharide export (OPX) protein. Mutants with single in-frame deletions in the five corresponding genes had defects in type IV pili-dependent motility and a conditional defect in fruiting body formation. Furthermore, all five mutants were deficient in type IV pili formation and genetic analyses suggest that EPS and/or the EPS biosynthetic machinery stimulates type IV pili extension. Additionally, we identify a polysaccharide biosynthesis gene cluster, which together with an orphan gene encoding an OPX protein make up a complete Wzx/Wzy-dependent pathway for synthesis of an unknown polysaccharide.ImportanceThe secreted polysaccharide referred to as exopolysaccharide (EPS) has important functions in the social life cycle of M. xanthus; however, little is known about how EPS is synthesized. Here, we characterized the EPS biosynthetic machinery and show that it makes up a Wzx/Wzy-dependent pathway for polysaccharide biosynthesis. Mutants lacking a component of this pathway had reduced type IV pili-dependent motility and a conditional defect in development. Also, these analysis suggest that EPS and/or the EPS biosynthetic machinery is important for type IV pili formation.

scholarly journals Cyclic Di-GMP Regulates Type IV Pilus-Dependent Motility in Myxococcus xanthus

2015 ◽  
Vol 198 (1) ◽  
pp. 77-90 ◽  
Author(s):  
Dorota Skotnicka ◽  
Tobias Petters ◽  
Jan Heering ◽  
Michael Hoppert ◽  
Volkhard Kaever ◽  
...  
Keyword(s):  
Myxococcus Xanthus ◽  
Lifestyle Changes ◽  
Gliding Motility ◽  
Cyclase Activity ◽  
Diguanylate Cyclase ◽  
Type Iv Pilus ◽  
Cell Agglutination ◽  
Content Type ◽  
Type Iv ◽  
Altered Cell

ABSTRACTThe 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 xanthusundergoes 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 inM. xanthusand show that this bacterium synthesizes c-di-GMP during growth. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase correlated with defects in type IV pilus (T4P)-dependent motility, whereas gliding motility was unaffected. An increased level of c-di-GMP correlated with reduced transcription of thepilAgene (which encodes the major pilin of T4P), reduced the assembly of T4P, and altered cell agglutination, whereas a decreased c-di-GMP level correlated with altered cell agglutination. The systematic inactivation of the 24 genes inM. xanthusencoding 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, whereas all mutants had normal gliding motility. Purified DmxA had diguanylate cyclase activity, whereas the hybrid histidine protein kinases TmoK and SgmT, each of which contains a GGDEF domain, did not have diguanylate cyclase activity. These results demonstrate that c-di-GMP is important for T4P-dependent motility inM. xanthus.IMPORTANCEWe provide the first direct evidence thatM. xanthussynthesizes c-di-GMP and demonstrate that c-di-GMP is important for T4P-dependent motility, whereas we did not obtain evidence that c-di-GMP regulates gliding motility. The data presented uncovered a novel mechanism for regulation of T4P-dependent motility, in which increased levels of c-di-GMP inhibit transcription of thepilAgene (which encodes the major pilin of T4P), ultimately resulting in the reduced assembly of T4P. Moreover, we identified an enzymatically active diguanylate cyclase that is important for T4P-dependent motility.

scholarly journals Identification of Functions Affecting Predator-Prey Interactions between Myxococcus xanthus and Bacillus subtilis

2016 ◽  
Vol 198 (24) ◽  
pp. 3335-3344 ◽  
Author(s):  
Susanne Müller ◽  
Sarah N. Strack ◽  
Sarah E. Ryan ◽  
Mary Shawgo ◽  
Abigail Walling ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Soil Bacteria ◽  
Myxococcus Xanthus ◽  
Soil Organism ◽  
Multiple Traits ◽  
Content Type ◽  
Predator Prey ◽  
Type Iv ◽  
Genes Encoding ◽  
Predation Capacity

ABSTRACTSoil bacteria engage each other in competitive and cooperative ways to determine their microenvironments. In this study, we report the identification of a large number of genes required forMyxococcus xanthusto engageBacillus subtilisin a predator-prey relationship. We generated and tested over 6,000 individual transposon insertion mutants ofM. xanthusand found many new factors required to promote efficient predation, including the specialized metabolite myxoprincomide, an ATP-binding cassette (ABC) transporter permease, and a clustered regularly interspaced short palindromic repeat (CRISPR) locus encoding bacterial immunity. We also identified genes known to be involved in predation, including those required for the production of exopolysaccharides and type IV pilus (T4P)-dependent motility, as well as chemosensory and two-component systems. Furthermore, deletion of these genes confirmed their role during predation. Overall,M. xanthuspredation appears to be a multifactorial process, with multiple determinants enhancing predation capacity.IMPORTANCESoil bacteria engage each other in complex environments and utilize multiple traits to ensure survival. Here, we report the identification of multiple traits that enable a common soil organism,Myxococcus xanthus, to prey upon and utilize nutrients from another common soil organism,Bacillus subtilis. We mutagenized the predator and carried out a screen to identify genes that were required to either enhance or diminish capacity to consume prey. We identified dozens of genes encoding factors that contribute to the overall repertoire for the predator to successfully engage its prey in the natural environment.

scholarly journals The Myxococcus xanthus Developmental Program Can Be Delayed by Inhibition of DNA Replication

2007 ◽  
Vol 189 (24) ◽  
pp. 8793-8800 ◽  
Author(s):  
Christopher J. Rosario ◽  
Mitchell Singer
Keyword(s):  
Dna Replication ◽  
Developmental Process ◽  
Myxococcus Xanthus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Temperature Growth ◽  
Developmental Program

ABSTRACT Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaBA116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaBA116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaBA116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.

scholarly journals Mutation of the Conserved Calcium-Binding Motif in Neisseria gonorrhoeae PilC1 Impacts Adhesion but Not Piliation

2013 ◽  
Vol 81 (11) ◽  
pp. 4280-4289 ◽  
Author(s):  
Yuan Cheng ◽  
Michael D. L. Johnson ◽  
Christine Burillo-Kirch ◽  
Jeffrey C. Mocny ◽  
James E. Anderson ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Calcium Binding ◽  
Critical Role ◽  
Full Length ◽  
Binding Motif ◽  
Type Iv Pilus ◽  
Kingella Kingae ◽  
Content Type ◽  
Terminal Domain ◽  
Type Iv

ABSTRACTNeisseria gonorrhoeaePilC1 is a member of the PilC family of type IV pilus-associated adhesins found inNeisseriaspecies and other type IV pilus-producing genera. Previously, a calcium-binding domain was described in the C-terminal domains of PilY1 ofPseudomonas aeruginosaand in PilC1 and PilC2 ofKingella kingae. Genetic analysis ofN. gonorrhoeaerevealed a similar calcium-binding motif in PilC1. To evaluate the potential significance of this calcium-binding region inN. gonorrhoeae, we produced recombinant full-length PilC1 and a PilC1 C-terminal domain fragment. We show that, while alterations of the calcium-binding motif disrupted the ability of PilC1 to bind calcium, they did not grossly affect the secondary structure of the protein. Furthermore, we demonstrate that both full-length wild-type PilC1 and full-length calcium-binding-deficient PilC1 inhibited gonococcal adherence to cultured human cervical epithelial cells, unlike the truncated PilC1 C-terminal domain. Similar to PilC1 inK. kingae, but in contrast to the calcium-binding mutant ofP. aeruginosaPilY1, an equivalent mutation inN. gonorrhoeaePilC1 produced normal amounts of pili. However, theN. gonorrhoeaePilC1 calcium-binding mutant still had partial defects in gonococcal adhesion to ME180 cells and genetic transformation, which are both essential virulence factors in this human pathogen. Thus, we conclude that calcium binding to PilC1 plays a critical role in pilus function inN. gonorrhoeae.

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