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

2006 ◽  
Vol 188 (12) ◽  
pp. 4384-4394 ◽  
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.

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.

Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis

Microbiology ◽  
2021 ◽  
Vol 167 (9) ◽  
Author(s):  
Marie Zachary ◽  
Susanne Bauer ◽  
Maximilian Klepsch ◽  
Katharina Wagler ◽  
Bruno Hüttel ◽  
...  
Keyword(s):  
Target Genes ◽  
Target Prediction ◽  
Type Iv Pilus ◽  
Content Type ◽  
Type Iv ◽  
Gene Expression Control ◽  
Initial Characterization ◽  
Pilus Biogenesis ◽  
Regulatory Rnas

Non-coding regulatory RNAs mediate post-transcriptional gene expression control by a variety of mechanisms relying mostly on base-pairing interactions with a target mRNA. Though a plethora of putative non-coding regulatory RNAs have been identified by global transcriptome analysis, knowledge about riboregulation in the pathogenic Neisseriae is still limited. Here we report the initial characterization of a pair of sRNAs of N. gonorrhoeae , TfpR1 and TfpR2, which exhibit a similar secondary structure and identical single-stranded seed regions, and therefore might be considered as sibling sRNAs. By combination of in silico target prediction and sRNA pulse expression followed by differential RNA sequencing we identified target genes of TfpR1 which are involved in type IV pilus biogenesis and DNA damage repair. We provide evidence that members of the TfpR1 regulon can also be targeted by the sibling TfpR2.

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 Three-Dimensional Observations of an Aperiodic Oscillatory Gliding Behavior in Myxococcus xanthus Using Confocal Interference Reflection Microscopy

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Liam M. Rooney ◽  
Lisa S. Kölln ◽  
Ross Scrimgeour ◽  
William B. Amos ◽  
Paul A. Hoskisson ◽  
...  
Keyword(s):  
Myxococcus Xanthus ◽  
Three Dimensional ◽  
Oscillatory Behavior ◽  
Gliding Motility ◽  
Bacterial Cells ◽  
Force Transmission ◽  
Content Type ◽  
Gliding Bacteria ◽  
Microscopy Techniques

ABSTRACT The deltaproteobacterium Myxococcus xanthus is a model for bacterial motility and has provided unprecedented insights into bacterial swarming behaviors. Fluorescence microscopy techniques have been invaluable in defining the mechanisms that are involved in gliding motility, but these have almost entirely been limited to two-dimensional (2D) studies, and there is currently no understanding of gliding motility in a three-dimensional (3D) context. We present here the first use of confocal interference reflection microscopy (IRM) to study gliding bacteria, revealing aperiodic oscillatory behavior with changes in the position of the basal membrane relative to the substrate on the order of 90 nm in vitro. First, we use a model planoconvex 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 bacteria and show that cells undergo previously unobserved changes in their adhesion profile as they glide. We compare the wild type with mutants that have reduced motility, which also exhibit the same changes in the adhesion profile during gliding. We find that the general gliding behavior is independent of the proton motive force-generating complex AglRQS and suggest that the novel behavior that 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. IMPORTANCE 3D imaging of live bacteria with optical microscopy techniques is a challenge due to the small size of bacterial cells, meaning that previous studies have been limited to observing motility behavior in 2D. We introduce the application of confocal multiwavelength interference reflection microscopy to bacteria, which enables visualization of 3D motility behaviors in a single 2D image. Using the model organism Myxococcus xanthus, we identified novel motility behaviors that are not explained by current motility models, where gliding bacteria exhibit aperiodic changes in their adhesion to an underlying solid surface. We concluded that the 3D behavior was not linked to canonical motility mechanisms and that IRM could be applied to study a range of microbiological specimens with minimal adaptation to a commercial microscope.

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 The Che4 pathway of Myxococcus xanthus regulates type IV pilus-mediated motility

2004 ◽  
Vol 52 (6) ◽  
pp. 1799-1811 ◽  
Author(s):  
Hera C. Vlamakis ◽  
John R. Kirby ◽  
David R. Zusman
Keyword(s):  
Myxococcus Xanthus ◽  
Type Iv Pilus ◽  
Type Iv

scholarly journals Discovery of a New Neisseria gonorrhoeae Type IV Pilus Assembly Factor, TfpC

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Linda I. Hu ◽  
Shaohui Yin ◽  
Egon A. Ozer ◽  
Lee Sewell ◽  
Saima Rehman ◽  
...  
Keyword(s):  
Cell Surface ◽  
Neisseria Gonorrhoeae ◽  
Bacterial Cell ◽  
Bacterial Species ◽  
Type Iv Pili ◽  
Type Iv Pilus ◽  
Transmitted Infection ◽  
Content Type ◽  
Sexually Transmitted ◽  
Type Iv

ABSTRACT Neisseria gonorrhoeae relies on type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the type IV pilus in its extended, nonretracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology, and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show that there are orthologues in numerous bacterial species, but not all type IV pilin-expressing bacteria contain orthologous genes. Coevolution and nuclear magnetic resonance (NMR) analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region, and a highly charged C-terminal coiled-coil domain. IMPORTANCE Most bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae type IV pilus is a major virulence and colonization factor for the sexually transmitted infection gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain type IV pili on the bacterial cell surface. There are similar proteins found in other members of the Neisseria genus and many other bacterial species important for human health.

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