Flavobacterium johnsoniae sprB is part of an operon spanning the additional gliding motility genes sprC, sprD, and sprF

Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as gliding motility. Gld proteins are thought to comprise the gliding motor that propels cell surface adhesins, such as the 669-kDa SprB. A novel protein secretion apparatus called the Por secretion system (PorSS) is required for assembly of SprB on the cell surface. Genetic and molecular analyses revealed that sprB is part of a seven-gene operon spanning 29.3 kbp of DNA. In addition to sprB, three other genes of this operon (sprC, sprD, and sprF) are involved in gliding. Mutations in sprB, sprC, sprD, and sprF resulted in cells that failed to form spreading colonies on agar but that exhibited some motility on glass in wet mounts. SprF exhibits some similarity to Porphyromonas gingivalis PorP, which is required for secretion of gingipain protease virulence factors via the P. gingivalis PorSS. F. johnsoniae sprF mutants produced SprB protein but were defective in localization of SprB to the cell surface, suggesting a role for SprF in secretion of SprB. The F. johnsoniae PorSS is involved in secretion of extracellular chitinase in addition to its role in secretion of SprB. SprF was not needed for chitinase secretion and may be specifically required for SprB secretion by the PorSS. Cells with nonpolar mutations in sprC or sprD produced and secreted SprB and propelled it rapidly along the cell surface. Multiple paralogs of sprB, sprC, sprD, and sprF are present in the genome, which may explain why mutations in sprB, sprC, sprD, and sprF do not result in complete loss of motility and suggests the possibility that semiredundant SprB-like adhesins may allow movement of cells over different surfaces.     

Mutations in Flavobacterium johnsoniae sprE result in defects in gliding motility and protein secretion

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces. Transposon mutagenesis was used to identify sprE, which is involved in gliding. Mutations in sprE resulted in the formation of nonspreading colonies on agar. sprE mutant cells in wet mounts were almost completely deficient in attachment to and movement on glass, but a small percentage of cells exhibited slight movements, indicating that the motility machinery was not completely disrupted. SprE is a predicted lipoprotein with a tetratricopeptide repeat domain. SprE is similar in sequence to Porphyromonas gingivalis PorW, which is required for secretion of gingipain protease virulence factors. Disruption of F. johnsoniae sprE resulted in decreased extracellular chitinase activity and decreased secretion of the cell surface motility protein SprB. Reduced secretion of cell surface components of the gliding machinery, such as SprB, may account for the defects in gliding. Orthologs of sprE are found in many gliding and nongliding members of the phylum Bacteroidetes, suggesting that similar protein secretion systems are common among members of this large and diverse group of bacteria.     

Dynamic proton-dependent motors power type IX secretion and gliding motility in Flavobacterium

Motile bacteria usually rely on external apparatus like flagella for swimming or pili for twitching. By contrast, gliding bacteria do not rely on obvious surface appendages to move on solid surfaces. Flavobacterium johnsoniae and other bacteria in the Bacteroidetes phylum use adhesins whose movement on the cell surface supports motility. In F. johnsoniae, secretion and helicoidal motion of the main adhesin SprB are intimately linked and depend on the type IX secretion system (T9SS). Both processes necessitate the proton motive force (PMF), which is thought to fuel a molecular motor that comprises the GldL and GldM cytoplasmic membrane proteins. Here, we show that F. johnsoniae gliding motility is powered by the pH gradient component of the PMF. We further delineate the interaction network between the GldLM transmembrane helices (TMHs) and show that conserved glutamate residues in GldL TMH2 are essential for gliding motility, although having distinct roles in SprB secretion and motion. We then demonstrate that the PMF and GldL trigger conformational changes in the GldM periplasmic domain. We finally show that multiple GldLM complexes are distributed in the membrane, suggesting that a network of motors may be present to move SprB along a helical path on the cell surface. Altogether, our results provide evidence that GldL and GldM assemble dynamic membrane channels that use the proton gradient to power both T9SS-dependent secretion of SprB and its motion at the cell surface.

Motile bacteria usually rely on external apparatus like flagella or pili, but gliding bacteria do not rely on obvious surface appendages for their movement. This study shows that bacteria in the phylum Bacteroidetes use proton-dependent motors to power protein secretion and gliding motility.     

Flavobacterium johnsoniae GldK,GldL, GldM,and SprA Are Required for Secretion of the Cell Surface Gliding Motility Adhesins SprB and RemA

Flavobacterium johnsoniae cells move rapidly over surfaces by gliding motility. Gliding results from the movement of adhesins such as SprB and RemA along the cell surface. These adhesins are delivered to the cell surface by a Bacteroidetes-specific secretion system referred to as the type IX secretion system (T9SS). GldN, SprE, SprF, and SprT are involved in secretion by this system. Here we demonstrate that GldK, GldL, GldM, and SprA are each also involved in secretion. Nonpolar deletions of gldK, gldL, or gldM resulted in the absence of gliding motility and in T9SS defects. The mutant cells produced SprB and RemA proteins but failed to secrete them to the cell surface. The mutants were resistant to phages that use SprB or RemA as a receptor, and they failed to attach to glass, presumably because of the absence of cell surface adhesins. Deletion of sprA resulted in similar but slightly less dramatic phenotypes. sprA mutant cells failed to secrete SprB and RemA, but cells remained susceptible to some phages and retained some limited ability to glide. The phenotype of the sprA mutant was similar to those previously described for sprE and sprT mutants. SprA, SprE, and SprT are needed for secretion of SprB and RemA but may not be needed for secretion of other proteins targeted to the T9SS. Genetic and molecular experiments demonstrate that gldK, gldL, gldM, and gldN form an operon and suggest that the proteins encoded by these genes may interact to form part of the F. johnsoniae T9SS.     

Flavobacterium johnsoniae RemA is a mobile cell surface lectin involved in gliding

Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as gliding motility. Gld proteins are thought to comprise the motor that propels the cell surface adhesin SprB. Cells with mutations in sprB are partially defective in motility and are also resistant to some bacteriophages. Transposon mutagenesis of a strain carrying a deletion spanning sprB identified eight mutants that were resistant to additional phages and exhibited reduced motility. Four of the mutants had transposon insertions in remA, which encodes a cell surface protein that has a lectin domain and appears to interact with polysaccharides. Three other genes identified in this screen (remC, wza, and wzc) encode proteins predicted to be involved in polysaccharide synthesis and secretion. Myc-tagged versions of RemA localized to the cell surface and were propelled rapidly along the cell at speeds of 1 to 2 μm/s. Deletion of gldN and gldO, which encode components of a bacteroidete protein secretion system, blocked the transport of RemA to the cell surface. Overexpression of RemA resulted in the formation of cell aggregates that were dispersed by the addition of galactose or rhamnose. Cells lacking RemC, Wza, and Wzc failed to aggregate. Cells of a remC mutant and cells of a remA mutant, neither of which formed aggregates in isolation, aggregated when they were mixed together, suggesting that polysaccharides secreted by one cell may interact with RemA on another cell. Fluorescently labeled lectin Ricinus communis agglutinin I detected polysaccharides secreted by F. johnsoniae. The polysaccharides bound to cells expressing RemA and were rapidly propelled on the cell surface. RemA appears to be a mobile cell surface adhesin, and secreted polysaccharides may interact with the lectin domain of RemA and enhance motility.     

Flavobacterium johnsoniae PorV Is Required for Secretion of a Subset of Proteins Targeted to the Type IX Secretion System

Flavobacterium johnsoniae exhibits gliding motility and digests many polysaccharides, including chitin. A novel protein secretion system, the type IX secretion system (T9SS), is required for gliding and chitin utilization. The T9SS secretes the cell surface motility adhesins SprB and RemA and the chitinase ChiA. Proteins involved in secretion by the T9SS include GldK, GldL, GldM, GldN, SprA, SprE, and SprT. Porphyromonas gingivalis has orthologs for each of these that are required for secretion of gingipain protease virulence factors by its T9SS. P. gingivalis porU and porV have also been linked to T9SS-mediated secretion, and F. johnsoniae has orthologs of these. Mutations in F. johnsoniae porU and porV were constructed to determine if they function in secretion. Cells of a porV deletion mutant were deficient in chitin utilization and failed to secrete ChiA. They were also deficient in secretion of the motility adhesin RemA but retained the ability to secrete SprB. SprB is involved in gliding motility and is needed for formation of spreading colonies on agar, and the porV mutant exhibited gliding motility and formed spreading colonies. However, the porV mutant was partially deficient in attachment to glass, apparently because of the absence of RemA and other adhesins on the cell surface. The porV mutant also appeared to be deficient in secretion of numerous other proteins that have carboxy-terminal domains associated with targeting to the T9SS. PorU was not required for secretion of ChiA, RemA, or SprB, indicating that it does not play an essential role in the F. johnsoniae T9SS.     

Cytophaga-flavobacterium gliding motility

Flavobacterium johnsoniae, like many other members of the Cytophaga-Flavobacterium-Bacteroides group, displays rapid gliding motility. Cells of F. johnsoniae glide over surfaces at rates of up to 10 microm/s. Latex spheres added to F. johnsoniae bind to and are rapidly propelled along cells, suggesting that adhesive molecules move laterally along the cell surface during gliding. Genetic analyses have identified a number of gld genes that are required for gliding. Three Gld proteins are thought to be components of an ATP-binding-cassette transporter. Five other Gld proteins are lipoproteins that localize to the cytoplasmic membrane or outer membrane. Disruption of gld genes results not only in loss of motility, but also in resistance to bacteriophages that infect wild-type cells, and loss of the ability to digest the insoluble polysaccharide chitin. Two models that attempt to incorporate the available data to explain the mechanism of F. johnsoniae gliding are presented.     

Development and use of a gene deletion strategy for Flavobacterium johnsoniae to identify the redundant gliding motility genes remF, remG, remH, and remI

Cells of Flavobacterium johnsoniae exhibit rapid gliding motility over surfaces. Cell movement is thought to involve motor complexes comprised of Gld proteins that propel the cell surface adhesin SprB. The four distal genes of the sprB operon (sprC, sprD, sprB, and sprF) are required for normal motility and for formation of spreading colonies, but the roles of the remaining three genes (remF, remG, and fjoh_0982) are unclear. A gene deletion strategy was developed to determine whether these genes are involved in gliding. A spontaneous streptomycin-resistant rpsL mutant of F. johnsoniae was isolated. Introduction of wild-type rpsL on a plasmid restored streptomycin sensitivity, demonstrating that wild-type rpsL is dominant to the mutant allele. The gene deletion strategy employed a suicide vector carrying wild-type rpsL and used streptomycin for counterselection. This approach was used to delete the region spanning remF, remG, and fjoh_0982. The mutant cells formed spreading colonies, demonstrating that these genes are not required for normal motility. Analysis of the genome revealed a paralog of remF (remH) and a paralog of remG (remI). Deletion of remH and remI had no effect on motility of wild-type cells, but cells lacking remF and remH, or cells lacking remG and remI, formed nonspreading colonies. The motility defects resulting from the combination of mutations suggest that the paralogous proteins perform redundant functions in motility. The rpsL counterselection strategy allows construction of unmarked mutations to determine the functions of individual motility proteins or to analyze other aspects of F. johnsoniae physiology.     

Gliding movement in Peranema trichophorum is powered by flagellar surface motility

A colorless euglenoid flagellate Peranema trichophorum shows unique unidirectional gliding cell locomotion on the substratum at velocities up to 30 micro m/s by an as yet unexplained mechanism. In this study, we found that (1) treatment with NiCl(2) inhibited flagellar beating without any effect on gliding movement; (2) water currents applied to a gliding cell from opposite sides caused detachment of the cell body from the substratum. With only the anterior flagellum adhering to the substratum, gliding movement continued along the direction of the anterior flagellum; (3) gentle pipetting induced flagellar severance into various lengths. In these cells, gliding velocity was proportional to the flagellar length; and (4) Polystyrene beads were translocated along the surface of the anterior flagellum. All of these results indicate that a cell surface motility system is present on the anterior flagellum, which is responsible for cell gliding in P. trichophorum.     

Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation

Capnocytophaga ochracea is a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces. C. ochracea possesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) in Flavobacterium johnsoniae was shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins in F. johnsoniae have been identified in the genome of C. ochracea; therefore, the T9SS may be involved in biofilm formation by C. ochracea. Here we constructed three ortholog-deficient C. ochracea mutants lacking sprB (which encodes a gliding motility adhesin) or gldK or sprT (which encode T9SS proteins in F. johnsoniae). Gliding motility was lost in each mutant, suggesting that, in C. ochracea, the proteins encoded by sprB, gldK, and sprT are necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprB strains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-type C. ochracea were denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation of C. ochracea.     

A phylogenetic analysis of the purple photosynthetic bacteria

It is proposed that gliding motility in bacteria is based on rotary assemblies located in the cell envelope and that these assemblies may be analogous to basal regions of bacterial flagella. This proposal rests on the following evidence: (i) Structures resembling flagellar basal regions have been demonstrated in cells ofCytophaga johnsonae andFlexibacter columnaris, and such structures are absent from one nonmotile mutant ofF. columnaris. (ii) The effects of inhibitors of energy metabolism on gliding motility are identical with their effects on prokaryotic fiagellar motility. (iii) The active movement of latex spheres along surfaces of gliding bacteria appears to depend on mechanisms responsible for motility and can be explained by the presence of rotary surface assemblies.     

Directed movements of ciliary and flagellar membrane components: a review.

The ability to rapidly translocate polystyrene microspheres attached to the surface of a plasma membrane domain reflects a unique form of cellular force transduction occurring in association with the plasma membrane of microtubule based cell extensions. This unusual form of cell motility can be utilized by protistan organisms for whole cell locomotion, the early events in mating, and transport of food organisms along the cell surface, and possibly intracellular transport of certain organelles. Since surface motility is observed in association with cilia and flagella of algae, sea urchin embryos and cultured mammalian cells, it is likely that it serves an additional role beyond those already cited; this is likely to be the transport of precursors for the assembly and turnover of ciliary and flagellar membranes and axonemes. In the case of the Chlamydomonas flagellum, where surface motility has been most extensively studied, it appears that cross-linking of flagellar surface exposed proteins induces a transmembrane signaling pathway that activates machinery for moving flagellar membrane proteins in the plane of the flagellar membrane. This signaling pathway in vegetative Chlamydomonas reinhardtii appears to involve an influx of calcium, a rise in intraflagellar free calcium concentration and a change in the level of phosphorylation of specific membrane-matrix proteins. It is hypothesized that flagellar surface contact with a solid substrate (during gliding), a polystyrene microsphere or another flagellum (during mating) will all activate a signaling pathway similar to the one artificially activated by the use of monoclonal antibodies to flagellar membrane glycoproteins. A somewhat different signaling pathway, involving a transient rise in intracellular cAMP level, may be associated with the mating of Chlamydomonas gametes, which is initiated by flagellum-flagellum contact. The hypothesis that the widespread observation of microsphere movements on various ciliary and flagellar surfaces may reflect a mechanism normally utilized to transport axonemal and membrane subunits along the internal surface of the organelle membrane presents a paradox in that one would expect this to be a constitutive mechanism, not one necessarily activated by a signaling pathway.     

Flavobacterium johnsoniae gldN and gldO Are Partially Redundant Genes Required for Gliding Motility and Surface Localization of SprB

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces. Mutations in gldN cause a partial defect in gliding. A novel bacteriophage selection strategy was used to aid construction of a strain with a deletion spanning gldN and the closely related gene gldO in an otherwise wild-type F. johnsoniae UW101 background. Bacteriophage transduction was used to move a gldN mutation into F. johnsoniae UW101 to allow phenotypic comparison with the gldNO deletion mutant. Cells of the gldN mutant formed nonspreading colonies on agar but retained some ability to glide in wet mounts. In contrast, cells of the gldNO deletion mutant were completely nonmotile, indicating that cells require GldN, or the GldN-like protein GldO, to glide. Recent results suggest that Porphyromonas gingivalis PorN, which is similar in sequence to GldN, has a role in protein secretion across the outer membrane. Cells of the F. johnsoniae gldNO deletion mutant were defective in localization of the motility protein SprB to the cell surface, suggesting that GldN may be involved in secretion of components of the motility machinery. Cells of the gldNO deletion mutant were also deficient in chitin utilization and were resistant to infection by bacteriophages, phenotypes that may also be related to defects in protein secretion.Cells of Flavobacterium johnsoniae, and of many other members of the phylum Bacteroidetes, crawl over surfaces at approximately 2 μm/s in a process called gliding motility. F. johnsoniae cells glide on agar, glass, polystyrene, Teflon, and many other surfaces (16, 22). Cells suspended in liquid also bind and propel added particles such as polystyrene latex spheres (23). The mechanism of this form of cell movement is not well understood despite decades of research (15). Genome analyses suggest that F. johnsoniae gliding is genetically unrelated to other well-studied forms of bacterial movement such as bacterial flagellar motility, type IV pilus-mediated twitching motility, myxobacterial gliding motility, and mycoplasma gliding motility (10, 20, 21). Genes and proteins required for F. johnsoniae motility have been identified (1-3, 7-9, 17, 18). GldA, GldF, and GldG appear to form an ATP-binding cassette transporter that is required for gliding (1, 7). Eight other Gld proteins (GldB, GldD, GldH, GldI, GldJ, GldK, GldL, and GldM) are also required for movement (2, 3, 8, 9, 17, 18). Many of these are unique to members of the phylum Bacteroidetes. Disruption of the genes encoding any of these 11 proteins results in complete loss of motility. The mutants form nonspreading colonies, and individual cells exhibit no movement on agar, glass, Teflon, and other surfaces tested. The Gld proteins are associated with the cell envelope and presumably constitute the gliding motor, but none of them appear to be exposed on the cell surface. Mutations in sprA and sprB, which encode cell surface proteins, result in partial motility defects. Cells form nonspreading colonies, but some of the cells exhibit limited movement in wet mounts. SprA is required for efficient attachment to glass (22), and SprB appears to be a mobile adhesin that is propelled along the cell surface by the gliding motor and thus transmits the force generated by the motor to the surface over which cells crawl (10, 21). The surface localization of SprA and SprB and the phenotypes of sprA and sprB mutants suggest that the gliding motor is at least partially functional in these mutants but that force is inefficiently transmitted to the substratum. Analysis of the F. johnsoniae genome revealed the presence of multiple paralogs of sprB, which may explain the residual motility of sprB mutants (20).gldN lies downstream of gldL and gldM, and the three genes constitute an operon (2). Cells with transposon insertions in gldN form nonspreading colonies that are indistinguishable from those of other gld mutants. However, unlike other gld mutants, gldN mutants exhibit some residual ability to glide in wet mounts (2). One possible explanation for this phenotype is that GldN may have a peripheral and nonessential role in gliding. Alternatively, GldN may perform a critical function in gliding, but in its absence another cellular protein may compensate for the missing GldN function. F. johnsoniae has a gldN paralog, gldO, that is located downstream of gldN but is transcribed independently (2). The GldN and GldO proteins are 85% identical over their entire lengths, making GldO a prime candidate for a protein that might compensate for lack of GldN.Recent results suggest that some of the F. johnsoniae Gld and Spr proteins, including GldN, may be components of a novel bacteroidete protein translocation apparatus referred to as the Por secretion system (PorSS) (28). This conclusion emerged from studies of gingipain protease secretion by the distantly related nonmotile bacteroidete Porphyromonas gingivalis. P. gingivalis is a human periodontal pathogen, and gingipain proteases are important virulence factors. Gingipains have signal peptides that allow export across the cytoplasmic membrane via the Sec machinery, but they rely on components of the PorSS for secretion across the outer membrane (27-29). P. gingivalis cells with mutations in genes homologous to F. johnsoniae gldK, gldL, gldM, gldN, and sprA are defective in gingipain secretion across the outer membrane (28). F. johnsoniae has a homologue to another P. gingivalis gene required for gingipain secretion, porT. Disruption of the F. johnsoniae porT homologue (referred to as sprT) results in motility defects and defects in surface localization of SprB (28).This study was designed to identify possible roles for GldN in motility and to determine whether GldN and GldO are partially redundant components of the motility apparatus. The results demonstrate that F. johnsoniae GldN has an important function in motility and that GldO can replace GldN in this role. They suggest that GldN is needed for efficient secretion of the cell surface motility protein SprB, which may explain some of the motility defects of the gldN mutants.     

Flavobacterium johnsoniae GldH is a lipoprotein that is required for gliding motility and chitin utilization

Cells of Flavobacterium johnsoniae move rapidly over surfaces by gliding motility. The mechanism of this form of motility is not known. Six genes (gldA, gldB, gldD, gldF, gldG, and ftsX) that are required for gliding have been described. Tn4351 mutagenesis was used to identify another gene, gldH, which is required for cell movement. GldH mutants formed nonspreading colonies, and individual cells lacked the cell movements and ability to propel latex spheres along their surfaces that are characteristic of wild-type cells. gldH mutants also failed to digest chitin and were resistant to bacteriophages that infect wild-type cells. Introduction of pMM293, which carries wild-type gldH, restored to the gldH mutants colony spreading, cell motility, the ability to move latex spheres, phage sensitivity, and the ability to digest chitin. gldH encodes a predicted 141-amino-acid protein that localized to the membrane fraction. Labeling studies with [3H]palmitate demonstrated that GldH is a lipoprotein. GldB and GldD, which were previously described, also appear to be lipoproteins. GldH does not exhibit significant amino acid similarity to proteins of known function in the databases. Putative homologs of gldH of unknown function are found in motile (Cytophaga hutchinsonii) and apparently nonmotile (Bacteroides thetaiotaomicron, Bacteroides fragilis, Tannerella forsythensis, Porphyromonas gingivalis, and Prevotella intermedia) members of the Cytophaga-Flavobacterium-Bacteroides group.     

The motors powering A-motility in Myxococcus xanthus are distributed along the cell body

Two models have been proposed to explain the adventurous gliding motility of Myxococcus xanthus: (i) polar secretion of slime and (ii) an unknown motor that uses cell surface adhesion complexes that form periodic attachments along the cell length. Gliding movements of the leading poles of cephalexin-treated filamentous cells were observed but not equivalent movements of the lagging poles. This demonstrates that the adventurous-motility motors are not confined to the rear of the cell.     

GldI is a lipoprotein that is required for Flavobacterium johnsoniae gliding motility and chitin utilization

Cells of Flavobacterium johnsoniae glide rapidly over surfaces by an unknown mechanism. Seven genes (gldA, gldB, gldD, gldF, gldG, gldH, and ftsX) that are required for gliding motility have been described. Complementation of the nonmotile mutants UW102-41, UW102-85, and UW102-92 identified another gene, gldI, that is required for gliding motility. gldI mutants formed nonspreading colonies, and individual cells were completely nonmotile. They were also resistant to bacteriophages that infect wild-type cells, and they failed to digest chitin. Introduction of wild-type gldI on a plasmid restored colony spreading, cell motility, phage sensitivity, and the ability to digest chitin to the gldI mutants. gldI encodes a predicted 199-amino-acid protein that localized to the membrane fraction. Labeling studies with [(3)H]palmitate indicated that GldI is a lipoprotein. GldI is similar to peptidyl-prolyl cis/trans-isomerases of the FK506-binding protein family and may be involved in folding cell envelope protein components of the motility machinery.     

Malaria sporozoites leave behind trails of circumsporozoite protein during gliding motility

As Plasmodium sporozoites undergo gliding motility in vitro, they leave behind trails of circumsporozoite (CS) protein that correspond to their patterns of movement. This light microscopic observation was made using Plasmodium berghei sporozoites, a monoclonal antibody (MAb H4) directed against the immunodominant repetitive epitope of the CS protein of P. berghei, and an immunogold-silver staining (IGSS) technique. Sporozoites pretreated with agents that inhibit sporozoite motility and invasiveness did not produce trails. Sporozoites that glided on microscope slides coated with MAb H4 left behind considerably longer CS protein trails than those on uncoated slides, and the staining of these trails was more intense. The fact that the CS protein is an exoantigen continuously released as trails by motile sporozoites, together with our previous finding that anti-CS protein antibodies inhibit sporozoite motility, strongly suggests that the CS protein plays a role in gliding motility. The sensitive IGSS technique used in this study may be a useful tool in the study of the translocation of surface proteins during gliding of other apicomplexans, other protists, and bacteria.     

Use of a novel Chlamydomonas mutant to demonstrate that flagellar glycoprotein movements are necessary for the expression of gliding motility

As an alternative to swimming through liquid medium by the coordinated bending activity of its two flagella, Chlamydomonas can exhibit whole cell gliding motility through the interaction of its flagellar surfaces with a solid substrate. The force transduction occurring at the flagellar surface can be visualized as the saltatory movements of polystyrene microspheres. Collectively, gliding motility and polystyrene microsphere movements are referred to as flagellar surface motility. The principal concanavalin A binding, surface-exposed glycoproteins of the Chlamydomonas reinhardtii flagellar surface are a pair of glycoproteins migrating with apparent molecular weight of 350 kDa. It has been hypothesized that these glycoproteins move within the plane of the flagellar membrane during the expression of flagellar surface motility. A novel mutant cell line of Chlamydomonas (designated L-23) that exhibits increased binding of concanavalin A to the flagellar surface has been utilized in order to restrict the mobility of the concanavalin A-binding flagellar glycoproteins. Under all conditions where the lateral mobility of the flagellar concanavalin A binding glycoproteins is restricted, the cells are unable to express whole cell gliding motility or polystyrene microsphere movements. Conversely, whenever cells can redistribute their concanavalin A binding glycoproteins in the plane of the flagellar membrane, they express flagellar surface motility. Since the 350 kDa glycoproteins are the major surface-exposed flagellar proteins, it is likely that most of the signal being followed using fluorescein isothiocyanate (FITC)-concanavalin A is attributable to these high molecular weight glycoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microsphere attachment induces glycoprotein redistribution and transmembrane signaling in theChlamydomonas flagellum

Summary The biflagellate green algaChlamydomonas can exhibit substrate-associated gliding motility in addition to its ability to swim through a liquid medium. The flagella are the organelles responsible for both forms of whole-cell locomotion although the mechanism in each case is very different. In this study, we demonstrate that the binding of polystyrene microspheres to the flagellar surface ofChlamydomonas initiates clustering of the major flagellar-membrane glycoprotein, which is known to be involved in motility-associated substrate adhesion. In addition, we demonstrate that microsphere binding to the flagellar surface initiates the same transmembrane signaling pathway that is initiated by antibody- or lectin-induced crosslinking of the major flagellar-membrane glycoprotein. In each case, the signaling pathway involves the activation of a calciumdependent protein phosphatase that dephosphorylates a flagellar phosphoprotein known to be associated with the major flagellar-membrane glycoprotein. Bound microspheres are translocated along the flagellar surface at approximately the same velocity as whole-cell gliding motility. Previous observations suggest that microsphere binding and translocation along the flagellar surface may be a reflection of the same force-transducing system responsible for whole-cell gliding motility. In which case, these observations suggest that the transmembrane signaling pathway initiated by crosslinking the major flagellar-membrane glycoprotein is the same one that is activated when the cell contacts a physiological substrate by its flagellar surface.     

Actin-based motility in the net slime mould Labyrinthula: evidence for the role of myosin in gliding movement

In contrast to crawling movement (e.g. in amoebae and tissue cells) the other major class of substratum-associated motility in eukaryotes, gliding, has received relatively little attention. The net slime mold Labyrinthula provides a useful laboratory model for studying this process since it exhibits a particular kind of gliding in its plasmodial stage. Here nucleated spindle cells glide along self-established cytoplasmic trackways in a predominantly unidirectional manner, at 1-2 microm/s. These trackways, upon which gliding is dependent, are held by filopodial tethers some distance off the well-developed reticulopodial mesh anchoring the plasmodium onto the substratum. Reflection interference microscopy resolves this matrix in live plasmodia. The axially disposed cytoskeletal elements of the trackways are revealed by rhodamine-labelled phalloidin to be rich in F-actin. A weft of peripheral, rapidly extending filopodia (50 microm/min) typifies the expanding regions of the plasmodium. Here spindle cells are recruited before emigrating into newly differentiated trackways. Immunoblotting whole plasmodia or a sucrose-soluble cytoplasmic extract reveals a single actin-positive band of Mr 48 kDa. Polyclonal antibodies to two distinct myosin peptide sequences identify a single myosin HC (Mr 96 kDa) in immunoblots. Gliding was reversibly blocked by 10 mM 2,3-butanedione-2-monoxime, a myosin ATPase inhibitor, but it was insensitive to the actin-binding drugs cytochalasin D and phalloidin. We suggest that the force (>50 pN) for gliding motility results from interaction of myosin molecules, associated with the spindle cells, with trackway F-actin via the bothrosomes.