Temporal sequence of the recovery of traits during phenotypic curing of a Cytophaga johnsonae motility mutant.

The lack of cell translocation and the resulting formation of nonspreading colonies of mutants of the gram-negative gliding bacterium Cytophaga johnsonae have been correlated with the loss of cell surface features of the organism. These cell surface traits include the ability to move polystyrene-latex beads over the cell surface and the ability to be infected by bacteriophages that infect the parent strain. In order to assess whether these traits reflect structures or functions that actually play a role in gliding, we studied a mutant (21A2I) selected for its inability to form spreading colonies; it is deficient in sulfonolipid, lacks bead movement ability, and is resistant to at least one bacteriophage. The provision of cysteate (a specific sulfonolipid precursor) restores lipid content and gliding to the mutant; hence, the lipids are necessary for motility. Growth with cysteate also restores bead movement and phage sensitivity. In order to determine the temporal relationship of these traits, we undertook a kinetic study of the appearance of them after addition of cysteate to the mutant. One predicts that appearance of a trait essential for cell translocation will either precede or accompany the appearance of this ability, while a nonessential trait need not do so. Sulfonolipid synthesis was the only trait that appeared before gliding; this is consistent with its established importance for motility. Bead movement and phage sensitivity first appeared only after gliding started, suggesting that the machinery involved in those processes is not necessary, at least for the initiation of gliding.     

Structural specificity of sugars that inhibit gliding motility of Cytophaga johnsonae

It is a common observation that gliding bacteria form raised, smooth-edged colonies on nutrient-rich media, and typical thin, spreading, uneven-edged colonies on nutrient-poor media. An earlier study of the effect of different sugars on colony spreading by Cytophaga johnsonae was expanded to include the effects of several sugars and other organic compounds on the motility of groups of cells (rafts), and latex bead movement on cells' surfaces. When the structures of those sugars that did, or did not, affect raft formation and colony spreading were compared, it was noted that those sugars that inhibited these two manifestations of gliding motility all possessed a common sub-structure, that found in the portion of glucopyranose comprising carbons 3, 4, 5, and 6. If these structural features were altered chemically or stereochemically, the resulting molecule had little to no effect on motility. The differential effects of some compounds on raft formation, colony spreading, and bead movement are noted. A regulatory mechanism that would turn off motility in the presence of an inhibitory sugar is implicated, and the relevance of such a system to the life of the organism is discussed. We report, as well, additional compounds that will serve as carbon and energy sources for C. johnsonae.     

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.     

Outer membrane polysaccharide deficiency in two nongliding mutants of Cytophaga johnsonae.

Phenol-extractable polysaccharides firmly associated with the outer membrane of the gliding bacterium Cytophaga johnsonae could be resolved by gel filtration in sodium dodecyl sulfate (SDS) or by SDS-polyacrylamide gel electrophoresis into a high-molecular-weight (H) fraction (excluded by Sephadex G-200) and a low-molecular-weight (L) fraction. Fraction L was rich in components typical of lipid A and the core region of lipopolysaccharide (P, 3-hydroxy fatty acids, and 2-keto-3-deoxyoctonate) and evidently was a lipopolysaccharide with a limited number of distal, repeating polysaccharide units, as judged by SDS-polyacrylamide gel electrophoresis. In relation to total carbohydrate, the H fraction was rich in amino sugar but poor in (possibly devoid of) the lipid A and core components. Two nongliding mutants were highly deficient in the H fraction; one of these was deficient in sulfonolipid but could be cured by provision of a specific sulfonolipid precursor, a process that also resulted in the return of both the H fraction and gliding, as well as the ability to move polystyrene latex spheres over the cell surface. Hence, the polysaccharide may be the component that is directly involved in motility, and the presence of sulfonolipids in the outer membrane is necessary for the synthesis or accumulation of the polysaccharide. This conclusion was reinforced by the fact that the second nongliding, polysaccharide-deficient mutant had a normal sulfonolipid content.     

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.     

Cloning and Characterization of the Flavobacterium johnsoniae Gliding Motility Genes gldD and gldE

Cells of Flavobacterium johnsoniae move over surfaces by a process known as gliding motility. The mechanism of this form of motility is not known. Cells of F. johnsoniae propel latex spheres along their surfaces, which is thought to be a manifestation of the motility machinery. Three of the genes that are required for F. johnsoniae gliding motility, gldA, gldB, and ftsX, have recently been described. Tn4351 mutagenesis was used to identify another gene, gldD, that is needed for gliding. Tn4351-induced gldD mutants formed nonspreading colonies, and cells failed to glide. They also lacked the ability to propel latex spheres and were resistant to bacteriophages that infect wild-type cells. Introduction of wild-type gldD into the mutants restored motility, ability to propel latex spheres, and sensitivity to bacteriophage infection. gldD codes for a cytoplasmic membrane protein that does not exhibit strong sequence similarity to proteins of known function. gldE, which lies immediately upstream of gldD, encodes another cytoplasmic membrane protein that may be involved in gliding motility. Overexpression of gldE partially suppressed the motility defects of a gldB point mutant, suggesting that GldB and GldE may interact. GldE exhibits sequence similarity to Borrelia burgdorferi TlyC and Salmonella enterica serovar Typhimurium CorC.     

Transposon mutagenesis identifies genes associated with Mycoplasma pneumoniae gliding motility

The wall-less prokaryote Mycoplasma pneumoniae, a common cause of chronic respiratory tract infections in humans, is considered to be among the smallest and simplest known cells capable of self-replication, yet it has a complex architecture with a novel cytoskeleton and a differentiated terminal organelle that function in adherence, cell division, and gliding motility. Recent findings have begun to elucidate the hierarchy of protein interactions required for terminal organelle assembly, but the engineering of its gliding machinery is largely unknown. In the current study, we assessed gliding in cytadherence mutants lacking terminal organelle proteins B, C, P1, and HMW1. Furthermore, we screened over 3,500 M. pneumoniae transposon mutants individually to identify genes associated with gliding but dispensable for cytadherence. Forty-seven transformants having motility defects were characterized further, with transposon insertions mapping to 32 different open reading frames widely distributed throughout the M. pneumoniae genome; 30 of these were dispensable for cytadherence. We confirmed the clonality of selected transformants by Southern blot hybridization and PCR analysis and characterized satellite growth and gliding by microcinematography. For some mutants, satellite growth was absent or developed more slowly than that of the wild type. Others produced lawn-like growth largely devoid of typical microcolonies, while still others had a dull, asymmetrical leading edge or a filamentous appearance of colony spreading. All mutants exhibited substantially reduced gliding velocities and/or frequencies. These findings significantly expand our understanding of the complexity of M. pneumoniae gliding and the identity of possible elements of the gliding machinery, providing a foundation for a detailed analysis of the engineering and regulation of motility in this unusual prokaryote.     

Gliding motility and polarized slime secretion

Myxococcus leaves a trail of slime on agar as it moves. A filament of slime can be seen attached to the end of a cell, but it is seen only at one end at any particular moment. To identify genes essential for A motility, transposon insertion mutations with defective A motility were studied. Fifteen of the 33 mutants had totally lost A motility. All these mutant cells had filaments of slime emerging from both ends, indicating that bipolar secretion prevents A motility. The remaining 18 A motility mutants, also produced by gene knockout, secreted slime only from one pole, but they swarmed at a lower rate than A(+) and are called 'partial' gliding mutants, or pgl. For each pgl mutant, the reduction in swarm expansion rate was directly proportional to the reduction in the coefficient of elasticotaxis. The pgl mutants have a normal reversal frequency and normal gliding speed when they move. But their probability of movement per unit time is lower than pgl(+) cells. Many of the pgl mutants are produced by transposon insertions in glycosyltransferase genes. It is proposed that these glycosyltransferases carry out the synthesis of a repeat unit polysaccharide that constitutes the slime.     

Cloning and characterization of the Flavobacterium johnsoniae gliding-motility genes gldB and gldC

The mechanism of bacterial gliding motility (active movement over surfaces without the aid of flagella) is not known. A large number of mutants of the gliding bacterium Flavobacterium johnsoniae (Cytophaga johnsonae) with defects in gliding motility have been previously isolated, and genetic techniques to analyze these mutants have recently been developed. We complemented a nongliding mutant of F. johnsoniae (UW102-99) with a library of wild-type DNA by using the shuttle cosmid pCP26. The complementing plasmid (pCP200) contained an insert of 26 kb and restored gliding motility to 4 of 50 independently isolated nongliding mutants. A 1.9-kb fragment which encompassed two genes, gldB and gldC, complemented all four mutants. An insertion mutation in gldB was polar on gldC, suggesting that the two genes form an operon. Disruption of the chromosomal copy of gldB in wild-type F. johnsoniae UW101 eliminated gliding motility. Introduction of the gldBC operon, or gldB alone, restored motility. gldB appears to be essential for F. johnsoniae gliding motility. It codes for a membrane protein that does not exhibit strong sequence similarity to other proteins in the databases. gldC is not absolutely required for gliding motility, but cells that do not produce GldC form colonies that spread less well than those of the wild type. GldC is a soluble protein and has weak sequence similarity to the fungal lectin AOL.     

Mycoplasma genitalium mg200 and mg386 genes are involved in gliding motility but not in cytadherence

Isolation and characterization of transposon-generated Mycoplasma genitalium gliding-deficient mutants has implicated mg200 and mg386 genes in gliding motility. The proposed role of these genes was confirmed by restoration of the gliding phenotype in deficient mutants through gene complementation with their respective mg386 or mg200 wild-type copies. mg200 and mg386 are the first reported gliding-associated mycoplasma genes not directly involved in cytadherence. Orthologues of MG200 and MG386 proteins are also found in the slow gliding mycoplasmas, Mycoplasma pneumoniae and Mycoplasma gallisepticum, suggesting the existence of a unique set of proteins involved in slow gliding motility. MG200 and MG386 proteins share common features, such as the presence of enriched in aromatic and glycine residues boxes and an acidic and proline-rich domain, suggesting that these motifs could play a significant role in gliding motility.     

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.     

Genetic requirements for potassium ion-dependent colony spreading in Bacillus subtilis

Undomesticated strains of Bacillus subtilis exhibit extensive colony spreading on certain soft agarose media: first the formation of dendritic clusters of cells, followed by spreading (pellicle-like) growth to cover the entire surface. These phases of colonization are dependent on the level of potassium ion (K(+)) but independent of flagella, as verified with a mutant with a hag gene replacement; this latter finding highlights the importance of sliding motility in colony spreading. Exploring the K(+) requirement, directed mutagenesis of the higher-affinity K(+) transporter KtrAB, but not the lower-affinity transporter KtrCD, was found to inhibit surface colonization unless sufficient KCl was added. To identify other genes involved in K(+)-dependent colony spreading, transposon insertion mutants in wild-type strain 3610 were screened. Disruption of genes for pyrimidine (pyrB) or purine (purD, purF, purH, purL, purM) biosynthetic pathways abolished the K(+)-dependent spreading phase. Consistent with a requirement for functional nucleic acid biosynthesis, disruption of purine synthesis with the folic acid antagonist sulfamethoxazole also inhibited spreading. Other transposon insertions disrupted acetoin biosynthesis (the alsS gene), acidifying the growth medium, glutamine synthetase (the glnA gene), and two surfactin biosynthetic genes (srfAA, srfAB). This work identified four classes of surface colonization mutants with defective (i) potassium transport, (ii) surfactin formation, (iii) growth rate or yield, or (iv) pH control. Overall, the ability of B. subtilis to colonize surfaces by spreading is highly dependent on balanced nucleotide biosynthesis and nutrient assimilation, which require sufficient K(+) ions, as well as growth conditions that promote sliding motility.     

Defects in motility and development of Myxococcus xanthus lipopolysaccharide mutants.

Five transposon Tn5 mutants of the procaryote Myxococcus xanthus had been shown previously to be defective in lipopolysaccharide biosynthesis (J. M. Fink,-M. Kalos, and J. F. Zissler, J. Bacteriol. 171:2033-2041, 1989). These mutants were studied for possible defects in gliding motility and multicellular development. Wild-type M. xanthus cells glide both as single cells and as groups of cells. We found that the Tn5 lipopolysaccharide O-antigen mutants were defective in single-cell motility but were unaltered in group motility. These mutant strains were slow to develop but eventually gave rise to normal, spore-filled fruiting bodies. We also had shown previously that 56 (ethyl methanesulfonate-induced and spontaneous) phage-resistant mutants were defective in lipopolysaccharide biosynthesis. We found that many of these lipopolysaccharide O-antigen mutants were defective in single-cell motility but were unaltered in group motility. These mutants also gave rise to normal, spore-filled fruiting bodies. We also studied several phage-resistant mutants which were lacking a side-chain carbohydrate on the lipopolysaccharide core. These mutants possessed both single-cell motility and group motility but were altered in the magnitude of gliding. These mutants were blocked early in development and could not form multicellular fruiting bodies. Several of the mutations in the developmentally aberrant strains were mapped to a single locus by using a collection of genetically linked transposons as genetic markers.     

Mutational analysis of flagellum-independent surface spreading of Serratia marcescens 274 on a low-agar medium.

In a previous study (J. O'Rear, L. Alberti, and R. M. Harshey, J. Bacteriol. 174:6125-6137, 1992) we reported the isolation of several transposon mutants of Serratia marcescens 274 that were defective either in swarming alone or in both swimming and swarming motility. All the nonflagellate (Fla-) mutants, while defective in both types of motility, were able to spread rapidly on the surface of low-agar (0.35%) media. We show here that some of the swarming-defective mutants are defective in the production of serrawettin W1, an extracellular cyclic lipopeptide produced by S. marcescens 274. When combined with a Fla defect, the serrawettin (Swt) mutants are deficient in spreading on low-agar media. The spreading deficiency can be overcome by serrawettin supplied extracellularly. Introduction of Fla defects into chemotaxis mutants does not affect this mode of surface translocation. These results suggest that spreading may be a passive form of translocation. We also report that swarming defects in all mutants showing a Dps phenotype (able to swarm within the inoculated area but unable to move outward) in the earlier study can be overcome by changing the commercial source of agar.     

Gliding mutants of Myxococcus xanthus with high reversal frequencies and small displacements

Myxococcus xanthus cells move on a solid surface by gliding motility. Several genes required for gliding motility have been identified, including those of the A- and S-motility systems as well as the mgl and frz genes. However, the cellular defects in gliding movement in many of these mutants were unknown. We conducted quantitative, high-resolution single-cell motility assays and found that mutants defective in mglAB or in cglB, an A-motility gene, reversed the direction of gliding at frequencies which were more than 1 order of magnitude higher than that of wild type cells (2.9 min-1 for DeltamglAB mutants and 2.7 min-1 for cglB mutants, compared to 0.17 min-1 for wild-type cells). The average gliding speed of DeltamglAB mutant cells was 40% of that of wild-type cells (on average 1.9 micrometers/min for DeltamglAB mutants, compared to 4.4 micrometers/min for wild-type cells). The mglA-dependent reversals and gliding speeds were dependent on the level of intracellular MglA protein: mglB mutant cells, which contain only 15 to 20% of the wild-type level of MglA protein, glided with an average reversal frequency of about 1.8 min-1 and an average speed of 2.6 micrometers/min. These values range between those exhibited by wild-type cells and by DeltamglAB mutant cells. Epistasis analysis of frz mutants, which are defective in aggregation and in single-cell reversals, showed that a frzD mutation, but not a frzE mutation, partially suppressed the mglA phenotype. In contrast to mgl mutants, cglB mutant cells were able to move with wild-type speeds only when in close proximity to each other. However, under those conditions, these mutant cells were found to glide less often with those speeds. By analyzing double mutants, the high reversing movements and gliding speeds of cglB cells were found to be strictly dependent on type IV pili, encoded by S-motility genes, whereas the high-reversal pattern of mglAB cells was only partially reduced by a pilR mutation. These results suggest that the MglA protein is required for both control of reversal frequency and gliding speed and that in the absence of A motility, type IV pilus-dependent cell movement includes reversals at high frequency. Furthermore, mglAB mutants behave as if they were severely defective in A motility but only partially defective in S motility.     

Lack of O‐polysaccharide enhances biofilm formation by Bradyrhizobium japonicum

Aims: To reveal the effects of the O‐polysaccharide antigen of Bradyrhizobium japonicum LPS on biofilm formation and motility. Methods and Results: Wild type and O‐antigen‐deficient mutant strains of B. japonicum were tested for biofilm formation on polyvinyl chloride (PVC) surfaces and motility on semi‐solid (0·3%) agar media. After 7 days of incubation, the amount of biofilms formed by the mutant was c. 3·5‐fold greater than that of the wild type. Unlike biofilm formation, the motility assay revealed that the mutant strain was less motile than the wild type. Conclusions: This study shows enhanced biofilm formation and decreased motility by the O‐antigen‐deficient mutant, suggesting that the lack of the O‐polysaccharide of the rhizobial LPS is associated with biofilm‐forming ability and movement. Significance and Impact of the Study: LPS plays an important role in both pathogenic and beneficial bacteria. It has also been reported that LPS deficiency negatively affects biofilm formation. However, our results demonstrate that the O‐antigen‐deficient mutant enhances biofilm formation, presumably through a significant increase in hydrophobicity. It is notable that the hydrophobicity of cell walls might be a key regulator in controlling biofilm development in B. japonicum.     

Mutations in Flavobacterium johnsoniae secDF result in defects in gliding motility and chitin utilization

secDF mutants of Flavobacterium johnsoniae were deficient in gliding motility and chitin utilization. Cells of the mutants had reduced levels of GldJ protein, which is required for both processes. SecDF is similar to Escherichia coli SecD and SecF, which are involved in protein secretion.     

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.     

Two continuum models for the spreading of myxobacteria swarms

We analyze the phenomenon of spreading of a Myxococcus xanthus bacterial colony on plates coated with nutrient. The bacteria spread by gliding on the surface. In the first few hours, cell growth is irrelevant to colony spread. In this case, bacteria spread through peninsular protrusions from the edge of the initial colony. We analyze the diffusion through the narrowing reticulum of cells on the surface mathematically and derive formulae for the spreading rates. On the time scale of tens of hours, effective diffusion of the bacteria, combined with cell division and growth, causes a constant linear increase in the colony's radius. Mathematical analysis and numerical solution of reaction-diffusion equations describing the bacterial and nutrient dynamics demonstrate that, in this regime, the spreading rate is proportional to the square root of both the effective diffusion coefficient and the nutrient concentration. The model predictions agree with the data on spreading rate dependence on the type of gliding motility.     

Type IV pili function upstream of the Dif chemotaxis pathway in Myxococcus xanthus EPS regulation

The developmental bacterium Myxococcus xanthus utilizes gliding motility to aggregate during the formation of multicellular fruiting bodies. The social (S) component of M. xanthus gliding motility requires at least two extracellular surface structures, type IV pili (Tfp) and the fibril polysaccharide or exopolysaccharide (EPS). Retraction of Tfp is proposed to power S motility and EPS from neighbouring cells is suggested to provide an anchor and trigger for Tfp retraction. The production of EPS in M. xanthus is regulated in part by the Dif chemosensory pathway; however, the input signal for the Dif pathway in EPS regulation remains to be uncovered. Using a genetic approach combined with quantitative and qualitative analysis, we demonstrate here that Tfp function upstream of the Dif proteins in regulating EPS production. The requirement of Tfp for the production of EPS was verified using various classes of Tfp mutants. Construction and examination of double and triple mutants indicated that mutations in dif are epistatic to those in pil. Furthermore, extracellular complementation between various Tfp and dif mutants suggests that Tfp, instead of being signals, may constitute the sensor or part of the sensor responsible for mediating signal input into the Dif pathway. We propose that S motility involves a regulatory loop in which EPS triggers Tfp retraction and Tfp provide proximity signals to the Dif pathway to modulate EPS production.