Search

Surface proteins of the gliding bacterium Cytophaga sp. strain U67 that make contact with glass substrata were radioiodinated, using a substratum-immobilized catalyst (Iodo-Gen). At least 15 polypeptides were iodinated, fewer than the number labeled by surface biotinylation of whole cells; these polypeptides define the set of possible candidates for the surface protein(s) that mediates gliding-associated substratum adhesion. The labeling of three adhesion-defective mutants exhibited two characteristic patterns of surface iodination which involved addition, loss, or alteration of several polypeptides of high molecular weight. An adhesion-competent revertant of mutant Adh3 and one of Adh2 exhibited the wild-type labeling pattern. Two other Adh2 revertants resembled their adhesion-defective parent. The labeling pattern of surface polypeptides of a nongliding but adhesive cell strain was similar to that of the wild type.

Gliding motility and flipping of 25 degrees C-adapted Cytophaga sp. strain U67 were inhibited when the bacteria were shifted to a less than or equal to 12 degrees C environment; motility was not blocked by a shift to 13 degrees C. Bacteria adapted to 4 degrees C were motile over the entire 4 to 25 degrees C temperature range tested. U67 adhesion to the substratum appeared to be unaffected by temperature shifts. Bacteria adapted to 4 degrees C had higher proportions of unsaturated and branched-chain fatty acids than did those grown at 25 degrees C. When 25 degrees C-adapted bacteria were subjected to a gradual temperature decline, the time of reappearance of gliding competence at 4 to 5 degrees C was correlated with these changes in fatty acid composition.

Two species of gliding bacteria were isolated from a marine biofilm. They were described and identified as members of the genus Cytophaga . One of them (RB1057) produced an extracellular inhibitor of colony expansion of the other (RB1058). The inhibitor was characterized as a glycoprotein with an apparent molecular mass of 60 kDa. It inhibited RB1058 adhesion to and gliding on substrata. Motility and adhesion of several other aquatic gliding bacteria were not measurably affected by this agent.

The cell surface hydrophobicities of a variety of aquatic and terrestrial gliding bacteria were measured by an assay of bacterial adherence to hydrocarbons (BATH), hydrophobic interaction chromatography, and the salt aggregation test. The bacteria demonstrated a broad range of hydrophobicities. Results among the three hydrophobicity assays performed on very hydrophilic strains were quite consistent. Bacterial adhesion to glass did not correlate with any particular measure of surface hydrophobicity. Several adhesion-defective mutants of Cytophaga sp. strain U67 were found to be more hydrophilic than the wild type, particularly by the BATH assay and hydrophobic interaction chromatography. The very limited adhesion of these mutants correlated well with hydrophilicity as determined by the BATH assay. The hydrophobicities of several adhesion-competent revertants ranged between those of the wild type and the mutants. As measured by the BATH assay, starvation increased hydrophobicity of both the wild type and an adhesion-defective mutant. During filament fragmentation of Flexibacter sp. strain FS-1, marked changes in hydrophobicity and adhesion were accompanied by changes in the arrays of surface-exposed proteins as detected by an immobilized radioiodination procedure.

The adhesion and motility of several aquatic and terrestrial gliding bacteria on slides differing in their critical surface energies have been examined. In general, adhesion was tenacious on low-critical surface energy (hydrophobic) surfaces and tenuous on hydrophilic surfaces. Gliding was inhibited on very hydrophobic substrata and skittish on very hydrophilic surfaces.

Sites of close contact between gliding Cytophaga sp. strain U67 cells and glass were examined by interference reflection microscopy. Site patterns changed during translocation and moved relative to the substratum, in contrast to previous interference reflection microscopy observations of fibroblast and amoeboid motility. Sinistral rotation around the long axis of the cell was coupled with gliding, except when curved cells traversed curvilinear pathways. Close contact was temporary, since cells flipped up off the substratum on one pole, pivoted, or were displaced laterally in collisions. Other members of the order Cytophagales and Myxococcus sp. demonstrated similar patterns of close association with substrata.

Motile bacteria move about in their environments by one of two means: swimming, powered by the rotation of one or more flagella (Silverman and Simon 1977), or gliding, the mechanism(s) of which is open to speculation. Gliding motility is a form of surface-associated, active, translocational movement by bacteria. No obvious changes of cell shape, other than occasional bending or flexing, are involved. Furthermore, gliding bacteria bear no resolvable, external organelles that are obviously involved in locomotion.

Two gliding motility mutants of Myxococcus xanthus are described. The semimotile mutant (SM) originated by high-frequency segregation from the motile FB t strain. Segregation was enhanced by acridine dye treatment. SM cells glide only when apposed to other cells in a swarm. The nonmotile strain (NM) originated by mutation from SM. NM cells neither glide individually nor cooperatively. FB t , SM, and NM are indistinguishable with respect to fine structure, vegetative growth rate, glycerol-induced microcyst formation, spheroplasting, bacteriophage sensitivity, and responses to light. The motility mutants are more resistant to penicillin and more sensitive to actinomycin D than is the gliding wild type. The NM mutant is also a morphogenetic mutant; it is unable to form fruiting bodies.

Burchard, Robert P. (University of Minnesota, Minneapolis), and Martin Dworkin . Light-induced lysis and carotenogenesis in Myxococcus xanthus . J. Bacteriol. 91: 535–545. 1966.— Myxococcus xanthus , grown vegetatively in the light, developed an orange carotenoid after the cells entered stationary phase of growth; pigment content increased with age. Cells grown in the dark did not develop carotenoid and could be photolysed by relatively low-intensity light only during stationary phase; rate of photolysis increased with age. Photolysis adhered to the reciprocity law, was temperature-independent and oxygen-dependent, and required the presence of nonspecific, monovalent cations; it was inhibited by one of several divalent cations. Logarithmic-phase cells were photosensitized by 100,000 × g pellet preparations of sonic-treated stationary-phase cells grown in the light and dark. A porphyrin with a Soret band at 408 mμ was isolated from photosensitive cells; logarithmic-phase cells contained about 1/16 the amount of porphyrin of stationary-phase cells. The purified material had spectral and chemical properties of protoporphyrin IX and photosensitized logarithmic-phase cells. Its spectrum was similar to the action spectrum for photolysis. We concluded that protoporphyrin IX is the natural endogenous photosensitizer. Carotenogenesis was stimulated by light in the blue-violet region of the visible spectrum and was inhibited by diphenylamine, resulting in photosensitivity of the cells. Photoprotection by carotenoid was lost in the cold. A mutant which synthesized carotenoid in the light and dark was photosensitive only after growth in diphenylamine. The ecological significance of these phenomena is discussed.

Burchard, Robert P. (University of Minnesota, Minneapolis), and M. Dworkin . A bacteriophage for Myxococcus xanthus : isolation, characterization and relation of infectivity to host morphogenesis. J. Bacteriol. 91: 1305–1313. 1966.—A bacteriophage (MX-1) infecting Myxococcus xanthus FB t has been isolated from cow dung. The bacteriophage particle is approximately 175 mμ long. A tail about 100 mμ in length is encased in a contractile sheath and terminates in a tail plate. The head is polyhedral with a width of about 75 mμ. The nucleic acid of the bacteriophage is deoxyribonucleic acid and has a guanine plus cytosine content of 55.5%. The bacteriophage requires 10 −3 m Ca ++ and 10 −2 m monovalent cation for optimal adsorption. Grown on vegetative cells of M. xanthus FB t at 30 C in 2% Casitone medium, the bacteriophage has a latent period of 120 min and a burst size of approximately 100. Host range studies indicate that three strains of M. xanthus including a morphogenetic mutant are sensitive to the bacteriophage, whereas M. fulvus, Cytophaga, Sporocytophaga myxococcoides , and a fourth strain of M. xanthus are not. Of the two cellular forms characteristic of the Myxococcus life cycle, the bacteriophage infect only the vegetative cells; they do not adsorb to microcysts. Ability to adsorb bacteriophage is lost between 65 and 75 min after initiation of the relatively synchronous conversion of vegetative cells to microcysts. The bacteriophage does not adsorb to spheroplasts. After the appearance of visible morphogenesis and before the loss of bacteriophage receptor sites, addition of bacteriophage results in the formation of microcysts which give rise to infective centers only upon germination. The possibility that the infected microcysts are harboring intact bacteriophages has been eliminated.

The gliding bacterium Flexibacter sp. strain FS-1 exhibits inducible resistance to chloramphenicol (Cmr) and tetracycline (Tcr). Either chloramphenicol or tetracycline alone induced a Cmr Tcr phenotype. The resistance is apparently not plasmid encoded.

In 1892 Thaxter described the myxobacteria as “possessing a power of slow locomotion ... a distinctly visible movement characterizes the active rods and consists in a sliding locomotion in conjunction with lateral bending.” Almost one century later there are still conflicting ideas about the mechanism of gliding. At a genomic level it is evident that Myxococcus gliding is a complex phenomenon (Chapter 8). Elucidation of the enigma of gliding motility has been much slower than that for swimming motility of flagellated bacteria, since myxobacteria (and other gliders) have no obvious organelles of motility protruding from their cell envelopes. Indeed, gliding has recently been described as a “backwater” in the motility field (Allen 1981).

Flexing movements have been described in a variety of gliding bacteria and cyanobacteria (blue-green algae)1–7. Flexing of trichomes (chains of cells) of the genera Oscillatoria and Beggiatoa was attributed8 to a “certain degree of elasticity”; it was concluded that there is no spontaneous flexibility. In contrast, observations of active flexing of free ends of otherwise immobilized Oscillatoria trichomes suggested9 that active contractions are involved. It has been proposed that flexing and gliding motility are powered by the same mechanism10. No organelles have been demonstrated irrefutably to be responsible for these movements, although several mechanistic hypotheses have been proposed11. I demonstrate here that flexing movements by the bacterium Flexibacter FS-1 require no more than one point of attachment by the cell to its substratum, which suggests that flexing may result from a unilateral, longitudinal contraction. As the same motility mechanism is thought to operate for both flexing and gliding, the latter may be powered by a contractile apparatus, at least in this species.

Derivatives of Myxococcus xanthus FB t resistant to chloramphenicol (25 μg/ml) arose spontaneously with a frequency of approximately 10 −7 . One of these organisms (FB t Cam 1 r ) was characterized. FB t Cam 1 r showed a unique type of phenotypic instability. After transfer from medium containing chloramphenicol to medium lacking the drug, resistance was lost after approximately one generation. The loss resulted in a sharp drop in the total number of chloramphenicol-resistant organisms and was not due to segregation of chloramphenicol-susceptible organisms during growth. Cell-free extracts of strain FB t Cam 1 r converted chloramphenicol to acetyl chloramphenicols in a fashion implicating activity of chloramphenicol acetyltransferase. This activity was lost simultaneously with the loss of chloramphenicol resistance after removal of the drug from cultures. Organisms with a similar phenotype to FB t Cam 1 r could be produced at high frequencies when strain FB t was exposed to low concentrations of chloramphenicol (2 to 5 μg/ml), to 3-acetylchloramphenicol (25 μg/ml), or to 1,3-diacetylchloramphenicol (25 μg/ml). Since strain FB t is capable of deacetylating acetyl chloramphenicols, these effects are probably all due to low concentrations of chloramphenicol. In the presence of chloramphenicol, FB t Cam 1 r produced fruiting bodies and myxospores on fruiting agar; however, glycerol-induced myxospore formation was inhibited. In the absence of the antibiotic, chloramphenicol resistance was maintained by glycerol-induced myxospores.

An action spectrum was measured for photoinduction of colored carotenoids in dark-grown, early stationary-phase cells of Myxococcus xanthus . Maximum activity was observed at 405 to 410 nm with subsidiary maxima at 512, 533, 548, 585, and 635 nm. These maxima correspond closely in position and magnitude with absorption maxima of protoporphyrin IX, which had previously been isolated from M. xanthus cells and had been shown to increase during the stationary phase of the culture. Late stationary-phase, dark-grown cells undergo photolysis which had been shown to have an action spectrum resembling the absorption spectrum of protoporphyrin IX. The similarity of the action spectra of photolysis and photoinduced carotenogenesis in M. xanthus and of other photoinduced biological phenomena is discussed.