Extracellular Lytic Enzymes of Myxococcus virescens

The aim of the investigation was to obtain large amounts of the bacteriolytic enzymes of Myxococcus virescens and to separate these enzymes from the non-bactcriolytic protemases produced by this organism. The bacteria were grown in Casitone broth. When the bacteriolytic activity had reached its maximal value, the cells were removed from the culture medium by centrifugation. Polyethylene glycol 4000 (20 g/1) and potassium phosphate (about 210 g/1) were added to the cell-free solution. The additions resulted in the formation of two liquid phases. The bottom layer was removed, and polyethylene glycol was added to it at a final concentration of 10 g/1. Again two liquid phases formed. The two top phases thus obtained were pooled and 1.6 volumes of cold acetone were added to the mixture. The precipitate formed was dissolved in water and desalted on a Sephadex G-25 column. The desalted protein solution was applied to a carboxymethyl-cellulose column equilibrated with 0.025 M sodium phosphate buffer of pH 6.0. Most of the proteins and the proteinases but none of the bacteriolytic enzymes passed the column unadsorbed. The column was washed with 0.05 M glycine-NaOH buffer of pH 8.8, whereupon the adsorbed bacteriolytic enzymes together with small amounts of proteinases and other proteins were eluted with 0.2 M ammonium carbonate. The material not adsorbed on the CM-cellulose column contained 22 % of the proteolytic activity of the initial cell-free solution and had a 26-fold higher specific activity. The enzyme solution eluted with carbonate contained 24 and 0.3 %, respectively, of the initial bacteriolytic and proteolytic activities. The specific activity of the bacteriolytic enzyme system was about 5000-fold higher than that of the original solution.     

Extracellular Lytic Enzymes of Myxococcus virescens

Two bacteriolytic hexosaminidases isolated from Myxococcus virescens were characterized. When acting on purified cell walls of Micrococcus lysodeikticus they liberated reducing groups and N-acetylhexosamines. By chromatography on Sephadex G-50 and G-25 columns disaccharides were isolated from degraded cell walls. After reduction of the disaccharides with sodium borohydride the acid hydrolysis products were identified by thin layer chromatography. Only pale spots of glucosamine appeared after this treatment but the spots of muramic acid remained unchanged. The enzymes were found to be devoid of exo-β-N-acetylglucosaminidase activity. These results are compatible with the action of endo-β-N-acetylglucosaminidases.     

Extracellular Lytic Enzymes of Myxococcus virescens II. Purification of Three Bacteriolytic Enzymes and Determination of their Molecular Weights and Isoelectric Points

The bacteriolytic enzymes produced by Myxococcus virescens and previously concentrated and separated from most of the non-bacteriolytic proteins have been further separated and purified. The bacteriolytic enzyme solution was concentrated by lyo-philization. When applied to a Sephadex G-100 column, three peaks of bacteriolytic activity were eluted. Polyacrylamide gel electrophoresis showed that all the three enzyme fractions were contaminated with at least four non-bacteriolytic proteins. In the first enzyme fraction the bacteriolytic enzymes could be freed from the contaminating proteolytic activity by adsorption on a hydroxylapatite column. The bacteriolytic enzymes could then be adsorbed on a CM-cellulose column. The remaining contaminating proteins passed the column un-adsorbed while the bacteriolytic enzymes could be eluted with a gradient of 0.02–0.10 M ammonium hydrogen carbonate solution. The second enzyme fraction was adsorbed on a CM-cellulose column and then eluted with 0.03–0.15 M NH4 HCO₃. After rechromatography on a new column under the same conditions, all of the contaminating proteins had disappeared. For purification of the third enzyme fraction chro-matography on one single CM-cellulose column was sufficient. The elution of the adsorbed enzymes was performed with a gradient of 0.15–0.30 M NH₄HCO₃. The recovery of activity for each of the ion-exchange chromatography separations was at least 90%. The purity of the enzymes was tested by polyacrylamid gel electrophoresis. Each of the purified enzymes gave only one coloured band which coincided with the enzyme activity assayed in sliced gels. The molecular weights of the enzymes were determined by electrophoresis on acryl-amide gels containing sodiumdodecylsulphate. The molecular weights determined in this way (about 40,000, 30,000 and 20,000, respectively) were about 10,000 daltons higher than those obtained by gel chromatography on Sephadex G-100. This discrepancy seems to depend on interactions between the enzymes and the dextran molecules probably caused by the strongly basic nature of the enzymes or by formation of enzyme-substrate complexes.     

Variants of Myxococcus virescens Exhibiting Dispersed Growth. Growth and Production of Extracellular Enzymes and Slime

The growth of two strains of Myxococcus virescens exhibiting dispersed growth was followed in casamino acids (N III-C) media and casitone media. The changes in optical density, pH, pigmentation as well as the secretion of bacteriolytic and proteolytic enzymes, DNA and polysaccharides during growth were recorded. In both media the bacteria grew exponentially with a generation time of 4 (casitone) and 20 hours (N III-C) respectively. The maximal cell mass was about 4 times higher in casitone than in casamino acids media. The amounts of bacteriolytic enzymes produced by the two strains in N III-C medium were different but in casitone medium they were about equal and considerably higher. The maximal values of proteolytic enzymes were about the same in both media and always occurred later than the bacteriolytic maxima. Both activity peaks appeared before the phase of decline. The polysaccharide production reached a maximum during the stationary growth phase in both media. A higher value was reached during growth in casitone medium than in N III-C medium. During the phase of decline a second increase of polysaccharide in the medium appeared. No DNA could be detected in the cell-free solutions until the beginning of the phase of decline.     

Production of Lytic Exoenzymes in Casamino Acids Media by Myxococcus virescens

The production of proteolytic enzymes and enzymes active on autoclaved aerobacter cells by Myxococcus virescens, strain B2, was studied In Casamino acids media. By modification of the N III–B media of Norén to a higher concentration of casamino acids and to a lower content of sodium chloride, the maximal production of lytic exoenzymes could be increased at least four times. Too high a concentration of casamino acids (more than 17 g/1) and sodium chloride (30 mM), as well as too low a concentration of sodium chloride, inhibited the enzyme production.     

Slime of Myxococcus virescens

The slime excreted by two strains of Myxococcus virescens during growth in liquid casitone medium was studied. Strain S1H, unable to grow in dispersion, excreted slime during growth later than strain D11, which grows in dispersion. Slime was precipitated from the cell-free culture solution with ethanol and the crude precipitate fractionately dissolved using first pH 5,4 and then pH 9.0 for the remainder of the precipitate. Comparatively more material from strain S1H than from strain D11 belonged to the pH 9.0 fraction. The fractions thus obtained were dialyzed and then lyophilized. The composition of the slime preparations varied with the density of the harvested cultures. The slime fraction dissolved at low pH contained 12–18 % (w/w) Folin reactive material, 2–4% lipid and 5–30% anthrone positive material (glucose equivalents). The fraction soluble at pH 9.0 was richer in Folin positive material. About 25% of the proteolytic activity in the culture solution was recovered in the slime preparations. No DNA was detected in the slime, unless the cultures were harvested daring the phase of decline. The high polymers of the slime were separated from material of low molecular weight and coprecipitated media constituents by gel filtration on Sepharose 2B. The relative amount of the high polymers increased during growth, although they seemed to be degraded in the culture during the phase of decline. The polymer had a molecular weight of about 20 million. In most preparations: it was Folin positive.     

Growth of Variants of Myxococcus virescens

Some observations on variant strains of Myxococcus virescens B2 with special emphasis on characteristics associated with the ability to grow in dispersion are reported. The isolated strains were divided into two major classes according to their mode of growth in shaken and static liquid cultures based on casitone and casamino acids media. Strains growing in dispersion were designated D⁺-strains and those growing in aggregates or as films, D^?-strains. Colony morphology, cell morphology, growth in liquid and on solid medium and morphogenesis were compared. The ability to grow in dispersion shown by D⁺-strains seemed to be associated with a smooth colony on casitone agar, inability to form typical fruiting bodies and a low linear growth rate of colonies on solid medium as compared with the D^?-strains. In contrast D^?-strains produced rough colonies on casitone agar, were able to fruit and evidently formed an adhesive slime in the form of fibrils extending from the cell surface. It is suggested that the observed differences depend on different envelopes of the cells in the two classes.     

Morphogenesis in Myxococcus xanthus and Myxococcus virescens (Myxobacterales)

1. Myxococcus xanthus B and M. virescens V2 were compared with a view to establishing the control of their morphogenetic cycles. Both organisms are typical myxococci and on solid media with low concentrations of nutrient they form fruiting bodies, within which vegetative cells convert to myxospores. Ultrathin sections of vegetative M. virescens resembled those of M. xanthus and contained prominent heavily stained bodies, presumed to be polyphosphate granules. Shadowed preparations showed fimbriae associated with M. xanthus but not with M. virescens. 2. M. xanthus B converted to myxospores in liquid medium in response to certain alcohols. M. virescens V2 produced phase-refractile spheres, which were not viable and had an unusual ultrastructure. 3. The distributions of fruiting bodies on solid media containing 0.02% Casitone were recorded for the two species and were compared with a Poisson distribution. Cells responded to differences in cell density in a manner suggestive of a response to a chemotactic attractant. Cells growing vegetatively and also cells forming fruiting bodies produced 3',5'-cyclic adenosine monophosphate (cAMP) as measured by the incorporation of exogeneous [3H] adenosine into cAMP. 4. The significance of these findings for theories of fruiting body formation are discussed.     

Growth and Enzyme Activity of Myxococcus virescens in Liquid Medium

The object of this work was to develop a method for determination of the growth of Myxocuccus virescens in liquid medium. The bacteria were grown in N III-B medium in 100 ml Kjeldahl flasks, which were fixed on a disc forming an angle of 50 degrees with the horizontal plane. The disc was rotated two full revolutions per minute. The total nitrogen content of the washed swarms, developing on the glass walls of the flasks, was used as an expression for the myxobacterial growth. After a lag the bacteria grown in total darkness had a growth phase, approximately exponential, of about 270 hours, which was followed by a steep phase of decline. When the bacteria were illuminated daily for a short period, a lag of 50–200 hours appeared in the middle of the exponential growth phase, after which a new exponential growth phase began. This second growth seemed to depend on variants insensitive to light induced lysis. The increase of enzymes, active on casein and autoclaved aerobacter cells, closely followed the first part of the growth curve. However both activities began to decrease before the growth maximum. No sign of proteolytic activity or lytic activity on autoclaved aerobacter cells could be detected after about 700 hours' incubation. In illuminated flasks it is shown that the production of yellow pigments in culture solution is sharply increased at the end of the exponential growth phase. The lytic enzymes of M. virescens seem to be extracellular, secreted during the exponential phase of growth. No activity was exhibited by washed cell swarms, even if they were sonically disintegrated.     

Purification and properties of an extracellular protease from Myxococcus virescens.

An extracellular protease from Myxococcus virescens was purified by phosphate precipitation, gel exclusion, and ion-exchange chromatography. The enzyme appeared homogeneous upon disc electrophoresis. The molecular weight of the protease was estimated to be 26,000. The enzyme was rapidly inactivated by ethylenediaminetetraacetate, but the activity could be partially restored by divalent cations. Diisopropylphosphorofluoridate inhibited enzyme activity completely. Michaelis-Menten kinetics were obeyed with casein and hemoglobin as substrates. First-order kinetics were obtained with elastin as the substrate, provided trypsin was in excess. Petidolytic activity indicated that the peptide bonds hydrolyzed by the enzyme were mainly those involving amino acids with nonpolar side chains.     

Occurrence of Cell Lytic Enzymes in Blue-Green Bacteria

Detergent extracts of three blue-green bacteria (Agmenellum quadruplicatum strain BG1, Anacystis nidulans strain TX20, and Nostoc sp. strain MAC) contained enzymes capable of lysing suspensions of Micrococcus lysodeikticus. The enzyme preparation from A. quadruplicatum released soluble reducing fragments from purified peptidoglycan. The lytic activity exhibited a pH optimum between 6 and 7, was relatively heat stable, and was susceptible to attack by proteolytic enzymes. These results extend the range of bacterial types exhibiting cell lytic activity as well as confirm the existence of the lytic system commonly observed in "water blooms".     

Transduction of Myxococcus virescens by coliphage P1CM: generation of plasmids containing both phage and Myxococcus genes.

Chloramphenicol-resistant Myxococcus virescens were obtained by infecting myxococci with Escherichia coli specialized transducing phage P1CM. The drug-resistant myxococci were phenotypically unstable. They contained more than one type of plasmid; these plasmids were not found in the parent strain. Chloramphenicol-resistant E. coli were obtained by transformation with either a fraction of myxococcal DNA containing the plasmids or with P1CM prophage DNA. These transformants contained plasmids. Escherichia coli transformed by DNA from the myxococci contained both P1CM and myxococcal genes. Individual transformant clones differed in the genetic make-up of their plasmids. Among the myxococcal genes expressed in these plasmid-harbouring E. coli strains were a capacity for self-transmissibility and a pattern of phage sensitivity characteristic of R factor incompatibility group W. Escherichia coli transformed with P1CM prophage contained incomplete P1CM genomes; none of the chloramphenicol-resistant transformants produced P1CM phage particles. The significance of these findings for an understanding of mechanisms for the generation of R factors is discussed.     

Purification and Properties of Lytic Enzymes from Streptomyces globisporus 1829

Two lytic enzymes capable of lysing Streptococcus mutans have been purified to give a single band on disc-gel electrophoresis, respectively. The M–1 and M–2 enzymes were both proved to be N-acetylmuramidases. However, these enzymes were entirely different on their enzymatic properties. The molecular weights were about 20,000 and 11,000 for M–1 and M–2 enzymes, respectively, The maximal lytic activity of M–1 enzyme was obtained at ionic strength 0.05, while lytic activity of M–2 enzyme did not change within the ionic strength range of 0 to 0.05. The M–1 enzyme constituted the majority of the total lytic activity against the cell walls of Streptococcus mutans BHT of cultured filtrate. The M–2 enzyme showed less specific lytic activity on the cell walls of Streptococcus mutans BHT than M–1 enzyme.     

Extracellular fibrils and contact-mediated cell interactions in Myxococcus xanthus.

Contact-mediated cell-cell interactions play an important role in the social life-style of Myxococcus xanthus. Previous investigations have demonstrated that fimbriae (also referred to as pili) and extracellular fibrils are involved in these social interactions (L. J. Shimkets, Microbiol. Rev. 54:473-501, 1990). We have used the relatively new technique of low-voltage scanning electron microscopy (an ultra-high-resolution scanning technique that allows for the nanometer resolution of biological materials) to observe the topological details of cell-cell interactions in M. xanthus. Our observations indicated that the fibrils (which measure approximately 30 nm in diameter) are produced most extensively by cells that are in close contact with each other and are aberrantly produced by the cohesion-deficient dsp mutants. Immunogold analysis identified an antigen which is located exclusively on the extracellular fibrils. Western blots (immunoblots) of this antigen (designated FA-1 for fibrillar antigen 1) indicated that it is composed of several immunoreactive bands (molecular size range, 90 to 14 kDa), all of which are sensitive to protease digestion. A technique for fibril isolation was developed by using FA-1 as a fibril-specific marker. Low-voltage scanning electron microscope observations of swarming cells demonstrated that the expression of fibrils is differentially regulated between adventurous (individual) and socially (group) motile cells. The differential expression of fibrils suggests the existence of a mechanism for the regulation of fibril biosynthesis that functions within the overall system governing social interactions in M. xanthus.     

Apparatus for Preparation of Bacterial Extracellular Enzymes

A method is described for the growth of bacteria within dialysis tubing to yield extracellular enzymes, or, possibly, other nondialyzable extracellular products, in concentrated form.