- Volume 80, Issue 2, 1974

Previous studies have shown that the insoluble glucan from the walls of baker’s yeast (Saccharomyces cerevisiae) is heterogeneous. It consists of about 85 % of an insoluble branched β-(1 → 3)-glucan and about 15 % of a soluble branched β-(1 → 6)-glucan. Glucan preparations from the walls of the yeasts Kloeckera apiculata, Schizosaccharomyces pombe, Saccharomyces fragilis and Saccharomyces fermentati have now been shown to be similarly heterogeneous. Quantitative periodate oxidation and enzymic degradation studies suggest that in these four yeasts, the amount of β-(1 → 6)-glucan is rather greater than in baker’s yeast.

SUMMARY: Yeast glutamine synthetase was purified and shown to be an octameric globular protein (s = 15·4, f/f 0 = 1·29, mol. wt = 390000). It consists of two weakly-bound half molecules (s = 8·7, f/f 0, = 1·35, mol. wt = 180500) and relatively harsh treatment is required to dissociate these octamers into component monomers (s = 3·8). Deactivation of the enzyme in vivo may include changes in the conformation of the native enzyme with its separation into two ‘ tight ’ tetramers, followed by their dissociation into component monomers and dimers. In the presence of Mg2+ and glutamate, monomers re-aggregate to oligomers which, although having transferase activity, are devoid of synthetase activity. The relevance of these observations in relation to control of glutamine synthetase activity in yeast is discussed.

Wall preparations from Corynebacterium poinsettiae and Corynebacterium betae contained no teichoic acids but phosphate-free polysaccharides were present and were extractable with trichloroacetic acid at 35 °C. The substance from C. poinsettiae contained rhamnose, glucuronic acid, galactose, mannose and pyruvic acid (molecular proportions 1:2:1:0·33: 0·35) and that from C. betae contained glucuronic acid, rhamnose, fucose and mannose (molecular proportions 1:2:0·5:0·3). In the former polymer, pyruvic acid appeared to be linked to galactose which was in turn linked to rhamnose. The residual peptidoglycans of these species contained much phosphorus. Some of this was present as muramic acid phosphate.

Summary: Axenic cyst cultures of Acanthamoeba castellanii (Neff strain) maintained in salt medium undergo excystment when transferred into enriched growth medium containing yeast extract and proteose peptone. Both the synchrony and extent of excystment induced in this manner are critically dependent upon the length of time cysts are aged in salt medium prior to the initiation of excystment. Cysts sustained in salt medium for 50 h or less characteristically show only about 10 % excystment even after 70 h in the enriched growth medium. With progressively longer periods of ageing the synchrony and degree of excystment improve such that for cysts of age ≥ 800 h, more than 85 % of the organisms germinate within a period of 12 h. Thus more complete and relatively synchronous excystment can be achieved by this simple experimental manipulation.

SUMMARY: Cultural conditions for forming and stabilizing protoplasts of Streptomyces griseus and S. venezuelae were studied by reference to the number of protoplasts formed, leakage from protoplasts and reversion rate. Effective formation and stabilization of the protoplasts was accomplished by using a hypertonic medium containing 10 mm-MgCl2 and 25 mm-CaCl2. Electron microscopy showed that the protoplasts of S. griseus, when prepared in the above medium, formed vesicles on the cytoplasmic membrane or out of the protoplasts, but when prepared in a medium with 3 mm-MgCl2 and 3 mm-CaCl2 they provided few such vesicles.

The reversion of protoplasts to the normal filamentous state was examined by the growth on various synthetic agar media. A high reversion rate was obtained by incubating the protoplasts on a hypertonic agar medium containing 20 or 50 mm-MgCl2, 50 or 20 mm-CaCl2, 0·44 or 0·22 mm-phosphate and 0·01 % Casamino acids. Combinations of appropriate concentrations of MgCl2 and CaCl2 were significantly effective in the reversion as well as in the stability of protoplasts. The concentration of phosphate should be adjusted to 0·44 or 0·22 mm. Casamino acids enhanced both growth and reversion rates.

The reversion of protoplasts of Streptomyces griseus was followed by using phase-contrast microscopy. The protoplasts incubated on medium R1 enlarged to about 40 µm and then generated filamentous hyphae from the periphery.

Electron microscopy of thin sections of Bacillus cereus 569 fixed under conditions that demonstrate Bacillus licheniformis 749c to have only one mesosome per bacterium has shown the former to possess consistently more than one. Treatment of outgrowing B. cereus 569 spores with actinomycin-D results in the appearance of extra membrane-like material lying between the plasma membrane and the new wall. Actinomycin-D causes almost immediate cessation of exponential growth in B. cereus 569 but extra membrane appears after a lag of approximately half a generation time. Autoradiography of thin sections of exponentially growing bacteria treated with actinomycin-D and labelled during inhibition with tritiated glycerol shows that the grain count is increased over areas of sections where extra membrane is present. Analysis of the lipids extracted shows that some of the labelled glycerol is incorporated during inhibition into material similar to or identical with the normal phospholipids. Virtually no incorporation of labelled amino acid occurred in trichloroacetic acid precipitates during actinomycin-D inhibition. These observations, which suggest that synthesis of new membrane occurs during actinomycin-D inhibition, are discussed in relation to a possible origin from mesosomes.

Pyocin sheaths treated with sodium dodecyl sulphate (SDS) assembled into long rod structures in saline phosphate buffer but not in tris-HCl buffer. For the effective formation of long rods, 0·05 m or more NaCl was required in the reaction mixture and the optimal concentration of SDS was 0·05 to 0·2 %. Morphological observation revealed that each long rod was formed by aggregation of several single sheaths.

The mechanism of long rod formation by SDS is discussed in relation to the interaction of soluble proteins with SDS.

SUMMARY

The phage reactions and pathogenicity of English isolates of Pseudomonas morsprunorum were compared with those of isolates from other countries. Nonindigenous cherry strains resembled English cherry strains pathologically, but not all were sensitive to A7, the type-determining phage for English cherry strains. Plum strains resistant to A7 adsorbed the phage as readily as cherry strains, indicating that phage receptor materials are not involved in host specificity. Cherry strain mutants resistant to specific and/or non-specific phages showed marked attenuation of virulence, although retaining the essential pathological characters of cherry strains. The resistance of all but one of the mutants was due to failure to adsorb the homologous phage, showing that the wall structure had changed and suggesting that wall components may function as general virulence determinants in P. morsprunorum.

The absorption of cells of a cherry strain of Pseudomonas morsprunorum through leaf scars into the leaf traces of cherry cultivar Napoleon was not affected when 10 × more cells of a plum strain were present, although this could inhibit the development of the disease. Ninety per cent of 14C-labelled cherry inoculum was deposited within 2 mm of the entry point, with small amounts extending 25 mm into the host in the presence or absence of the plum strain. Bacteria were observed in regular arrays inside the vessels of the leaf traces, and there was evidence that bacteria migrated from these to other tissues within 5 days of inoculation. No visible damage or changes in the phenolic content of the host were observed during this period.

The phenomenon of paradoxical inhibition (p.i.) manifests itself as well-defined growth of a sensitive bacterium in the centre of the zone of inhibition overlying an area of growth of the inhibitory bacterium. Bacteriocin typing medium (BTM) for Vibrio cholerae elicited p.i. for this group only. A medium for paradoxical inhibition (MPI) was devised which supported the development of p.i. among members of Enterobacteriaceae, Vibrio and Alcaligenes spp. at high frequency. The reacting pair of strains could belong either to the same or to different genera. Colonies of non-bacteriocinogenic strains derived from the central area of p.i. showed acquisition of bacteriocinogeny at high frequency. Acquisition was rapid in the beginning (up to 4 h) followed by a decline and virtual disappearance of bacteriocinogeny by 18 h on MPI, but was substantially retained on BTM. The overall frequency of acquisition among cell populations of different strains varied between 10−1 and 10−4, and the acquired characters were stable. Acquisition did not require either cell contact or participation of phages, but depended upon extracellular diffusable agents that were DNase sensitive and RNase resistant. A mutational basis for such acquisition could be ruled out, and the process of gene transfer was considered to be transformation. The extra-chromosomal nature of the determinants of bacteriocinogeny in the bacteria studied was suggested by effective elimination of these markers by sodium dodecyl sulphate and acridine orange.

Two representative lipoamide dehydrogenase mutants (lpd1 and lpd4) were used to locate the lpd gene in the linkage map of Escherichia coli by conjugation and detailed transductional analysis with phage P1. Time of entry mapping indicated that the lpd gene was between leu and proC and very close to leu. Average cotransduction frequencies between lpd and other markers were: aceF (97 %), aceE (93 %), aroP (84 %), nadC (71 %), azi (64 %), leu (8 to 41 %), ara (38 %), pan (21 %), tonA (17 %) and thr, nad and gal (< 1 %). These values, together with the contransduction frequencies for many other pairs of markers in this region and the results of three-factor crosses, established the gene order leu-azi-nadC-aroP-aceE-aceF-lpd-pan. The very close proximity of aceF and lpd suggested that they may be contiguous and that the lpd gene may be the distal gene in the pyruvate dehydrogenase operon. In this case, the expression of the lpd gene may be governed by a secondary transcriptional promoter in the aceF-lpd region in order to generate lipoamide dehydrogenase components for assembly of the α-ketoglutarate dehydrogenase complex. Other possible mechanisms are also discussed.

SUMMARY: The addition of amphotericin methyl ester (AME) to suspensions of Candida albicans is followed by a progressively increasing rate of release of K+ from within the cell. The time taken for a given rate of release to be reached depends upon the AME concentration (the product of AME concentration and time being approximately constant), the pH and concentration of buffer, the temperature, the suspension density, and the phase of growth of the organisms. The sensitivity of C. albicans to AME decreases during the growth of a batch culture, rapid decrease occurring after the organisms have reached the stationary phase of growth. The induction of K+ release does not occur at 0 °C and shows a high temperature coefficient over the range 15 to 30 °C.

Suspensions of mouse LS cells are 10 to 30 times less sensitive than Candida albicans towards AME, amphotericin B and nystatin; the two types of cell display little difference in sensitivity towards perimycin and candicidin, while mouse LS cells are more sensitive than C. albicans towards filipin.

Addition of sterols at the same time as AME increases the time taken for the release of K+ to reach a given rate; of the sterols tested, zymosterol was the most effective in antagonizing AME, while ergosterol was approximately twice as effective as cholesterol on a molar basis. When organisms were exposed to AME for a short time, removed and resuspended in the absence of drug, the rate of K+ release continued to increase; this increase was prevented by ergosterol: 2 × 10−5 m-ergosterol prevented further damage after organisms had been exposed to 10−6 m AME.

SUMMARY: The localization of total and newly synthesized [3H]prenol in Lactobacillus casei was investigated by autoradiography of electron micrographs of bacteria grown with [3H]mevalonic acid. The distribution of radioactive grains differed from a random distribution in being concentrated in the membrane; this confirmed earlier chemical studies. The distribution of total [3H]prenol along the cell, after continuous growth with [3H]mevalonic acid, did not indicate any specific localization. Newly synthesized prenol, located after a 20 min pulse with [3H]mevalonic acid, was also uniformly distributed except in organisms which had spent the previous 20 min forming septa. In these bacteria there was a concentration of prenol in the septal region.

SUMMARY: Chondroitinase was induced in Proteus vulgaris by chondroitin sulphate or by N-acetylgalactosamine. With batch cultures growing on chondroitin sulphate or N-acetylgalactosamine the differential rate of chondroitinase synthesis increased throughout the exponential growth phase. Induction of chondroitinase by chondroitin sulphate was prevented by glucose, glycerol, lactate, pyruvate or succinate. The rate of uptake of chondroitin sulphate by Proteus vulgaris suspensions was not altered by the presence of glucose. With steady state chemostat cultures, limited either by the supply of chondroitin sulphate or nicotinic acid, the specific activity of chondroitinase was maximal at diffusion coefficient (D) = 0·18h−1. Addition of glucose to nicotinic acid-limited chemostat cultures resulted in an exponential reduction in chondroitinase specific activity; this could be partially prevented by the simultaneous addition of 5 mm-adenosine cyclic-3′,5′-monophosphate.

SUMMARY: The growth rate of Vibrio fetus in laboratory media could be stimulated or inhibited by the addition of appropriate concentrations of widely different potential nutrients and salts. The effect appeared to depend on the osmotic activity of the supplements since, with electrolytes, growth was maximal at about the same osmolal concentration irrespective of the chemical nature of the electrolyte.With sucrose a higher osmolality was required. In comparative tests the growth rate of Escherichia coli was much less affected by variation in solute concentration.V. fetus appears unusually sensitive to change in the osmolality of the growth medium and this may account for the general difficulty of obtaining satisfactory growth of the organism.