- Volume 56, Issue 3, 1969

p-Chloromercuric benzoate (p-CMB) caused marked delay in excystment of Schizopyrenus russelli cysts when incorporated in the aqueous extract of Escherichia coli, an excystment agent. Cysts treated with p-CMB first and then with reduced glutathione (GSH), excysted readily in the presence of bacterial extract, after the removal of p-CMB and GSH.

Penicillium chrysogenum utilized the purines hypoxanthine, xanthine, uric acid and adenine as sole nitrogen sources but not the methylated purines caffeine and theobromine. Cell-free extracts of this organism contained the enzymes xanthine dehydrogenase, uricase, allantoinase, allantoicase and urease. Uric acid was degraded to allantoic acid by way of allantoin; allantoin was degraded to glyoxylic acid by way of allantoic acid. Xanthine dehydrogenase, uricase, allantoinase and urease were constitutive whereas allantoicase was inducible by xanthine or allantoin.

Electrophoretic measurements indicated that with increasing concentrations of illite or montmorillonite the surfaces of bacteria became progressively enveloped by a layer of adsorbed clay. The amount of clay adsorbed per unit area of cell surface depended mainly on the surface ionogenic properties of the isolates of the Rhizobium species used. For any particular isolate, almost identical amounts of illite and montmorillonite were adsorbed per bacterium. Microscopic studies confirmed the presence of a clay envelope around the bacteria and provided some information on the mode of adsorption of the clays.

Azotobacter chroococcum (ncib 8003) organisms, grown in continuous culture without fixed nitrogen, had chemical compositions at various dilution rates characteristic of nitrogen-limited populations. Fast-growing variants were selected for at high dilution rates; the efficiency of nitrogen fixation decreased with decreasing growth rate. In suitable media, carbon- and phosphate-limited populations were obtained and showed different compositions; they were very sensitive to inhibition by oxygen. Carbon-limited populations utilizing NH4 under argon were not oxygen sensitive; they formed nitrogenase when they were N-limited. The chemical compositions of the various populations corresponded to theory for the nutritional state considered. Nitrogen fixation entrained a maintenance coefficient of 1·06 g. substrate/g. organism/hr compared with about 0·40 for ammonia assimilation. Assuming most of this maintenance was directed to respiratory protection of nitrogenase, an extrapolated maximum requirement of 4 moles ATP/mole N2 fixed was observed. Attempts to repeat reports of (1) dependence of cytochrome pattern on nitrogen fixation and (2) increased efficiency of fixation with ultraviolet-irradiated N2 were not successful with the strain of A. chroococcum used.

Histochemical evidence showed that infection of tuber tissues and tissue-culture cells of Solanum tuberosum by Phytophthora erythroseptica, Phytophthora infestans and Fusarium caeruleum caused swelling and disruption of host cytoplasmic particles containing acid phosphatase, esterases and proteases. Heavy diffuse cytoplasmic staining for acid phosphatase was a consistent feature of infection by all three fungi, but staining reactions for esterases and proteases showed much less diffuse staining and a lesser degree of particle swelling. Biochemical assays showed that acid phosphatase was liberated from the particulate fraction to the supernatant fluid fraction of infected callus cells; acid ribonuclease behaved similarly. The biochemical evidence suggested that esterase activity of infected cells remained associated with a sedimentable fraction from cell homogenates. Assay methods for proteolytic enzymes did not confirm the histochemical evidence of a particulate localization of these enzymes in healthy or infected callus tissues. An excess recovery of ribonuclease from tissues infected with P. infestans and F. caeruleum was found; the significance of this is discussed. Attempts to separate a particulate fraction rich in hydrolases from homogenates of healthy or infected potato tubers were unsuccessful.

Four hypocholesteremic compounds, SKF 3301-A, SKF 525-A, SKF 16467-A and AY 9944, inhibited the growth of Sordaria fimicola; SKF 2314, SKF 7732-A3 and SKF 7997-A3 did not. All seven compounds inhibited the growth of Phytophthora cactorum. The inhibition of growth of S. fimicola by SKF 3301-A and AY 9944 was annulled by certain unsaturated fatty acids but not by sterols; with P. cactorum the inhibition was annulled by oleic acid + cholesterol.

With Sordaria fimicola sub-inhibitory concentrations of SKF 3301-A caused growth which showed two phases, distinguished by an abrupt change in growth rate. At the position of the hyphal front when growth rate changed a ring of perithecia was formed. During the second period, when growth was slower, perithecia were formed in rings. The number of perithecia formed could be at least twice that in the drug-free controls. With AY 9944 no perithecia were produced at concentrations sub-inhibitory to vegetative growth. On media with SKF 3301-A or AY 9944 and oleic acid, few perithecia were formed despite good vegetative growth; with SKF 3301-A, but not AY 9944, the addition of cholesterol as well increased perithecial production.

Phenol-purified RNA was prepared from Rhodopseudomonas spheroides and characterized by sucrose-gradient centrifugation. The patterns obtained indicate that R. spheroides possesses a ribosomal RNA complement similar to that of Escherichia coli. This is contrary to previous claims that the 23S component is missing. This species of ribosomal RNA was demonstrated to be more labile than the corresponding component in E. coli and variations in yields produced by changes in extraction procedures are described.

The sera of 11 ruminants and one horse contained relatively high titres of agglutinating antibodies against strains of anaerobic bacteria isolated from the bovine rumen. They were not detected in the sera of non-ruminants, i.e. pig, rabbit, guinea pig, rat and man. A close relationship existed between motility of rumen organisms and their ability to evoke detectable antibodies, although antibodies to both flagellar and somatic antigens were produced. The antibodies to the rumen organisms were highly specific and of the IgM type. They occurred in bovine colostrum at the same level as in the blood serum, and were transferred to the calf via the colostrum. After 1 month they had disappeared from the calf serum but between 5 and 8 months later antibodies of a similar activity were detected. In addition, antibodies to the non-motile Bifidobacterium bifidus were found in the sera of the young calf.

Proteins which inhibited the growth of two strains of Staphylococcus aureus and one strain of Streptococcus agalactiae were isolated from the teat canal keratin of the cow. Electrophoresis of the proteins on polyacryl-amide gels suggested that they were basic. They were separated into six bands at pH 3·0 but gave only two bands at pH 4·5 and two lines of precipitin after gel diffusion and immunoelectrophoresis at pH values between 4·5 and 8·5. In the presence of the anionic polymers DNA or heparin, complexes were formed which were not inhibitory of bacterial growth. The isolated proteins and the whole teat canal keratin were completely free from lysozyme. Their role is discussed in relation to the natural defence mechanisms of the teat canal.

Changes in the morphology of staphylococci incubated with antimicrobial cationic proteins isolated from the teat canal of the cow were studied with the electron microscope. Morphological changes in similar organisms deposited in the teat canals of a healthy cow for 24hr and subsequently recovered were also examined. In both instances changes were observed in the cell wall and plasma membrane. Accumulation of a dense layer of finely fibrillar material on the surface of the organism was also observed.

Calf thymus histone, lysozyme, protamine sulphate and poly-l-lysine also produced morphological changes in staphylococci in vitro. The changes induced in staphylococcal morphology following incubation with calf thymus histone closely resembled those produced by the materials of teat canal origin. Incubation with lysozyme induced lysis of limited regions of the cell wall; incubation with protamine sulphate and poly-l-lysine produced an agranular cytoplasm and the loss of nuclear areas.

Cell-free extracts of trophozoites of Hartmannella (Culbertson strain a-1), grown axenically, catalysed transamination between 2-oxoglutarate and a number of amino acids. Aspartic acid, alanine and serine showed the maximum activity. The l-alanine-2-oxoglutarate aminotransferase (E.C. 2.6.1.2) was purified about 15-fold by adsorption with calcium phosphate gel and elution with 0·2 m-phosphate buffer. Stability during storage, pH optima in different buffers K m values for alanine and 2-oxoglutarate, and the role of pyridoxal-5-phosphate are reported. p-Chloromercuribenzoate, cyanide and metal-chelating agents markedly inhibited the enzyme. Among dicarboxylic acids, maleic acid had the maximum inhibitory effect. Amoe-bicidal drugs and antibiotics had no marked inhibitory effect on the enzyme.

Capsular transformation of Haemophilus influenzae strain rd was examined at the cellular level by a semi-quantitative method. Type b capsular antigen synthesized by transformants was detected by its reaction with type b antibody conjugated to fluorescein isothiocyanate. Deoxyribonucleate (DNA) preparations from type b capsular transformants of strain rd elicited somewhat higher frequencies of transformation than preparations from type b clinical isolates. Str-r markers on these DNAs conferred resistance to a concentration of 500μg. dihydrostreptomycin sulphate/ml. For a given DNA, the frequency of transformation to streptomycin resistance was at least 100 times higher than that to capsular synthesis. The time course of expression of the two phenotypes by DNA-treated bacteria was examined in broth in which the generation time of the total population was about 50 min. Resistant transformants began to appear 25 min. after initiation of treatment and capsulated bacteria were observed after 40 min. Both properties continued to be expressed until about 120 min. Thereafter, the behaviour of recipients of the two kinds of genetic markers differed. The rate of increase of the str-r transformants became equal to that of the total population, and in a subculture diluted 1/20 they increased 20-fold during subsequent incubation for 150 min. The number of capsulated transformants, on the other hand, increased less than two-fold over the period 120–330 min.

Although bacteria are visible in Gram-stained smears of rumen entodinomorphid protozoa, no successful attempts to culture these bacteria have been reported. Gutierrez & Davis (1959), using oligotrich protozoa from the calf rumen, cultured 3 to 10 Streptococcus bovis per protozoon, after disruption by crushing between glass slides. The purpose of the present study was to investigate the viable bacterial flora of Entodinium caudatum which had originated from the sheep rumen, and had been maintained under constant conditions in vitro for over 6 years (Coleman, 1958, 1960). Part of the work has been reported previously (White, 1966).