- Volume 43, Issue 1, 1966

With a view to an improved taxonomy of the genus Agrobacterium, 45 strains. including representatives of all nomen-species, were used to investigate the following features: base composition and compositional distribution of pure deoxyribonucleic acid (DNA); type of flagellation; 3-ketoglycoside formation; phytopathogenicity for tomato and Datura. It is proposed to limit the genus Agrobacterium to two, or possibly three, species: (I) Agrobacterium radiobacter and its phytopathogenic variety A. radiobacter var. rumefaciens; (2) A. rhizogenes; and (3) possibly A. pseudotsugae. More work on the latter two species is required before they can definitely be accepted as separate species of this genus. The DNA of all the strains of the former two species has a Tm value in the narrow range of 93.8°-95. The corresponding with an average molar guanine + cytosine (GC) content of 59.5–62.8%. The variance σ of the compositional distribution of the DNA molecules ranges between 0 and 0.88% with an average of GC). The only available strain of A. pseudotsugae, with 67.7% GC, was mpletely out of this range and its chromosomal DNA was clearly different from that of the other two species. All strains of Agrobacterium proper were peritrichous, frequently with 5–6 flagella. All 8 strains of A. radiobacter and 24 of 28 strains of the variety ‘tumefaciens’ converted lactose into 3-ketolactose; all the other strains were negative in this respet. Several arguments are advanced to include the strain A. stellulatum and A. gypsophilae from this genus. The relationship between Agrobacterium and some other genera is shown graphically in a plot of mean similarity vermus DNA base composition.

Electron micrographs of negatively stained Semliki Forest virus were made before and after treatment of the virus with caseinase C from Streptomyces albus G. The virus appeared as roughly spherical (diameter 550–590 Å), covered with projections and sometimes bearing an appendix, which seemed to be a fold of the envelope. Treated with caseinase C in tris buffered saline, the virus lost its projections and its haemagglutinating activity but remained infectious. The site of haemagglutination, then, is probably located on the projections. Treated with caseinase C in phosphate buffer, the virus lost its infectivity as well as its haemagglutinating activity; the number of the remaining viral particles was not significantly modified. These structures had no projections; their envelope was degraded and sometimes completely destroyed. In this case, they had a smaller diameter (385–390 Å) than the projectionless particles obtained by treatment in tris buffered saline (410–460 Å). As the virus particles with degraded envelope still contained as much infectious RNA as the controls, it was thought that some degree of integrity of the envelope was necessary for the particle infectivity. Thus the site for infectivity appeared to be different from that for haemagglutination.

A chemically defined culture medium was used to study the effect of Parified lactoperoxidase and thiocyanate on the growth of several cultures of Streptococcus pyogenes and S. agalactiae. While not inhibited by either component alone, S. pyogenes growth was completely inhibited when both components were present in the medium. The growth inhibition was annulled completely by glutathione, thioglycollic (mercaptoacetic) acid of catalase. S. pyogenes glyceraldehyde phosphate dehydrogenase was inhibited by lactoperoxidase when hydrogen peroxide was present. The inhibition was annulled with cysteine and glutathione which suggested this dehydrogenase to be a possible site of inhibition. The inhibition was Postulated to be a peroxidatic conversion of essential enzymic sulphydryl groups to inactive disulphide groups, thus interfering with the energy metabolism up of S. pyogenes. With S. agalactiae cultures a delay in growth inhibition up to 6 hr resulted instead of complete growth inhibition. Catalase neutralized this effect. The extent of growth inhibition was greatest in those strains which were unable to adapt to an oxidative pathway for their energy supply. In becoming independent of the fermentative pathway, the cultures were no longer as sensitive to peroxidase, thioeysnate and hydrogen peroxide. The necessity for thiocyanate in the is inhibitory system is not yet clear. Thiouracil and thiourea were ineffective replacements for thiocyanate.

Acriflavine-induced photosensitivity has been used to measure resistance in trypanosomes to certain aromatic arsenical drugs. Trypanosomes were incubated in a logarithmic dilution series of acriflavine and slides were prepared. After a standard exposure to light the % mortality as judged by mobility was calculated and converted into probits. Mean sensitivity and its standard deviation were estimated from graphs of probits vs log concentration of acriflavine. It is possible by a graphical method of analysis to distinguish between two or three populations even when these occur in the same infection. Numerical estimates of replicate analyses and analyses made on the same strain over a period of 2 1/2 years show that the system of measurement is stable. A range of 0.12 log units has been found in all measurements made over the period on the normal strain of Trypanosoma brucei. This may be compared with the lowest degree of stable resistance which is an increase of log unit from the normal strain. Some factors which affect photosensitivity were examined, including temperature, action spectrum, concentration of drug, concentration of trypanosomes, time of incubation and the effect of oxygen. The method described is thought to be a compromise between that which is theoretically desirable and that which is simple to perform while still having an accuracy necessary for detailed studies of the development of drug resistance and of inheritance in trypanosomes.

A plaque assay method is described for the titration of the infective ribonucleic acid (RNA) of foot-and-mouth disease (FMD)> virus. The method depends on the dilution of the RNA in salt solutions of high molarity and the treatment of the BHK 21 cell monolayers used with M-NaCl. The method is reproducible and is at least as sensitive as other methods of titration. The extraction of RNA in the presence of EDTA gave titres corresponding to 0.1 % of the titre of the intact virus. RNA prepared from virus suspensions containing EDTA retained about 50% of initial infectivity for 7 days at 4° but for longer periods it was best preserved in 70 % (v/v) ethanol at 20°.

Addition of any of the chelating agents ethylenediaminetetra-acetic acid, diamino-cyclohexane-N, N-tetra-acetic acid or diethylenetriamine-penta-acetic acid at about 1.0 mM to a sugar+NH4NO3 +salts medium stimulated citric acid production by Aspergillus niger. The greatest stimulation (about tenfold) was obtained with EDTA (1.0 mM). In contrast, potassium ferrocyanide at 84 μM stimulated citric acid production about 20-fold. Ferrocyanide was effective only when present at the beginning of the mycelial growth period, whereas the chelating agents were effective at any time during the growth period. Stimulation of citric acid production by ferrocyanide could be repressed by transferring the mycelium to fresh medium without ferrocyanide, or by removal of the ferrocyanide precipitate in the medium. Analyses of the iron, copper and zinc contents of the mould mycelium are given. Ferrocyanide did not affect the copper and zinc contents of the mycelium: EDTA did not affect the iron, copper and zinc contents of the mycelium.

Shigella dysenteriae organisms of a very young culture released neurotoxin into the medium within 1 hr after it was infected with T-phages. Two types of phage-associated enzymes were considered to be involved in this process. The first type was phage-bound enzyme, which was bound firmly with phage, worked together with phage, and depolymerized neurotoxin into smaller molecules. The second type enzyme was phage-induced lysin, so-called ‘endolysin’, which was separable from phage and had lytic activity on dead bacteria. The latter enzyme, however, had no direct effect on the amount of neurotoxin released from acetone-powder preparations of the bacteria into phosphate buffer, although endolysin lysed 25 % of the bacteria at 30° in 30 min. Young living S. dysenteriae organisms did not release neurotoxin into the medium under normal conditions, whereas the acetone-powder preparations of S. dysenteriae, i.e. dead organisms, released neurotoxin into the medium freely under the same conditions.

Conidia of Glomerella cingulata did not germinate under crowded conditions. This was not due to limiting effects of concentration of oxygen or carbon dioxide. The ill effect of crowding on germination was alleviated by adding large amounts of twice-crystallized bovine serum albumin. Nearly 88 % conidia germinated in redistilled water when they were present in amounts less than 100/mm.2; but on addition of the washings and exudates of conidia into such suspensions, germination as well as length of germ tubes was markedly decreased. Germination of conidia was increased by prolonged leaching of the conidia by soaking in redistilled water. More than 30% of the thoroughly leached conidia germinated in redistilled water under crowded conditions (3000/mm.2), whereas less than 2% of the conidia sampled before leaching germinated under similar conditions. It is concluded that diffusible inhibitory compounds from the conidia appear to be responsible for the inhibition of germination. Several solubility classes of inhibitory compounds have been extracted from the cultures of G. cingulata. Among them, a basic fraction was relatively more toxic to G. cingulata than to Bacillus subtilis.

Desulfovibrio gigas and several strains of D. desulfuricans grew by fumarate dismutation in a sulphate-free medium. Two strains of D. desulfuricans grown in a chemically defined medium formed sueeinate, malate and acetate during fumarate dismutation. Sulphate reduction by these strains, though not by D. gigas, was almost completely inhibited in presence of fumarate as alternative electron acceptor. The anomalous behaviour of D. gigas was reflected to some extent by the hydrogen absorption coefficients for fumarate and sulphate reduction. Effects of fumarate media on the morphology of one strain are recorded.

The addition of either sulphide or thiosulphate to an aerobic suspension of a Thiobacillus was followed within 10 sec. by the synthesis of intracellular ATP. The ATP formation accompanying sulphide oxidation was strongly inhibited by 0.1mM-2,4-dinitrophenol, but that accompanying thiosulphate oxidation was unaffected. The presence of carbon dioxide had no effect on ATP formation or on the steady-state concentration attained. 2,4-Dinitro-phenol did not significantly affect oxygen uptake nor the kinetics of thiosulphate oxidation but it inhibited carbon dioxide fixation with either sulphide or thiosulphate as substrate. It is concluded that sulphurcompound oxidation can be coupled to two different types of phosphorylation, only one of which is sensitive to 2,4-dinitrophenol. In the presence of 2,4-dinitrophenol and with thiosulphate as substrate, carbon dioxide fixation was not limited by the availability of ATP but might be limited by the availability of reduced nicotinamide adenine dinucleotides.

Capsules of Bacillus anthracis only begin to be formed towards the end of exponential growth and appear first at the extremities of the cells. Once begun, capsule formation is not inhibited by tetracycline, so that capsular polypeptide is not synthesized like protein. Cultures were grown in broth + albumin in the presence of 0.015 M-HCO3—and 5 vol. CO2 + 95 vol. air until capsulation began, and were then incubated in air in tetracycline broth (to inhibit subsequent enzyme formation): capsules continued to increase in size, which suggested that HCO3—made capsular synthesis possible but was not required for the formation of the polypeptide itself. Cultures transferred from air to adequate concentrations of CO2 did not immediately become capsulated, whatever their stage of growth.

Mutants with altered nutritional requirements for capsulation were selected by phage α from wild-type strains grown either (1) on charcoal agar in air, so selecting for CO2-independence (D mutants), or (2) on bicarbonate agar incubated in CO2. so selecting for independence of an absorbent (F mutants). Both classes formed capsules in air (≃ 0.0001 M-HCO3-) and the capsular polymer apparently had the same chemical structure as that of their parent. No evidence was found that the mutants differed from their parent in being able to fix HCO3—more efficiently or in being able to utilize compounds normally derived from HCO3—that were present in the medium. They may therefore arise following mutation in regulatory genes controlling capsular synthesis.

Some D mutants grew very slowly in CO2 but yielded CO2-resistant mutants of which 66 were examined: 44 resembled the parental strain 2160s, and 22 were rough (C—). CO2-sensitive D mutants also gave rise to derivatives which formed rough colonies in air: some resembled strain 2160s; others were C—; but some had a new phenotype in being rough in air, fully capsulated in 0.006-0-015 M-HCO3—and inhibited by 0.03 M-HCO3 -.

Absorbents are required by wild-type strains to inactivate the long-chain fatty acids that occur in nutrient media. These acids are thought to interfere with the uptake or utilization of HCO3—, rather than with a later stage in capsular synthesis, because (1) they do not inhibit capsulation of D or F mutants, (2) they enable CO2-sensitive D mutants to grow in CO2, and (3) they probably interfere with toxin formation, which also requires CO2. As toxin is protein it must be synthesized differently from capsular polypeptide, and the only stage common to both pathways is therefore likely to be HCO3—uptake.

The consumption and utilization of a series of carbohydrates by a strain of Chlorella pyrenoidosa under different conditions of culture was examined. Only glucose, fructose and mannose were avidly consumed; galactose was consumed in small quantities. Light was highly stimulatory for the consumption of sugars. The carbohydrates assimilated were polymerized to starch; this induced gigantism in the organisms, a considerable increase in the dry weight, and inhibition of division.

Extracts of Streptococcus faecalis cultures made with 0.01 N-HCI gave a precipitin reaction with type antisera. With 0.05 N-HCI, however, some cultures gave inactive extracts. Part of the type-specific polysaccharide is converted by the strong acid to a form in which it does not precipitate with homologous sera, but which specifically inhibits the reaction between antibody and unaltered antigen.

The biochemical and serological characters of 36 unclassified cultures are described, and according to these characters the organisms belong to the genera Citrobacter, Aerobacter and Hafnia. While studying the antigenic relationships of the O antigens the known antigenic relationship between some serotypes of Citrobacter and Salmonella was confirmed; new antigenic relationships with S. locarno, S. kentucky, S. uccle and S. tranoroa are described. The antigenic relationships of the Hafnia cultures with Shigella flexneri 4a and with two cultures with a provisional O group of Citrobacter were confirmed.