- Volume 31, Issue 2, 1963

Foot-and-mouth disease (FMD) virus was partially inactivated by several methods: incubation with dilute formaldehyde or acetylethylene-imine, ultraviolet (U.V.) irradiation, heating, or by mixing with type specific antiserum, trypsin or extracts from cells susceptible to the virus. The serological properties of the treated virus preparations were studied by complement-fixation and agar diffusion tests and their content of infective ribonucleic acid (RNA) determined by phenol extraction. The ability of the treated preparations to attach to susceptible cultivated pig kidney cells was examined. The decrease in viral infectivity when FMD virus was treated with formaldehyde or acetylethyleneimine, U.V., or heat at 25° or 37° was proportional to the loss of infective RNA, with little impairment of its serological properties or its ability to attach to susceptible cells. In contrast, loss of viral infectivity on mixing with antiserum, trypsin or cell extracts was due to the failure of the virus to attach to susceptible cells; the viral RNA is still present in an infective form in these mixtures.

A method for the quantitative isolation of pure highly polymerized DNA from Serratia marcescens, based on Kirby's phenol extraction procedure (Kirby, 1956, 1957), has been developed. No statistically significant differences were detected in the base composition or sequence between the DNA of a wild and a mutant strain of the organism. 6-Methyl-aminopurine was found in the DNA of Serratia marcescens. The properties of this base and its N-nitroso derivative were compared with those of a synthetic sample.

Vitamin B12 is an essential nutrient for the flagellate Prymnesium parvum when growing in axenic culture and apparently can be replaced only by a few related ‘incomplete’ analogues, i.e. only those devoid of one or both benzimidazole-methyl groups; and, of the substituted analogues, some (e.g. the mono-acid, the dicarboxylic acid and the ethylamide of B12) can replace vitamin B12, but some others of this group, e.g. pseudovitamin B12, 2-methylmercaptoadenine cobamide, the ethylamide mono-acid, methylamide and anilide of the vitamin B12, act as competitive inhibitors of the growth of P. parvum.

Methionine or ethionine, although capable of serving as sole nitrogen source for Prymnesium parvum, cannot replace or spare the vitamin B12 nutrient requirement for the growth of this flagellate. In the presence of vitamin B12 methionine counteracted the inhibition of growth exerted by some vitamin B12 analogues which are substituted at the benzimidazole part of the molecule. No such effect was observed against the inhibition by certain other analogues. Analogue FIII (α-(5-hydroxybenzimidazolyl)-cobamide cyanide) replaced vitamin B12 in the presence of methionine, and to a lesser degree also in the presence of other methyl donors such as methylmethionine sulphonium iodide, dimethylpropiothetin, choline chloride and betaine. The conclusion drawn is that for P. parvum analogue FIII is capable of replacing vitamin B12 in all the metabolic pathways other than methyl-group synthesis. For the latter process, the benzimidazole methyl groups present in vitamin B12 and analogue FIIIm (α-(5-methoxy-benzimidazolyl)-cobamide cyanide) are indispensable.

Transducing phage PBS1 and a clear-plaque type mutant PBS2 derived from PBS1 show similar growth characteristics with a relatively small burst size (23 to 24), and are related serologically. They are morphologically indistinguishable and both have a dense head of about 100 mμ in diameter and a tail about 250 mμ in length. PBS 1 is more efficient in trans-duction and its plaque-forming activity is more sensitive to ultraviolet (u.v.) irradiation than that of PBS2. The host range of the phages is limited to strains of Bacillus subtilis. All Marburg strains tested were sensitive to the lytic action of the phages. Prophages of PBS1 and PBS 2 were not u.v.-inducible and the irradiation of lysogenic cultures with a relatively high dose of u.v. radiation resulted in considerable decrease of the free phage titre. However, transducing activities of lysates treated with a high dose of u.v. radiation were not affected significantly. Organisms lysogenic either for PBS 1 or to PBS 2 were not stable and reverted to the sensitive state.

Twenty-two different phage-types of Proteus rettgeri were investigated for lysogeny by growing strains singly, in mixtures and by ultraviolet induction. Five of the strains were lysogenic for other members of the group. The phages differed from one another in host-ranges. Eighteen different lytic phages were isolated from sewage with action on one or more of the strains. The host-range of all 23 phages was tested against a number of P. hauseri, P. morganii, Providence and other intestinal organisms. Twelve of the phages had productive cycles of infection on 24 strains of P. hauseri; 21 of the phages attacked 19 Providence strains. Two of the phages lysed the same 2 out of 40 strains of Escherichia coli. The efficiencies of plating varied from 10-4 to unity. No action was demonstrated on any of the P. morganii or other strains tested. The host-range of 23 P. hauseri phages previously isolated from sewage and from lysogenic P. hauseri was tested against P. rettgeri, P. morganii and Providence strains. Three Providence and 2 P. morganii strains proved susceptible to productive infection by a number of the P. hauseri phages; 15 P. rettgeri strains were lysed by one or more of 18 P. hauseri phages. Some of the P. hauseri phages also caused abortive infection associated with cell death in some P. morganii and P. rettgeri organisms. The results indicate close ties between the P. hauseri and P. rettgeri groups, and between P. rettgeri and the Providence strains.

The ‘intracellular’ teichoic acids from two strains of group D streptococci (strains 8191, 39) were purified and their serological and chemical properties examined. Both compounds reacted serologically with group D streptococcal antiserum. Chemical analysis showed them to be polymers of glycerol phosphate containing a high proportion of glucose and bearing alanine ester residues on the sugar; it is likely that the glucose comprises di- and tri-saccharide residues attached to most or all of the glycerol units. Analysis of group antigen preparations from three other strains of group D streptococci previously studied indicated that they are serologically identical with and chemically similar to the teichoic acid from strain 39. The difference in serological activity between the intracellular teichoic acid from strain 8191 and that from the other four strains is probably associated with small differences in chemical composition, particularly with respect to glucose content.

Mutants of Aerobacter aerogenes blocked in the accumulation of inorganic polyphosphate were isolated by the following method. Organisms previously subjected to ultraviolet (u.v.) irradiation were induced to accumulate polyphosphate in medium containing 32P. Upon subsequent growth in medium devoid of phosphate, RNA and DNA synthesis took place at the expense of polyphosphate. The organisms containing radioactive DNA were inactivated by decay of the 32P during prolonged cold storage. From the survivors, mutants unable to form volutin granules were selected by microscopic examination. The mutants fell into two classes. The majority did not accumulate polyphosphate when phosphate was added to a phosphate-starved culture, but did so upon prolonged sulphur starvation. The remainder of the mutants did not accumulate polyphosphate under either condition. Both kinds of mutant grew well in a defined medium and showed no obvious physiological disabilities.

A study of the accumulation by a brewer's yeast of six sulphur-containing amino acids labelled with sulphur-35 showed several distinct patterns of uptake. L-Methionine was accumulated rapidly and completely from the medium. L-cysteine was taken up slowly, and L-cystine not at all. DL-Ethionine accumulation was incomplete. S-Methyl-L-cysteine and S-ethyl-L-cysteine were taken up rapidly for a short period, and part of the sulphur of these compounds was then released into the medium. Accumulation of the sulphur amino acids was inhibited by certain other amino acids with a close structural relationship to them and when present in high concentration. Cysteine uptake was enhanced by the presence of reduced glutathione. These effects on accumulation were shown to be primarily changes in the transport of the sulphur amino acids into the yeast, although subsequent incorporation into protein may also be affected.

The function of serum in the composition of culture media for trypanosomes was investigated with Trypanosoma cruzi. Serum supplies stearic acid, essential for the growth of this trypanosome. A liquid heat-sterilizable medium, composed of peptone and known chemicals is described. The growth of T. cruzi in this medium reached 70 x 106 trypanosomes/ml.

When the NaCl concentration of a suspension of Halobacterium salinarium is gradually lowered by adding water, the rod-shaped organisms are converted to spheres which lyse. The organisms do not change in size when transformed from rods to spheres. Chemicals which do not possess a strong net charge in aqueous solutions do not protect the structure of the organism when they replace NaCl in iso-osmolar concentions. The rod shape of the organisms is only maintained by very high concentrations of ions which interact weakly with common proteins; lithium and ammonium ions are exceptions to this rule. Ions which display strong interactions with common proteins, and chemicals which are believed to break secondary bonds between protein molecules, effect a transformation of rods to spheres, and frequently a lysis of the spheres, when brought in contact with organisms suspended in strong NaCl solution. The changes in structure in hypotonic and isotonic solutions are not affected by metabolic inhibitors. Isolated cell-wall fragments disintegrate into smaller units when exposed to conditions under which whole organisms lyse. It is concluded that the structural transformations and lysis of H. salinarium in hypotonic solutions are not caused by the action of enzymes and that osmotic phenomena play no, or only a minor, role. The observations support the contention that the globular lipoprotein particles, which constitute the bulk of the material of the cell wall of these bacteria, are bound together mainly by electrostatic forces and secondary bonds. When the cells are exposed to hypotonic solutions, or to ions which bind strongly to proteins, or to chemicals which are believed to break secondary bonds between protein molecules, the linkages binding the lipoprotein particles together are weakened so that the wall structure disintegrates. Only in the presence of high concentrations of sodium and chloride ions, or other ions which bind loosely to proteins, is it possible for the proteinaceous particles of the cell wall to associate in an orderly array.

The production of swarmers by Bacillus cereus occurs naturally, especially in the case of ‘mycoides’ (i.e. morphologically rough) variants, but can also be induced by growth on media of low nitrogen content containing low concentrations of basic dyes. Very small coccobacilli, usually with one or two polar flagella, are released from the interior of the cells of the mother bacillus. The swarmers are Gram-negative or weakly Gram-positive. On further growth, or on subculture, they increase in size, and may simulate typical Gram-negative bacteria, or in some cases resemble granular, Gram-positive corynebacteria or mycobacteria. Growth of isolated swarmers is extremely slow. On continued subculture, a proportion of strains gradually revert to a Bacillus-like form, complete with heat-resistant endospore. Certain implications of this phenomenon are discussed.

Methods are described for the growth of Aspergillus nidulans in submerged culture. In experiments with intact organisms D-galactose was oxidized by an inducible enzyme system; D-fucose was a poor inducer of this system. Mutants isolated by their failure to grow on galactose as sole carbon source were defective in the oxidation of galactose.

Infection of Escherichia coli by phage λ22 in 0.02M-Sr(NO3)2, CaCl2 or MnSO4 occurred as readily as in 0.02M-MgSO4. The response of bacteria infected in the presence of Sr2-, Ca2+ and Mg2+ was predominantly lysogenic, but infection in 0.02 M-Mg2+ gave a higher lysogenic response (90%) than with the other cations tested. With Mn2+ in optimal conditions, at least 25% of the infected bacteria always gave the lytic response. When the newly infected bacteria were transferred to a growth medium, a period of about 40 min. had to elapse before the prophage could be detected by zygotic induction by mating with a non-lysogenic F - organism. A period of about 100 min. was required before all the bacteria giving the lysogenic response could be induced by ultraviolet radiation.

The addition of metal ions to the suspending medium of vaccinia virus preparations made them more resistant to heat. Monovalent metals were more effective than divalent, but a mixture of the two kinds was the most effective. Heavy metals rapidly destroyed virus infectivity. The protective effect is probably due to the formation of metal-protein complexes with increased resistance to heat denaturation.

Measurements of individual generation times were carried out on three species of bacteria, each in two different growth media. They confirmed in every respect conclusions already reached about the broad features of the generation time pattern. In addition, they supported two important inferences the existing evidence for which was no more than suggestive: (i) complex media give rise to a greater dispersion of generation time than do simple media; (ii) there is positive association between the generation times of second cousins, i.e. organisms whose nearest common ancestor is three generations removed.

A lethal mixture of the three factors (I, II, III) of anthrax toxin had none of the following enzyme activities: adenosine triphosphatase, alkaline or acid phosphatase, catalase, collagenase, ribonuclease, desoxyribonuclease, gelatinase, hyaluronidase, lecithinase, lipase, proteinase. The mode of action of anthrax toxin is unknown. Although factor II is the only factor which is immunogenic when injected alone, the addition of factor I results in an increased immunizing activity; however, the activity of this mixture can be decreased by adding factor III. The possible effect of these results on preparations used to immunize against anthrax is discussed.