- Volume 30, Issue 1, 1963

Form 2 of a strain of Mycobacterium tuberculosis var. hominis was isolated. The cellular morphology of this organism when growing in glycerol agar slide culture is described. The form 2 strain grew by the initial production of septate filaments which soon ramified as a result of pseudobranching. The filaments fragmented early into bacillary elements and much later into coccoid elements. Endospores were formed within some of the bacillary elements. The young cells were Gram negative and the older cells Gram positive. The cells were never acid-fast. Growth occurred in aerobic and anaerobic culture, but the morphological changes progressed more rapidly under anaerobic conditions. The strain has many characteristics also found in some members of the Actinomycetaceae; however there are also differences, the most important of which is endospore formation. Thus the strain cannot yet be classified.

Mutants with small colonies and impaired respiration were induced by 5-fluorouracil in cultures of Staphylococcus afermentans, nctc 7503. The consumption of oxygen in colourless mutants attained 66-77%, and in mutants with orange colonies 68-77% of normal values. Inhibitors of protein synthesis (chloramphenicol, tetracycline, chlortetracycline), inhibitors of cell-wall synthesis (penicillin G, phenoxymethylpenicillin), as well as an inhibitor of RNA formation (actinomycin C) affected the growth of the parent and mutant staphylococci to the same extent. On the other hand, inhibitors which affect DNA—namely, degranol, myleran-mannitol and mitomycin C—selectively inhibited the growth of mutant staphylococci with impaired respiration. Mitomycin C induced irreversible depolymerization of DNA in bacterial cells; colourless mutants were 2·7-3·5 times, and mutants with orange colonies 33-45 times more vulnerable to its action than the parent culture. The extraction of DNA from bacterial cells showed that the capacity of DNA for extraction from a complex with proteins was decreased in colourless mutants by 2·8-2·9 times, and in mutants with orange colonies by 5·0-7·3 times.

Various transamination reactions were observed in germinating uredospores of Puccinia helianthi Schw. The localization and kinetic properties of glutamate-aspartate transaminase were examined. The presence of these transaminase enzymes in the uredospores suggests that the uredospore could synthesize a large number of amino acids.

The effects of intranasal inoculation of mice more than 6 weeks old with the TE 55 strain of trachoma virus were studied. The histology and course of the pulmonary lesions produced are described. Infected mice showed a poor response of complement-fixing antibodies after infection. Precipitating antigens were detected in infected mouse lungs.

When water-washed organisms of a strain of Pseudomonas aeruginosa were exposed to solutions of NaCl or sucrose, they shrank in size and became more dense. The optical density of suspensions increased rapidly. Pre-treatment of the organisms with various cations, surface-active agents and heat and cold, affected the reaction, and the organisms then behaved differently toward NaCl and sucrose. Some but not all of the effects observed with P. aeruginosa were also observed with a strain of Escherichia coli.

Propagation of the trachoma agent in human cell cultures has made possible the observation by electron microscopy of sequential stages in the replication of the micro-organism. Scattered reticular foci with incomplete limiting membranes were present in the cytoplasm of HeLa cells 12 hr. after exposure to the infectious agent in high multiplicity. After 24 hr. the foci had coalesced to give a single reticular mass. Within it there appeared large and discrete developmental forms from which, by progressive subdivision and diminution in size, typical elementary bodies were formed. The elementary bodies had a prominent limiting membrane, and a dense core within which a fibrillar nucleoid could be recognized. The controversial taxonomic status of the trachoma agent is considered briefly in the light of ultrastructural evidence.

Small samples of hay were shaken in a perforated drum in a wind of 4·2 m./sec.; the liberated dust cloud was sampled with the cascade impactor for microscopical examination, and with the Andersen sampler for identification of organisms in culture. The results of testing batches of hay showed large differences in microbial content. Twenty-eight batches classed as ‘good hay’ gave up to 3 million spores/g. dry wt. hay, mainly Aspergillus glaucus with Cladosporium spp. and Hemispora stellata. Dust blown from seventeen batches of ‘mouldy hay’ differed greatly in composition and had from 5 to 250 million spores/g.: abundant forms included A. glaucus, A. fumigatus, A. nidulans, Penicillium spp., Absidia spp., Mucor pusillus, bacteria, many actinomycetes. Fourteen batches of mouldy hay associated with cases of ‘farmer's lung’ disease were generally similar in mould content to the batches of mouldy hay, but had many more spores of Humicola (Monotospora) lanuginosa and A. fumigatus, and were specially characterized by very many actinomycetes. Farmer's lung hays tended to neutrality (averaging pH 7·0, as compared with pH 5 to 6 of other hays), and were rich in thermophilic organisms, commonly with hundreds of millions of actinomycete spores/g., a large proportion of which grew readily at 60°. Hays associated with farmer's lung appear to have heated spontaneously to a higher temperature during maturation than the other hays. Dust from all hays contained particles of higher plants, but there were not obviously more in farmer's lung than in other hays. All types of spores were shown to be potentially able to penetrate to the deeper parts of the lung (especially the spores of actinomycetes, A. fumigatus and H. stellata), but whether these organisms play any part in the aetiology of the disease is unknown.

The transforming activity for an indole locus of DNA extracted from a methionine-requiring strain of Bacillus subtilis was decreased about tenfold per unit of DNA by treatment of organisms with 0·025 m-methylmethane-sulphonate for 30 min. at 37°. The treatment decreased the viable count to one tenth of the original and induced a reversion frequency of 10-5. Transforming activity of DNA prepared from alkylated organisms was sensitive, to heating at 50° as compared to DNA extracted from non-alkylated organisms. Methylation and heating of DNA in vitro did not inhibit its uptake by competent organisms. Incubation of treated and non-treated organisms in amino acid supplemented medium led to an increase in extractable transforming activity. A period of incubation which led to a 34% increase in DNA content more than doubled the transforming activity obtainable from methyl methanesulphonate treated organisms. The heat sensitivity of transforming DNA from alkylated organisms decreased after incubation of the organisms. Synthesis of DNA in the absence of a net DNA increase was demonstrated in methyl methanesulphonate treated organisms since thymidine-2-14C was incorporated without lag from the start of incubation into an alkali-stable hot acid-soluble form. The experiments are interpreted to mean that alkylated DNA can serve as a template for the replication of DNA of normal activity.

All strains of Bordetella pertussis, B. bronchiseptica and B. parapertussis tested, were found to contain a blue protein; this has been named ‘Azurin’. It has been extracted from the cells of all three species and it has also been isolated from culture supernatant fluids of B. pertussis. The azurin was purified by chromatography on ion-exchange cellulose derivatives and obtained in physically pure form; the materials from each species were identical. In one strain of B. bronchiseptica azurin comprised 0·1% of the bacterial dry weight. Azurin is a water-soluble autoxidizable protein of high oxidation-reduction potential (+395 mV.). It has an absorption maximum at 625 mμ in the oxidized form, with a millimolar extinction coefficient of 3·5 × 103. It contains 0·45% copper which can be completely removed by dialysis against cyanide. The molecular weight is 14,600 (S 20,w = 1·58 × 10-13, D 20, w = 10·6 × 10-7). Azurin can be reduced by cysteine, glutathione and other reducing agents; the blue colour disappears on reduction. Azurin undergoes reduction in the presence of concentrated cell-free extracts of B. bronchiseptica and succinate, and is reoxidized on aeration.

Macroconidia of Trichophyton mentagrophytes harvested from a culture grown on a medium rich in glucose contained 33% acid-soluble carbohydrate. Paper chromatography of hydrolysates showed glucose as the main sugar in this fraction; there was also a trace of an unidentified sugar. Macroconidia from the same strain grown on the same medium but deficient in glucose contained 19% carbohydrate; hydrolysates of this fraction contained glucose, with traces of galactose and the unidentified sugar. Microconidia harvested from a second strain grown on a medium rich in glucose contained 11% acid-soluble carbohydrate; hydrolysates of this fraction also contained glucose with traces of galactose and the unidentified sugar. These differences were reflected in whole cultures induced for macroconidial formation with carbon dioxide. Strains which responded well increased in acid-soluble carbohydrate; glucose was the predominating sugar in hydrolysates of these fractions. Strains which responded poorly did not increase in carbohydrate content; glucose, and traces of galactose and an unidentified sugar, were found in hydrolysates of these fractions.

An inducible UDPG pyrophosphorylase (UTP:α-d-glucose-1-phosphate uridyltransferase, EC 2.7.7.9) has been demonstrated in cell-free extracts from Trichophyton mentagrophytes incubated in increasing atmospheric tensions of carbon dioxide. Assays of related enzymes have shown that UDPG pyrophosphorylase is not the only enzyme induced by CO2. Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glucose-phosphate isomerase (EC 5.3.1.7) were more strongly stimulated than UDPG pyrophosphorylase. Hexokinase (EC 2.7.1.1), phosphoglucomutase (EC 2.7.5.1) and 6-phosphogluconate dehydrogenase (EC 1.1.1.43) were more weakly stimulated than UDPG pyrophosphorylase. The stimulation of UDPG pyrophosphorylase does not occur as a result of sequential induction.

The cell wall of Salmonella gallinarum, like that of other Gram-negative organisms, was found to contain a separate continuous rigid mucopolymer layer. This structure was rapidly broken down by the cell's own enzymes during wall preparation when suitable precaution against this action was not taken. Possible errors in the interpretation of cell-wall structure, deriving from this and other causes, are discussed.

Ascites tumour cells were homogenized and fractionated at various times after infection with encephalomyocarditis virus in vitro. Infective ribonucleic acid (RNA) could be obtained from the nuclear fraction soon after infection and the amount of infective RNA recovered from this fraction increased until a maximum was reached at about 4 1/2 hr. From 4 1/2 hr. onwards the amount of infective RNA associated with the nuclear fraction fell while that from the mitochondrial fraction increased, reaching a maximum at about 5 1/2 hr. The amount of haemagglutinin and plaque-forming virus began to increase at about 4 1/2 hr. and continued to rise until 8 hr. Most of this virus activity was found in the mitochondrial fraction, although from 6 hr. onwards an increasing amount was found in the microsomal fraction.

A study of the nutritional requirements of Mycoplasma laidlawii has led to the development of a growth medium containing inorganic salts, amino acids, glucose, nucleosides, vitamins and 1% (w/v) bovine serum albumin fraction V. No cholesterol was detected in this medium. Potassium, magnesium and phosphate ions were essential growth requirements. Sodium chloride provided the high tonicity required by the growth medium. The addition of an ammonium salt to the medium enabled the growth of the test organism in the presence of 13 amino acids. Resting organisms of M. laidlawii did not catabolize any of the 22 different amino acids tested under conditions which allowed amino acid degradation by Mycoplasma hominis. Glucose was essential for growth and could be replaced by maltose but not galactose, fructose, mannose, lactose or sucrose. The requirement for nucleic acid precursors was satisfied by adenosine, guanosine and cytidine, and that for vitamins by nicotinic acid, riboflavin, folinic acid, pyridoxine, pyridoxal and thiamine. Cholesterol was not necessary for growth of the test organism.

The susceptibility to lysis of several Mycoplasma organisms, bacterial protoplasts, spheroplasts, L-forms and intact bacteria was compared. The Mycoplasma and L-forms were much more resistant to lysis by osmotic shock and to alternate freezing and thawing than were the bacterial protoplasts and spheroplasts. Like bacterial protoplasts, the mycoplasmas were very sensitive to lysis by surface-active substances, primary alcohols and alkali; bacterial spheroplasts and L-forms were less sensitive to lysis by these agents. All the organisms tested, except intact bacteria, showed various degrees of sensitivity to lysis by pancreatic lipase. The mycoplasmas and the L-forms were completely resistant to the lytic action of lysozyme. The mycoplasmas differed from all other microorganisms tested in being sensitive to lysis by digitonin and saponin. This sensitivity to digitonin was considerably smaller when the organism was grown in a cholesterol-free defined medium. The structure and properties of the Mycoplasma cell envelope are discussed on the basis of the present findings.