- Volume 40, Issue 2, 1965

After incubation for 1 hr at 37° with caseinase C (a purified fraction of actinomycetin), the neuraminidase activity of concentrated purified influenza virus pr 8 suspension was unchanged, whereas its infectivity and haemagglutinating activity were considerably decreased. After 4 hr, infectivity and haemagglutinating activity were still more decreased and most of the neuraminidase activity was destroyed. The ability to fix complement in the presence of specific antibodies was slightly decreased, whereas the ability to neutralize haemagglutination-inhibiting antibodies was not affected.

Influenza virus pr 8 suspension treated with enzyme caseinase C contained material sedimentable by centrifugation for 1 hr at 31,000 g and material which remained in the supernatant fluid under these conditions. Both materials fixed complement in the presence of control pr 8 virus antiserum. The ability of control pr 8 virus suspension to neutralize haemagglutination-inhibiting antibodies and its ability to react with strain-specific complement-fixing antibodies were related to material sedimentable by centrifugation for 1 hr at 31,000 g. Treatment of PR8 virus suspension by caseinase C destroyed its ability to produce antibody-fixing complement in the presence of control influenza virus pr 8. But this treatment did not suppress ability to produce specific haemagglutination-inhibiting antibodies. Following this treatment of pr 8 virus a new antigenic activity was shown: the antiserum to enzyme-treated virus not only fixed complement in the presence of enzyme-treated pr 8 virus, but also in the presence of enzyme-treated Asian virus. Material which fixed complement in the presence of antiserum to enzyme-treated virus was sedimentable from enzyme-treated pr 8 virus suspension by centrifugation at 31,000 g.

A study was made of the kinetics of killing by ultraviolet (u.v.) radiation (2537 Å) and subsequent photo-reactivation (with black light) of Escherichia coli strain 15 t ‒ a ‒ u ‒ (requires thymine, arginine, uracil) by using (a) logarithmic-phase cultures, (b) early-stationary-phase cultures, (c) logarithmic-phase cultures after 90 min. of incubation in the absence of arginine and uracil (– AU cultures). The stationary and – AU cultures showed the same enhanced resistance to u.v. killing. In all three cultures (1) the photo-reactivable sectors were the same, (2) the rate of photoreactivation was the same function of u.v. dose, (3) the amount of light required for maximum photo-reactivation was a linear function of the u.v. dose. We conclude that the initial lethal lesions produced by a given u.v. dose were qualitatively and quantitatively the same in all three cultures, despite differences in u.v. survival. This implies that the stationary-phase cultures and the – AU cultures were more radiation resistant solely because they were better able to cope with the initial lethal damage. The survival curves can be satisfactorily described mathematically in terms of this model.

A logarithmic-phase culture of Escherichia coli strain 15 t – a – u –, in which protein and RNA syntheses were inhibited for 90min. by withdrawal of arginine and uracil, was compared with a culture grown with all required nutrients present until 90–120 min. after the end of exponential increase in extinction (early stationary phase). The two cultures were indistinguishable by tests of radiation (ultraviolet, gamma) sensitivity, growth kinetics and morphology. It is concluded that the interruption of protein and RNA syntheses during active logarithmic growth forced the culture into the stationary phase.

The life-cycle of actinophage MSP 2 in host Streptomyces venezuelae s 13 was divisible into distinct stages by manipulating the cultural conditions and by the use of certain chemical compounds. Two types of attachment were observed: reversible and irreversible. Reversible attachment occurred instantaneously and was relatively independent of temperature and phage concentration. The controlling variables in extent of adsorption were the amount of host mycelium and ionic content of the medium. Irreversible attachment was time dependent and temperature dependent. Addition of acriflavine allowed phage attachment and injection to occur, but suppressed some stage of intracellular growth or maturation. Acriflavine prevented formation of viable phage, but phage components were demonstrated immunologically and electron-microscopically. Chilling of phage-infected cultures allowed lysis to proceed, with release of preformed phages; but no new phages were formed. These results indicate that the life-cycle of actinophage MSP2 is experimentally separable into attachment, injection, growth, maturation and lysis.

Lysostaphin attacked both viable staphylococci and the mucopeptide portion of the staphylococcal cell wall. In the absence of salts, lysostaphin activity could only be recovered from the particulate portion of the lysed cell after centrifugation, whereas in the absence of salts its action on the mucopeptide resulted in a recovery of active material in both the sediment and the supernatant fluid. It appears from these observations that lysostaphin is complexed with its substrate and that NaCl is required to break the complex.

The inhibitory effect of high-titre preparations of phage 47 on certain strains of Staphylococcus aureus belonging to phage group III was produced with a phage: cell ratio of approximately 1 : 1. Staphylococci showing inhibition by preparations of phage 47 were converted to phage sensitivity by lysogenization with a phage derived from a strain sensitive to phage 47. Multiplication of phage 47 in lysogenized clones was compared with that in the original inhibited strains. This showed that inhibition was due to a phage/cell interaction in which most of the infected cocci were killed; a minority produced a few phage particles in smaller numbers with a longer latent period.

In a non-growing population of an arginine-requiring auxotroph of Escherichia coli, the uptake of [14C]arginine and [14C]glucose by the whole organisms, and the incorporation of [14C]arginine in the acid-soluble fraction and total protein of the organisms, decreased with increasing bacterial concentration in a wide range (0·1–3·0 mg. dry wt. bacteria/ml.). It is concluded that the rate of transport/organism of arginine and glucose decreased with increasing bacterial concentration.

The morphology of Mycoplasma mycoides was well preserved after washing and suspension in buffered 0·4 m-sucrose solutions, but the survival of viable particles was no better, and the loss of ultraviolet (u.v.)-absorbing substances and the decrease of turbidity was no less than in hypotonic solution (0·01 m-tris HCl or 0·01 m-Na2HPO + KH4PO4). The addition of Mg2+, Ca2+, spermidine or spermine (increasing order of activity) decreased the decrease of turbidity and loss of u.v-absorbing substances. Ca2+ and Mg2+, but not spermine, increased the degree of survival of viable particles. Ethylenediaminetetra-acetate (EDTA; 0·01 m) increased the loss of u.v.-absorbing substances, and decreased the turbidity and degree of survival. Ca2+, Mg2+ and spermine annulled the effects of EDTA on loss of u.v.-absorbing material and on the turbidity, but only Ca2+ prevented the lethal effect of EDTA. Filaments disappeared and cell volume increased when the organisms were transferred from hypertonic to hypotonic solutions ; the shape changes were reversible.

Strains of Saccharomyces cereυisiae heteroallelic for the adenine-2 locus, heterozygous for outside markers and homozygous for tryptophan-1, were ultraviolet (u.v.)-irradiated and the effect of post-treatment with white light determined. Mitotic gene conversion to adenine independence and mutation to tryptophan independence and to adenine independence were both decreased by post-u.v.-irradiation treatment with white light, whereas mitotic reciprocal recombination of outside markers was unaffected. The effect of varying the treatment with white light after a constant degree of exposure to u.v.-radiation produced a decrease in reversion frequency to a constant value at light exposures of 5 min. and more. The differing responses of reciprocal recombination and gene conversion are discussed.

The synthesis of p-aminobenzoic acid and folic acid was investigated in three strains of Staphylococcus sensitive to sulphonamide drugs, and in four substrains resistant to sulphonamides. Another strain (Staphylococcus aureus r122) which was resistant to sulphonamides when isolated was also examined. A microbiological assay was used to measure the synthesis of p-aminobenzoic acid during the growth of organisms in a partially defined medium. S. aureus r122 formed about twenty times as much p-aminobenzoic acid as did any of the other strains, among which the synthesis showed only small variations and was not greatly affected by growth of the organisms in presence of sulphathiazole.

Folic acid was assayed as Lactobacillus casei factor. The response curve of the assay organism to extracts of every strain of Staphylococcus examined was the same; but it differed from the response to known forms of folic acid. The active material had little growth-promoting activity for Leuconostoc citroυorum or for Streptococcus faecalis. Washed suspensions of Staphylococcus lactis 2102r formed about 10 times as much folic acid as the sensitive parent strain (2102) from glucose, p-aminobenzoate and glutamate; the sulphonamide-resistant substrain (2102r) also formed more folic acid during growth. However, a second resistant S. lactis substrain (2102r2) formed little more folic acid than did the parent strain. S. aureus r122 synthesized an amount of folic acid similar to that formed by S. lactis 2102r. With two other strains (S. aureus h and jhm) the synthesis of folic acid was not increased when resistance was acquired. Washed suspensions of all the sulphonamide resistant substrains were able to synthesize folic acid in the presence of higher concentrations of sulphathiazole than were the sensitive strains.

Four staphylococcal substrains resistant to sulphonamides were developed from sensitive parent strains by subcultivation in increasing concentrations of sulphathiazole. As compared with the parent strains, the growth of the resistant strains was 20–200-fold less sensitive to sulphathiazole in a semi-defined medium based on acid hydrolyzed casein than were the parent strains. Inhibition of growth by sulphathiazole was overcome competitively by p-aminobenzoic acid. The inhibition indices of the sulphathiazole-sensitive strains were about 10, and 100–1000 for the resistant substrains; the change in index was about proportional to the degree of resistance. The sulphathiazole-resistant strains were cross-resistant to other sulphonamides but were not resistant to p-nitrobenzoic acid. Not one of the sensitive or resistant strains was inhibited by aminopterin.

No change in the inhibitory effect of sulphathiazole occurred during growth of cultures in its presence, though the drug was partly converted to a second substance (of unknown structure). Resistant and sensitive pairs of strains formed similar amounts of unknown compound, so that it is unlikely that its formation has to do with resistance. Concentrated suspensions of organisms of all strains took up similar amounts of [35S]-sulphathiazole when incubated in the growth medium. The attachment of the drug to the organisms was weak, but the uptake was not altered by the presence of p-aminobenzoic acid. The rates of aerobic growth and the nutritional requirements for anaerobic growth of sensitive and resistant strains were generally similar. The nutritional requirements for vitamins and amino acids varied among the sensitive strains, but there were only slight differences between resistant and sensitive pairs of strains.

Resistant strains of Aspergillus nidulans were obtained by one-step selection on high concentrations of various inhibitors; each strain was mutant in a different gene conferring resistance to actidione (Act), p-fluoro-phenylalanine (pf), teoquil (te), iodoacetate (Iod) or malachite green (mg). Some mutant alleles have been firmly, others tentatively, located. For comparative purposes attempts were made to find instances of multi-step or non-genic increases in resistance to malachite green and to teoquil, by prolonged exposure to low concentrations of inhibitor. No such increases were found.

Iod1 is fully dominant, Act1 is semidominant, pf21, tel and mg1 are recessive. mg1 confers resistance to acriflavine to about the same degree as the non-allelic acr2 but these two mutant alleles do not show additivity. pf21 confers resistance to iodoacetate and also suppresses requirement for nicotinic acid (nic8). Iod1 strains, which are not resistant to fluoroacetate, are able to use acetate as sole carbon source.

Nutritionally balanced heterokaryons, between Act1 and sensitive strains, show a gradual (and reversible) increase of the Act component on increasing actidione concentrations. Ultimately a plateau is reached; this presumably represents the nutritional limits of each particular combination of nutritional markers.

Two phospholipase enzymes have been identified in toxic preparations from Staphylococcus aureus, each having a mode of action like that of phospholipase C. One enzyme hydrolysed phosphatidyl inositol and lysophosphatidyl inositol, while the other hydrolysed sphingomyelin and lysophosphatidyl choline. The latter enzyme was always associated with β-haemolysin activity and it is concluded that β-haemolysin, sphingomyelinase and lysophospholipase are activities of one protein. A phospholipase A was also detected in a toxic preparation from an αβ strain. A study of cultural conditions showed that this enzyme was produced under a variety of conditions, but only when the β-haemolysin activity was high.