- Volume 30, Issue 2, 1963

The adsorption of a tryptophan-requiring (tryp+) hydroxymethylcytosine (HMC) phage (Ox 6) to bacteria has been compared with that to kaolin. In a system free from monovalent cations other than tris (2-amino-2-hydroxymethylpropane-1, 3-diol) buffer, adsorption in both cases was temperature-dependent and required not only tryptophan but an optimum concentration of NaCl. The adsorption to kaolin came to an equilibrium which was determined by temperature since it depended on a preliminary activation of the phage, which process was temperature-dependent. Elution of phage adsorbed to kaolin took place to a greater or less degree on altering any one of the three requirements, NaCl, tryptophan or temperature. In experiments with bacteria no equilibrium was reached because adsorption was followed rapidly by inactivation; no elution was demonstrable.

Spheroplast suspensions containing as few as one rod in a thousand were made with Escherichia coli (15 his -) by the use of penicillin. These spheroplasts could be reverted to the rod state with efficiency approaching 100%. Spontaneous mutation from histidine requirement to non-requirement was observed in these spheroplasts, in which cell division did not take place.

In 18 hr. incubation of broth inoculated with a majority of bacteria of Salmonella typhimurium LT2 col -, i.e. non-colicinogenic, and a minority of strain LT2 (colI), i.e. carrying colicine factor colI, 30--70% of the col - bacteria acquired colI; this increased to 50--90% after 2 hr. of secondary incubation after tenfold dilution with broth. These cultures, containing a high proportion of bacteria newly infected by colI, transmitted colI to about 50% of the bacteria of a col - strain in 1 hr. (whereas pure cultures of LT2 (colI) transmitted to only 0.01%); they are termed HFC (high-frequency colicinogeny-transferring). An inoculum containing 1--3 recently infected bacteria from an HFC preparation of a streptomycin-sensitive strain sufficed to transmit colI to a resistant strain in streptomycin broth. We infer that 30--100% of bacteria newly infected by colI are ‘competent donors’, able to transmit colI. By the same test the proportion of competent donors in LT2 (colI) strains was only 0.02%. Inoculum size, ratio of inoculum components, motility, aeration and secondary incubation affected the HFC property of mixed cultures in a way explicable by the need for the epidemic spread of colI in the col - component to reach a peak at the time of testing. The rate of loss of the HFC property on further growth indicated that for 3--7 generations the progeny of newly infected bacteria are competent donors. Transmission was associated with clumping and pairing. Bacteria of an HFC preparation pair with 10% of an acceptor population within 2 min. of mixing; completion of transfer requires 2 1/2--30 min. Non-availability of nutrients and some growth inhibitors interfere with transfer.

We conclude that colI multiplies autonomously in newly infected bacteria and their immediate progeny and enables them to conjugate, but does not confer ability to conjugate in established colicinogenic strains; perhaps it is then integrated into the host chromosome.

None of the classes of major chemical constituents of the walls of bacteria and yeasts is exclusive to organisms which give a Gram-positive reaction. Walls of Gram-positive bacteria are richer in mucopeptide than are those of Gram-negative bacteria. The latter are characterized by high lipid contents. Yeast walls are rich in polysaccharide complexes. There appears to be a broad correlation between the Gram reaction and the decreased leakage of 32P compounds from labelled cells on exposure to increasing ethanol concentrations in the range 50--100% (v/v) ethanol. The data on the release of 32P compounds are in accord with the cell-wall ‘permeability’ mechanism for the Gram reaction and imply quantitative rather than all or none differences in Gram behaviour. Mechanical rupture of the cell wall or digestion of the wall with lysozyme rendered organisms which had been previously Gram stained in suspension by the technique of Wensinck & Boevé (1957), susceptible to decolorization with 96% (v/v) ethanol in water.

An Adansonian analysis of the features of 62 yellow pigmented marine bacteria, all but one of which were identified as Flavobacterium species by the workers who isolated them, revealed that 34 fell into one of two large pleista or groups; the rest fell into smaller pleista, or were the sole representative of their pleiston present. The possibility that these pleista approximate to various established genera is discussed.

The vitamin requirements of 63 strains of Rhizobium and 18 of Agrobacterium were examined. R. trifolii, R. leguminosarum and R. phaseoli responded in most cases to thiamine, biotin, and calcium pantothenate. Strains of R. meliloti and the slow-growing rhizobia sometimes responded to biotin, but not to the other vitamins. An unknown growth factor, active for the slow-growing rhizobia, was detected. Three strains of Agrobacterium required thiamine.

Certain of the colonies that arise when spores from a mixed culture of two complementary auxotrophic strains of Streptomyces coelicolor A3(2) are plated on a selective medium give rise to a mixture of spores with parental and recombinant genotypes. These heterogeneous colonies (heteroclones) probably develop from spores containing heterozygous nuclei which segregate during the development of the colonies. The heterozygous nuclei probably undergo a number of equational divisions before segregating. The markers in two linkage groups segregate independently. Large imbalances in the allele ratios and in the frequencies of complementary genotypes have been attributed to terminal chromosome deletions which affect both chromosome pairs. Absence (or homozygosity of the markers) of one whole chromosome is observed very often in heteroclones selected on media which make one chromosome dispensable. The heteroclones provide an efficient system for studying the genetics of the organism without recourse to selective methods.

The mechanism by which 11-deoxycorticosterone (DOC) inhibits the growth of Trichophyton rubrum was studied by examining the effects of this steroid upon metabolism by the intact mycelium. This compound inhibited the rate of endogenous respiration and the rate of uptake of glucose from the medium, but no evidence was found for the release of intracellular components from the mycelium. Incubation of mycelium in the presence of [14C]-glucose resulted in the incorporation of [14C] into 37 components within 3 min. in aqueous ethanol extracts of mycelium, although 98% of the [14C] in these extracts was contained in fructose, alanine, phosphorylated compounds and an unidentified compound. Incubation in the presence of 11-deoxycorticosterone resulted in a marked change in distribution, with decrease of the labelling of the phosphorylated compounds and fructose and increases in the labelling of the alanine and unidentified component. The results show considerable similarity to the effects of agents such as 2, 4-dinitrophenol on yeast metabolism and suggest that the uncoupling of phosphorylation from oxidation may be one result of the action of 11-deoxycorticosterone on T. rubrum.

Quantitative methods are described for estimating the activity of antisera against Paramecium aurelia, variety 1, and purified preparations of the immobilizing antigen derived from the organisms. Confirmation was obtained that the relation between the serum concentration and the reciprocal of the immobilization time is approximately linear, for times between 3 and 10 min. Titration of antigen preparations by a method based on inhibition of the immobilization reaction gave somewhat higher values than did titration by a gel-diffusion method. Cross-reactions between antigens controlled by genes at different loci and their various specific antisera were in all cases very small. The cross-reactions between antigens controlled by three genes at the D locus and their antisera varied according to the method used to measure them. Extensive cross-reactions were observed when the antisera were absorbed with heterologous paramecia and when precipitin formation in agar gel was followed. Only slight cross-reactions were observed by two methods based on the immobilization reaction.

Curves for the absolute yield of revertants as a function of ultraviolet (uv) radiation dose were obtained after post-irradiation incubation in saline and after exposure to reactivating light. The curves exhibited plateau regions and final slopes which were equal to the slopes of the survival curves for the auxotrophic Escherichia coli parent. These features may be due to a saturation of the mutagenic effect at low doses. This saturation would be the result of (a) heterogeneity in the unirradiated population with respect to the amount of protein and RNA synthesis which can occur before DNA replicates in the irradiated organisms, combined with (b) limitation of the observable yield by the selection method. The observable yield would be derived from the fraction of the population most resistant to the lethal effect of radiation.

Spontaneous and u.v.-induced revertants were found to be unstable in serial subculture. Instability was manifested by (a) replacement of the original revertant by revertants with slower growth rates on minimal medium, and (b) the appearance of auxotrophic organisms which overgrew in some cases but not others. In one case, the instability was found to be more pronounced when the organisms were maintained in the logarithmic phase of growth. This evidence, along with the previous radiological evidence, was interpreted according to the following hypothesis. The auxotrophic organism has an episomic element located at the suppressor locus. The damage inflicted by radiation consists in the detachment of this episome. The prototrophic cell may again become auxotrophic by reattachment.

A penicillinase-positive variant was isolated from a penicillinase-negative strain of Escherichia coli during ‘training’ towards ampicillin resistance. The penicillinase activity of the variant was increased tenfold when the organisms were disrupted in a Milner press or treated with toluene, suggesting the presence of a permeability barrier. The enzyme released by disrupting the organisms was not precipitated by centrifugation nor removed by filtration. The penicillinase activity of toluene-treated organisms was easily removed by centrifugation. Disruption of the organisms released enzyme into solution whereas it remained intracellular in toluene-treated organisms. The variant strain, although resistant to over 200 μg. ampicillin/ml., did not appreciably destroy it; of other penicillins tested only penicillins G and V were readily destroyed. The enzyme was a ‘penicillinase’ (β-lactamase) since the product was penicilloic acid. The enzyme was not inducible: exposure of organisms to 6-amino-penicillanic acid or phenoxybenzylpenicillin produced a threefold increase in penicillinase activity not due to true induction but to an increased permeability. The penicillinase of this strain of E. coli thus differs qualitatively and quantitatively from other penicillinases.

Damage to living organisms by X-rays (but not by ultraviolet radiation) is generally enhanced by the presence of oxygen, which probably acts at a physico-chemical level. Sensitivity to both u.v.- and X-radiation may also be related to the presence of oxygen acting as a metabolic factor during the growth of micro-organisms before or after irradiation.

Anaerobic growth of Escherichia coli strain B after u.v. or X-rays decreased lethal damage, the treatment being most effective after u.v.-and more effective after X-rays delivered in the absence than in the presence of oxygen. In this respect anaerobic growth corresponded with a variety of inhibitory conditions after irradiation, and rescue by anaerobic growth was about as effective as chloramphenicol treatment; but when these two treatments were used together, the effectiveness of each was decreased.

The effects of u.v.- and X-radiation on Escherichia coli B/r were unchanged by anaerobic growth after irradiation; but anaerobic growth beforehand resulted in survival curves which were much more sigmoid in shape than those pertaining to aerobically grown organisms. This tendency was noted with all strains tested. Anaerobic growth before X-irradiation did not alter the effect of oxygen during irradiation.

Forward mutations at the pyr locus in Neurospora, and back mutations at arg, were scored by using the ‘suppressor method’. This method is efficient and well suited for mutagen screening. Nitrous acid, ethyl-methane sulphonate and ultraviolet radiation were effective as mutagens; 8-ethoxycaffeine yielded inconclusive results. The same pattern of forward mutations at pyr, but different frequencies of back mutations at the arg locus, were obtained with the three agents. In this Neurospora system, nitrous acid appeared to induce mutations which affected simultaneously both DNA strands.

About 200 forward mutants of Neurospora obtained as suppressors of arg were studied. All fell in the pyr region, and all appeared deficient in aspartic transcarbamylase (ATC) to various degrees: extreme deficiencies in ATC (pyr-N alleles) resulted in pyrimidine-dependence as well as suppression of arg, while partial ATC deficiencies (pyr su-arg lleles) led only to suppression. Five different complementation groups were represented among the pyr-N alleles studied. Evidence is presented which suggests that pyr-N and pyr su-arg lleles affect ATC via different mechanisms.

Ampicillin sensitivity tests and tests for ampicillin inactivation were made with 148 strains of coliform bacilli. A correlation was found in strains of Proteus mirabilis and Klebsiella between ampicillin resistance and inactivation. No correlation was obtained with Escherichia coli, whether sensitive or resistant to ampicillin, although small amounts of penicillinase were produced. Ampicillin-sensitive strains of P. mirabilis showed even lower amounts of penicillinase. The strains of Pseudomonas pyocyanea (aeruginosa) were resistant to ampicillin, but also showed low penicillinase levels. No evidence of induction of penicillinase was obtained with E. coli or Klebsiella in the presence of methicillin, ampicillin or cephalosporin C. The ampicillin-inactivating enzyme is a β-lactamase, not an amidase. Resistance of ampicillin-sensitive strains of E. coli was increased rapidly by serial subcultures but was not associated with higher penicillinase values. Ampicillin was inactivated less rapidly than was benzylpenicillin by penicillinase from an E. coli strain.