- Volume 50, Issue 1, 1968

Washed myxamoebae of Dictyostelium discoideum exhibit a transitory accumulation of 2′,3′-mononucleotides in the soluble pool. The accumulation is correlated with the time of transition from the vegetative stage to the initiation of morphogenesis. Materials which stimulate the rate of morphogenesis and prevent the efflux of RNA and protein from washed amoebae, also enhance the accumulation of mononucleotides many fold.

The aerobic growth of Klebsiella aerogenes adapted to a chemically defined glucose ammonium sulphate medium was studied in the same medium containing triethylene-melamine (TEM). In liquid medium containing TEM up to 205 mg./l. growth and division were only slightly retarded. With concentrations of TEM greater than about 255 mg./l. division was strongly inhibited while growth continued slowly, giving filaments the longest of which later separated from the medium as a white pellicle. Further growth and division occurred in the same culture after a lag. During continued subculture in liquid medium containing a constant concentration of TEM the lag was negligible but growth on return to drug-free liquid or solid medium was impaired. At first filament formation increased to a maximum, the growth rate fluctuated and adaptation was rapidly lost during subculture in drug-free medium. The progressive impairment of division was later opposed by a gradual adaptation which eventually produced bacteria of a more nearly normal size, with a steady growth rate and with resistance to precipitation and to higher concentrations of TEM. TEM was less active in solid medium than in liquid medium. The proportion of filaments and the size of the lenticular areas observed in colonies increased with increasing concentration of TEM but decreased with increasing age and size of the colonies.

Thin sections of Staphylococcus aureus were examined in the electron microscope, before and after treatments which released part of their cell-bound penicillinase. In all cases, such treatment resulted in alteration of the mesosome structure: the mesosome was replaced by a series of pockets or invaginations in the cytoplasmic membrane. The converse situation was not always true; cocci treated in such a way as to alter the mesosome structure did not necessarily release penicillinase. This leads to the conclusion that structural alteration of the mesosome is only one of a number of steps in the release of penicillinase.

Mycoplasma laidlawii organisms are capable of binding in deoxyribonuclease-resistant form high molecular weight double- or single-stranded homologous or heterologous deoxyribonculeate in amounts of 0·1 – 1·0 × 10–15 g./organism. in a temperature-dependent mode. The maximum amounts of bound DNA ranged from 25 to 125 molecules of 5 × 106 d. Transformation of streptomycin-sensitive recipients to streptomycin resistance under conditions of maximum DNA uptake was relatively rare, being about 2-4-fold higher than spontaneous mutation. Ultraviolet (u.v.) inactivation studies showed that no caffeine-inhibitable u.v. dark-repair mechanism was present. Photoreactivation was demonstrable; the PR cross-sector was 0·39. From these results it was inferred that M. laidlawii is unable to be genetically transformed because of the absence of one or more steps which occur between irreversible DNA uptake and genetic integration. A model system which incorporates the above findings and suggests that donor DNA functions transiently in recipients is presented.

Mutants resistant to high levels of chloramphenicol can be obtained in Escherichia coli b by one or two mutational events. All of 144 high-level resistant mutant clones examined were powerful inactivators of the drug. Growth of this kind of mutants in nutrient media containing chloramphenicol 100 μg./ml. or more depended on the inoculum size, the composition of the medium, and the concentration of the drug. No growth was observed with lactose as sole energy source unless the organisms had been previously induced for β-galactosidase production.

With a strain of Escherichia coli k 12 an entirely different type of resistant mutants occurred. High-level resistant derivatives were obtainable only through several serial mutations. None of 36 high-level resistant mutants was able to inactivate the drug. Growth of these bacteria was extremely slow, even in the absence of drug. This resistance was due to a decreased rate of permeation, which probably was non-specific and concerned many species of micromolecules.

Experiments tracing the kinetics of the hyperimmune response in rabbits injected with several lower trypanosomatids (Crithidia fasciculata (Anopheles strain), C. fasciculata (Culex strain, Wallace isolate), C. fasciculata (Culex strain, Nöller isolate), Crithidia sp. from Euryophthalmus davisi, Blastocrithidia leptocoridis, Leptomonas sp. from Dysdercus and L. mirabilis) mixed with Freund’s complete adjuvant revealed a slow increase in homologous agglutinating titre, followed by a lasting plateau titre. Cross-reactions tested at various homologous titres showed that cross-reacting specificity increased as the titre increased. At peak agglutinating titre, sera were at their maximum specificity and remained like that during the lasting plateau period. Precipitins appeared before agglutinins and faded as the homologous agglutinating titres increased. Precipitins, in contrast to agglutinins, were specific even when the agglutinating titres were low and non-specific. By analogy with work done by other investigators on identification of serum fractions associated with non-protozoan antigens in which immune kinetics, globulin generation, or globulin specificity were traced, we suggest that the early-appearing nonspecific agglutinins may be composed of 19S globulins and the late- appearing specific agglutinins and specific precipitins may be composed of 7S globulins.

Spores of Bacillus cereus strain PX which were exposed to 0·02, 0·06, 0·18, 0·54 and 1·08 Mrad of γ-radiation became progressively more activated, i.e. they germinated more rapidly in the presence of germinants (e.g. l-alanine, inosine, n-dodecylamine, calcium dipicolinate) than did unirradiated spores. Heat- and radiation-activation differed in that irradiated spores but not heated spores germinated faster than untreated control spores in n-dodecylamine. The two methods of activation were similar in that both sorts of activated spores could be de-activated, and heated and irradiated spores both had increased contents of titratable thiol groups as compared with untreated spores. Germination-like changes still occurred rapidly in spores which had been subjected to doses of γ-radiation sufficient to render them non-viable. Ionizing radiation, heat and other agents which activate spores probably do so by changing the tertiary structure of spore macromolecules and thereby exposing previously masked reactive sites which are important in germination.

By repeated subcultivation of the parent strain Pediococcus cerevisiae atcc 8081 in the presence of methicillin, a substrain P. cerevisiae 8081 CRD was developed which grew only when the partly defined medium was supplemented with methicillin or certain other penicillins. The methicillin-dependent strain was not highly resistant to methicillin and grew only in the presence of a limited range of concentrations of it (about 10–300 μg./ml.). Even with an optimal growth concentration of methicillin (50–100 μg./ml.), the dependent organisms grew less well than did the parent strain without methicillin and showed a longer lag period before growth became visible.

Although other derivatives of 6-aminopenicillanic acid (but not of 7-aminocephalosporanic acid) supported moderate growth of Pediococcus cerevisiae 8081 crd, none was as effective as methicillin, nor was there marked cross-resistance to any of these other derivatives. The more potent the penicillin as inhibitor of growth of the parent strain, the smaller was the optimal concentration needed to support growth of the dependent substrain. Several other antibiotics were ineffective as growth factors.

When methicillin was hydrolysed with acid, alkali or pencillinase, activity as a growth factor was lost. During growth of the parent strain and of the methicillin-dependent strain at pH 6·5 material was produced in the medium which was able to destroy the antibiotic potency of methicillin or other penicillins. The substance was not an enzyme, and the presence of methicillin was not necessary to induce its formation by the parent strain.

The methicillin-dependent strain did not grow, with or without methicillin, when sodium acetate was omitted from the medium. No substance of known chemical structure was found which could replace acetate for growth. Pediococcus cerevisiae 8081 CRD grew rapidly in the absence of both acetate and methicillin when the medium was supplemented with yeast extract. However, when acetate was present as well as yeast extract, methicillin again became necessary for growth of the dependent organisms.

Cell walls were prepared from: (1) Pediococcus cerevisiae 8081 grown (in partly defined medium) without methicillin; (2) P. cerevisiae 8081 grown with a sub-inhibitory concentration of methicillin (10 μg./ml.); (3) P. cerevisiae 8081 CRD (methicillin-dependent substrain) grown with an optimal concentration of methicillin (100 μg./ml.). All three preparations contained glucosamine, muramic acid, N-acetyl groups (no O-acetyl groups), alanine, glutamic acid, lysine and aspartic acid, in proportions which suggested that they were components of a mucopeptide polymer; small amounts of serine, glycine and threonine were also present. The mucopeptide components made up about 50 % by weight of walls (1) and (2) but about 80 % in walls (3). Glucose and phosphorus were present in the walls, in greater amounts in walls (1) and (2) than in walls (3). No free amino groups were detected in any of the walls, and most of the lysine was released as ϵ-(aminosuccinyl)-lysine when mucopeptide from the parent strain was hydrolysed for a short period. About 35 % of walls (1) and (2) was removed by extraction at 2° with trichloroacetic acid; only 16 % was removed from walls (3). Nearly all the glucose and most of the phosphorus was removed from all the walls by such extraction; the residues after extraction all contained about 90% of mucopeptide components. Teichoic acids were isolated from each trichloroacetic acid extract: phosphorus, glucose, alanine and glycerol were found in all. Teichoic acid from walls (3) contained, in addition, about 7 % of mucopeptide components. Walls and trichoroacetic acid insoluble residues from the methicillin-dependent substrain were hydrolysed much more rapidly by lysozyme than were walls or residues of the parent strain, whether grown in absence or presence of methicillin.

The main characteristics of the previously described proline-specific transport mechanism (permease) of Escherichia coli were confirmed in strain c4. The same permease was responsible for entry of a number of proline analogues, including 3,4-dehydroproline, 4-methyleneproline, cis- and trans-4-chloroprolines, thiazolidine-4-carboxylic acid (thioproline) and the lower homologue, azetidine-2-carboxylic acid. These analogues also entered the cells by an exchange reaction between extracellular analogue and previously accumulated intracellular proline. Growth of the parent (c4) strain was inhibited by 3,4-dehydroproline and azetidine-2-carboxylic acid, both of which were incorporated into cellular protein. Several classes of mutants, selected for resistance to either dehydroproline or azetidine, failed to incorporate one or both analogues into protein. Some of these mutants owed their resistance to failure to produce a functional proline permease. At least one strain, resistant to azetidine but not to dehydroproline, possessed an altered permease with little affinity for azetidine-2-carboxylic acid but still capable of transporting proline and 3,4-dehydroproline ; the permease of this strain could no longer promote exchange between intracellular proline and extracellular proline or proline analogues.

The survival of Escherichia coli strain b sprayed from distilled water into atmospheres of nitrogen, argon and helium, as a function of relative humidity (RH) at an aerosol age of 30 min. was good at low RH, while at high RH values regions of marked instability occurred. At high RH differences in survival were observed, indicating that the gas atmospheres were not completely biologically inert. The results indicated that the initial evaporation rates of the aerosol droplets did not influence the long-term survival of E. coli b in the aerosol. An alternative reason for the importance of RH is discussed, together with considerations of death mechanisms.

Computers were used for numerical studies as an aid to the classification of streptococci. The results obtained with 75 biochemical and physiological tests of 216 strains were examined by three different computer programmes. Two of these programmes formed clusters in general agreement with the well-established species and also formed three clusters among the viridans-like streptococci. These 3 clusters seem to provide a basis for the recognition of 3 divisions among the streptococci which are at present usually described as lacking adequate distinguishing characters.

R factors, responsible for transmissible drug resistance in Enterobacteriaceae, convert their hosts into donors of genetic material, but fewer recombinants for chromosomal genes are produced than with F. For one R factor, at least, this was merely a consequence of less frequent conjugation. With a mutant where conjugation was not repressed in contrast to the wild type, this R factor, R 1, was shown to behave like an F′ factor and to transfer the bacterial chromosome at high frequency from a fixed origin near the try genes.

Bacteria carrying an R factor conferring resistance to low concentrations of streptomycin frequently give highly resistant variants. Resistance to high concentrations of streptomycin can arise in a sensitive bacterium either from a single mutation or as the result of successive mutations in different genes (Demerec, 1948; Hsu & Herriott, 1961). The resistance genes may be either in the chromosome or in an R factor.