Intracellular Antibiotic Research Articles

Changes in the extracellular accumulation of antibiotics during growth and sporulation of Bacillus subtilis in liquid culture.

Ten antimicrobial metabolites, produced by Bacillus subtilis NCIB 8872, have been categorized into 3 groups on the basis of their antimicrobial spectra, chromatographic mobility and solvent solubility. The maximum concentration of the different groups occurred at different times during fermentation. The accumulation of one antibiotic group was characteristic of the logarithmic growth phase. Groups also differed in their persistence. The intracellular antibiotics contributed little to the total antibiotic activity of the culture. The onset of production and the maintenance of the extracellular accumulation of the 3 antibiotic groups were linked to sporulation and associated specific changes in the intracellular and extracellular protein complement. Production of the antibiotics was not controlled by glucose catabolite repression, since the presence or absence of glucose in the medium did not affect the pattern of antibiotic accumulation.

Effects of dihydrostreptomycin on ribosome function in vivo: lack of correlation between changes in ribosome patterns and growth.

In vivo treatment of susceptible Escherichia coli cultures with low concentrations of dihydrostreptomycin leads to a decline in polysomes and a corresponding increase in 70S particles which behave as run-off ribosomes, as well as free 30S and 50S subunits. We have examined the timing and extent of these effects on ribosomes and compared them to the effects of this antibiotic on growth and protein synthesis. We have shown that no changes in ribosome distribution are observed until growth inhibition by dihydrostreptomycin is almost complete. Thus, intracellular dihydrostreptomycin can inhibit growth and net protein synthesis without apparently affecting the ribosome cycle. Since it is known that the antibiotic combines with free 30S subunits, the question is how such combination can bring about the observed inhibition of protein synthesis and growth. We suggest that specific interaction of intracellular antibiotic with proteins of the 30S subunits allows repeated use of the ribosome cycle by such affected particles, but with selective misreading of certain amino acid codons as terminator codons, so that they produce incomplete polypeptide chains. The cumulative effect of such a mechanism would lead to eventual cessation of protein synthesis and growth.

UPTAKE AND RELEASE OF 3H‐MITOMYCIN C IN MOUSE P‐388 CELL CULTURES

Mouse P‐388 cells were grown in suspension culture and exposed to 3H‐labelled mitomycin C for 1 hour. With concentrations from 0.5 to 50 μg of 3H‐mitomycin C per ml cell culture, about 0.01 per cent of the radioactivity added was found in washed cells. Most of the intracellular antibiotic was present as cold acid soluble material, but significant amounts of tritium were found in the nucleic acid‐ and protein fractions. The ratio of 3H per weight unit was of the same order of magnitude for DNA and RNA, but substantially lower for cell protein. Double labelling experiments using 14C‐thymidine and 3H‐mitomycin C were carried out to study the specific loss of antibiotic from the cells during post‐treatment incubation. Cold TCA soluble 3H‐mitomycin C was rapidly released from the cells during the first hours after they had been transferred to non‐radioactive growth medium. Loss of 3H‐radioactivity from the RNA‐ and protein fractions roughly paralleled the loss of 14C radioactivity from the DNA fraction over a period of 3 days. The content of 3H‐radioactivity in the DNA containing cell fraction increased during the first day of post‐treatment incubation, followed by a substantial drop during the second day. This drop was far more extensive than the corresponding loss of 14C radioactivity. During the third day after exposure to mitomycin C the drops in 3H and 14C in the DNA fraction were not significantly different.

Actinomycete variants with impaired differentiation: production in high yield, recognition and phenotypic characterization in seven species.

Summary. When plated on mineral synthetic media with D‐fructose as a sole carbon and energy source 7 strains of actinomycetes belonging to different streptomycete species regularly produced secondary variant colonies in high yield. Beside the ability of strepto‐mycetes to utilize fructose as a sole carbon source, the following factors were shown to be of importance in the control of variational events: the state of the parent culture used for platings; the composition of fructose‐containing media used for the production of variants. Indole, anthranilate and phenol inhibited formation of secondary colonies by most strains. Phenotypically all the variants obtained shared in common the loss of ability to form secondary (aerial) mycelium and spores as well as a tendency of the substrate mycelium to fragment. These traits were shown to be: hereditarily stable on all media tested for variants derived from Streptcmyces ruber, Str. roseolus, Str. lateritius and Str. roseoflavus var. roseofungini; less stable and nutritionally affected for variants derived from Str. albocyaneus, Str. roseoflavus and Str. anthocyaneus; unstable on all except fructose‐containing mineral medium for the variants of Str. flavofungini. Vegetative growth of some of the variants obtained was not inferior to that of parent cultures; some variants produced increased amounts of intracellular antibiotics. Some implications of the reported findings are discussed.

Cultivation and antibiotic activity of mycorrhizal basidiomycetes.

In 17 species (35 strains) of basidiomycetes which form mycorrhizas with Scotch pine (Pinus silvestris L.) the influence of media and method of cultivation on growth and manifestation of antibiotic activity were investigated under laboratory conditions. The antibiotic activity was influenced by the composition of the nutrient media as well as by mode of cultivation, while the ability and intensity of growth depended first of all on the composition of the media.Amanita citrina, Clitopilus prunulus, Lactarius helvus, Rhizopogon roseolus, Russula fragilis, Tricholoma albobrunneum, Tricholoma imbricatum, andTricholoma saponaceum exhibited antibiotic activity. The intracellular antibiotics ofTricholoma saponaceum andRhizopogon roseolus exerted the highest activity. It has been confirmed that the antibiotic activity of the cultures corresponds in most cases to the activity of the fruit-bodies grown in nature.

Incidence and possibilities of screening antifungal non-polyene antibiotics in Actinomycetes.

The author studied 114 strains of actinomycetes recovered from different geographical localities with different vegetation. Under submerse cultivation conditions 57% of the strains displayed antibiotic activity—48% antibacterial, 56% antifungal. As distinct from intracellular antibiotics, production of extracellular antibiotics was manifestly influenced by the composition of the fermentation medium. The commonest antifungal antibiotics were polyenes and non-polyenes, in the ratio 2∶1 Twenty strains produced non-polyene antifungal antibiotics, 14 of which corresponded to substances described in the literature. Only three strains produced antifungal non-polyenes singly; the others simultaneously formed basic antibacterial antibiotics of the type of streptomycin, streptothricin and neomycin or (also) polyene antibiotics, as a third component. Non-polyenes and polyenes did not occur together in the mycelium only or in fermentation fluid filtrate only. No correlation was found between the formation of non-polyenes and the place of origin of the strain. The possibility of screening new antibiotics is discussed.

Mixed Bacterial Infections in Relation to Antibiotic Activities

Summary A high degree of protection was obtained by antibiotic-producing Micrococcus pyogenes var. albus strains of the human ocular microflora in mixed infections of mice with Clostridium septicum. Protection was obtained against at least 1,000 LD50 doses of this latter organism. The protection is most probably associated with in vivo secretion of functioning antibiotics. Strong correlation of in vitro and in vivo results with strains of varying antibiotic properties points to this conclusion. Failure to observe any differences in the abilities of these strains to survive in the tissues tends to rule out the possibility of nonspecific factors being operative. Solid protection was obtained both against the Clostridium septicum spores and vegetative cells. Living micrococci were more protective than heat-killed organisms. The latter apparently owe their small to moderate degree of prophylactic abilities to strongly adsorbed or intracellular antibiotic. This could not be washed off, and attempts to grow these organisms without antibiotic production were failures. Antibiotic-producing micrococci injected at sites removed from the Cl. septicum inoculation site, also seemed to confer a small degree of protection against lethal outcome. Delay in the injection of the antibiotic-producing micrococci until 2 or 5 hours after the Cl. septicum inoculum, greatly reduced the effectiveness of the protection, but did not completely eliminate it. Some of the animals injected after 5 hours had already developed local gas gangrene. The data presented strongly indicate that the human bacterial flora may represent a rich source of potentially valuable antibiotics for certain limited infections.

The binding of penicillin in relation to its cytotoxic action. II. The reactivity with penicillin of resistant variants of streptococci, pneumococci, and staphylococci.

1. In a previous study, the differing sensitivity of bacterial strains as they occur in nature appeared to be correlated with their correspondingly differing reactivity with penicillin. Presumably, the over-all reactivity of the cell with penicillin paralleled that of the vulnerable cell component(s). However, when penicillin-resistant variants of these strains (Streptococcus pyogenes, Micrococcus pyogenes, Diplococcus pneumoniae, and Streptococcus faecalis) were produced by serial passage through increasing concentrations of antibiotic, this correlation between resistance and the ability of the cell to bind penicillin was no longer apparent. Some resistant variants bound more penicillin than the parent, sensitive cell (Streptococcus faecalis, Micrococcus pyogenes), some were unchanged in their reactivity (Diplococcus pneumoniae, Micrococcus pyogenes), and some bound less (Streptococcus pyogenes, Micrococcus pyogenes). One resistant variant of Micrococcus pyogenes at first showed enhanced reactivity with penicillin; on continued passage through antibiotic, there was a further increase in resistance, but now associated with a significantly decreased reactivity. In the case of Diplococcus pneumoniae, a resistant variant at first reacted normally with penicillin; on continued passage in antibiotic, its binding affinity for penicillin gradually decreased, but with no associated further increase in resistance. 2. The reactivity with penicillin of cell-free sonic extracts of the resistant variants paralleled that of the intact organisms. Permeability considerations therefore did not seem involved in the increased resistance produced by serial passage in antibiotic. 3. The penicillin-resistant variants did not have an enhanced capacity to degrade the free intracellular antibiotic. 4. Possible alternative explanations are discussed in the text.