- Volume 57, Issue 2, 1969

Of 20 unidentified Streptomyces isolates tested, all inhibited Mucor ramannianus and 18 inhibited Glomerella cingulata in agar. Agar discs from inhibition zones, or paper discs placed beneath inhibition zones, of nine of the Streptomyces isolates caused new inhibition zones when transferred to fresh, seeded agar plates. Transferable inhibition zones were not produced by the other 11 isolates. Inhibitory substances were produced in liquid cultures by eight of the nine isolates which produced them in agar media, whereas no antibiotics were detected in liquid cultures of the other 11 isolates. Glomerella cingulata conidia, which do not require exogenous nutrients for germination, germinated in liquid cultures of non-antibiotic-producing streptomycetes, but failed to germinate in cultures of antibiotic-producing streptomycetes. Inhibition zones produced by non-antibiotic streptomycetes decreased in size with increasing concentration of nutrients, whereas those of antibiotic streptomycetes were unchanged. Glucose and glutamic acid levels in agar rapidly decreased adjacent to streptomycete colonies. Agar, leached of nutrients by sterilized distilled water running slowly through a groove cut in the agar, developed clear inhibition zones. Therefore, inhibition of fungi by streptomycetes in agar, in some cases, appears to be due to nutrient deprivation.

At high concentrations, self-inhibition of germination of pycnidiospores of Mycosphaerella ligulicola increased with rise in temperature at which the spores were formed.

Spores formed at 15° (15° spores), which showed little self-inhibition at high concentrations, and 26° (26° spores), which showed marked self-inhibition at high concentrations, were used for experiments. Mixing 15° spores equally with 26° spores increased inhibition of the latter but the former were unaffected. Leachates from 15° and 26° cultures did not affect germination of 26° and 15° spores respectively, but germ tubes of 15° spores showed increased growth. Inhibition of 26° spores was overcome by washing ten times with deionized water and germ tube growth from washed spores was increased in culture leachates. Diffusates collected from dense suspensions of 26° spores did not affect germination. Diffusates from 15° spores prevented germination of 26° spores but not 15° spores where growth of germ tubes was increased. The inhibitory substance from 15° spores was not readily volatile and not affected by high temperature in solution. Volatile inhibitors were not detected from either 15° or 26° spores. Pfeffer 1 % glucose solution overcame self-inhibition of 26° spores but 1 % glucose or Pfeffer solution alone were ineffective.

Klebsiella (Aerobacter) aerogenes (ncib 418, Bacterium aerogenes no. 240) was grown at dilution rates between 0·1 and 1·0 hr–1 in a variety of nutrient-limited chemostats and the activities of dehydrogenases, particularly glucose (RG), fructose (RF), sucrose (RS), maltose (RM) and gluconate (RGN) dehydrogenases, were determined in intact organisms. Their activities varied from system to system, but, with a few exceptions, were largely independent of the dilution rate. RG was generally high when sugars provided the carbon for growth but when intermediates of the tricarboxylic acid cycle were used it was often low. RGN behaved like RG but RF, RS, and RM were more variable. In general the activities were higher in C-limitation than in other nutrient-limited conditions.

Specific activities of α- and β-glucosidase were also determined in some of the systems. Organisms grown in a maltose-limited system had the highest α-glucosidase activity but 40% of this level was observed in those from a cellobiose-limited system although cellobiose is a β-glucoside. Glucose, fructose, sucrose, lactose or disaccharides containing an α 1 → 6 linkage as carbon sources induced little activity. As expected, growth in cellobiose-limited conditions led to high β-glucosidase activity but melibiose-grown cells were about 50% as active. Replacing NH4 +, the usual N source in the medium, by NO2 – in a glucose-limited system increased the β-glucosidase activity five- to sixfold, while NO3 – led to a tenfold decrease. α-Glucosi-dase was less affected.

The two components of virginiamycin, M and S, separately exerted a reversible bacteriostatic activity on Bacillus subtilis. Their combination increased by a hundredfold the inhibitory activity of each factor and induced a loss of viability of bacteria. Such an irreversible step was preceded by a reversible phase, which was characterized by a long lag in colony formation.

Very short incubation with single virginiamycin components and their combination suddenly and completely blocked protein synthesis, whereas the rate of incorporation of labelled bases and nucleosides into polynucleotides was not altered appreciably unless protein formation was halted completely.

Nevertheless, some alterations of ribosomal RNA metabolism occurred very early after treatment with virginiamycin. The synthesis of 23S rRNA was specifically inhibited. Moreover, the degree of methylation of the rRNA which was made in the presence of the drug was lower than that of the controls. Also, the rRNA labelled in virginiamycin-treated cells was metabolically unstable. This indicates that formation and stability of rRNA, as well as the balance among rRNA species, depend on virginiamycin-sensitive protein synthesis.

Metabolism of pulse-labelled RNA was also altered in the presence of virginiamycin: its half-life was prolonged about sixfold by single components and eightfold by their combination. This was due to an increased turnover of rRNA and to prevention of messenger RNA decay.

It is concluded that peptide chain formation is the primary target of virginiamycins M and S (hence their synergistic antibiotic activity), that translation—not transcription—is prevented by these inhibitors, and that the alterations of nucleic acid metabolism are due to the halt of protein synthesis.

Addition of virginiamycin M or S before the virulent phage 2 C prevented Bacillus subtilis from lysing, and this effect was increased 100-fold by simultaneous addition of both factors. When the antibiotics were administered at the end of the eclipse phase, the viral cycle was shortened by virginiamycin S, prolonged by factor M and halted by the two inhibitors together. Virginia-mycins also prevented the accumulation of phage particles by inhibiting the synthesis of viral precursors during the eclipse period as well as during the maturation phase.

Synthesis of cellular macromolecules was decreased (not suppressed) in Bacillus subtilis after infection with phage 2C, and the degree of repression was a function of the input multiplicity. Viral inhibition of thymidine and uridine incorporation into host DNA and RNA was prevented when virginiamycin was added before infection but unaffected when addition was made at the end of the eclipse phase. Virus and virginiamycin had additive non-overlapping effects on protein synthesis. Moreover, virginia-mycins interfered with the function, not the formation, of RNA pulse-labelled after infection, and prevented its decay.

It can be concluded that virginiamycin blocks (a) the preferential translation of viral message, (b) the mechanism by which virus halts host-macro-molecule formation, and (c) the synthesis of viral DNA. This can be explained by an inhibitory action of virginiamycin on the synthesis and function of virus-dictated proteins.

Aerobic enrichment at 30° in a medium containing either cis, cis- or cis, trans-muconic acid as the sole source of carbon and energy is a highly specific method for the isolation of Pseudomonas acidovorans from soil. Nutritional studies with previously isolated strains of P. acidovorans and P. testosteroni, none of which was selected for the ability to utilize muconates, show that nearly all strains of both these species grow readily and promptly with both cis isomers of muconic acid. This ability is absent from all fluorescent pseudomonads examined, despite the fact that they possess the requisite enzymic machinery. The fluorescent pseudomonads appear to be impermeable to the muconic acids, and can use them as substrates for growth only through a mutation that alters the permeability of the cell.

Studies with one strain of Pseudomonas acidovorans show that it synthesizes inducibly all the enzymes responsible for the conversion of catechol to β-ketoadipate, their synthesis being elicited by growth with either cis, cis- or cis, trans-muconate. Mutants of P. acidovorans defective in the synthesis of either β-ketoadipate enol-lactone hydrolase or β-ketoadipate succinyl-CoA transferase are unable to grow at the expense of cis, cis-or cis, trans-muconate; this confirms the role of the β-ketoadipate pathway in the dissimilation of these two substrates by P. acidovorans.

The lysis of Escherichia coli following exposure to EDTA in tris buffer is similar for most F+, F– and Hfr strains. Survival and release of acid soluble nucleotide material was identical in male and female strains. Following osmotic shock by the EDTA + tris method, regrowth patterns of F–, F+ and Hfr strains showed no significant differences. Physiological buffers—Tes, Hepes, Bicine—cannot replace tris in EDTA + lysozyme lysis of E. coli or in osmotic shock to release surface enzymes. Tes, Hepes and Bicine can replace tris in causing lysis of Pseudomonas aeruginosa either with EDTA alone or with lysozyme.

Thin sections of an anaerobic filamentous oral organism possibly related to Leptotrichia buccalis have been examined in the electron microscope. The organism exhibited a cell wall profile characteristic of Gram-negative bacteria, viz. a solid membrane and an outer double-layered membrane. The outer membrane could be removed by treating intact cells with trypsin or phenol-water. The organism contained numerous intracytoplasmic vacuoles.

Perithecial initiation is strongly inhibited by visible light in less than 18-hr-old hyphae of Sordaria fimicola which are grown in the presence of 1 μ m-6-thioguanine. It is known that 6-thioguanine is destroyed when exposed to light in vitro with oxygen and a sensitizing dye like methylene blue is present. The most effective wavelength region for perithecial inhibition is the blue region, indicating that possibly flavins are the photosensitizing compounds in this case. The synthesis of carotenoid and melanin pigments is also affected by blue light in this organism. Another, transient, inhibitory effect on perithecial production is found when light-grown cultures are transferred to darkness. As a consequence of the two light effects, mycelia exposed to alternating light and dark periods in growth tubes develop well-defined zones of perithecia. A simple state-input model is presented in the Appendix to demonstrate how such zonation patterns can be generated on the basis of the light effects and a few other assumptions.

The ease of isolation of two mutants of Streptococcus faecalis 8043 appeared to be the result of the presence in the parent strain of a system for slow synthesis of lysine. The mutation to lysine independence occurred when the population became large enough for a significant number of spontaneous mutational events to occur. The slow growth and lysine depletion lysis of the parent strain selected for the new strain. The hydroxylysine-resistant mutant can arise from the parent strain or from the lysine-independent strain and is selected for by growth in a medium containing hydroxylysine. The three strains appeared to be identical by all tests employed except in their responses to lysine and hydroxylysine.

Previous work has suggested that there are six divisions among the viridans-like streptococci of man. Reciprocal transformations were carried out between strains classified as Streptococcus milleri agg., S. sanguis agg. and S. viridans agg. The results of the quantitative transformation reactions confirmed a classification of these strains arrived at partly by traditional methods and partly by numerical methods.

Cells of Haemophilus influenzae grown with or without competence promoting factors, inosine and lactate, were compared in their ability to bind deoxyribonucleic acid (DNA) and to undergo a genetic transformation. It was found that cells grown with the above factors bound 70% of the 14C-labelled DNA and transformed to a relatively high level (1·4 to 2·5 %). Cells grown without the factors bound only 8 % of the 14C-labelled DNA and transformed to a low level (0·01 % or less).

It is suggested from these studies that during growth cells of Haemophilus influenzae use inosine and lactate either to form or modify that portion of the cell surface that can later become involved in binding DNA.

The growth of some strains of Mycoplasma hyorhinis on solid medium was inhibited by rabbit antisera incorporated in filter-paper discs. Other strains were resistant to the same antisera. Rings of precipitate were observed in the agar around the discs. The largest number of precipitation rings and the most intense ones occurred with antiserum-sensitive strains. The antiserum-sensitive strains were also more sensitive than the resistant strains to colistin and polymyxin B when these antibiotics were incorporated in paper discs. Colonies of antiserum-resistant strains developed on solid medium containing low concentrations of horse serum, while colonies of sensitive strains developed only on solid medium containing higher concentrations of serum. The growth of antiserum-sensitive strains, but not resistant ones, was suppressed on solid medium at pH 6·5. All these differences between sensitive and resistant strains were only expressed on solid medium; the phenomena were not observed when the organisms were grown in liquid medium. It is suggested that the differences between sensitive and resistant strains are due to changes in the composition of the mycoplasma membranes.

Mutants of Pseudomonas aeruginosa 8602 were isolated which, unlike the wild type, were able to grow with butyramide as a carbon source. Six mutants derived from the constitutive strain c 11 were shown to produce an enzyme (B amidase) with altered electrophoretic mobility and altered substrate specificity. The apparent K m for butyramide of the B amidase was about a tenth of that of the A amidase and the V max was about ten-fold greater. A further mutation produced mutants able to grow on valeramide.