- Volume 47, Issue 3, 1967

As well as the known sporidesmolides I, II and III, Pithomyces chartarum produces sporidesmolides or similar compounds which contain residues of erythro-isoleucine and α-hydroxyisocaproic acid. When erythro-L-isoleucine was added to the growth medium, synthesis of sporidesmolides I and III was inhibited and synthesis of isoleucine-containing sporidesmolides, mainly Sporidesmolide II, was promoted. Threo-L-isoleucine in the medium was poorly utilized, but its presence resulted in production of a very complex mixture of sporidesmolides containing a higher proportion of isoleucine residues than the control. Epimerization of erythro-D-isoleucine occurred in the medium. Neither epimer was well utilized, and this isomer had little effect on sporidesmolide production or composition; it promoted some increase in the synthesis of isoleucine-containing sporidesmolides. Threo-D-isoleucine had effects qualitatively similar to but less pronounced than those of erythro-L-isoleucine, probably because of extracellular epimerization. The use of epimeric pairs of amino acids as tools for investigating biochemical processes involving inversion of configuration at an amino acid α-carbon atom is suggested.

The catabolic pathways for the utilization of glucose and gluconate by one strain of each of five species of the genus Corynebacterium were examined. The relative participation of concurrent glucose pathways in each of these five strains was estimated. The findings indicated that Corynebacterium hoagii ATCC 7005 and C. tritici ATCC 11408 displayed catabolic behaviour similar to that of Arthrobacter globiformis ATCC 8010 in that the glycolytic pathway played a major role in glucose catabolism. In contrast, in C. equi ATCC 7698 and C. sepedonicum ATCC 9850, the glycolytic pathway and the pentose cycle pathway played almost equally important roles in glucose utilization. The catabolic pathway functioning in C. xerosis ATCC 7084 was distinct from these other strains of Corynebacteria and resemble that of some Acetobacter strains in that the pentose cycle pathway played a predominant role in the utilization of glucose and gluconate.

Swarming long forms of Proteus arose from short forms at the edge of a colony on nutrient agar. Specialized small pre-swarmer forms were not seen. The long forms contained many nuclear units and did not have cross walls while actively swarming. Long forms were not found in liquid media. Chemical analysis of short and long forms showed that cell walls of the two forms had qualitatively the same amino acid composition and that inhibition of DNA synthesis was not responsible for the formation of long forms. Long forms differed from short forms in having no detectable amino acid pool, a characteristic that may be associated with excessive flagellar synthesis by developing long forms.

The ability of various compounds to support swarming of Proteus was determined by making additions to a minimal medium agar on which swarming did not occur. Swarming occurred when alanine, asparagine aspartic acid, glutamic acid, glutamine, proline or serine were present, either individually or together. It did not occur when certain other amino acids were added individually to minimal medium agar but did occur when these were added together. The ability of a compound to support swarming was correlated with its ability to serve as a carbon+energy source and with the stimulation of growth rate. The swarming phenomenon is discussed in the light of these findings.

Radioactive incorporation techniques have been employed to investigate the effect of isonicotinic acid hydrazide (INH) on biosynthetic reactions in Mycobacterium tuberculosis var. bovis BCG. INH has no measurable effect on incorporation of 32P phosphate, 35S sulphate or 14C glycine into whole cells, although it inhibits by 50% the incorporation of 14C acetate. Incorporation of 32P into trichloracetic acid (TCA) insoluble components of supernatant solutions from centrifuged disrupted bacteria appears to be inhibited soon after the cells are exposed to INH. Similar experiments with 35S show a lag of 30–40 hr before incorporation is influenced by the drug. Chemical fractionation of organisms grown in the presence of 32P show that the major effect of INH is exerted on incorporation into the ribonucleic acid fraction.

The incorporation of 14C INH into sensitive and resistant bacteria has been investigated. Only organisms susceptible to INH took up the drug. Uptake was irreversible and became saturated at higher INH concentrations. Uptake was heat-labile, KCN-sensitive, partially inhibited by dinitrophenol and stimulated by SH group reagents. The activation energy of binding was of the same order as the activation energy of an enzyme catalysed reaction. Uptake at 37° was rapidly inhibited although at 4° it was not. The INH binding mechanism had many of the properties of catalase and peroxidase. It seems likely that all these functions were part of one enzyme which was missing from INH-resistant BCG.

Chromatographic studies on hot-water extracts of INH-treated BCG indicated that INH was converted into several unidentified products within the organism.

The extracellular production of ‘free endotoxin’ from Escherichia coli sero-type O78K80 is discussed. Isolation of purified toxic and non-toxic fractions from crude extracellular material and the physico-chemical properties of these fractions are described. Purified free endotoxin possessed similar properties to endotoxins extracted from the bacteria by conventional procedures; a purified non-toxic extracellular fraction corresponded to the so-called ‘native hapten’. When dissolved in buffers containing sodium dodecylsulphate, the non-toxic fraction was not disaggregated, but the endotoxin produced sub-units of a smaller particle size than the non-toxic material. From these experiments it was concluded that the non-toxic material did not constitute simple sub-units of endotoxin.

Tris and EDTA combined to form a homologue which has far greater chelating ability than EDTA alone. The weaker serine and NH4Cl homo-logues of EDTA replaced EDTA + tris in causing toxicity and permitting lysozyme lysis in Azotobacter vinelandii. Serine could effectively substitute for tris in these systems for Pseudomonas aeruginosa. These compounds had relatively little effect on Escherichia coli. The toxicity of EDTA + tris for these organisms could be alleviated by pre-incubation in physiological saline (0–15 M-NaCl). Subsequent lysozyme lysis was prevented by this treatment. The removal of metals from A. vinelandii cysts by EDTA + tris was also inhibited. The EDTA + tris + metallo chelate complexes were inactivated (dissociated) by saline.

Washed suspensions of Entodinium caudatum, grown in vitro, incubated anaerobically in the presence of penicillin and neomycin, incorporated single amino acids into the cell protein without conversion to any other amino acid. The 14C-labelled components of the protozoal ‘pool’ and the medium were also investigated but no extensive catabolism of the amino acids observed. At low external concentrations the amino acids were taken up by an ‘active’ process, but above a critical concentration the amino acids entered the cell by passive diffusion and they have been divided into two groups depending on whether this critical concentration was approximately 0–0001 M or 0–001 M. The rate of amino acid uptake was not altered by the presence of inert particulate matter. Of the 75% of the cell volume occupied by liquid, approximately two-thirds was freely available to amino acids.

Non-multiplying and growing cultures of Entodinium caudatum incubated anaerobically engulfed Escherichia coli bacteria specifically labelled with individual 14C-amino acids and incorporated the amino acids into protozoal protein without conversion to any other amino acid. The protozoal cisternae ‘pool’ and the medium contained the free amino acid and with some amino acids the N-acetyl or N-formyl derivative in addition. The constituents of the pool were probably by-products of the metabolism of the bacteria and not intermediates between bacterial and protozoal proteins. There was no extensive catabolism of the bacterial amino acids by the protozoa, although some of the bacterial leucine, isoleucine and valine was broken down to isovaleric acid, α-methylbutyric acid and isobutyric acid, respectively. The addition to the medium of the 12C-form of the 14C-amino acid present in the E. coli decreased the incorporation of 14C into the protozoa with half of the amino acids tested. The rate of loss of viability of various other bacterial species after engulfment by the protozoa was found to be independent of Gram-reaction, size or natural habitat.