- Volume 64, Issue 3, 1970

SUMMARY: Staphylococcus aureus synthesizes pyrimidines using a pathway similar to that established in Escherichia coli. Soluble and bound forms of dihydro-orotate dehydrogenase are present. Both forms are active in 2,6-dichloro-phenolindophenol reduction assays; that the particulate enzyme normally links to oxygen via a cytochrome system was shown in studies of a haem-requiring mutant.

Activities of the enzymes of pyrimidine synthesis are six- to 20-fold greater in anaerobically or semi-anaerobically grown bacteria than in those grown in air, unless uracil is present. Aerobic cultures rapidly accumulate dihydroorotate in the medium after transfer to anaerobic conditions; derepression of the dehydrogenase also ensues.

These findings show that the requirement for uracil displayed by Staphylococcus aureus when growing anaerobically is due to its inability to dehydrogenate dihydroorotate in the absence of oxygen: they cannot explain the ability of Escherichia coli to grow anaerobically without uracil.

SUMMARY: The effect of increasing concentrations of glucose and galactose on the physiology and composition of Saccharomyces cerevisiae was studied in continuous culture. Glucose, at concentrations ranging from 1 to 50 g./l. was degraded predominantly by aerobic fermentation. Galactose catabolism was mainly aerobic at concentrations up to 5 g./l. but became increasingly fermentative at higher sugar concentrations. The fatty-acid content varied with the type of metabolism. Thus at low concentrations of sugar, fatty acids accounted for 8 % dry wt but about 5 % dry wt at high sugar concentrations. High sugar concentrations may be associated with the repression of mitochondrial structures.

The size of the amino acid pool was a function of the availability of ammonia in the medium. Such ‘pools’ contained appreciable amounts of arginine when NH4+ was in excess, whereas in the absence of NH4+ from the culture filtrate only trace amounts of arginine were detected. The content of glutamic dehydrogenase in the yeast varied directly with the availability of ammonia and the size of the amino acid pool.

SUMMARY: Spores of Bacillus cereus were inactivated by γ-radiation more effectively in the presence than in the absence of a variety of halogen compounds. Where homologous series of compounds were used, activity decreased in the order I > Br > CI and F. Activity did not depend upon affinity of the compounds for thiol groups. Potassium iodate was the most active compound tested. Iodate and iodoacetamide (IAM), and to a lesser extent iodide, were most effective at low pH values, but potentiation by 3,5-diiodo-L-tyrosine was hardly affected by pH value. Survivor curves of spores irradiated in water plus IAM or iodate were convex, suggesting that potentiation increased with increase in γ-radiation dose. The compounds tested were all more effective potentiators of radiation inactivation of spores in water than in sodium phosphate buffers or in complex medium, and potentiation was greater with low than with high spore concentrations. Histidine antagonized the potentiation of radiation inactivation by iodate, iodide and iodoacetamide. Potentiation decreased with decrease in temperature, and was not shown at - 15°. The results suggest that potentiation resulted from toxicity to spores of halogen-free radicals, and to a lesser extent free halogen, formed by reaction of the compounds with the radiolytic products formed from water during irradiation.

SUMMARY: Whereas γ-radiation alone either did not affect or stimulated spore germination, spores which had been irradiated in the presence of iodoacetamide (IAM) or potassium iodate were inhibited from germinating in germinants like L-alanine, inosine or yeast glucose broth. Activity of a lytic enzyme which may be involved in germination was mostly lost in spores irradiated in IAM, and a phosphomonoesterase was about 60 % inhibited. Spore alanine racemase was partly inactivated by γ-radiation alone but, with spore protease, survived irradiation in IAM without much further loss of activity. Although spores made non-germinable by γ-radiation plus IAM were non-viable as measured by colony counts, viability could be partly restored by chemical treatments known to by-pass the normal germination reactions. It was concluded that spores were inactivated by γ-radiation in the presence of the potentiators through inactivation of enzyme(s) essential in the degradative reactions of germination. The enzyme inactivation was caused by iodine-containing free radicals rather than directly by the γ-radiation.

SUMMARY: Spontaneous mutants with altered flagellar antigen were isolated from a phase-1 stable Salmonella strain, tr 6, whose phase-1 antigen consists of at least three antigenic factors, g3, g4 and f. Three kinds of antisera reacting with each of these factors were used as the selective agents. The mutants were isolated by picking from swarms in semisolid medium containing the respective antisera. Mutants were classified into two groups. A group comprising four mutants selected with antiserum to g3 and two mutants selected with antiserum to g4, in which all three antigenic factors examined were simultaneously altered; and a second group comprising all the other mutants, in which only a single factor corresponding to the selective antiserum was altered. All the altered factors retained weak cross-reactivity with the original factors. In all cross-absorption-agglutination tests between each of the mutant antigens and the wild-type antigen, activity of the antisera was fully absorbed, showing that the mutant antigens had neither lost any antigenic specificities nor gained any new specificity. Transductional analysis showed that the sites of mutation were inseparable from H 1, the structural gene for phase-1 flagellin. Tryptic peptide patterns of flagellins of two mutants were compared with that of the wild-type strain and a difference of one or two peptides was detected. From these results it is concluded that these mutants possessed a minor change in primary structure of flagellin which resulted from a mutation in the H 1 gene.

SUMMARY: Tryptophan auxotrophs were isolated from Staphylococcus aureus strain 655 following mutagenesis with ultraviolet radiation, N-methyl-N'-nitro-N-nitrosoguanidine or ethyl methanesulphonate. The mutants were placed in five mutant classes on the basis of auxotrophic response to and accumulations of tryptophan intermediates or derivatives. The tryptophan loci were mapped by comparing relative distances of each locus from two reference loci, trp B and trp E. Relative distances of loci were determined by ratio test analysis of cotransduction data. The tryptophan loci were shown to be contained on a cluster on the S. aureus genome; the mapping data suggested the gene order: trpE, trp(DF), trpC, trp(DF), trpB, trpA. The genetically distinct trp(DF) regions presumably control two enzymic functions in the conversion of anthranilic acid to 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate.

SUMMARY: Mutants of Pseudomonas aeruginosa 8602 were isolated which, unlike the wild-type strain, utilized butyramide as a growth substrate. One group produced amidases with altered substrate specificities, indicating that the mutations may be in the amidase structural gene. The second group of butyramide-utilizing mutants produced wild-type enzyme and were all constitutive. It was concluded that such mutants possessed mutations in an amidase regulator gene which allowed them to synthesize large amounts of amidase in the presence of butyramide.

Induction of amidase synthesis by N-acetylacetamide in the wild-type strain was repressed in a competitive manner by butyramide and other amide analogues, e.g. cyanoacetamide. The constitutive mutant c11, which could not utilize butyramide for growth, was subject to severe repression of amidase synthesis by butyramide and cyanoacetamide. The regulator mutants which were able to grow on butyramide were all less sensitive to repression by butyramide. Mutant cb 2, a butyramide-utilizing regulator mutant, was relatively insensitive to repression of amidase synthesis by cyanoacetamide whereas strain b6, a butyramide-utilizing mutant producing an altered enzyme, was as sensitive to repression by cyanoacetamide as its parent strain the constitutive mutant c11. The contribution of mutations in structural and regulator genes to utilization of novel growth substrates is discussed.

SUMMARY: Eight strains of Pseudomonas aeruginosa, induced by penicillin G, produced different amounts of β-lactamase. Significant enzyme activity appeared within 5 min. and its highest value was obtained 3 h. after the addition of penicillin G. The production of the enzyme was arrested shortly after removal of the inducer. High concentrations of penicillin G, other penicillins or cephalosporins were necessary for optimal induction. 6-Amino-penicillanic acid (6-APA) was the best inducer tested, while cephalothin was the worst. Most of the enzyme was intracellular after conversion of the cells to spheroplasts in the presence of carbenicillin.

The structure of both the nucleus and side chain of the penicillins and cephalosporins determined the rate of their hydrolysis by the β-lactamase. Generally, the enzyme was more active against 7-aminocephalosporanic acid (7-ACA) derivatives than against 6-APA derivatives, except cephalexin and cephaloglycin, which were relatively resistant to hydrolysis.

No relation was found between the sensitivity of the pseudomonads to penicillin G and the amount of the enzyme in their cell-free preparations. The sensitivity of the different antibiotics to hydrolysis by the enzyme was not a major determinant in the resistance of Pseudomonas aeruginosa 1978 to them. These results indicate that the resistance of the bacteria to the β-lactamase-sensitive penicillins and to cephalosporins is dependent on a combined effect of β-lactamase and on an intrinsic resistance, while the resistance of the bacteria to the β-lactamase-resistant penicillins depends on the intrinsic resistance alone.

SUMMARY: Single-step erythromycin-resistant strains of Streptococcus pyogenes 56188 fell into two classes: whereas class A mutants were sensitive to lincomycin and carried mutations transducible by phage A 25, class B mutations conferred lincomycin cross-resistance and failed to be transducible under the conditions employed. Recombination and cotransduction experiments allowed the assigning of at least 10 of 13 class A mutations to the same linkage group. From interclass crosses recombinants emerged at frequencies comparable with those at which the donor markers were integrated into wild type. The resistance to three macrolides and lincomycin of interclass recombinants, carrying mutations which were not cotransducible, was related to the resistance levels determined by the individual mutations. The pheno-typic properties of the ery mutants together with their genetic relationship lead to the conclusion that there are at least two loci governing resistance to erythromycin in strain 56188 of S. pyogenes.

SUMMARY: Diploid isolates of Hansenula holstii Wick, and H. wingei Wick. usually produce two or three ascospores per ascus. An unusual nuclear event during meiosis might account for less than the expected number of four. When acridine orange or Feulgen staining was used, meiosis in both species followed the pattern established in higher plants and animals. Deoxyribonuclease treatment destroyed nuclear staining material. Six chromosomes were seen during prophase I. Three bivalents seen during metaphase I divided and moved to two poles with loss of distinct chromosomes for the remainder of meiosis. When only two or three spores were formed in an ascus, apparently normal nuclei, not enclosed in spore walls, were pushed against the ascus wall. The haploid (n) chromosome count in both H. holstii and H. wingei was shown to be three.