- Volume 35, Issue 1, 1964

The ability of some α-haemolytic micrococci described by Clausen (1964) to form lactic acid from glucose has been determined by means of ‘resting’ bacterial cells. The total quantity of lactic acid was measured in each individual test. Reference organisms used were a strain of Aerococcus viridans, 2 enterococcus strains, and 1 strain of Staphylococcus epidermidis. Glucose conversion was found to be in large measure dependent upon the concentration of active bacterial cells in the suspension. All the α-haemolytic micrococcus strains tested, together with the reference strains, proved to be homofermentative or near homofermentative formers of lactic acid, provided that the number of active organisms was brought to a sufficiently high level in the suspension when converting the glucose.

Some lactic acid bacteria formed detectable H2O2 and some did not, regardless of their preference or requirement for aerobic or anaerobic conditions. Whether or not H2O2 was formed depended in some instances on the substrate used as energy source. Two H2O2-splitting activities were encountered though never in the same organism. One, named pseudo-catalase activity, was insensitive to 0·01 m-azide or 0∙01 m-cyanide and appeared to be the action of an acid-sensitive non-haem-containing enzyme detectable in some leuconostocs and pediococci when grown in media containing a low concentration of glucose. The second, named catalase activity, was detected in a number of lactobacilli, leuconostocs, streptococci and pediococci grown on media containing haematin or heated blood; presumably these organisms are able to synthesize the apoenzyme but not the prosthetic group of catalase. This activity was inhibited by 0∙01 m-azide or 0∙01 m-cyanide; it was not acid-sensitive. There was little correlation between H2O2-splitting activity and the preference or requirement of the organisms for aerobic or anaerobic conditions, or between H2O2-splitting activity and H2O2 formation. Of a few organisms examined, some appeared capable of forming cytochromes when grown in media containing heated blood. One showed traces of a cytochrome whether grown in the presence or absence of heated blood.

A sensitive and simply calculated complement-fixation test is described in which viral antigen and antibody react to completion for up to 90 min. at 37° before the addition of one 70 % haemolysis unit of complement. The test has been applied to reactions between the components of the virus system of foot-and-mouth disease, tomato bushy stunt virus, bovine plasma albumin and their rabbit or guinea-pig hyperimmune sera. In the restricted region of proportional fixation each μl. of complement represents about 3 × 109 virus particles and 6 × 1011 molecules of albumin. In suspensions of foot-and-mouth disease virus of highest infectivity (1010 mouse ID50/ml.), probably less than 1 in 30 of the characteristic 25 mμ particles are infective.

Quantitative data define the regions of antibody excess, equivalence and antigen excess, and show that complement is bound as a secondary process to that of antigen/antibody complex formation, that complement is bound only by relatively massive complexes and that the independent formation of unrelated complexes in the same system may be sensitively detected. The location of the optimum reaction and the confirmation of equivalence allow fixation data to be related closely to parallel data on the neutralization of infectivity. Since the concentrations of antigen and of antibody for optimal fixation are almost independent, it is concluded that the maintenance of equivalence in terms of a constant antigen/antibody ratio is not a valid principle for the interpretation of such data.

The point in the growth cycle at which a strain of brewer’s yeast became potentially flocculent could be delayed by supplementing the medium with ammonia, basic amino acids, glutamine, asparagine, γ-aminobutyric acid or urea. Other amino acids were ineffective. β-Alanine and 2-chloro-4-aminobenzoic acid led to an abnormally early appearance of potential flocculence. No development of flocculence occurred in the absence of glucose. It is suggested that the maintenance of non-flocculence is dependent upon the presence in the cell wall of a nitrogenous compound; potential flocculence will develop when this compound is not synthesized at a rate sufficient to maintain its concentration in the wall. The nitrogenous nutrients which delay flocculation would then act by enhancing this rate of synthesis.

The flocculation of a strain of brewers' yeast was absolutely dependent upon the presence of calcium; a concentration of 200 mm-CaCl2 was sufficient to ensure almost complete flocculation. No other metal could replace calcium; several metals aggregated potentially flocculent cells but also aggregated non-flocculent cells. Sodium ions antagonized the action of the calcium. The effects of pH value and esterification suggested that carboxyl groups were involved. The flocs had a ‘melting temperature’ of 50–60° and were dispersed by urea, suggesting that hydrogen bonds were also present.

Non-flocculent yeast was aggregated when the dielectric constant of the medium was decreased by the addition of organic solvents, but this aggregation was also dependent on the presence of traces of calcium. Conversely, increase of the dielectric constant of the medium, by adding formamide, dispersed flocculent yeast. Certain specific sugars also dispersed flocculent yeast. It is suggested that flocculent yeast cells are linked by salt bridges formed by calcium atoms joined with two carboxyl groups in the surfaces of different cells and that this structure is stabilized by hydrogen bonds formed between complementary patterns of carbohydrate hydrogens and hydroxyls in the cell surfaces.

A temperate bacteriophage named ‘tau’ which forms plaques on F- strains but not on F+strains of Escherichia colik 12 has been isolated. This phage adsorbs equally well to F+ and F-strains, which indicates that some later step of the growth cycle is inhibited by the presence of the sex-factor F. The frequency of lysogenization of the phage is also affected by the F factor. The genetic material of phage tau may be DNA. The phage is 40–50 mμ in diameter.

Ascorbic acid undergoing auto-oxidation inactivated vaccinia virus. Copper ion was shown to have a catalytic effect on the inactivation. Neither unoxidized ascorbic acid nor its oxidation product, dehydroascorbic acid, were inhibitory. When ascorbic acid was oxidized at high pH in the absence of copper ion no inactivation took place. Similarly, enzymic oxidation of ascorbic acid in the absence of copper was without effect on the virus. Catalase prevented inactivation but not the oxidation of ascorbic acid. Glutathione prevented both inactivation and the oxidation of ascorbic acid. Inhibition experiments with ascorbic acid under anaerobic conditions were inconclusive. The mechanism of ascorbic acid inactivation is discussed in the light of these data and that of other authors with different viruses.

Addition of orthophosphate to Aerobacter aerogenes strain a3(0) organisms previously subjected to phosphate starvation induced accumulation of inorganic polyphosphate within the organisms. With resumption of growth the polyphosphate was degraded and served as a source of nucleic acid phosphorus. During phosphate starvation the specific activity of polyphosphate kinase and inorganic polyphosphatase increased five- to tenfold while the amount of alkaline phosphatase increased 50 times. The results suggest that synthesis of polyphosphate kinase and alkaline phosphatase was subject to repression by exogenous orthophosphate. Two mutant strains blocked in polyphosphate accumulation were found to carry defects in the synthesis of these enzymes. Mutants of class Pn-1 contained normal amounts of all three enzymes, but repression of their synthesis was not annulled by phosphate starvation. Mutants of class Pn-2 contained no polyphosphate kinase. It is suggested that synthesis of polyphosphate kinase is controlled by two genes, a structural gene and a regulator gene; the latter gene also appears to control the synthesis of alkaline phosphatase and perhaps polyphosphatase. The patterns of polyphosphate accumulation under various nutritional conditions are discussed in relation to the amounts and activities of the enzymes of polyphosphate synthesis and degradation.

Washed suspensions of Entodinium caudatum incubated anaerobically in the presence of penicillin and neomycin incorporated 14C-glycine in the cell ‘pool’ as N-acetylglycine and into the cell protein as glycine without conversion to any other amino acid. At low external concentrations the glycine was present in the protozoa themselves and was not free in the gastric sac or present solely in the intracellular or extracellular bacteria. The incorporation reached a constant value after incubation for 24 hr; after this time the uptake of glycine was balanced by an equal loss of N-acetylglycine from the organisms. These suspensions also incorporated 14C from NaH14CO3 into carbon atoms 3 and 4 of protozoal glucose which was probably present as starch in the intact protozoa.

A group of twelve ultraviolet-sensitive mutants has been isolated after u.v. irradiation of Escherichia coli strain b. The use of crystal violet during irradiation and subsequent growth appears to increase the frequency of such mutants among colonies formed by surviving cells. This effect may be due to post-irradiation selection. Eleven of the mutants were more crystal violet resistant than their parent.

The mutants were compared by determining their u.v. survival curves, the extent of elongation after u.v. irradiation, their ability to repair u.v. induced damage to bacteriophage T1, and their resistance to furacin. They comprised nine different phenotypes. In all but one case, the mutants differed from the parent in more than one property.

Numerical methods were applied to an analysis of the relationships among Salmonella serotypes listed in the Kauffmann-White schema. Although the result suggested a possible new basis for schematic arrangement of these serotypes, a complete and satisfactory classification could not be derived entirely from the computer results. Examination of this outcome suggests some cautions to be observed in the design and interpretation of experiments in numerical taxonomy.

The protein-synthesizing activity of cell-free preparations of Escherichia coli was estimated by adding radioactive amino acids according to the system of Matthei & Nirenberg (1961). Cell-free systems were prepared from antibiotic-sensitive strains of E. coli and from resistant variants, and the sensitivity of amino acid incorporation to inhibition by chloramphenicol and tetracycline determined. The same sensitivity was shown for the sensitive as for the resistant strains.

A complex tissue culture medium supplemented with swine serum and peptone supported optimal growth of Mycoplasma gallisepticum strain 293. Media lacking any of these components supported little or no growth. However, when 5′-monophosphate nucleotides replaced the peptone growth was supported. The minimal nucleotides necessary to support good growth were adenylic, cytidylic, guanylic and thymidylic acids. In some cases the addition of the four ribonucleotides and the four deoxyribonucleotides in place of peptone improved growth; the four ribonucleotides alone supported poor growth. Thymidylic acid seemed essential for growth, and uridylic acid appeared to be inhibitory.

The mixture of ribonucleosides and deoxyribonucleosides, but not of purines and pyrimidines, when substituted for the peptone, also supported good growth. The concentrations of nucleosides and nucleotides had a significant effect on growth. Although the nutritional factors of swine serum were not defined, the effects of different sera on growth were investigated. Rabbit, horse, turkey and swine serum supported optimum growth; human serum supported less, whereas PPLO serum fraction (Difco) or bovine serum supported poor growth. Dog serum did not support growth.

Cystathionase catalyses the formation of homocysteine from cystathionine; its formation in cultures of Escherichia coli is repressed by the presence of methionine in the growth medium, and to a similar extent to that shown with homocysteine methylase (the enzyme complex which catalyses the conversion homocysteine → methionine). Cystathionase, again like homocysteine methylase, is formed rapidly without concomitant growth when repressed organisms are transferred to a medium free from methionine; such enzyme formation is prevented by chloramphenicol, suggesting that de novo synthesis of protein is required. The co-repression by methionine of the two enzyme systems fortifies the evidence that cystathionase is a component of the normal pathway of methionine synthesis by E. coli and consequently that its substrate, cystathionine, is a normal intermediate. Cystathionase preparations also formed pyruvate from cysteine; this activity paralleled that of cystathionase itself when the methionine status of the medium was changed, and it is concluded that the same protein is responsible for both activities.

When a sensitive population of Mycobacterium fortuitum is exposed to streptomycin, two effects of the drug on viability can be experimentally differentiated: (1) a sublethal injury from which the cells recover once streptomycin is removed, and (2) a lethal injury from which no recovery occurs. Protein synthesis is markedly inhibited by streptomycin before sublethal or lethal effects on viability are noted. The effect on protein synthesis of exposure to streptomycin cannot be reversed by washing the cells by centrifugation, but protein synthesis recovers its original rate on further incubation after streptomycin is removed. It is postulated that streptomycin causes both sublethal and lethal injury to cells by irreversibly blocking protein synthesis, possibly by inactivating ribosomes.