Search

AFTER capsulated B. anthracis had grown in a modified synthetic medium of Brewer et al.1, a mixture of growth products was isolated from the glass-filtered medium which gave a precipitate with copper sulphate. Crude polyglutamyl peptide was obtained from this precipitate2. An attempt was made to separate the components of the original mixture of growth products by paper electrophoresis using a tank as described by Flynn and Mayo3, and it was found that the peptide could not be detected using naphthalene black, bromo-phenol blue, ninhydrin or ultra-violet light4. We found it could be detected by taking advantage of the acidic nature of the peptide, using the following technique : (1) paper strips with the peptide applied are run in a suitable buffer, for example, veronal/veronal sodium at pH. 8.6 or acetic acid/sodium acetate at pH 4.5, and dried at 100°; (2) the buffer is washed out with two changes of 80 per cent ethanol; (3) the strips are treated with N/50 hydrochloric acid in 90 per cent ethanol; (4) the acid is thoroughly removed with ethanol.; (5) after drying, the strips are dipped into N/300 alcoholic sodium hydroxide containing 0.04 per cent bromo-cresol purple, which is repeated if necessary until colour differentiation is obtained, and the strips are blotted.

WHEN certain bacteria are suspended in sucrose solution of suitable concentration and incubated with lysozyme, the rigid cell-walls are dissolved away, leaving relatively stable spherical protoplasts1. Lytic enzymes which dissolve the isolated cell-walls of vegetative Bacillus cereus have been found in extracts of mechanically disintegrated resting spores2 and partial autolysates of sporulating cells3 of this organism. When heat-treated, intact vegetative cells were treated with preparations of these enzymes, the walls were dissolved, leaving the coagulated cell-contents apparently unchanged. It has now been found that when viable organisms are suspended in sucrose solution and treated with these enzymes, relatively stable protoplasts are obtained in good yield.

Bacillus anthracis contains a polysaccharide which appears to be part of the cell wall1. Ivanovics2 first isolated the polysaccharide which contained galactose 34 per cent, glucosamine 34 per cent, nitrogen 4.2 per cent and α-carboxyl amino-nitrogen 0.8 per cent. Strange and Belton3 obtained from culture filtrates of B. anthracis a polysaccharide which contained galactose 39–43 per cent, glucosamine 34 per cent, nitrogen 3.9 per cent and α-carboxyl amino-nitrogen 1.26 per cent. The differences between the total nitrogen and glucosamine nitrogen values together with the figures for α-carboxyl amino-nitrogen in these preparations indicated the presence of 5–10 per cent of amino-acid residues. Smith and Zwartouw4 obtained a polysaccharide from B. anthracisgrown in vivo. It contained galactose 38–43 per cent, glucosamine 38–44 per cent, nitrogen 4.0 per cent and α-carboxyl amino-nitrogen 0.3 per cent. Since this preparation satisfied a number of rigorous tests for homogeneity it was concluded that a small peptide moiety (2–3 per cent) was firmly attached to the polysaccharide. We are concerned here with the constitution of this peptide moiety.

THE lethal effect of chilling on certain exponential phase Gram-negative bacteria (‘cold shock’)1 may be due to interference with bacterial permeability control mechanisms2. Evidence supporting this view is that, on chilling, susceptible bacteria release normal endocellular constituents into the environment3 and are more permeable than similar unchilled bacteria to various substances including ribonuclease4. Cold shock also affects the stability of endogenous RNA. Thus, if susceptible bacteria are chilled and then incubated in fresh diluent at 37°, the rate of RNA-degradation increases with the duration of chilling4. In view of these symptoms of cold shock, the protection afforded to chilled bacteria by magnesium ions3 may be due to the metal's stabilizing effect on bacterial membranes concerned with osmotic control and/or endogenous RNA. In this connexion it is well known that magnesium stabilizes isolated protoplast membranes5, spheroplasts6 and ribosomes7. This note reports the use of the ‘optical effect’8 to determine the influence of magnesium on permeability control in exponential phase Aerobacter aerogenes during chilling.

THE endogenous metabolism of micro-organisms in aqueous suspension in the absence of added nutrients may involve the utilization of endogenous protein and RNA1, as well as amino-acid pool constituents and reserve substances such as glycogen and polyhydroxybutyric acid (see reviews by Dawes and Ribbons2). Investigations with Aerobacter aerogenes1, Escherichia coli and Pseudomonas fluorescens have shown that starvation in aerated saline buffer at 37° results in a considerable loss of protein during a period when viability of the populations remains at 95–100 per cent. Since analyses of ultracentrifugal fractions separated from A. aerogenes after various periods of starvation showed decreases in the protein content of the insoluble, ribosomal and soluble fractions3, the loss apparently represents a depletion of various cell proteins. It is interesting, therefore, that induced β-galactosidase in Escherichia coli was found to be relatively stable while other proteins were being rapidly degraded during turnover4. The relative stability of induced β-galactosidase in a wild-type strain of E. coli during starvation has been investigated and this communication records the relevant data.

IN certain conditions, bacteria accumulate relatively large amounts of polyglucose compounds with properties similar to those of animal glycogen1. An interpretation of bacterial “glycogen” production is that it provides a food and/or energy reserve for the organisms in unfavourable environments; in other words, bacteria rich in glycogen should survive longer than bacteria without such reserves. Experimental results apparently supporting this teleological interpretation were obtained with Aerobacter aerogenes2 and Escherichia coli3 but not with Sarcina lutea4; glycogen-rich S. lutea died at a faster rate than cells without glycogen during starvation in aerated phosphate buffer at 37° C. A feature of glycogen reserves in bacteria is their rapid depletion during starvation which suggests that any contribution glycogen makes towards maintenance and survival is of short duration5. It is possible that growth conditions which stimulate the accumulation of glycogen give rise to bacteria better able to resist stress for reasons not concerned with their glycogen content. The effect of glycogen reserves on bacterial survival was examined with E. coli; cells containing different amounts of glycogen were grown in a chemostat and their survival properties were determined in aerated saline phosphate buffer with and without magnesium at 37° and 48° C.

AN enzyme system catalysing the synthesis of glucosamine-6-phosphate from glutamine and fructose-6-phosphate or glucose-6-phosphate has been demonstrated in Neurospora crassa 1,3, rat liver2,3 and Escherichia coli 3. Ghosh et al. 3, who purified the enzyme from these various sources and studied its properties, have called it L-glutamine-D-fructose-6-phosphate transamidase. We have determined transamidase activity in fresh, cell-free extracts of Bacillus cereus, Micrococcus lysodeikticus, Aerobacter aerogenes and Escherichia coli. Results indicate that, for a given weight of cells, activity is higher in Gram-positive than in Gram-negative bacteria, and for a given organism, activity increases markedly during the lag and early exponential phases of growth, but decreases during the stationary phase. The high transamidase activity associated with dividing cells is probably related to the synthesis of cell-wall basal structure4, which contains amino-sugars. During these investigations, using a system with standard conditions of pH, substrate concentrations and reaction time at 37°, it was found that the amount of glucos-amine-6-phosphate synthesized rarely exceeded 25 per cent theoretical, and frequently was not proportional to enzyme concentration when different volumes of the same cell extract were tested in parallel. An attempt was made to explain these findings.

CEBTAIN bacteria in the logarithmic growth phase become non-viable when chilled in aqueous suspension. The phenomenon has been observed with strains of Escherichia coli1, Pseudomonas pyocyanea2 and Aerobacter aerogenes3. Loss of viability of logarithmic phase A. aerogenes suspended in chilled phosphate-saline is accompanied by the leakage of endogenous solutes, and the leakage products, in sufficient concentration, protect the bacteria against the lethal effect3. Thus, loss of viability is greater the sparser the population and is insignificant in chilled suspensions containing more than 5 × 109 organisms/ml. Correlation between loss of viability and leakage of endogenous solutes has now been obtained by observing the effects of chilling on three Gram-negative bacteria.