- Volume 49, Issue 1, 1967

Application of the anomalous diffraction theory of van de Hulst to turbidity measurements of Gram-positive bacteria is discussed. It is shown that this method is capable of detecting characteristic morphological changes during growth. Comparison with the cell mass method and direct observations of Micrococcus freudenreichii suggest that these changes are primarily related to cluster size. Initial application of the method in the study of osmotic change in Bacillus megaterium is described.

‘Whole’ and autolysed motile-phase cells of Dermatophilus congolensis were examined after negative staining with phosphotungstate. Motile-phase cells and cells in other developmental stages were examined in thin sections. The integument of motile-phase cells was homogeneous and about 30 mμ thick and was associated with an outer diffuse capsule-like matrix. Three types of intracytoplasmic organelles and occasionally a large fluid-filled vesicle were present. Flagella varied from a few to more than 50 per cell; their diameter was estimated to be approximately 8–9 mμ. In autolysed cells, the flagella were seen to be attached to differentiated regions of the cytoplasmic membrane. The peripheral zone of the integument of cells in all developmental stages appeared to undergo sloughing. In ‘young hyphal’ and ‘packet’ cells, stratification of the integument was frequently observed. Germinating cells of D. congolensis were larger than motile-phase cells and contained characteristic translucent membraneless cytoplasmic inclusions. The general anatomical features of D. congolensis, especially of germinating cells, closely resembled those of Streptomyces violaceoruber.

Haemin dependent mutants were isolated from Bacillus subtilis after (a) ultraviolet irradiation and selection by streptomycin, (b) exposure of the organisms to copper ions and (c) treatment with N-methyl-N′-nitro-N-nitro-soguanidine. The isolates were grown in a synthetic medium containing haemin when substances able to neutralize inhibitory factors and to stabilize the medium were included. Two of the mutants were capable of growth in media supplemented only with δ-aminolaevulinic acid, the first intermediate of porphyrin synthesis. Transformation of the mutants to haemin independence was accomplished using deoxyribonucleic acid.

By subcultivating a streptococcal z3 III strain in medium containing anti-III serum a strain lacking the type antigen was isolated. Evidence is given that this strain possesses only z3 antigen. From partial acid hydrolysates of formamide extracts of both z3 and z3 III bacteria the same disaccharide was isolated. The most probable structure of the disaccharide is 3-O-α-N-acetyl-d-glucosaminoyl-N-acetyl-d-galactosamine. It was 250 times more active than a-methyl-JV-acetylglucosamine in the inhibition of the z3/anti-z3 system. This denotes that it is an important part of the determinant group of the z3 antigen. By ethanol fractionation of a formamide extract of z3 III bacteria two distinct fractions were isolated. The first fraction reacted only with type III antiserum and consisted of glucose, galactose and rhamnose in the ratio 5: 3: 1. The second polymer was composed of rhaninose, glucosamine and galactosamine in relative amounts of about 2 : 1 : 1. Chemical and serological evidence suggests very strongly that this is the z3 antigen. The similarities between z and true group antigens are discussed.

The carotenoid pigments of Sarcina flava (Staphylococcus afermentans) and S. lutea were extracted, purified and separated by thin-layer chromato-graphy into seven fractions. These fractions were hydrocarbon or polar materials. The two bacteria appeared to synthesize identical pigments which could be complexed in vivo to different proteins or to different amounts of a protein, which would explain the apparent differences in the colour of colonies of the two bacteria when grown on nutrient agar.

A mutant growing as long filaments was regularly observed in continuous cultures of Bacillus cereus strain t. The filaments had approximately the same diameter as the parent bacilli and septa were observed at regular intervals, corresponding to the length of single bacilli of the parental type. The links between the individual organisms in the filaments appeared to possess high mechanical strength, as shown by their resistance to ultrasonic treatment. Lysozyme treatment resulted in a complete fragmentation of the filaments into bacilli of the same size as single normal organisms. Electron microscopy showed that the septa of the filaments were thinner than those displayed by dividing normal organisms. A zone of lower electron density, which developed in the contact zones between dividing normal bacilli in the early stages of division, was not observed in the links between the filamentous bacilli.

Thirteen strains of motile enterococci showed more similarity, in their physiological reactions, to Streptococcus faecium than to S. faecalis. A serological study of the type antigens divided the motile strains into four sets ; (1) three strains previously described as S. faecium serotype 29, (2) one strain reacting as S. faecium serotype 38, (3) five strains showing a specific reaction with antiserum prepared to one of them (‘serotype 4725’), (4) four untyped strains. Esterase and protein patterns from the soluble fractions of the motile strains were examined by electrophoresis in polyacrylamide gel and were different from those of non-motile strains of S. faecalis, S. faecium and S. durans. Extracts of the three motile strains of S. faecium type 29 showed a common esterase pattern, extracts from five strains of serotype 4725 showed three different esterase patterns. The motile strain of S. faecium serotype 38 gave extracts with a strong esterase band which differed from the very weak bands shown by extracts of non-motile strains. Differences in esterase pattern could be found between motile strains whether untyped, of different serotypes, or of the same serotype. Comparison of the ‘protein patterns’ of motile strains gave some indication that major protein bands occurred at similar positions after electrophoresis.

A mutant of Salmonella typhimurium produced straight flagella in phase 2 (antigen-1,2) and normal flagella in phase 1 (antigen-i). The straight flagella were observed by light microscopy and electron microscopy either with or without formalin fixation. Flagellar bundles of the mutant bacteria prepared in 0·25% methylcellulose (w/v) and examined by dark-field microscopy were also found to be straight. It was shown by electron microscopy that the component flagella of the straight flagellar bundle were in most instances irregularly twisted about each other. Heteromorphous bacteria which had straight flagella and either normal or mini-small-amplitude flagella were seen at a frequency of 10–13 % among the bacterial clones in phase 2. The bacteria with straight flagella were non-motile but they were sensitive to bacteriophage χ, which is known to infect motile bacteria of Salmonella species. In transduction, using phage P22 grown on a normal flagellar strain and the phase 2 straight strain as recipient, transductional clones with normal flagella in both phase 1 and phase 2 were obtained. The transductional clones showed the antigen of the recipient in phase 1 and that of the donor in phase 2. This indicated that the straight mutant originated by a mutation of the structural gene of phase 2 flagellin. In absorption-agglutination experiments with antisera prepared against flagella of either normal-1,2 or straight-1,2 no antigenic difference between normal and straight flagella could be detected.

Production of pili directed either by the sex factor F or by the drug-resistance factor R 100 in Escherichia coli K 12 is regulated by a gene which acts by producing a repressor, and the mutant R 100–1 no longer produces this repressor. The specific pilus determined by R 100–1 resembled the F pilus morphologically, but differed in its affinity for F-specific RNA phages. Mutants of F and R 100–1 which had lost the ability to synthesize pili could each restore to the other the ability to produce pili on mixed infection of the same host.

The expression of R-gene resistance in tissue cultures of Solanum species was investigated. Tissue aggregates (4 mm. diam.) of Solanum tuberosum var. Majestic (rr) and Orion (R I) and other R-gene Solanum lines behaved in a similar way to the intact plants from which they were derived, in response to Phytophthora infestans race 4. Tissue aggregates of S. tuberosum var. Orion supported good growth of P. infestans race I, to which the intact Orion plant is susceptible, but did not support more than rudimentary growth of race 4, to which the intact Orion plant is resistant. Sectioning revealed that P. infestans race 4 made general growth in Majestic tissues, but was almost totally excluded from Orion tissues. A slide technique showed that Orion tissue suspensions, unlike those of Majestic, inhibited the growth of P. infestans race 4 germ tubes. This led to a demonstration that toxicity to P. infestans zoospores was developed in Orion but not in Majestic tissue cultures in response to infection with P. infestans race 4. It was concluded that R-genes may be expressed in tissue culture, that the failure of the Orion tissue to support growth of P. infestans may be due to expression of the R 1 gene, and that this may operate through the development of post infectional toxicity.

The aerosol survival of Escherichia coli jepp, a particularly sensitive strain, has been studied in a nitrogen atmosphere (< 99·9%) when sprayed from suspension in solutions of sodium glutamate or glycerol, and collected into phosphate buffer with or without m-sucrose. The response caused by changes in relative humidity (RH) to 30% and 100% before collection was also examined. These two protecting agents were selected because glycerol readily permeated the cytoplasmic membrane, whereas sodium glutamate did not. E. coli jepp sprayed as a suspension in sodium glutamate and collected in phosphate buffer showed two minima in the survival versus RH curves: (i) a comparatively narrow zone between 90 and 82% RH; (ii) a broad instability zone between 82 and 50% RH. The presence of sucrose in the collecting fluid increased the survival for storage above 50% RH, but decreased it below 50% RH. For storage above 50% RH a change to 100 % RH before collection increased the survival for collection in phosphate buffer but not in phosphate buffer + sucrose; below 50% RH this change decreased the survival. A change to 30% RH before collection had little effect. In similar experiments with glycerol a gradual decline in survival occurred as the RH value was decreased and no critical minima were apparent. Addition of sucrose to the phosphate buffer collecting fluid increased survival. A similar improvement was obtained by changing the RH to 100% before collection into phosphate buffer. The combined effect of having sucrose in the collecting fluid and an RH change were not additive, and were somewhat antagonistic for aerosols stored at low RH values. A change to 30% RH before collection decreased the survival. These results are compared with those for E. coli jepp sprayed from suspension in distilled water and in solutions of raffinose, and show that at high RH values glycerol provided the best protection. At low RH values glycerol was inferior to sodium glutamate and raffinose, which were similar. The importance of the manner in which water re-enters the bacterium on collection is discussed.

At high relative humidity (RH) similar survivals were obtained for storage in oxygen, air or nitrogen. At low RH the survival in nitrogen was much greater than that in air or in a 20 % (v/v) oxygen + 80 % (v/v) nitrogen mixture, in which the survivals were similar. In oxygen alone, survival was even lower than in air. Hence oxygen, or a trace contaminant in it, was responsible for the poorer survival in air than in nitrogen. The mechanism of death caused by oxygen is discussed.

Three strains of group G streptococci isolated from a community in which glomerulonephritis is common were found to have an M-protein antigen indistinguishable from the type 12 M-protein of group A, type 12, streptococci.

When grown in broth cultures containing sodium chloride and glucose, Staphylococcus aureus JHM produced protein, RNA and DNA in the culture fluid. Maximum yields were obtained with about 4 % (w/v) sodium chloride and 0·1 % (w/v) glucose. Accumulation of macromolecules began at the end of exponential growth and was accompanied by a decrease in culture turbidity. Electron microscopy revealed considerable cellular lysis, arising from rupture of newly formed septa. Before lysis occurred, the cocci exhibited many morphological abnormalities. After incubation for about 15 hr the culture showed evidence of fresh growth and the resulting cocci were characterized by abormally thick and uneven walls.

The results suggest that there was interference with the regulation of cell-wall synthesis, perhaps due to loss of mesosomes, which were never seen in the cytoplasm of organisms grown in sodium chloride glucose broth.

The influence of Mg2+-limitation on the growth of a typical Gram-positive organism—Bacillus subtilis—was investigated and the data compared with that obtained with Aerobacter aerogenes grown under similar conditions. The magnesium contents of both organisms varied with growth rate but were very similar at corresponding growth rates. With Mg2+-limited chemostat cultures of each organism, uptake of Mg2+ was almost complete at specific growth rates less than 0·7 × maximum. Cellular Mg2+ was tightly bound, none being removed by suspension of the organisms at 20° in 0·85% (w/v) NaCl. When Mg2+-limited organisms were suspended in environments containing Mg2+, this ion was rapidly adsorbed; the amount adsorbed varied with both the initial extracellular Mg2+ concentration and the composition of the diluent. B. subtilis had a greater capacity for Mg2+ adsorption than A. aerogenes but its affinity for this ion was less. The latter difference correlated with the ability of A. aerogenes to outgrow B. subtilis rapidly in Mg2+-limited chemostat cultures containing both organisms. The significance of these results is discussed in relation to the reports from other laboratories concerning differences between Gram-positive and Gram-negative bacteria in Mg2+ content, uptake of Mg2+ and ability to grow in media of low Mg2+ content.

The frequency of spontaneous and ultraviolet-induced, recessive, lethal mutations was estimated in two diploid strains of Aspergillus nidulans. The parent diploids were heterozygous for recessive and semi-dominant markers; each recessive lethal resulted in failure to recover haploid strains carrying the allele linked in coupling to that lethal. Diploids 1 and 2 carried markers permitting direct assay of lethals on 4 and 8 chromosomes, respectively, out of the total diploid complement of 16. No lethals were detected in 100 isolates from each untreated diploid. One hundred isolates from each treated parent yielded 6 and 16 lethals, respectively, suggesting a frequency for the whole genome, at 5 % survival, of about 28 % lethals. The lethals were nutritionally irreparable and were not temperature-sensitive. Several of them have been located roughly by mitotic crossing-over.

One isolate was heterozygous for two unlinked lethals. It segregated to give a stable heterokaryon bearing two classes of non-viable haploid conidia. Meiotic analysis, via a three-component heterokaryon, showed that the stable heterokaryon was balanced by the non-allelic lethals, one in each haploid component. Another isolate had a balanced lethal system resulting from linked, non-allelic lethals in trans.

Six isolates from treatment were stable diploids which produced no haploid sectors.

The carotenoids of Sarcina flava were examined; four main fractions were separated. These were hydrocarbons, mono- and di-hydroxylated compounds and a very polar fraction. Further separation of the hydrocarbon fraction showed the presence of nine compounds and of the very polar fraction which showed an all-trans form and three cis isomers. Data for these fractions is presented; at least two of the fractions are C50 carotenoids.

Unusual swollen and branched cell forms were produced in a thymineless mutant of Escherichia coli strain c by treatment with mitomycin C, and to lesser extents, by incubation without thymine and by exposure to ultraviolet light. In the case of mitomycin C treatment the morphological changes were accompanied at first by degradation of deoxyribonucleic acid (DNA), later by degradation of ribonucleic acid (RNA) and inhibition of protein synthesis, and throughout by inhibition of DNA synthesis and extensive loss in viability. Thymineless incubation prevented DNA synthesis and also resulted in extensive killing. Cultures incubated after exposure to ultraviolet light exhibited a small amount of DNA degradation and a lag in DNA synthesis. Upon prolonged incubation with mitomycin C or without thymine many of the abnormal forms became very enlarged and eventually lysed. No evidence of bacteriophage or bacteriocin could be detected in the treated cells.