- Volume 55, Issue 1, 1969

SUMMARY Explants of young tumour tissue from several Brassica species infected with Plasmodiophora brassicae plasmodia gave rapid callus growth on medium containing 2,4-dichlorophenoxyacetic acid and coconut milk. Degeneration of the callus followed as resting spores were formed. The resting spores germinated in situ and primary plasmodia and zoosporangia developed in some cells, then secondary plasmodia reappeared and normal callus growth was resumed; thereafter all stages of the parasite life-cycle were present in active cells. This situation has been maintaned for over 18 months by 8weekly transfer of calluses to new medium. Callus clones containing only vegetative plasmodia were established on a coconut-milk + 2, 4-dichloro-phenoxyacetic acid medium. Sporogenesis did not occur readily in these clones unless callus was transferred to a kinetin + α-naphthylacetic acid medium or to other media which did not contain 2,4-dichlorophenoxyacetic acid. Stages of the P. brassicae life-cycle found in callus appeared similar to those in intact hosts, while zoosporangia were formed in the root hairs of Sinapis alba organ cultures infected with callus-produced resting spores. It was concluded that on suitable media P. brassicae may be maintained in complete balance with host callus.

SUMMARY The gas mixtures from cultures of Saccharomyces cerevisiae inhibited growth and sporulation of Aspergillus niger and germination of seeds of Lepidium sativum. In test conditions seven volatile organic metabolites in the culture gases were identified by gas-liquid chromatography (GLC) as acetaldehyde, ethyl acetate, ethanol, n-propanol, isobutanol, and a mixture of isopentanols (1 part 2-methyl-butan-1-ol to 2 parts 3-methyl-butan-1-ol); changes in the concentrations of CO2 and O2 were also measured. Pure samples of each of these components were tested at the concentrations found in the culture gases in order to identify the inhibitory substances.

Inhibition of growth of Aspergillus niger could be produced by these culture gas concentrations of acetaldehyde and of ethanol. Inhibition by this concentration of CO2 was just significant in these tests. The effect on sporulation could be produced by the CO2, but not by these concentrations of any of the other identified components.

The effect of Lepidium sativum seed germination could be produced by these culture gas concentrations of ethanol and of 3-methyl-butan-1-ol to a lesser extent. Slight effects were also observed with a lowered O2 concentration and with a raised CO2 concentration but not with the other constituents.

SUMMARY The capillary tube method is found to be better than the hanging drop method for examining the motility of cultures of Clostridia in different media.In the capillary tube, Clostridium butyricum and C. sporogenes moved away from air and oxygen gas-phases at the meniscus. Reasons for the failure to show this negative aerotaxis in C. septicum are discussed. The motility of C. sporogenes was activated by l-arginine.

SUMMARY: The effect of streptomycin, penicillin, polymyxin, chloramphenicol, novobiocin, erythromycin, d-cycloserine, 8-azaguanine, and 6-azauracil on competence development in Haemophilus influenzae was studied.

d-cycloserine and 6-azauracil had no inhibitory effects on either competence development or cell viability in concentrations that were inhibitory to growth. Penicillin and polymyxin reduced cell viability and competence development proportionately thereby maintaining a high percentage of transformation in the treated cultures. Chloramphenicol, novobiocin, 8- azaguanine, erythromycin, and streptomycin inhibited markedly competence development with little or no effect on cell viability.

SUMMARY: The antigenic properties of the soluble and membrane-bound proteins of Mycoplasma organisms were studied by a variety of gel-diffusion techniques. The strains tested belonged to several distinct serological groups, showing no cross-reactions. One group consisted of the Mycoplasma laidlawii strains and the other of caprine and bovine strains, which were closely related to Mycoplasma mycoides var. mycoides. The avian strains tested were serologically heterogenous. After Mycoplasma membranes were solubilized by sodium dodecyl sulphate (SDS) the antigenic properties of their membrane proteins were analysed. In immunodiffusion tests they showed a serological specificity similar to the soluble cell proteins. This specificity was not found in lipids of M. laidlawii membranes extracted with chloroform + methanol. In gel diffusion tests the hydrophobic protein fraction isolated from M. laidlawii membranes by detergent action and (NH4)SO4 precipitation reacted with antiserum to whole organisms, was antigenic in rabbits and there produced an antiserum which reacted with it, but not with the soluble cell proteins or membrane material solubilized in SDS.

Summary: Using non-flagellate (fla −) mutants of gn-complex antigenic derivatives of a stable phase-1 strain of Salmonella abortusequi, the positions of the antigenic specificity-determining sections on the genetic map of H1, a structural gene for flagellar protein, were located by establishing a system for selecting intra-H1 recombinants. Before the recombination experiments, a factor analysis of the gn-complex antigens of the strains was made by cross-absorption tests: nine factors were detected.

P22 phage-mediated transductions were carried out between different pairs of H1-linked fla − mutants, whose H1-alleles were different and whose fla − shes were on opposite sides of H1 The fla + transductants, present as swarms in semi-solid medium, were isolated and the composition of their flagellar antigens examined with specific antisera. Among 1366 fla + transductants obtained, 26 clones were shown to have ‘recombinant antigens’ carrying some factors of one or both of the parental-type antigens. With several ‘recombinant antigens’, some new specificities with weak antigenicity were detected. The flagellar protein, ‘flagellin’, of an ‘antigen recombinant’ was proved to be a recombinant of the two parental flagellins by finger-printing analysis of tryptic digests.

On the assumption that these ‘antigen recombinants’ resulted from a single cross-over within H1, the relative positions of the antigenic specificitydetermining sections within donor and recipient H1 with regard to the crossover point were obtained for individual ‘antigen recombinants’. Summarizing the relative positions thus inferred, it was shown that each of the antigenic specificity-determining sections maps as a unit; and that as a whole they form a linear array within H1. Sections specifying some tryptic peptides of flagellins were also mapped within H1.

Summary: Polygalacturonase (PG) was produced by Penicillium expansum in rotted apples and in liquid culture medium containing pectin; acetone precipitates of the enzyme were prepared from both sources. The PG extracted from apples (in vivo PG) behaved as an endo-PG and degraded sodium polypectate to tetragalacturonic acid. PG from culture filtrates (in vitro PG) before purification had quite different properties, the final hydrolysis product being mono-galacturonic acid. PG from both sources was purified by ion-exchange chromatography. The properties of some purified PG preparations were quite different from those of the crude material. None of the purified in vitro PG released monogalacturonic acid from sodium polypectate, whilst some preparations from both sources had identical endo-PG properties after purification. Changes in the properties of endo-PG produced in vivo and in vitro were apparent during purification and a series of distinct endo-PG’s were obtained which yielded unresolved oligomers, tri- or tetragalacturonic acid as final hydrolysis products. It is suggested that the changes in behaviour of the various PG preparations during purification were brought about by changes in the molecular configuration of the enzyme, and that such changes could account for some of the variability between enzymes produced in vivo and in vitro.

Summary: The action of the first enzyme in the argmine pathway of Proteus mirabilis strain 13 is controlled by feed-back inhibition and the formation of arginine-synthesizing enzymes is influenced by the concentration of the end-product. Concentrations of arginine below 10 μg./ml. induce their formation, but higher concentrations result in repression. The variations in enzyme synthesis caused by changes in the arginine content of the medium are markedly smaller than those observed in Escherichia coli. The carbon source affects the response of enzyme synthesis to arginine. Two mutants argR-1 and argR-2 which excrete arginine have higher enzyme levels than the wild type. In one of these the enzymes are repressible and in the other mutant the enzymes are de-repressed. The sites of these two mutants map closely in a locus argR which is linked to a his marker but not to any of the structural arginine genes. The de-repression of enzyme synthesis in these mutants is limited and less than in argR mutants of E. coli. The derepressed state of argR-2 gives it a growth advantage over the wild type if arginine is withdrawn. The derepressed mutants also exhibit a changed response to feedback inhibition.

SUMMARY: Pairs of non-tetracycline-producing mutants of Streptomyces rimosus or S. aureofaciens were grown side by side on agar. Their ability to produce antibiotic by cosynthesis was tested by placing a strip of agar cut from the combined culture on plates containing Bacillus subtilis. The activity was revealed as an inhibition halo formed on B. subtilis, opposite one or other mutant strain. The strain surrounded by the halo was considered as a converter of an intermediate product secreted by the other strain. Two types of mutants were observed: a rare type probably affecting the main pathway of antibiotic biosynthesis, and a more frequent type probably affecting some regulatory process.

Summary: Methods by which the R-factor 1818 was transferred from Escherichia coli K12 to different species of Gram-negative bacteria are described. Possession of 1818 resulted in the production of similar amounts of R-factor specific penicillinase per organism in E. coli, Serratia marcescens, Alkalescens sp. and Aerobacter aerogenes. The Aerobacter strain produced additionally a ‘chromosomal’ penicillinase which had no influence on the level of R-factor enzyme.

Two different strains of Proteus mirabilis containing R-factor 1818 produced only about th of this penicillinase activity per bacterium Two further R-factors 7268 and TEM also specifying penicillinase were introduced into theP. mirabilis strains and enzyme activities per bacterium of about th and th, respectively, were obtained compared with those in E. coli. All organisms in cultures of P. mirabilis exhibited properties of R-factor possession and thus R-factor instability cannot explain these results. It would seem that the phenotypic expression of R-factor penicillinase genes is impaired in this species.

SUMMARY Optimal conditions, defined by the use of a known auxotrophic strain, are described for the mutagenesis of Coprinus lagopus oidia by N-methyl-N′- nitro-N-nitrosoguanidine (NTG), which is a very effective mutagen for bacteria and yeast. The oidia were best treated in phosphate buffer (pH 6·8) with a concentration of 15 μg. NTG/ml. for about 70 min. The yield of auxo- trophs was considerably less than might be expected from a comparison of other systems in which NTG has been used as a mutagen.

SUMMARY: Antisera to nine strains of Mycoplasma gallisepticumwere prepared in rabbits, chickens and turkeys, the response of individual rabbits or birds varying widely. Minor antigenic differences between some of the strains were shown in metabolic inhibition (m.i.) tests with rabbit antisera. The strains could be divided into at least three subtypes. In general, antibody diminished so that it was barely detectable 6 months after inoculation.

Antibody titres measured by m.i. were lower in chicken and turkey antisera than in rabbit antisera. The choice of the mycoplasma strain to be used in the m.i. test for the measurement of antibody in the bird sera was of fundamental importance since not all strains were capable of detecting antibody; strain T37 was the most useful.

Antibody measured by m.i. was not destroyed by repeated freezing and thawing of antisera, and the antibody titres were not, in general, increased by the addition of unheated guinea-pig serum to the tests. The titres of antibody which inhibited metabolism of the mycoplasma strains were closely similar to those which inhibited their growth in liquid medium. On solid medium, however, some rabbit antisera with high m.i. antibody titres did not inhibit or inhibited poorly the development of colonies of the homologous strains. Only one of the nine rabbit antisera inhibited the development of colonies of all the strains, a feature of importance in mycoplasma identification.

SUMMARY: The amount of haemolysin produced by Escherichia coli grown under various gas phases was determined by the amount of growth obtained under these conditions. Calcium or strontium was required for activation of haemolysin. The haemolytic reaction was stopped by sodium citrate. The loss of haemolytic activity after incubation with trypsin and chymotrypsin indicated that at least the active group of the haemolytic molecule is protein or a peptide.

SUMMARY Some factors affecting induction and repression of steroid-transforming enzymes were studied in single and mixed cultures of Arthrobacter simplex, Nocardia restrictus and Streptomyces roseochromogenes. In single or mixed cultures induction and repression of i-dehydrogenase, 16α-hydroxylase, and 20-ketoreductase were found to be dependent on the type of steroid substrate and the composition of the medium. In mixed cultures patterns of growth as well as enzyme induction and repression were altered by culture interaction. An observation having practical significance was that 16α- hydroxylation and i-dehydrogenation of 9α-fluorohydrocortisone to triamcinolone could be done, in a single fermentation operation, by a mixed culture of A. simplex and S. roseochromogenes. The practicality of this transformation derives from the fact that 20-ketoreductase, an enzyme responsible for the production of undesirable by-products in cultures of A. simplex ,was completely repressed in the mixed culture system. Although growth of A. simplex was suppressed by metabolites of S. roseochromogenes, conversion of steroids with this mixed culture was reproducible and controllable within a wide range of ratios of organisms in the starting mixtures.

SUMMARY: Bacitracin at concentrations of 6·5–13·0 µg./ml. inhibited the growth of Mycobacterium smegmatis atcc 607. Concentrations 10 times higher were required to produce a similar effect on M. tuberculosis bcg. Assays of the bacitracin effect in Sauton medium showed no significant drug inhibition due to the cancelling effect of citrate ions present in this medium. Low concentrations of bacitracin produced smooth-surface colonies consisting of bacteria which showed a modified cell-wall appearance, in contrast to the rough colonies formed by control bacteria. Ultrastructure studies indicated that the main target of bacitracin action on mycobacteria may be a direct effect on the membrane system.