- Volume 130, Issue 4, 1984

The effect of antimicrobial agents on the intracellular multiplication of Legionella pneumophila in cultured guinea-pig peritoneal macrophages was measured. Beta-lactam antibiotics at concentrations 5 to 400 times the MIC in vitro did not inhibit the intracellular growth of the organism. Gentamicin inhibited the growth considerably but failed to eliminate the organism from the phagocytic mixture. Chloramphenicol or tetracycline at 10 μg ml−1 (40 or 5 times the MIC in vitro respectively) did not eliminate the organism. At a higher concentration (30 μg ml−1), however, these drugs eliminated the bacterium from the mixture. Only erythromycin and rifampin were effective in killing the organism at very low concentration (1 μg ml−1). Intracellular multiplication of L. pneumophila was observed clearly by light microscopy using Wright-Giemsa staining.

Summary: Isotopically-labelled Protein A from Staphylococcus aureus was used as a marker to quantify binding of IgG molecules to surface antigens of Aspergillus fumigatus mycelium. The IgG class antibodies were obtained from rabbits inoculated with mycelial fractions and from patients suffering from aspergillosis and aspergillus-related diseases. The highest incorporation of label was obtained with an antiserum from rabbits inoculated with A. fumigatus wall material. Antibodies raised to other antigenic fractions of A. fumigatus and antibodies from patients infected with aspergillus gave lower levels of incorporation of Protein A. In competitive binding experiments, pre-incubation of antibodies with partially purified aspergillus antigens depressed subsequent binding to the mycelial surface by 20-30% of that of the control values. Low molecular weight disaccharides and oligosaccharides were without effect in this system. Preincubation of A. fumigatus with lectins having specificities for defined sugar residues did not reduce subsequent antigen/antibody interaction. Treatment of the mycelial surface with certain proteolytic or polysaccharolytic enzymes led to a decrease in antibody binding, while pretreatment of A. fumigatus with the hydrolytic enzyme mixture of Trichoderma harzianum culture filtrate gave increased antibody binding. Aspergillus species showed different susceptibilities to enzyme action and their surface structures could be differentiated from A. fumigatus on this basis. These differences were not obvious in direct binding experiments where antibodies raised to A. fumigatus wall bound with equal facility to antigenic sites located on the walls of other Aspergillusspecies.

A water-soluble mycelial extract of Aspergillus fumigatus has been fractionated by preparative isoelectric focusing using carrier ampholytes in a layer of granulated gel. The separated components were located by staining paper prints from the gel. Within a narrow pH range of 2·5 units, multiple protein bands were visualized with Coomassie Brilliant Blue G. Periodate-Schiff-positive material was generally associated with the major protein zones. When these fractions were eluted the total recovery, calculated on the basis of protein and carbohydrate analyses of the isolated fractions, varied between 20 and 60% of the applied material. Low recoveries were associated with low recoveries of protein; recoveries of carbohydrate were higher and less variable. The immunological activity and specificity of the eluted fractions were assessed in an enzyme-linked immunosorbent assay for the detection of IgG antibodies to A. fumigatus.

The effect of polycationic polypeptides (polylysine, polyarginine and polyhistidine) on the invasion of mammalian cells and plant protoplasts by Toxoplasma gondii was studied. In JM cells, a human lymphoblastoid cell line with T cell characteristics, all polycationic polypeptides used increased the invasion rate in a concentration-dependent manner. This effect and the morphological changes revealed by electron microscopy resembled the action of the penetration-enhancing factor previously described by E. Lycke and co-workers. Plant protoplasts of Catharanthus roseus, which are resistant to T. gondii invasion, showed the same morphological changes in the presence of polycationic polypeptides as observed for JM cells, but were not invaded.

Heterocysts of the cyanobacterium Cylindrospermum licheniforme occur at the ends of the filaments. These cells, specialized for aerobic N2 fixation, synchronously differentiate after the fragmentation of filaments grown in a medium free of combined nitrogen. This study has examined the role of transcription during and after heterocyst differentiation. Autoradiography of intact filaments pre-labelled for 3 h with [3H]uracil revealed that RNA synthesis occurred at similar rates in vegetative cells, developing (pro)heterocysts and mature heterocysts. Since mature heterocysts no longer divide and contain a similar amount of DNA as vegetative cells, transcription must continue under conditions where replication no longer occurs. Rifampicin, when added to fragmented filaments at concentrations that reduced RNA synthesis by 95% in all cell types, inhibited proheterocyst formation but not the final morphological step of differentiation (pore plug deposition). However, chloramphenicol blocked all stages of heterocyst differentiation. Since the average mRNA half-life was 29 min, and the rifampicin-insensitive stage lasted 3–6 h, the later stages of differentiation may depend on long-lived mRNAs.

The stimulation by betaine of vitamin B12 and extracellular coproporphyrin production in the industrial species Pseudomonas denitrificans GY57/2 was confirmed using a replacement culture technique; biosynthesis of haem was also found to be stimulated. In the absence of betaine, δ-aminolaevulinic acid synthase decreased in specific activity from time zero. In the presence of betaine, the enzyme activity increased markedly after 3 h and then decreased after exhaustion of extracellular betaine. The peak specific activity of δ-aminolaevulinic acid synthase increased in a dose-dependent manner. The enzyme was inhibited by haem. δ-Aminolaevulinic acid dehydratase was present in considerable excess over δ-aminolaevulinic acid synthase under all conditions studied and its specific activity was not affected by the addition of betaine. Our tentative conclusion is that the betaine effect is exerted via some positive effect on δ-aminolaevulinic acid synthase, the initial, rate-limiting and very labile enzyme of porphyrin and corrin biosynthesis.

A strain of Rhizobium sp. (ORS571), isolated from stem nodules on Sesbania rostrata, was grown as a N2-fixing continuous culture for up to five weeks (> 80 culture replacements). Under optimum conditions of O2 supply [< 9 m dissolved O2, 100160 nmol O2 (mg dry wt)−1 min−1], N2 fixation rates were 300400 nmol N2 (mg dry wt)−1 h−1 and nitrogenase activity in culture samples reached 2000 nmol C2H4 (mg dry wt)−1 h−1, the highest values yet recorded for any Rhizobium species. Other properties of the continuous cultures are described and a comparison is made with previous reports of N2 fixation by cultures of Rhizobium spp. Significant features were the greater tolerance to O2 of ORS571 and the retention of fixed N in the cells of this strain.

Rhizobium leguminosarum WU235 expresses aspartase (EC 4.3.1.1) when grown on aspartate or asparagine as the sole carbon source, but not on glucose or fumarate. Cells grown on glucose plus aspartate, or fumarate plus aspartate, do not express aspartase. Although these results are reminiscent of catabolite control of an inducible enzyme, induction of aspartase cannot be demonstrated in this strain. Aspartase-producing cells synthesize the enzyme after repeated subculture on glucose plus NH4Cl. Cells grown in glucose plus NH4Cl and plated onto aspartate produce different colony sizes; the larger (0·1% of the total) express aspartase, while the smaller do not. At dilutions sufficient to exclude the large aspartase-producing colonies, a single-sized, aspartase-negative colony was found initially. Such colonies later developed papillae or became cluster colonies; aspartase was produced with papillae formation. The aspartase producing strains were shown, by analysis of native plasmids and periplasmic proteins and by the use of antibiotic resistant strains, to be derived from the parental type. The data suggest that strain WU235 is unable to produce aspartase unless a mutation occurs which leads to constitutive enzyme synthesis. The significance of these observations for studies claiming catabolite repression in Rhizobium is discussed.

Summary: Methods for the direct visualization of F and type 1 pili of Escherichia coli in the light microscope are described. The method for visualizing F pili is based on the specific adsorption of fluorescent dye-labelled RNA phages to F pili. The best results were obtained with MS2 phages labelled with rhodamine B. Semi-quantitative determination of the amount of F pili is possible. Type 1 pili can be visualized rapidly and specifically by indirect immunofluorescence. Other structures on the cell surface are neither detected by, nor interfere with these assays. By using different fluorescent dyes the two methods can be combined and both F and type 1 pili can be determined in the same sample.

Summary: F and type 1 piliation of various Escherichia coli K12 Hfr and F+ strains was re-examined by using a new visualization assay. In anaerobic cultures in rich media, bacteria were well F piliated throughout all growth phases. In aerobic cultures in rich media, F piliation reached a maximum in the mid-exponential phase. The yield of pili was up to 15 mg 1−1. In aerated cultures. F pili disappeared in the late exponential phase. In rich media F pili were on average 10-20 μm long; in synthetic media they were an order of magnitude shorter, and less numerous. Addition of metabolic poisons (NaCN, NaN3, Na3 AsO4, phenylethyl alcohol) and starving the bacteria caused rapid disappearance of F pili under aerobic conditions, but had no influence on F piliation under anaerobic conditions. Type 1 piliation was not influenced by these drugs or by an alteration of growth conditions.

DNA preparations from two reference (H37Ra and H37Rv) and two wild strains of Mycobacterium tuberculosis and one re-isolated strain of Mycobacterium bovis BCG were analysed using 17 restriction endonucleases. The enzyme BstEII revealed the greatest differences between strains. Electrophoretic DNA patterns from the wild M. tuberculosis strains differed from each other and from the reference strains at relatively few positions. At the highest resolution attained, patterns from the two reference strains remained indistinguishable from each other. The pattern of the M. bovis BCG strain was substantially different from, but had many bands in common with, the M. tuberculosis patterns.