- Volume 135, Issue 5, 1989

The dynamic nature of Chlamydia trachomatis inclusions was studied by video and 35 mm timelapse photomicrography of live cells, and by immunolocalization of inclusions in fixed cells. A serotype E isolate was used to infect the McCoy cell line and endometrial epithelia. Then resulting inclusions were observed over 4 d. They appeared as slowly expanding fluid-filled membrane vesicles whose growth varied considerably, and which were subject to great physical distortion by the host cell during interphase and mitosis. When this distortion became extreme the inclusion was observed to divide. However, as inclusions were mobile within the cytoplasm and thus able to come into contact with each other, there was a net tendency for the opposite process of inclusion fusion to occur when cells contained more than one inclusion. The proportion of infected cells decreased with time as a result of host cell proliferation, despite transmission of inclusions to progeny at the time of mitosis. Inclusion growth physically disrupted karyokinesis and cytokinesis so that host cell division became distorted or blocked on the second or third day of infection. Cell death eventually occurred by a very rapid lysis event.

The mechanism of adhesion of Lactobacillus fermentum strain 737 to mouse stomach squamous epithelium was investigated. Adhesion inhibition tests involving chelators, monosaccharides, periodate and concanavalin A and the use of bacteria grown in the presence of tunicamycin failed to clarify the adhesive mechanism. Washed bacterial cells had reduced adhesive capacity, except in the presence of spent broth culture supernatant fraction or cell washings. Spent culture supernatant fractions of erythrosine-supplemented broth did not enhance adhesion of washed cells. The adhesion-promoting factor(s) in the spent broth culture supernatant fractions and cell washings bound to both bacterial and epithelial cell surfaces, but did not promote adhesion of two other Lactobacillus strains which were not of mouse origin, thereby indicating host specificity for the adhesion-promoting activity. Chemical characteristics of the adhesion-promoting factor were determined by pretreatment of the dialysis retentate of spent broth culture supernatant fractions with proteolytic enzymes, concanavalin A-Sepharose or periodate before the adhesion assay. The adhesin was non-dialysable, pronase-sensitive, heat sensitive at 100 C, had no affinity for concanavalin A-Sepharose and contained no carbohydrate groups active in the adhesion process. The protein profiles of dialysis retentates of spent broth culture supernatant fractions after bacterial growth in the absence and presence of erythrosine were determined by 2-dimensional SDS-PAGE. Gel filtration by HPLC was used for purification of an adhesion-promoting fraction. The host-specific adhesion of L. fermentum strain 737 was mediated by a protein, with an M r of 12–13000, that was not detectable in cells grown in the presence of erythrosine. A model for the mode of binding of the adhesin to host epithelia and bacterial surfaces is proposed.

We have used salicylhydroxamic acid (SHAM) to inhibit intraphagosomal myeloperoxidase activity in order to evaluate the role of this enzyme in the killing of Staphylococcus aureus by human neutrophils. 50 m-SHAM reduced the luminol-dependent chemiluminescence response stimulated during phagocytosis of unopsonized latex beads and opsonized S. aureus by over 80% and 60%, respectively. When opsonized S. aureus were incubated with neutrophils, 45% were killed within 15 min incubation and 60% by 1 h. However, in neutrophil suspensions incubated with 50 m-SHAM, only 13% were killed by 15 min whilst 71% still remained viable after 1 h. This inhibitor had no effect upon the number of bacteria phagocytosed or upon degranulation. In a cell-free system, 25 m-H2O2 alone killed 55% of the bacteria, whereas in the presence of myeloperoxidase (i.e. 10 mU myeloperoxidase and 25 m-H2O2) virtually all of the bacteria were killed: the addition of 50 m-SHAM abolished this myeloperoxidase-enhanced killing but did not affect the H2O2-dependent killing. We therefore conclude that in normal neutrophils whilst H2O2 is required for killing of this pathogen, both myeloperoxidase-dependent and -independent pathways exist.

Outer membranes were isolated from iron-limited and iron-sufficient Azomonas macrocytogenes ATCC 12334 by sucrose density centrifugation or treatment with Sarkosyl. Analysis of the outer membranes by sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that iron-limited cells produced greater amounts of two proteins of apparent molecular mass 74 kDa and 70 kDa than iron-sufficient cells. The enhanced production of these two proteins was evident when media contained less than 9·7 μm added iron with maximum production occurring when media contained less than 1·1 μm added iron. Iron-limited growth conditions also caused A. macrocytogenes ATCC 12334 to excrete a visible yellow, blue-white fluorescent compound into culture supernatant fluids with maximum levels being produced in media that contained less than 1·1 μm added iron. This fluorescent compound had a distinctive pH-dependent absorption maximum characteristic of pyoverdin-type siderophores and appeared to bind iron. Furthermore, culture supernatant fluids containing the fluorescent compound promoted the uptake of 55Fe in A. macrocytogenes ATCC 12334 cells. Three other A. macrocytogenes strains, NCIB 10958, NCIB 8700 and NCIB 9129, produced iron-repressible outer-membrane proteins but only the former two strains produced the iron-repressible fluorescent compound. It is proposed that the iron-regulated proteins and fluorescent compound produced by A. macrocytogenes may function in high-affinity iron uptake.

Using purine auxotrophic strains of Escherichia coli with additional genetic lesions in the pathways of interconversion and salvage of purine compounds, we demonstrated the in vivo function of guanosine kinase and inosine kinase. Mutants with increased ability to utilize guanosine were isolated by plating cells on medium with guanosine as the sole purine source. These mutants had altered guanosine kinase activity and the mutations were mapped in the gene encoding guanosine kinase, gsk. Some of the mutants had acquired an additional genetic lesion in the purine de novo biosynthetic pathway, namely a purF, a purL or a purM mutation. A rev-recd map location of the gsk gene is presented and the gene order established as proC-acrA-apt-adk-gsk-purE.

Cells of mucoid and non-mucoid Pseudomonas aeruginosa in colonies were at least one-thousandfold less sensitive to the antibiotics tobramycin or cefsulodin than were cells of the same bacteria in dispersed suspension. We did not detect any difference between the mucoid form and the non-mucoid form in the antibiotic sensitivity of colonies, from which we infer that the exopolysaccharide of the mucoid form does not contribute to colony-resistance by forming a barrier to antibiotic diffusion. Mathematical models were constructed in order to estimate time-courses of penetration of tobramycin and cefsolodin into biofilms and microcolonies of mucoid and non-mucoid P. aeruginosa. For tobramyen penetration, adsorption of antibiotic to the exopolysaccharide of the glycocalyx and antibiotic uptake by cells were taken into account in the calculations. The longest time-period for the concentration of tobramycin at the base of a biofilm 100 m deep to rise to 90% of the concentration outside the biofilm was predicted to be 24 h. For cefsulodin penetration, irreversible hydrolysis catalysed by lactamase was taken into account, using lactamase levels taken from the literature. The calculations predicted that the cefsulodin concentration at the base of antifilm 100 m deep would rise to 90% of the external concentration in 29 s when the -lactamase was synthesized at the basal level. For a similar biofilm of bacteria synthesizing enhanced levels of lactamase (derepressed), the concentration of cefsulodin at the base was calculated to rise to 41% of the external concentration in about 50s and then remain at that level. This was despite the fact that cefsulodin is a poor substrate for this -lactamase.

Changes in polyamine content and in the activity of ornithine decarboxylase (ODC) were followed during growth and differentiation of the plant pathogenic fungus Sclerotium rolfsii, in submerged mycelium liquid cultures and on solid agar cultures. Mycelial growth in submerged cultures was characterized by high putrescine content and ODC activity. Growth cessation, resulting from glucose exhaustion in the medium, was accompanied by a sharp decrease in putrescine content and ODC activity. Spermine, the level of which was initially low, was detected in high amounts after all of the glucose was consumed and when the fungus developed the potential for sclerotium formation. A decrease in spermidine, and especially putrescine content, and an increase in spermine content, were observed during the transition from mycelium to mature sclerotia on solid agar medium. Addition of spermine to solid agar medium increased the number of sclerotia by 40%. The changes in the content of the three polyamines were reversed when sclerotia were allowed to germinate. Moreover, α-difluoromethylornithine, the enzyme-activated inhibitor of ODC, greatly inhibited mycelium growth, sclerotium germination and ODC activity, and this inhibition was completely reversed by the addition of putrescine. Cycloheximide delayed sclerotium germination and initially inhibited ODC activity, but ODC inhibition was relieved as soon as sclerotia began to germinate. The data indicate that specific changes in polyamines are linked with two distinct developmental events in S. rolfsii. Mycelial growth and sclerotium germination are positively correlated, and possibly causally linked, with a marked increase in putrescine content and biosynthesis (while spermine cannot be detected). Differentiation (sclerotium formation), however, is accompanied by a major increase in spermine content.

Proteinase activity was detected in culture filtrates of eight wood-decaying basidiomycete fungi and compared in terms of ability to clear skimmed milk agar. All the fungi were proteolytic, but to differing extents. Five were compared using azocasein hydrolysis as a measure of proteolytic activity at the centres and margins of agar-grown colonies and it was found that in Coriolus versicolor the marginal mycelium was the more strongly proteolytic, while in all the other fungi proteolysis by central mycelium was greater. The time course of changes in proteolytic activity in culture filtrates of C. versicolor and Serpula lacrimans grown on the surface of liquid media was compared over 4 weeks, and differences were found which suggested that C. versicolor mycelium inactivates its proteinases after secreting them, but that S. lacrimans does not. The results are interpreted in terms of the likely role of proteinases in the nitrogen economy of these fungi when growing on their wood substrates.

A wall-less ‘slime’ strain of Neurospora crassa was shown to be pleiotropically altered in the production and secretion of several enzymes related to carbon catabolism, including invertase, alkaline protease, aryl-β-glucosidase and cellobiase. Invertase and protease were produced constitutively. The two β-glucosidases required induction by cellobiose but were poorly affected by glucose repression. Exoenzymes were released in larger amounts by the ‘slime’ strain than by the wild-type. Two other carbon-regulated enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were normally regulated in the ‘slime’ strain. A study of recombinants from a cross of a ‘slime’-containing heterokaryon and wild-type suggested that the anomalies observed were inherent in the ‘slime’ phenotype.

Uptake rates of benzoic and propanoic acid into Zygosaccharomyces bailii were proportional to the concentration of undissociated acid and showed no indication of limitation at high concentrations. Benzoic acid permeated 27 times faster than propanoic acid. Other low-M r fatty acids were taken up at rates approximately related to their lipophilicity. Glucose stimulated uptake rate and inhibitors of glucose transport or metabolism removed the stimulation. It was concluded that the principal mechanism of uptake was diffusion of undissociated acid. The Z. bailii membrane had lower permeabilities than reported for other cell types and lipid bilayers. Growth in the presence of benzoic acid reduced permeability to benzoic acid further. Preservative-resistant yeast species had lower uptake rates of propanoic acid than sensitive ones. Degradation of the cell wall did not change the permeability to propanoic acid.