- Volume 145, Issue 3, 1999

An Aspergillus niger strain expressing a red-shifted green fluorescent protein (GFP) in the cytoplasm under the control of the glucoamylase promoter (PglaA) was characterized with respect to its physiology and morphology. Although xylose acted as a repressor carbon source during batch cultivations, PglaA-driven GFP expression by the glucoamylase promoter could be demonstrated in xylose-limited continuous cultures. In these cultivations, the xylose concentration was therefore too low to cause repression. Transient experiments initiated with a maltose pulse did not further induce red-shifted GFP production in xylose-limited continuous cultures. Maltose induction under conditions of xylose repression was microscopically observed and quantified in a flow-through chamber. Red-shifted GFP was first produced after 5 h induction. Finally the strain was characterized in glucose-limited continuous cultures, and here the area of the mycelium stained with cytoplasmic GFP increased with increasing specific growth rate, indicating that GFP can be used as a marker of cellular activity in this type of cultivation.

To identify genes responsible for the utilization of haem as an iron source in Helicobacter pylori, a siderophore synthesis mutant of Escherichia coli was transformed with an ordered cosmid library of H. pylori NCTC 11638. Four independent cosmids were found that were able to complement this mutant on iron-restrictive solid media containing different haem compounds as the sole source of iron. Hybridization experiments revealed that the four cosmids contained unrelated DNA fragments. No major differences were observed in the growth of the four transformants on iron-restrictive solid media to which different haem compounds had been added. None of the cosmids could confer the ability to use haem as an iron source to an E. coli aroB tonB mutant, which means that transport of iron and/or haem across the outer membrane requires a functional TonB protein. Further characterization of the cosmids revealed that one of them was also able to complement E. coli aroB hemA, indicating that the haem molecule is taken up as a whole by this haem-biosynthesis mutant. Expression of this haem-uptake system could not be repressed by excess iron. Another cosmid expressed two polypeptides in E. coli which were specifically immunoreactive with a polyclonal antiserum raised against whole cells of H. pylori. The production of these proteins appeared to be iron repressible. One of these proteins has the same molecular mass as a previously described 77 kDa haem-binding iron-repressible outer-membrane protein (IROMP) of H. pylori.

The culture supernatant of Candida albicans promoted the disruption of human red blood cells (RBCs). The haemolytic activity was detected in a sugar-rich fraction (about 200 kDa) from Sephacryl S-100 chromatography. As the haemolytic activity was adsorbed by concanavalin A-Sepharose, the haemolytic factor may be a mannoprotein. The activity was inactivated by periodate oxidation, indicating that the sugar moiety of the mannoprotein played an important role in the haemolysis. The structure of the sugar moiety of the mannoprotein was identified as a cell-wall mannan by 1H-NMR analysis, and purified C. albicans mannan promoted the disruption of RBCs. The binding of mannan to RBCs was demonstrated by flow cytometric analysis and was inhibited by the addition of band 3 protein inhibitor, 4,4′-diisothio-cyanatostilbene-2,2′-disulfonic acid (DIDS). The haemolysis caused by mannan was inhibited by DIDS, SITS (4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid) and bis(sulfosuccinimidyl) suberate, but not by pyridoxal 5-phosphate. These results indicated that a mannoprotein released from C. albicans bound to the band 3 protein on RBCs, thereby promoting their disruption.

A number of experimental methods in biology depend on the kinetics of diffusion of a substance through a gel. This paper reviews the diffusion equations, gives the experimental limitations for some useful cases, and presents computer simulations for cases that cannot be treated analytically. While double diffusion is not considered, three single-diffusion situations are treated. (1) Systems for the study of chemotaxis in the gliding bacterium Myxococcus xanthus. Experimental designs used for this in many cases in the literature were inappropriate and mathematical analysis of these is presented. (2) The development of gradient plates. The time necessary for vertical diffusion to become substantially complete and before diffusion in the direction of the original slant has proceeded significantly is calculated. (3) The application to antimicrobial disk susceptibility tests. The basis of the measurement of antibiotic sensitivities with disks containing antimicrobial agents, as routinely used in clinical microbiological and testing laboratories, is analysed and the limitations are assessed and improvements suggested.

Levels of mRNAs encoding metabolic enzymes and their cellular activities were measured on continuous culture samples of the parasitic protists Trypanosoma brucei and Trichomonas vaginalis. The organisms were grown in chemostats at varying growth rates under glucose limitation or in the presence of excess glucose (EG), resulting in extensive adaptation of the cellular activities of glycolytic enzymes. rRNA and mRNA for β-tubulin were monitored as controls. In Trypanosoma brucei levels of all RNAs showed a biphasic dependence on growth rate (= dilution rate D), with a sharp increase at higher D values. Cellular RNA levels of Trichomonas vaginalis rate-limited by glucose decreased slightly with increasing D. In EG-grown cells the opposite trend was observed. Equal levels for both carbon regimes were observed at intermediate D values. In both species the ratio between rRNA and mRNA encoding β-tubulin was constant, independent of the carbon regime. mRNA encoding metabolic enzymes showed varying degrees of correlation with rRNA and β-tubulin mRNA. In contrast, there was little to no correlation between mRNA levels and the activities of the enzymes they encode, even though only one of these is allosterically regulated. The data indicate that RNA levels in Trypanosoma brucei and Trichomonas vaginalis are determined by growth rate and in the latter species by the availability of the carbon and energy source. Rates of synthesis of metabolic enzymes are most likely regulated at the post-transcriptional level.