BACTOX, a rapid bioassay that uses protozoa to assess the toxicity of bacteria.

A new type of toxicity test based on the protozoan Tetrahymena pyriformis has been developed to assess the overall toxicity of bacterial strains given as prey. This simple and rapid test is able to detect toxicant-producing bacteria, which may present a biohazard. It can also be used for the risk assessment of microbes designed for deliberate release.

Genetic transformation in freshwater: Escherichia coli is able to develop natural competence.

Until now, Escherichia coli was thought to be unable to develop natural competence, i.e., genetic transformation could be achieved only artificially with the aid of nonphysiological concentrations of calcium ions or by other treatments. We have tested the competence development of E. coli through transformation under natural conditions in river water, springwater, and mineral water which contained between 0 and 11 mM Ca2+, using pUC18 DNA. The presence of calcium ions at concentrations as low as 1 to 2 mM was sufficient to obtain transformants. Variations in the temperature of incubation were not required for competence development but had an influence on the transformation frequency. Using water from mineral springs originating from calcareous regions, we have obtained transformation frequencies with laboratory strains of E. coli similar to those reported for other gram-negative bacteria known to develop natural competence. The competence development of E. coli is most probably internally regulated (as for the other gram-negative bacteria), and inadequate conditions chosen for the transformation tests in the laboratory might impair the detection of higher natural transformation frequencies. The results will enhance our knowledge about the fate of laboratory or production strains of E. coli cells reaching natural aquatic ecosystems.

Properties of the histones and functional aspects of the soluble chromatin of epimastigote Trypanosoma cruzi.

The amino acid composition of all histones of Trypanosoma cruzi was analyzed, and the terminology of the histones of higher eukaryotes adopted. One chromatin associated protein, previously considered to be a variant of histone H1, could not be clearly identified, and shows features of core histones as well as of histone H1. An improved method for the isolation of intact nuclei and the production of soluble chromatin in T. cruzi was established. The chromatin of T. cruzi is relatively instable and histone H1 is easily lost during experimental manipulations. Histone H1 dissociates completely at a relatively low NaCl concentration of 380 mM, leading to an open nucleosome filament which does not condense. The influence of histone H1 of T. cruzi and of rat liver on the compaction pattern of the chromatin was investigated by homologous and heterologous reconstitution experiments, and analysed by electron microscopy. It could be shown that histone H1 of T. cruzi induces nucleosome filaments of T. cruzi as well as those of rat liver to condense. The same is true for histone H1 of rats. It can be concluded that T. cruzi has a functional histone H1.

An agarose slide method to follow the fate of bacteria within digestive vacuoles of protozoa.

A method to follow the fate of ingested bacteria within digestive vacuoles of protozoa is presented. Tetrahymena pyriformis, previously fed with bacteria, is deposited onto glass microscope slides covered with a film of nutritive agarose. The protozoa lyse and the digestive vacuoles containing the bacteria stay undamaged and can be observed microscopically. After incubation, microcolonies reveal those vacuoles which contained living bacteria. The method can be used to study the survival ability of the ingested bacteria. It is a potentially valuable technique for studies on digestion efficacy, virulence ability, or escape mechanisms of bacteria from digestion.

Partial amino acid sequence and functional aspects of histone H1 proteins in Trypanosoma brucei brucei.

Trypanosoma brucei brucei, a protozoan parasite of wild and domestic animals in Africa, is related to the pathogenic agent of human sleeping sickness. Four H1 histone proteins were isolated from nuclei of procyclic culture forms and cleaved with proteases. Amino acid sequence analysis of purified fragments indicated the presence of variants which displayed sequence identities as compared to the C-terminal domain of human H1. Substitutions of amino acids and posttranslational modifications of the histones in T b brucei H1 may influence protein conformation and histone-histone as well as histone-DNA interactions in the chromatin of the parasite. Digestion of soluble chromatin with immobilized trypsin at low and high ionic strengths indicated an internal localization of H1 in the condensed chromatin. The influence of histone H1 of T b brucei on the compaction pattern of the chromatin was investigated by dissociation and reconstitution experiments. Electron microscopy revealed that trypanosome H1 was able to induce condensation of the chromatin of the parasite and of rat liver into dense tangles. After dephosphorylation of H1, 30 nm fibers were induced in rat liver chromatin, while the resulting fibers were distinctly thinner in T b brucei. It can be concluded that the absence of 30 nm fibers in T b brucei chromatin cannot be explained by the divergent variants and posttranslational phosphorylations of H1 only but rather by the influence of both, the divergent core histones, previously described, and H1 properties.

Characterization of the histones of Trypanosoma brucei brucei bloodstream forms.

Five groups of histones were shown in Trypanosoma brucei brucei, displaying qualitative and quantitative differences between two stages of the parasite's life-cycle. The influence of the histones of T. b. brucei bloodstream forms on the compaction pattern of the chromatin was investigated and their extractability in diluted acids and their amino acid composition were analysed. While nonhistone proteins barely influenced the formation of higher-order chromatin structures, the histone H1-like proteins were essential for the regular spacing of the nucleosomes and the salt-dependent condensation of the nucleosome filament. Differences were seen in the amino-acid composition of histones of bloodstream forms as compared to procyclic culture forms and higher eukaryotes which may explain the disparities seen in the condensation of the chromatin between the two stages of the life cycle as well as the lack of a salt-dependent condensation into a 30 nm fiber. They point to an alternative method of organizing and processing the genetic information in the nucleus of the trypanosome as compared to higher eukaryotes, the possible hosts of the parasite.

The chromatin of trypanosomes.

The nuclear chromatin of trypanosomes is organised in the form of nucleosome filaments. When soluble chromatin is prepared under suitable conditions, a regular array of nucleosomes can be shown by electron microscopy. Chromatin of blood stream as well as procyclic culture forms of Trypanosoma brucei brucei and of T. cruzi shows limited compaction at salt concentrations increasing from 1 to 100 mM. No 30 nm fibres, typical for higher eukaryotes, are formed. Digestion of the nuclear chromatin with micrococcal nuclease and analysis of the histone proteins with various techniques reveal that the basic organisation of the trypanosome chromatin is similar but not identical as compared to that of higher eukaryotes. Distinct differences are present with respect to biochemical properties of the histones as well as to their interaction with the DNA. The primary structure of the histones also differs significantly from that found in other lower and higher eukaryotes. The function of the recently described H1-like proteins in trypanosomes is currently being investigated. The differences that have already been found in the structure and compaction of the trypanosome chromatin compared to that of higher eukaryotes lead us to expect differences of gene expression which, in turn, might offer targets for the control of trypanosomiasis.

Comparative chromatin analysis of Trypanosoma congolense.

The chromatin of Trypanosoma congolense was analyzed by electron microscopy. The chromatin is organized as nucleosome filaments but does not form a 30 nm fiber. There are five groups of histones, including a histone H1-like protein, which as a molecular weight within the range of the core histones, and is extremely hydrophilic. Weak histone-histone interaction, a typical feature of trypanosome chromatin, was found. These results are similar to those for T. cruzi and T. b. brucei, but differ significantly from those for higher eukaryotes. The results confirm the model of trypanosome chromatin, and support the theory of their early separation from the other eukaryotes during the evolution. T. congolensis is an excellent model for chromatin research on trypanosomes, because it is easy to cultivate and its chromatin has, a relatively high stability, compared to that of other trypanosomes.

Trypanosoma brucei brucei: differences in the nuclear chromatin of bloodstream forms and procyclic culture forms.

Nucleosome filaments of two stages of the life-cycle of Trypanosoma brucei brucei, namely bloodstream forms and procyclic culture forms, were investigated by electron microscopy. Chromatin of bloodstream forms showed a salt-dependent condensation. The level of condensation was higher than that shown by chromatin from procyclic culture forms, but 30 nm fibres as formed in rat liver chromatin preparations were not found. Analysis of histones provided new evidence for the existence of H1-like proteins, which comigrated in the region of the core histones in SDS-PAGE and in front of the core histones in Triton acid urea gels. Differences were found between the H1-like proteins of the two trypanosome stages as well as between the core histones in their amount, number of bands and banding pattern. It can be concluded that T. b. brucei contains a full set of histones, including H1-like proteins, and that the poor condensation of its chromatin is not due to the absence of H1, but most probably due to histone-DNA interaction being weak. It is obvious that structural and functional differences of the chromatin exist not only between T. b. brucei and higher eukaryotes, but also between various stages of the life-cycle of the parasite. It is therefore not adequate to investigate the chromatin only of the procyclic culture forms as a model for all stages of the life-cycle of T. b. brucei.

Biochemical and functional characterization of histone H1-like proteins in procyclic Trypanosoma brucei brucei.

Four variants and/or posttranslational modifications of histone H1-like proteins of Trypanosoma brucei brucei procyclic culture forms were extracted with 0.25 N HCl from isolated nuclei and analyzed by two-dimensional gel electrophoresis. The amino acid composition of these proteins, their ability to space nucleosomes regularly and to induce salt-dependent condensation of the chromatin indicated their histone H1 nature. On the other hand, the histone H1-like proteins clearly differed from their higher-eukaryote counterparts by their weak interaction with DNA under low-salt conditions. As a consequence, intact nucleosome filaments were prepared according to a new preparation protocol especially adapted to the unstable chromatin of T. b. brucei. Our results indicate that the biochemical properties of the histone H1-like proteins contribute to the structural and functional differences between the chromatin of procyclic T. b. brucei and that of higher eukaryotes.