An analysis of the small subunit ribosomal RNA (16S-like rRNA) from the protozoan Giardia lamblia provided a new perspective on the evolution of nucleated cells. Evolutionary distances estimated from sequence comparisons between the 16S-like rRNAs of Giardia lamblia and other eukaryotes exceed similar estimates of evolutionary diversity between archaebacteria and eubacteria and challenge the phylogenetic significance of multiple eukaryotic kingdoms. The Giardia lamblia 16S-like rRNA has retained many of the features that may have been present in the common ancestor of eukaryotes and prokaryotes.

The small-subunit rRNA gene sequences of the flagellated protists Euglena gracilis and Trypanosoma brucei were determined and compared to those of other eukaryotes. A phylogenetic tree was constructed in which the earliest branching among the eukaryotes is represented by E. gracilis. The E. gracils divergence far antedates a period of massive evolutionary radiation that gave rise to the plants, animals, fungi, and certain groups of protists such as ciliates and the acanthamoebae. The genetic diversity in this collection of eukaryotes is seen to exceed that displayed within either the eubacterial or the archaebacterial lines of descent.The eukaryotes can be represented as four major divisions: the Protista, the Fungi, the Plantae, and the Animalia (1). The order of succession for these major eukaryotic lineages is an unsettled question, but it is generally accepted that the first eukaryotes were most similar to certain members of the present-day Protista. Efforts to reconstruct the evolutionary history of protists, and hence the phylogenetic origins of the major eukaryotic divisions, have been frustrated by the enormous physiological, cytological, and biochemical diversity exhibited by these "simple" microorganisms. Traditional phylogenetic analyses-i.e., examination of the fossil record and comparative studies of phenotypes-are suitable for defining relationships between multicellular organisms, but they are of limited value in determining relationships among the protists. These soft-body organisms are not well represented in the fossil record, and there is little agreement regarding which phenotypic characters are most useful for inferring protist relationships. Furthermore, comparative studies of phenotypes do not provide quantitative measures of genetic similarity that can be used to deduce the order of branching for the major eukaryotic lines of descent.As an alternative, comparisons of the nucleotide or amino acid sequences of functionally equivalent macromolecules can be used to infer quantitative phylogenetic relationships between diverse organisms (2). Amino acid sequence homologies among cytochrome c molecules (3, 4), ferredoxins (5-7), and superoxide dismutases (8-10) have been useful for refining classical eukaryotic phylogenies, but they are of limited value in resolving the deepest phylogenetic branching patterns. The use of cytochrome c and other genes that are defined by plastid and mitochondrial genomes as "molecular chronometers" for measuring eukaryotic relationships is further complicated by their non-nuclear origin. It is now apparent that plastids and mitochondria arose as bacterial endosymbionts within some ancestral eukaryotes (11-13). Therefore, genes that are contained within the plastid or mitochondrial genomes or genes that were present in the original bacterial endosymbionts and then transferred to the nucleus-e.g., cytochrome c genes-should be regarded as markers of prokaryotic rather than eukaryotic evolution.By comparison, the rRNAs are better suited for defining evolutiona...

The phylogenetic relationships among the chlorophyte Chlamydomnonas reinhardtii, the chrysophyte Ochromonas danica, and the oomycete Achlya bisexualis were explored by comparing the sequences of their small-subunit ribosomal RNA coding regions. Comparisons of similarity values or inspection of phylogenetic trees constructed by distance matrix methods reveal a very close relationship between oomycetes and chrysophytes. The separation of chrysophytes from chlorophytes is comparable to that of plants from animals, and both separations are far antedated by the divergence of a number of other protist groups.Traditional analyses based on phenotypic criteria frequently depict chlorophytes (green algae) and chrysophytes (goldenbrown algae) as representatives of lineages that diverged soon after the appearance of the earliest protists (1, 2). Comparisons of numerous characteristics such as features of nuclear division, chloroplast structure and pigment types, kinetid ultrastructure, the nature of the cell wall, and mitochondrial crista structure suggest a long and separate evolutionary history for these algal lineages. The presence of tubular mitochondrial cristae in chrysophytes and lamellar mitochondria cristae in chlorophytes (3) may be particularly significant. Since no recognizably monophyletic protistan groups are split with respect to crista type, and related groups seem to be connected by this characteristic, fundamental differences in mitochondrial structure are thought to reflect ancient divergences and to be of significance at a high taxonomic level.Certain colorless protistan lineages may be more closely related to the chrysophytes than to the chlorophytes. Among these groups are the "lower" fungi. These forms are widely regarded as a polyphyletic assemblage of uncertain relationships to "higher" fungi (4). For example, the oomycetes have traditionally been grouped with the "true" fungi but comparisons of several biochemical and morphological characteristics suggest a close relationship to xanthophytes and chrysophytes (4-7). Taxonomic placement of the oomycetes is still controversial; in some schemes they are grouped with the higher fungi (8), and in others they are grouped with the chrysophytes and their relatives (9, 10).Since there is little agreement about which phenotypic characteristics are most reliable for inferring evolutionary relationships, a consensus phylogeny for protists has never emerged. A phylogenetic tree, which reflects true genotypic similarity, can be inferred from comparisons of macromolecular sequences. Ribosomal RNAs have been used extensively for measuring evolutionary distances (11, 12) and these can be converted into phylogenetic trees by parsimony or distance matrix analyses. Because of their large size (relative to that of 5S and 5.8S rRNAs) and the existence of highly conserved and partially conserved sequence elements, the 16S-like or small-subunit rRNAs have been particularly useful for measuring both close and distant phylogenetic relationships (13,14). As part of a...

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The survival of Salmonella enterica was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of Tetrahymena. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce and to examine their interactions with S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Glaucoma sp., Colpoda steinii, and Acanthamoeba palestinensis were cultured from store-bought spinach and lettuce and used in our study. A strain of Tetrahymena pyriformis previously isolated from spinach and a soil-borne Tetrahymena sp. were also used. Washed protozoa were allowed to graze on green fluorescent proteinor red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by Glaucoma with all of the bacterial strains, although L. monocytogenes resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of Tetrahymena on E. coli O157:H7 and S. enterica but not during grazing on L. monocytogenes, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast, C. steinii and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of E. coli O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.