The evolution of microbial parasites involves the counterplay between natural selection forcing p... more The evolution of microbial parasites involves the counterplay between natural selection forcing parasites to improve and genetic drifts forcing parasites to lose genes and accumulate deleterious mutations. Here, to understand how this counterplay occurs at the scale of individual macromolecules, we describe cryo-EM structure of ribosomes from Encephalitozoon cuniculi, a eukaryote with one of the smallest genomes in nature. The extreme rRNA reduction in E. cuniculi ribosomes is accompanied with unparalleled structural changes, such as the evolution of previously unknown molten rRNA linkers and bulgeless rRNA. Furthermore, E. cuniculi ribosomes withstand the loss of rRNA and protein segments by evolving an ability to use small molecules as structural mimics of degenerated rRNA and protein segments. Overall, we show that the molecular structures long viewed as reduced, degenerated, and suffering from debilitating mutations possess an array of compensatory mechanisms that allow them to ...
Microsporidians are obligate intracellular parasites that have minimized their genome content and... more Microsporidians are obligate intracellular parasites that have minimized their genome content and sub-cellular structures by reductive evolution. Here, we demonstrate that cristae-deficient mitochondria (mitosomes) of Trachipleistophora hominis are the functional site of iron-sulfur cluster (ISC) assembly, which we suggest is the essential task of these organelles. Cell fractionation, fluorescence imaging and immunoelectron microscopy demonstrate that mitosomes contain a complete pathway for [2Fe-2S] cluster biosynthesis that we biochemically reconstituted using purified mitosomal ISC proteins. The T. hominis cytosolic iron-sulfur protein assembly (CIA) pathway includes the essential Cfd1-Nbp35 scaffold complex that assembles a [4Fe-4S] cluster as shown by spectroscopic methods in vitro. Phylogenetic analyses reveal that the ISC and CIA pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of F...
Proceedings of the National Academy of Sciences, 2008
The origin of the eukaryotic genetic apparatus is thought to be central to understanding the evol... more The origin of the eukaryotic genetic apparatus is thought to be central to understanding the evolution of the eukaryotic cell. Disagreement about the source of the relevant genes has spawned competing hypotheses for the origins of the eukaryote nuclear lineage. The iconic rooted 3-domains tree of life shows eukaryotes and archaebacteria as separate groups that share a common ancestor to the exclusion of eubacteria. By contrast, the eocyte hypothesis has eukaryotes originating within the archaebacteria and sharing a common ancestor with a particular group called the Crenarchaeota or eocytes. Here, we have investigated the relative support for each hypothesis from analysis of 53 genes spanning the 3 domains, including essential components of the eukaryotic nucleic acid replication, transcription, and translation apparatus. As an important component of our analysis, we investigated the fit between model and data with respect to composition. Compositional heterogeneity is a pervasive pr...
Trichomonas vaginalis is one of the most widespread, sexually transmitted pathogens. The infectio... more Trichomonas vaginalis is one of the most widespread, sexually transmitted pathogens. The infection involves a morphological switch from a free-swimming pyriform trophozoite to an amoeboid cell upon adhesion to host epithelial cells. While details on how the switch is induced and to what proteins of the host surface the parasite adheres remain poorly characterized, several surface proteins of the parasite itself have been identified as potential candidates. Among those are two expanded protein families that harbor domains that share similarity to functionally investigated surface proteins of prokaryotic oral pathogens; these are the BspA proteins of Bacteroidales and Spirochaetales, and the Pmp proteins of Chlamydiales. We sequenced the transcriptomes of five Trichomonads and screened for the presence of BspA and Pmp domain-containing proteins and tested the ability of individual T. vaginalis candidates to mediate adhesion. Here we demonstrate that (i) BspA and Pmp domain-containing ...
Lateral gene transfer (LGT) in eukaryotes from non-organellar sources is a controversial subject ... more Lateral gene transfer (LGT) in eukaryotes from non-organellar sources is a controversial subject in need of further study. Here we present gene distribution and phylogenetic analyses of the genes encoding the hybrid-cluster protein, A-type flavoprotein, glucosamine-6-phosphate isomerase, and alcohol dehydrogenase E. These four genes have a limited distribution among sequenced prokaryotic and eukaryotic genomes and were previously implicated in gene transfer events affecting eukaryotes. If our previous contention that these genes were introduced by LGT independently into the diplomonad and Entamoeba lineages were true, we expect that the number of putative transfers and the phylogenetic signal supporting LGT should be stable or increase, rather than decrease, when novel eukaryotic and prokaryotic homologs are added to the analyses. The addition of homologs from phagotrophic protists, including several Entamoeba species, the pelobiont Mastigamoeba balamuthi, and the parabasalid Tricho...
A glucocorticoid-responsive vector is described which allows for the highly inducible expression ... more A glucocorticoid-responsive vector is described which allows for the highly inducible expression of complementary DNAs (cDNAs) in stably transfected mammalian cell lines. This vector, pLK-neo, composed of a variant mouse mammary tumor virus long terminal repeat promoter, containing a hormone regulatory element, a Geneticin resistance-encoding gene in a simian virus 40 transcription unit, and a polylinker insertion site for heterologous cDNAs, was used to express the polymeric immunoglobulin (poly-Ig) receptor and the thymocyte marker, Thy-1, in Madin-Darby canine kidney (MDCK) cells and in murine fibroblast L cells. A high level of poly-Ig receptor or Thy-1 mRNA accumulation was observed in MDCK cells in response to dexamethasone with a parallel ten- to 200-fold increase in protein synthesis depending on the recombinant protein and the transfected cell clone.
The evolution of microbial parasites involves the counterplay between natural selection forcing p... more The evolution of microbial parasites involves the counterplay between natural selection forcing parasites to improve and genetic drifts forcing parasites to lose genes and accumulate deleterious mutations. Here, to understand how this counterplay occurs at the scale of individual macromolecules, we describe cryo-EM structure of ribosomes from Encephalitozoon cuniculi, a eukaryote with one of the smallest genomes in nature. The extreme rRNA reduction in E. cuniculi ribosomes is accompanied with unparalleled structural changes, such as the evolution of previously unknown molten rRNA linkers and bulgeless rRNA. Furthermore, E. cuniculi ribosomes withstand the loss of rRNA and protein segments by evolving an ability to use small molecules as structural mimics of degenerated rRNA and protein segments. Overall, we show that the molecular structures long viewed as reduced, degenerated, and suffering from debilitating mutations possess an array of compensatory mechanisms that allow them to ...
Microsporidians are obligate intracellular parasites that have minimized their genome content and... more Microsporidians are obligate intracellular parasites that have minimized their genome content and sub-cellular structures by reductive evolution. Here, we demonstrate that cristae-deficient mitochondria (mitosomes) of Trachipleistophora hominis are the functional site of iron-sulfur cluster (ISC) assembly, which we suggest is the essential task of these organelles. Cell fractionation, fluorescence imaging and immunoelectron microscopy demonstrate that mitosomes contain a complete pathway for [2Fe-2S] cluster biosynthesis that we biochemically reconstituted using purified mitosomal ISC proteins. The T. hominis cytosolic iron-sulfur protein assembly (CIA) pathway includes the essential Cfd1-Nbp35 scaffold complex that assembles a [4Fe-4S] cluster as shown by spectroscopic methods in vitro. Phylogenetic analyses reveal that the ISC and CIA pathways are predominantly bacterial, but their cytosolic and nuclear target Fe/S proteins are mainly archaeal. This mixed evolutionary history of F...
Proceedings of the National Academy of Sciences, 2008
The origin of the eukaryotic genetic apparatus is thought to be central to understanding the evol... more The origin of the eukaryotic genetic apparatus is thought to be central to understanding the evolution of the eukaryotic cell. Disagreement about the source of the relevant genes has spawned competing hypotheses for the origins of the eukaryote nuclear lineage. The iconic rooted 3-domains tree of life shows eukaryotes and archaebacteria as separate groups that share a common ancestor to the exclusion of eubacteria. By contrast, the eocyte hypothesis has eukaryotes originating within the archaebacteria and sharing a common ancestor with a particular group called the Crenarchaeota or eocytes. Here, we have investigated the relative support for each hypothesis from analysis of 53 genes spanning the 3 domains, including essential components of the eukaryotic nucleic acid replication, transcription, and translation apparatus. As an important component of our analysis, we investigated the fit between model and data with respect to composition. Compositional heterogeneity is a pervasive pr...
Trichomonas vaginalis is one of the most widespread, sexually transmitted pathogens. The infectio... more Trichomonas vaginalis is one of the most widespread, sexually transmitted pathogens. The infection involves a morphological switch from a free-swimming pyriform trophozoite to an amoeboid cell upon adhesion to host epithelial cells. While details on how the switch is induced and to what proteins of the host surface the parasite adheres remain poorly characterized, several surface proteins of the parasite itself have been identified as potential candidates. Among those are two expanded protein families that harbor domains that share similarity to functionally investigated surface proteins of prokaryotic oral pathogens; these are the BspA proteins of Bacteroidales and Spirochaetales, and the Pmp proteins of Chlamydiales. We sequenced the transcriptomes of five Trichomonads and screened for the presence of BspA and Pmp domain-containing proteins and tested the ability of individual T. vaginalis candidates to mediate adhesion. Here we demonstrate that (i) BspA and Pmp domain-containing ...
Lateral gene transfer (LGT) in eukaryotes from non-organellar sources is a controversial subject ... more Lateral gene transfer (LGT) in eukaryotes from non-organellar sources is a controversial subject in need of further study. Here we present gene distribution and phylogenetic analyses of the genes encoding the hybrid-cluster protein, A-type flavoprotein, glucosamine-6-phosphate isomerase, and alcohol dehydrogenase E. These four genes have a limited distribution among sequenced prokaryotic and eukaryotic genomes and were previously implicated in gene transfer events affecting eukaryotes. If our previous contention that these genes were introduced by LGT independently into the diplomonad and Entamoeba lineages were true, we expect that the number of putative transfers and the phylogenetic signal supporting LGT should be stable or increase, rather than decrease, when novel eukaryotic and prokaryotic homologs are added to the analyses. The addition of homologs from phagotrophic protists, including several Entamoeba species, the pelobiont Mastigamoeba balamuthi, and the parabasalid Tricho...
A glucocorticoid-responsive vector is described which allows for the highly inducible expression ... more A glucocorticoid-responsive vector is described which allows for the highly inducible expression of complementary DNAs (cDNAs) in stably transfected mammalian cell lines. This vector, pLK-neo, composed of a variant mouse mammary tumor virus long terminal repeat promoter, containing a hormone regulatory element, a Geneticin resistance-encoding gene in a simian virus 40 transcription unit, and a polylinker insertion site for heterologous cDNAs, was used to express the polymeric immunoglobulin (poly-Ig) receptor and the thymocyte marker, Thy-1, in Madin-Darby canine kidney (MDCK) cells and in murine fibroblast L cells. A high level of poly-Ig receptor or Thy-1 mRNA accumulation was observed in MDCK cells in response to dexamethasone with a parallel ten- to 200-fold increase in protein synthesis depending on the recombinant protein and the transfected cell clone.
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Papers by R. Hirt