We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodiumproton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.
SummaryTo facilitate the functional genomic analysis of an archaeon, we have developed a homologous gene replacement strategy for Halobacterium salinarum based on ura3, which encodes the pyrimidine biosynthetic enzyme orotidine-58-monophosphate decarboxylase. H. salinarum was shown to be sensitive to 5-¯uoroorotic acid (5-FOA), which can select for mutations in ura3. A spontaneous 5-FOA-resistant mutant was found to contain an insertion in ura3 and was a uracil auxotroph. Integration of ura3 at the bacterioopsin locus (bop ) of this mutant restored 5-FOA sensitivity and uracil prototrophy. Parallel results were obtained with a Dura3 strain constructed by gene replacement and with derivatives of this strain in which ura3 replaced bop. These results show that H. salinarum ura3 encodes functional orotidine-58-monophosphate decarboxylase. To demonstrate ura3-based gene replacement, a Dbop strain was constructed by transforming a Dura3 host with a bop deletion plasmid containing a mevinolin resistance marker. In one approach, the host contained intact ura3 at the chromosomal bop locus; in another, ura3 was included in the plasmid. Plasmid integrants selected with mevinolin were resolved with 5-FOA, yielding Dbop recombinants at a frequency of > 10 À2 in both approaches. These studies establish an ef®cient new genetic strategy towards the systematic knockout of genes in an archaeon.
SummaryRNAi knockdown was employed to study the function of p67, a lysosome-associated membrane protein (LAMP)-like type I transmembrane lysosomal glycoprotein in African trypanosomes. Conditional induction of p67 dsRNA resulted in specific~90% reductions in de novo p67 synthesis in both mammalian bloodstream and procyclic insect-stage parasites. Bloodstream cell growth was severely retarded with extensive death after > 24 h of induction. Biosynthetic trafficking of residual p67, and of the soluble lysosomal protease trypanopain, were unimpaired. Endocytosis of tomato lectin, a surrogate receptor-mediated cargo, was only mildly impaired (~20%), but proper lysosomal targeting was unaffected. p67 ablation had dramatic effects on lysosomal morphology with gross enlargement (four-to fivefold) and internal membrane profiles reminiscent of autophagic vacuoles. Ablation of p67 expression rendered bloodstream trypanosomes refractory to lysis by human trypanolytic factor (TLF), a lysosomally activated host innate immune mediator. Similar effects on lysosomal morphology and TLF sensitivity were also obtained by two pharmacological agents that neutralize lysosomal pH -chloroquine and bafilomycin A1. Surprisingly, however, lysosomal pH was not affected in ablated cells suggesting that other physiological alterations must account for increased resistance to TLF. These results indicate p67 plays an essential role in maintenance of normal lysosomal structure and physiology in bloodstream-stage African trypanosomes.
We conducted a controlled investigation to examine whether a combination of computer imagery and tactile tools helps introductory cell biology laboratory undergraduate students better learn about protein structure/function relationships as compared with computer imagery alone. In all five laboratory sections, students used the molecular imaging program, Protein Explorer (PE). In the three experimental sections, three-dimensional physical models were made available to the students, in addition to PE. Student learning was assessed via oral and written research summaries and videotaped interviews. Differences between the experimental and control group students were not found in our typical course assessments such as research papers, but rather were revealed during one-on-one interviews with students at the end of the semester. A subset of students in the experimental group produced superior answers to some higher-order interview questions as compared with students in the control group. During the interview, students in both groups preferred to use either the hand-held models alone or in combination with the PE imaging program. Students typically did not use any tools when answering knowledge (lowerlevel thinking) questions, but when challenged with higher-level thinking questions, students in both the control and experimental groups elected to use the models.
Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the ⌬brp strain, bacteriorhodopsin levels are decreased ϳ4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, -carotene, is increased ϳ3.8-fold, whereas retinal is decreased by ϳ3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the ⌬brp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of -carotene increased ϳ5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of -carotene to retinal.Rhodopsins are integral membrane proteins containing seven transmembrane ␣-helices and a covalently bound molecule of retinal. Two distinct rhodopsin families are known: the visual rhodopsins, which bind 11-cis retinal or related compounds and function as photoreceptors in vertebrates (1) and invertebrates (2), and the archaeal rhodopsins, which bind all-trans-retinal and function as light-driven ion pumps and phototaxis receptors in archaea (3). Archaeal rhodopsin orthologs have been found recently in bacteria (4) and fungi (5), suggesting that retinal-based pigments are of widespread significance. Despite their importance, the biogenesis of these molecules is not fully understood. In particular, relatively little is known about how retinal is assembled with the opsin apoprotein in vivo. Thus, a goal in elucidating rhodopsin biogenesis is to identify the cellular factors that mediate the biosynthesis or uptake of retinal, the transport of retinal in the cell, and the binding of retinal to the corresponding opsin.To this end, we have studied the biogenesis of bacteriorhodopsin (BR), 1 a light-driven proton pump in the archaeon Halobacterium salinarum. BR consists of the membrane protein bacterioopsin (BO) and all-trans-retinal. Under microaerobic conditions, BR is induced ϳ50-fold (6) and forms a twodimensional crystal known as the purple membrane. This system has served as a model for studying key steps in membrane protein biogenesis, including protein insertion into the membrane (7,8) and the assembly of protein-lipid complexes (9, 10). H. salinarum is genetically tractable, and the genome sequence of a closely related organism, Halo...
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