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The cobY Gene of the Archaeon Halobacterium sp . Strain NRC-1 Is Required for De Novo Cobamide Synthesis. J. D. Woodson, 2003.Genetic and nutritional analyses of mutants of the extremely halophilic archaeon Halobacterium sp . strain NRC-1 showed that open reading frame (ORF) Vng1581C encodes a protein with nucleoside triphosphate:adenosylcobinamide-phosphate nucleotidyltransferase enzyme activity . This activity was previously associated with the cobY gene of the methanogenic archaeon Methanobacterium thermoautotrophicum strain  H, but no evidence was obtained to demonstrate the direct involvement of this protein in cobamide biosynthesis in archaea . Computer analysis of the Halobacterium sp . strain NRC-1 ORF Vng1581C gene and the cobY gene of M . thermoautotrophicum strain
H showed the primary amino acid sequence of the proteins encoded by these two genes to be 35% identical and 48% similar . A strain of Halobacterium sp . strain NRC-1 carrying a null allele of the cobY gene was auxotrophic for cobinamide-GDP, a known intermediate of the late steps of cobamide biosynthesis . The auxotrophic requirement for cobinamide-GDP was corrected when a wild-type allele of cobY was introduced into the mutant strain, demonstrating that the lack of cobY function was solely responsible for the observed block in cobamide biosynthesis in this archaeon . The data also show that Halobacterium sp . strain NRC-1 possesses a high-affinity transport system for corrinoids and that this archaeon can synthesize cobamides de novo under aerobic growth conditions . To the best of our knowledge this is the first genetic and nutritional analysis of cobalamin biosynthetic mutants in archaea .
Assembly of TolC, a Structurally Unique and Multifunctional Outer Membrane Protein of Escherichia coli K-12. John Werner, 2003.TolC is a multifunctional outer membrane protein of Escherichia coli that folds into a novel  -ß-barrel conformation absent in the other model outer membrane proteins used in assembly studies . The data presented in this work show that the unique folded structure of TolC reflects a unique assembly pathway . During its assembly, the newly translocated nascent TolC monomers are released in the periplasm . Maturation of these nascent monomers, and possibly their oligomerization, in the periplasm precedes their insertion in the outer membrane . The completion of the assembly process is signaled by the development of a characteristic proteinase K-resistant fragment generated by cleavage at a single, periplasmically exposed, protease-sensitive site of the membrane-anchored trimer . None of the assembly steps of TolC is affected by known folding factors, such as SurA, Skp, and lipopolysaccharide, which have profound effects on the assembly of other model trimeric outer membrane proteins . Two assembly-defective TolC mutants were isolated and characterized . One of the mutants (TolCI106N) was defective in the folding of nascent monomers, while the other (TolCS350F) was impaired in steps involving trimerization and membrane insertion of folded monomers .
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