Genome sequence of the model hyperthermophilic archaeon Thermococcus litoralis NS-C

The hyperthermophilic archaeon Thermococcus litoralis strain NS-C, first isolated in 1985, has been a foundational organism for archaeal research in biocatalysis, DNA replication, metabolism, and the discovery of inteins. Here, we present the genome sequence of T. litoralis with a focus on the replication machinery and inteins.     

Properties of the C-terminal domain of enzyme I of the Escherichia coli phosphotransferase system

The bacterial phosphoenolpyruvate (PEP):glycose phosphotransferase system (PTS) mediates uptake/phosphorylation of sugars. The transport of all PTS sugars requires Enzyme I (EI) and a phosphocarrier histidine protein of the PTS (HPr). The PTS is stringently regulated, and a potential mechanism is the monomer/dimer transition of EI, because only the dimer accepts the phosphoryl group from PEP. EI monomer consists of two major domains, at the N and C termini (EI-N and EI-C, respectively). EI-N accepts the phosphoryl group from phospho-HPr but not PEP. However, it is phosphorylated by PEP(Mg(2+)) when complemented with EI-C. Here we report that the phosphotransfer rate increases approximately 25-fold when HPr is added to a mixture of EI-N, EI-C, and PEP(Mg(2+)). A model to explain this effect is offered. Sedimentation equilibrium results show that the association constant for dimerization of EI-C monomers is 260-fold greater than the K(a) for native EI. The ligands have no detectable effect on the secondary structure of the dimer (far UV CD) but have profound effects on the tertiary structure as determined by near UV CD spectroscopy, thermal denaturation, sedimentation equilibrium and velocity, and intrinsic fluorescence of the 2 Trp residues. The binding of PEP requires Mg(2+). For example, there is no effect of PEP on the T(m), an increase of 7 degrees C in the presence of Mg(2+), and approximately 14 degrees C when both are present. Interestingly, the dissociation constants for each of the ligands from EI-C are approximately the same as the kinetic (K(m)) constants for the ligands in the complete PTS sugar phosphorylation assays.     

The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

Inteins are the protein equivalent of introns. They are remarkable and robust single turnover enzymes that splice out of precursor proteins during post‐translational maturation of the host protein (extein). The Deinococcus radiodurans Snf2 intein is the second member of the recently discovered Class 3 subfamily of inteins to be characterized. Class 3 inteins have a unique sequence signature: (a) they start with residues other than the standard Class 1 Cys, Ser or Thr, (b) have a noncontiguous, centrally located Trp/Cys/Thr triplet, and (c) all but one have Ser or Thr at the start of the C‐extein instead of the more common Cys. We previously proposed that Class 3 inteins splice by a variation in the standard intein‐mediated protein splicing mechanism that includes a novel initiating step leading to the formation of a previously unrecognized branched intermediate. In this mechanism defined with the Class 3 prototypic Mycobacteriophage Bethlehem DnaB intein, the triplet Cys attacks the peptide bond at the N‐terminal splice junction to form the class specific branched intermediate after which the N‐extein is transferred to the side chain of the Ser, Thr, or Cys at the C‐terminal splice junction to form the standard intein branched intermediate. Analysis of the Deinococcus radiodurans Snf2 intein confirms this splicing mechanism. Moreover, the Class 3 specific Block F branched intermediate was isolated, providing the first direct proof of its existence.     

Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation.

Protein splicing involves the excision of an internal domain from a precursor protein and the ligation of the external domains so as to generate two new proteins. Study of this process has recently been facilitated by the isolation of a precursor and a branched intermediate from a thermophilic protein splicing element expressed in a foreign protein context. Two aspects of protein splicing are examined in this paper. We demonstrate a succinimide at the C-terminus of the spliced internal protein, implicating cyclization of asparagine in resolution of the branched intermediate, and we identify an alkali-labile bond in the branched intermediate. A revised protein splicing model based on these experimental results is presented.     

Identification of surrogate rodent hosts for larval Onchocerca lienalis and induction of protective immunity in a model system.

The objectives of this project were to screen a variety of inbred rodent species and strains to determine their usefulness as surrogate hosts for the study of the early larval development of Onchocerca lienalis and then to use a selected model to study the induction of protective immunity. In the primary screen, 6 strains of mice, 5 strains of rats, jirds, and multimammate rats were tested. Animals were infected with fresh O. lienalis by subcutaneous implantation of third-stage larvae (L3) contained in diffusion chambers covered with 5.0-microns pore-size membranes. After 7 days the chambers were recovered, and larval viability and growth were assessed. Approximately one-half of inoculated larvae were recovered alive regardless of the host tested. Larvae were implanted in CBA/J and DBA/2J mice in chambers covered with membranes that prevented host cells from entering; survival and growth rates of the larvae were not altered by the absence of cells from the chambers. Cryopreserved larvae were implanted in chambers with 5.0-microns pore-size membranes in CBA/J and DBA/2J mice and Wistar Furth rats for 3-28 days. No statistically significant difference was seen in the larval recoveries on days 3-28 in all 3 hosts. Statistically significant increases in length were seen in the 3 strains from day 3 to day 14, after which growth appeared to cease. Molting from L3 to fourth-stage larvae was observed in all 3 hosts beginning on day 3, with most larvae completing the molt by day 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

InBase, the New England Biolabs Intein Database.

Inteins are intervening sequences that splice as proteins, not RNA. InBase, the New England Biolabs Intein Database (http://www.neb. com/neb/inteins.html), is a comprehensive on-line database that includes the Intein Registry, along with detailed information about each intein and its host protein, tabulated comparisons and a comprehensive bibliography including papers in press.