Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.

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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.

The activation mechanism of glycosylasparaginase of Flavobacterium meningosepticum has been analyzed by site-directed mutagenesis and activation of purified precursors in vitro. Mutation of Thr-152 to Ser or Cys leads to gene products that are not activated in vivo but are activated in vitro because processing of the mutant precursors is inhibited by certain amino acids in the cell. Kinetic studies reveal that activation is an intramolecular autoproteolytic process. The involvement of His-150 and Thr/Ser/Cys-152 in activation suggests that autoproteolysis resembles proteolysis by serine/cysteine proteases. Multiple functions of the highly conserved active threonine residue are implicated.

The DNA polymerase gene from the Archaea In 1985, a species of extreme thermophile was isolated from a submarine thermal vent near Naples, Italy (1). This organism, Thermococcus litoralis, can be cultured at up to 980C and contains a heat-stable DNA polymerase that we call Vent DNA polymerase (New England Biolabs). This paper describes the cloning, sequencing, and expression of the Vent DNA polymerase gene ¶ and the finding of two intervening sequences (IVSs) that make up 55% of the polymerase gene, one of which, IVS2, encodes the I-Tli I (I, intron) endonuclease. To our knowledge, this is the first report of introns in protein coding genes of Archaea or eubacteria, although introns have been found in protein coding genes of eubacteriophage (2). Previously described Archaea or eubacterial introns are mainly pre-tRNA or self-splicing introns in stable .Introns often contain open reading frames (ORFs) that are in-frame with either the 5' or 3' exon, but not with both exons. An intron in the Saccharomyces cerevisiae TFP1 gene forms a single ORF with the surrounding exons; the authors proposed (6, 7) that this intron is spliced at the protein, not the mRNA, level. In the present study, we describe two introns that form a single ORF with the surrounding exons. Furthermore, we present evidence indicating that the Vent DNA polymerase IVSs are removed either by protein splicing or by RNA splicing that requires I-Tli I as a maturase.

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 ϳ25-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°C in the presence of Mg 2؉ , and ϳ14°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 best characterized function of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) 4 is in the uptake of sugars by bacterial cells, which are phosphorylated during their translocation across the cytoplasmic membrane (for reviews see Refs. 1-3). There are many variants of the PTS, but all of them require the first two proteins in the phosphotransfer process, Enzyme I (EI) and the low molecular weight phosphocarrier protein, HPr. The first two steps in the reaction sequence are depicted in Reactions 1 and 2.The phosphoryl group is then transferred from P-HPr to the sugarspecific proteins. The properties of EI have been reviewed previously (4, 5).As discussed in an earlier report (6), the phosphotransfer potential of PEP is so high that the PTS must be stringently regulated or cells would burst with accumulated sugar phosphate. One potential mechanism for this regulation is dependent on the properties of the monomer/dimer transition of EI. The dimer accepts the phosphoryl group from PEP, whereas the monomer does not, and association/dissociation is a very slow process relative to the phosphotransfer reactions. A report on the kinetics of EI phosphorylation and dimerization has recently been published (7), and the hydrodynamic properties of the monomer/dimer transition are described in the companion paper (36).Early work on thermal unfolding of EI, using high sensitivity scanning calorimetry and partial proteolysis,...

These experiments indicate that vitamin Blz affected the conversion of formate to choline, and therefore presumably to methyl groups, more than any other reaction, suggesting a function in an oxidation-reduction system related directly or indirectly to the metabolism of formate.

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