An analysis of the organization and evolution of type 4 fimbrial (MePhe) subunit proteins

Summary We have analyzed and compared the amino acid sequences of the type 4 fimbrial subunits fromPseudomonas aeruginosa, Moraxella bovis, M. nonliquefaciens, Bacteroides nodosus, Neisseria gonorrhoeae, andN. meningitidis. We propose a consensus sequence for the highly conserved aminoterminal regions of these proteins. In the variable regions, a domain corresponding to an epitope common toN. gonorrhoeae andN. meningitidis fimbriae is conserved, both in sequence and in environment, in fimbrial subunits fromB. nodosus. The subunits fromM. bovis andP. aeruginosa do not show any homologies to this sequence. In all of the subunits, the carboxy-terminal half of the molecule consists of a series of fairly hydrophobic domains. The last three domains, two of which include the cysteines of the disulfide bridge inN. gonorrhoeae, P. aeruginosa, andM. bovis, are more or less conserved in sequence in all of the proteins including that ofB. nodosus. We propose that these conserved hydrophobic regions, which have the potential to form a series of beta-sheets, form a structural framework around which more variable hydrophilic sequences determining immunological profile are arranged. The evolutionary relationships of the contemporary proteins and the distribution of type 4 fimbriae are also discussed.     

Cloning and expression in Escherichia coli of the gene encoding the structural subunit of Bacteroides nodosus fimbriae.

Bacteroides nodosus is the primary causative agent of ovine foot rot. Virulent isolates of this bacterium contain fimbriae which appear to play a major role in both infectivity and protective immunity. This paper presents the cloning and expression in Escherichia coli of the gene encoding the structural subunit of the fimbriae of B. nodosus. Total DNA was isolated from B. nodosus VCS 1001 (serogroup A), digested with HindIII, and inserted into the positive-selection vector pTR262. Recombinant E. coli clones were screened directly with anti-fimbrial antiserum by using a colony immunoassay. Several positive colonies were identified, each of which contained the same 5.5-kilobase HindIII insert. The prototype has been designated pBA101. Some clones also contained additional flanking sequences from the B. nodosus genome. Western transfer analyses verified that the positive clones were producing the B. nodosus fimbrial structural subunit, molecular weight ca. 17,500. The level of expression of the antigen in E. coli was comparable to that in B. nodosus itself and was unaffected by the insertion site or orientation of the cloned fragment, indicating that synthesis was being directed from an internal promoter. Restriction mapping and deletion analyses localized the fimbrial subunit gene to the vicinity of a PvuII site near the central region of the original HindIII insert. The expressed antigen was located in the membrane-cell wall fraction and may be exposed on the surface of the recombinant E. coli cells.     

The architecture of the animal fatty acid synthetase. III. Isolation and characterization of beta-ketoacyl reductase

Sequential treatment of the chicken liver fatty acid synthetase by trypsin and subtilisin cleaved the Mr 267,000 subunit to 6-8 polypeptides, ranging in molecular weights from 15,000 to 94,000. Fractionation of the digest by ammonium sulfate and chromatography on a Procion Red HE3B affinity column permitted the isolation of a polypeptide (Mr = 94,000) containing the beta-ketoacyl reductase activity but no other partial activities normally associated with the synthetase. The specific activity of the beta-ketoacyl reductase increased 2 to 3 times in this fraction, an increase that is within the expected range based on relative molecular weight. The kinetic parameters of this fraction towards NADPH and N-acetyl-S-acetoacetyl cysteamine were essentially the same as the beta-ketoacyl reductase component of the intact synthetase. However, the purified fragment did not catalyze the reduction of acetoacetyl-S-CoA derivative, a substrate that is readily reduced by the intact synthetase. Fluorescence measurements with etheno-NADP+ indicate the binding of about 1 mol of NADP+/94,000 daltons, a value which is in agreement with the results obtained from fluorescence measurements with NADPH and the binding of a radiolabeled photoaffinity analog of NADP+. Phenylglyoxal inhibits the beta-ketoacyl reductase activity of either the intact synthetase or the isolated fragment, suggesting the involvement of an essential arginine at or near the active site. Another fragment (Mr 36,000) containing beta-ketoacyl reductase activity was isolated from the synthetase after kallikrein/subtilisin double digestion. Previous mapping studies had shown that this fragment lies adjacent to the COOH-terminal thioesterase domain and overlaps the tryptic Mr 94,000 peptide by approximately 21 daltons. This fragment, but not the Mr 94,000 fragment, was found to contain the phosphopantetheine prosthetic group, indicating that the acyl carrier protein moiety is located in the 15,000-dalton segment that separates the beta-ketoacyl reductase from the thioesterase domain.     

Lipid Alterations During the Fermentation of Dill Pickles

Analyses of various lipid fractions of sections of cucumbers and of good and bloated dill pickles showed that marked changes occur in all lipid fractions during fermentation. The most striking difference noted was the decrease in the phospholipid fraction. A nearly fourfold increase in free fatty acid, as well as a marked increase in the neutral fat fatty acids and unsaponifiables, occurred. Gas chromatographic analyses of the methyl esters of fatty acids from the various lipid fractions yielded further interesting data. From the analyses, 41 esters were identified; however, 16 of the esters accounted for at least 95% of the acids. Among the marked changes were the increases in linoleic and linolenic acids in good pickles, in contrast to the increase in oleic acid in the bloated pickles. The presence of tridecenoic acid in cucumbers, and its absence in pickles; and the absence of caproic, caprylic, and capric acids in cucumbers, and its presence in pickles, were interesting. The data demonstrated that the lipid alterations that occur during fermentation of cucumbers are analogous to those previously reported for the sauerkraut fermentation.     

Identification of two genes with prepilin-like leader sequences involved in type 4 fimbrial biogenesis in Pseudomonas aeruginosa.

Type 4 fimbriae are surface filaments produced by a range of bacterial pathogens for colonization of host epithelial surfaces. In Pseudomonas aeruginosa, they are involved in adhesion as well as in a form of surface translocation called twitching motility, and sensitivity to infection by fimbria-specific bacteriophage. Analysis of the 2.5-kb intergenic region between the previously defined pilR and pilV genes on P. aeruginosa genomic SpeI fragment E has identified three new genes, fimT, fimU, and dadA*. The predicted 18.5-kDa products of the fimT and fimU genes contain prepilin-like leader sequences, whereas the third gene, dadA*, encodes a protein similar to the D-amino acid dehydrogenase of Escherichia coli. Isogenic mutants constructed by allelic exchange demonstrated that the fimU gene was required for fimbrial biogenesis and twitching motility, whereas the fimT and dada* mutants retained wild-type phenotypes. However, overexpression of the fimT gene was found to be able to functionally replace the lack of a fimU gene product, suggesting a subtle role in fimbrial biogenesis. The identification of these proteins increases the similarity between type 4 fimbrial biogenesis and the supersystems involved in macromolecular traffic, such as extracellular protein secretion and DNA uptake, all of which now possess multiple protein species that possess prepilin-like leader sequences.     

Expression of active yeast pyruvate decarboxylase in Escherichia coli.

We have shown by appropriate modification of the translational signals and using the strong T7 RNA polymerase promoter phi 10, that a cloned Saccharomyces cerevisiae pyruvate decarboxylase gene (pdc1) can be expressed in Escherichia coli. This protein, which migrated as a single band on SDS-polyacrylamide gels, was found to have a subunit molecular mass of approximately 62 kDa, similar to that of the enzyme produced by yeast. Polyclonal antibodies raised against purified yeast pyruvate decarboxylase recognized this bacterially produced protein. We found that this recombinant enzyme is active, indicating that the homotetramer encoded by the pdc1 gene is functional.