Purification and properties of a protein linked to the soluble hydrogenase of hydrogen-oxidizing bacteria.

In Alcaligenes eutrophus, the formation of the hydrogenases and of five new peptides is subject to the hydrogenase control system. Of these, the B peptide was purified to homogeneity. This protein (Mr, 37,500) was composed of two identical subunits (Mr, 18,800). Antibodies against the B protein were used for its quantification by rocket immunoelectrophoresis. About 4% of the total protein consisted of the B protein; its molar ratio to the NAD-linked hydrogenase was about 4:1. The B protein appeared to be associated with the NAD-linked hydrogenase, as shown by gel filtration analysis with Sephadex G-200. The B protein was not detected in cells that had not expressed the hydrogenase proteins or that lacked the genetic information of the hydrogen-oxidizing character; it was also not detected in Tn5 insertional mutants that were unable to form soluble hydrogenase antigens. Immunochemical analysis of other species and genera than A. eutrophus revealed that only strains able to form a NAD-linked hydrogenase also formed B-protein antigens. The B protein is not required for the catalytic activity of soluble hydrogenase in vitro; its function is at present unknown.     

Coordinate regulation of phospholipid biosynthesis by serine in Saccharomyces cerevisiae.

The addition of L-serine to inositol-containing growth medium repressed membrane-associated CDPdiacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) and phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activities and subunit levels in wild-type Saccharomyces cerevisiae. Enzyme activities and subunit levels were not repressed when inositol was absent from the growth medium. The addition of L-serine to the growth medium did not affect the phospholipid composition of wild-type cells. CDPdiacylglycerol synthase and phosphatidylserine synthase were not regulated in the S. cerevisiae inositol biosynthesis ino2, ino4, and opi1 regulatory mutants, suggesting that regulation by inositol plus L-serine is coupled to inositol synthesis. Inositol and L-serine did not affect the activities of purified CDPdiacylglycerol synthase and phosphatidylserine synthase. The addition of compounds structurally related to L-serine to the growth medium of wild-type cells also resulted in a repression of CDPdiacylglycerol synthase and phosphatidylserine synthase but only in the presence of inositol. Phosphatidylinositol synthase (CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was not regulated by inositol plus L-serine.     

Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure.

Methionine aminopeptidase (MAP) catalyzes the removal of amino-terminal methionine from proteins. The Escherichia coli map gene encoding this enzyme was cloned; it consists of 264 codons and encodes a monomeric enzyme of 29,333 daltons. In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine. The methionine residues from the amino-terminal end of the recombinant proteins interleukin-2 (Met-Ala-Pro-IL-2) and ricin A (Met-Ile-Phe-ricin A) could be removed either in vitro with purified MAP enzyme or in vivo in MAP-hyperproducing strains of E. coli. In vitro analyses of the substrate preference of the E. coli MAP indicated that the residues adjacent to the initiation methionine could significantly influence the methionine cleavage process. This conclusion is consistent, in general, with the deduced specificity of the enzyme based on the analysis of known amino-terminal sequences of intracellular proteins (S. Tsunasawa, J. W. Stewart, and F. Sherman, J. Biol. Chem. 260:5382-5391, 1985).     

Cell wall and lipid composition of Isosphaera pallida, a budding eubacterium from hot springs.

Isosphaera pallida is an unusual gliding, budding eubacterium recently isolated from North American hot springs. Electron micrographs of ultrathin sections revealed a cell wall atypical of eubacteria: two electrondense layers separated by an electron-transparent layer, with no evident peptidoglycan layer. Growth was not inhibited by penicillin. Cell walls were isolated from sheared cells by velocity sedimentation. The rigid-layer fraction, prepared from cell walls by treatment with boiling 10% sodium dodecyl sulfate, was hydrolyzed and chemically analyzed for muramic acid. This essential component of peptidoglycan was absent. Amino acid analysis demonstrated a proteinaceous wall structure. Pitlike surface structures seen in negatively stained whole cells and thin sections were correlated with periodically spaced perforations of the rigid sacculus. An analysis of the lipid composition of I. pallida revealed typical ester-linked lipids with unbranched fatty acids, in contrast to the isoprenyl ether-linked lipids of archaebacteria, which also have proteinaceous cell walls. Capnoids, unusual sulfonolipids which are present in gliding bacteria of the Cytophaga-Flexibacter group, were absent.     

Assembly of a chemically synthesized peptide of Escherichia coli type 1 fimbriae into fimbria-like antigenic structures.

Escherichia coli type 1 fimbriae are composed of subunits, each of which comprises 158 amino acids. We synthesized a copy of a 13-residue peptide, located near the NH2 terminus of the fimbrial subunit, that assumed some of the properties of type 1 fimbriae. At pH 5.5 the synthetic peptide autoassembled into fibrillar structures that resembled type 1 fimbriae except that they appeared less rigid and rodlike. A quaternary structure-specific monoclonal antibody against type 1 fimbriae recognized the synthetic peptide in the assembled but not the unassembled state. Furthermore, when the synthetic peptide was injected in its fimbrial conformation into rabbits, it evoked antibodies that reacted with type 1 fimbriae isolated from E. coli.     

Purification and properties of two isozymes of pyruvate kinase from Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was found to contain up to five electrophoretic forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) depending on growth conditions. M. racemosus hyphal cells grown on glutamic acid as the carbon source contained only the fastest electrophoretic form, designated PK1, while yeast cells grown on glucose contained only the slowest electrophoretic form, PK5. Intermediate electrophoretic forms PK2, PK3, and PK4 as well as PK1 and PK5 were found in hyphal cells grown on media containing fructose or cellibiose. All five electrophoretic forms had molecular weights of ca. 230,000 as determined from plots of log Rm versus acrylamide gel concentration. Both PK1 and PK5 were purified to homogeneity and determined to be homotetramers, with subunit molecular weights of 54,000 and 58,100, respectively. The amino acid content of PK1 and PK5 was determined and found to be similar but not identical. Analysis of limited tryptic digests and cyanogen bromide cleavage fragments of PK1 and PK5 indicate that the subunits of the two isozymes are significantly different.     

Regulated nitrate transport in the cyanobacterium Anacystis nidulans.

Intracellular accumulation of nitrate, indicative of the operation of an active nitrate transport system, has been measured in intact cells of the cyanobacterium Anacystis nidulans. The ability of the cells to accumulate nitrate was effectively hindered by either ammonium addition or selective inhibition of CO2 fixation by DL-glyceraldehyde, with the effect of either compound being prevented by previously blocking ammonium assimilation. The results support the contention that nitrate utilization in cyanobacteria is regulated at the level of nitrate transport through the concerted action of ammonium assimilation and CO2 fixation.     

Nucleotide sequence of the gene for the hole-forming toxin aerolysin of Aeromonas hydrophila.

The gene for the hole-forming toxin aerolysin from Aeromonas hydrophila was sequenced. Although most of the sequence seems unrelated to that of Staphylococcus aureus alpha-toxin, both proteins are very hydrophilic, and they each contain a nearly identical string of 10 amino acids.