Transductional Analysis of Pseudomonas aeruginosa Methionineless Auxotrophs

Seventy-one methionineless and cysteineless auxotrophs of Pseudomonas aeruginosa were placed into nine groups on the basis of their growth on methionine precursors and the cross-feeding response. Transduction experiments with bacteriophage F116 indicated the presence of four linkage groups among the methionineless mutants and at least three among the cysteineless mutants. These studies suggested that the biosynthesis of methionine in P. aeruginosa is similar to that described in other microorganisms, although none of the mutants lacking the ability to methylate homocysteine grew with vitamin B(12) or S-adenosylmethionine.     

Rab proteins in gastric parietal cells: evidence for the membrane recycling hypothesis.

The gastric parietal cell secretes large quantities of HCl into the lumen of the gastric gland in response to secretagogues such as histamine. In the membrane recycling hypothesis, this secretory activity requires the trafficking of the gastric H+/K(+)-ATPase to the cell surface from intracellular tubulovesicles. The Rab subclass of small GTP-binding proteins is thought to confer specificity to vesicle transport throughout the secretory pathway, and previous investigations established that Rab11 is highly expressed in gastric parietal cells. Recent discoveries in intra-Golgi transport and neuronal synaptic vesicle fusion have fortuitously converged on an evolutionarily conserved protein complex involved in vesicle docking and fusion. Recent results indicate that Rab11 is involved in the apical targeting of vesicles in parietal cells and other epithelial cells throughout the gastrointestinal tract. In support of the membrane recycling hypothesis, Rab co-segregates with H+/K(+)-ATPase in parietal cells. The presence of Rab11 on tubulovesicles supports a role for this Rab protein in recycling vesicle trafficking.     

Positive selection is a general phenomenon in the evolution of abalone sperm lysin

Lysin is a 16kDa acrosomal protein used by abalone sperm to create a hole in the egg vitelline envelope (VE). The interaction of lysin with the VE is species-selective and is one step in the multistep fertilization process that restricts heterospecific (cross-species) fertilization. For this reason, the evolution of lysin could play a role in establishing prezygotic reproductive isolation between species. Previously, we sequenced sperm lysin cDNAs from seven California abalone species and showed that positive Darwinian selection promotes their divergence. In this paper an additional 13 lysin sequences are presented representing species from Japan, Taiwan, Australia, New Zealand, South Africa, and Europe. The total of 20 sequences represents the most extensive analysis of a fertilization protein to date. The phylogenetic analysis divides the sequences into two major clades, one composed of species from the northern Pacific (California and Japan) and the other composed of species from other parts of the world. Analysis of nucleotide substitution demonstrates that positive selection is a general process in the evolution of this fertilization protein. Analysis of nucleotide and codon usage bias shows that neither parameter can account for the robust data supporting positive selection. The selection pressure responsible for the positive selection on lysin remains unknown.      

Suppression of B-cell and T-cell responses by the prostaglandin-induced T-cell-derived suppressor (PITS). I. Analysis of the PITS beta factor

Within populations of mitogenically (PWM) stimulated normal human lymphocytes, the proliferation of B lymphocytes is terminated by T cells. In contrast, T cells limit their own proliferation. T cells thus apparently measure and terminate the proliferation of B cells as well as themselves, suggesting an important role for them in limiting amplification during immune response. Under the culture conditions employed, PWM-induced B- and T-cell proliferation was uncoupled from B-cell differentiation into plasmacytes. Termination of B-cell proliferation in this in vitro model of humoral immune response is independent of B-cell differentiation.     

Identification of the protein products of the rrnC, ilv, rho region of the Escherichia coli K-12 chromosome

Summary Two methods have been used to identify the protein products of the Escherichia coli K-12 ilv region at 84 min and the flanking rrnC (counterclockwise) and rho (clockwise) loci. First, a set of dilv specialized transducing phages, including some phages that carry rho and others that carry part of rrnC, was used to infect UV irradiated cells. The proteins produced by the infecting dilv phage were selectively labelled with radioactive amino acids and identified by SDS gel electrophoresis and autoradiography. Second, restriction enzyme fragments were cloned from the dilv phage into pBR322 and the plasmid specific gene products produced in maxicells were similarly identified by SDS gel electrophoresis and autoradiography. The proteins produced were correlated with specific genes and restriction enzyme fragments present in the dilv phage and the pBR322 derivatives. Several ilv gene products that have previously been refractory to protein purification attempts have been identified for the first time by this technique. The presence of mutations at the ilvO site is shown to activate the cryptic ilvG gene and to result in the production of a 62,000 dalton protein. A 15,000 dalton protein of unknown function is synthesized from a DNA segment between ilv and rrnC. The rho gene was cloned from dilv phage into pBR322 and shown to be dominant to a rho mutation on the host cromosome. The rho gene product and four additional proteins coded by genes near or between rho and ilv have been detected.     

Sphingomyelin--lecithin bilayers and their interaction with cholesterol.

Utilizing X-ray diffraction and differential scanning calorimetry (DSC), we have studied (1) the structure and thermotropic properties of hydrated N-palmitoylsphingomyelin, (2) the interaction of N-palmitoylsphingomyelin with dimyristoyllecithin, and (3) the interaction of cholesterol with N-palmitoylsphingomyelin and dimyristoyllecithin, both individually and in a 50:50 (mol/mol) mixture. N-Palmitoylsphingomyelin forms bilayers which undergo a thermotropic order--disorder (gel--liquid crystalline) transition at 40.5 degrees C (delta H = 5.8 kcal/mol). The bilayer repeat distance is 66.8 A at 10 degrees C and 61.6 A at 50 degrees C. N-Palmitoylsphingomyelin exhibits miscibility with dimyristoylecithin in both the gel and liquid-crystalline phases, and no lateral phase separation occurs. Scanning calorimetry indicates that interaction with cholesterol is similar for both N-palmitoylsphingomyelin and dimyristoyllecithin and that in a 50:50 (mol/mol) mixture cholesterol shows no preferential affinity for either phospholipid.     

Threonine deaminase from Escherichia coli. II. Maturation and physical properties of the enzyme from a mutant altered in its regulation of gene expression.

The biosynthetic L-threonine deaminase (L-threonine hydrolase deaminating, EC 4.2.1.16) has been purified from Escherichia coli K12 regulatory mutant CU18. This mutant has properties that follow the predictions of the autogregulatory model previously proposed for the control of synthesis of the isoleucine-valine biosynthetic enzymes. The autoregulatory model specifies that L-threonine deaminase participates in the control of the expression of the ilv ADE gene cluster as well as the ilv B gene and ilv C gene, which constitute three separate units of regulation. The single mutation in strain CU18 results in altered regulation of ilv gene expression and in the production of an altered L-threonine deaminase. The immature form of the enzyme purified from mutant CU18 exhibits an altered response to L-valine, a maturation-inducing ligand. The native form of the mutant is altered in its apparent Km for L-threonine and in its response to the effects of L-valine and L-isoleucine upon catalytic activity. The mutant and wild type L-threonine deaminases differ in the apoenzyme formed as a consequence of alkaline dialysis. Dialysis of the mutant enzyme yields an apoenzyme mixture, apparently of dimers and monomers, while the wild type enzyme yields only dimers. The CU18 L-threonine deaminase, is however, indistinguishable from the wild type enzyme in molecular weight and subunit composition.