Cloning and expression of the pediocin operon in Streptococcus thermophilus and other lactic fermentation bacteria.

Production of pediocin in Pediococcus acidilactici is associated with pMBR1.0, which encodes prepediocin, a pediocin immunity protein, and two proteins involved in secretion and precursor processing. These four genes are organized as an operon under control of a single promoter. We have constructed shuttle vectors that contain all four structural genes, the chromosomal promoter ST(P2201) from Streptococcus thermophilus, and repA from the 2-kbp S. thermophilus plasmid pER8. The recombinant plasmid, pPC318, expressed and secreted active pediocin in Escherichia coli. Streptococcus thermophilus, Lactococcus lactis subsp. lactis, and Enterococcus faecalis were electrotransformed with pPC418, a modified vector fitted with an erythromycin resistance tracking gene. Pediocin was produced and secreted in each of the lactic acid bacteria, and production was stable for up to ten passages. The expression of pediocin in dairy fermentation microbes has important implications for bacteriocins as food preservatives in dairy products.

Creating health with chronic illness.

Within the current context of health care, health promotion for individuals with chronic illness often reflects the priorities of disease-specific preventive care needs and related physical, social, emotional, and spiritual well-being. This article reports a phenomenological study of how older people with chronic illness experience health and health promotion and illuminates a different perspective of health resources and strategies. The findings have profound implications for nursing practice and theory, suggesting the need for restructuring work assignments and refocusing nursing care more clearly away from the medical model.

Glucose transporter function is controlled by transporter oligomeric structure. A single, intramolecular disulfide promotes GLUT1 tetramerization.

The human erythrocyte glucose transporter is an allosteric complex of four GLUT1 proteins whose structure and substrate binding properties are stabilized by reductant-sensitive, noncovalent subunit interactions [Hebert, D. N., & Carruthers, A. (1992) J. Biol. Chem. 267, 23829-23838]. In the present study, we use biochemical and molecular approaches to isolate specific determinants of transporter oligomeric structure and transport function. When unfolded in denaturant, each subunit (GLUT1 protein) of the transporter complex exposes two sulfhydryl groups. Four additional thiol groups are accessible following subunit exposure to reductant. Assays of subunit disulfide bridge content suggest that two inaccessible sulfhydryl groups form an internal disulfide bridge. Differential alkylation/peptide mapping/N-terminal sequence analyses show that a GLUT1 carboxyl-terminal peptide (residues 232-492) contains three inaccessible sulfhydryl groups and that an N-terminal GLUT1 peptide (residues 147-261/299) contains two accessible thiols. The carboxyl-terminal peptide most likely contains the intramolecular disulfide bridge since neither its yield nor its electrophoretic mobility is altered by addition of reductant. Each GLUT1 cysteine was changed to serine by oligonucleotide-directed, in vitro mutagenesis. The resulting transport proteins were expressed in CHO cells and screened by immunofluorescence microscopy for their ability to expose tetrameric GLUT1-specific epitopes. Serine substitution at cysteine residues 133, 201, 207, and 429 does not inhibit exposure of tetrameric GLUT1-specific epitopes. Serine substitution at cysteines 347 or 421 prevents exposure of tetrameric GLUT1-specific epitopes. Hydrodynamic analysis of GLUT1/GLUT4 chimeras expressed in and subsequently solubilized from CHO cells indicates that GLUT1 residues 1-199 promote chimera dimerization and permit GLUT1/chimera heterotetramerization. This GLUT1 N-terminal domain is insufficient for chimera tetramerization which additionally requires GLUT1 residues 200-463. Extracellular reductants (dithiothreitol, beta-mercaptoethanol, or glutathione) reduce erythrocyte 3-O-methylglucose uptake by up to 15-fold. This noncompetitive inhibition of sugar uptake is reversed by the cell-impermeant, oxidized glutathione. Reductant is without effect on sugar exit from erythrocytes. Dithiothreitol doubles the cytochalasin B binding capacity of erythrocyte-resident glucose transporter, abolishes allosteric interactions between substrate binding sites on adjacent subunits, and occludes tetrameric GLUT1-specific GLUT1 epitopes in situ. CHO cell-resident GLUT1 structure and transport function are similarly affected by extracellular reductant. We conclude that each subunit of the glucose transporter contains an extracellular disulfide bridge (Cys347 and Cys421) that stabilizes transporter oligomeric structure and thereby accelerates transport function.

Rapid substrate translocation by the multisubunit, erythroid glucose transporter requires subunit associations but not cooperative ligand binding.

The human erythroid glucose transporter is a GLUT1 homotetramer whose structure and function are stabilized by noncovalent, cooperative subunit interactions. The present study demonstrates that exofacial tryptic digestion of GLUT1 abolishes cooperative interactions between substrate binding sites on adjacent subunits under circumstances where subunit associations and high catalytic turnover are maintained. Extracellular trypsin produces rapid, quantitative cleavage of the human red cell-resident sugar transport protein, GLUT1. One major carboxyl-terminal peptide of M(r)(app) 25,000 is detected by immunoblot analysis. Endofacial tryptic digestion of GLUT1 results in the complete loss of GLUT1 carboxyl-terminal structure. GLUT1-mediated erythrocyte sugar uptake, transport inhibition by cytochalasin B, and GLUT1 oligomeric structure are unaffected by exofacial GLUT1 proteolysis. In contrast, the cytochalasin B binding capacity of GLUT1 and the Kd(app) for cytochalasin B binding to the transporter are doubled following exofacial tryptic digestion of GLUT1. Photoaffinity labeling experiments show that increased cytochalasin B binding results from increased ligand binding to the 25 kDa carboxyl-terminal GLUT1 peptide. Proteolysis abolishes allosteric interactions between sugar import (maltose binding) and sugar export (cytochalasin B binding) sites that normally exist on adjacent subunits within the transporter complex, but interact with negative cooperativity. Following exofacial proteolysis, these sites become mutually exclusive. Dithiothreitol disrupts GLUT1 quaternary structure, inhibits 3-O-methylglucose transport, and abolishes cooperative interactions between sugar import and export sites in control cells. Studies with reconstituted purified GLUT1 confirm that the action of trypsin on cytochalasin B binding is direct, show that proteolysis increases the apparent affinity of the sugar efflux site for transported sugars, and suggest that the membrane bilayer stabilizes GLUT1 noncovalent structure and catalytic function following GLUT1 proteolysis. Collectively, these findings demonstrate that GLUT1 does not require an intact polypeptide backbone for catalytic function. They show that the multisite sugar transporter mechanism is converted to a simple ping-pong carrier mechanism following exofacial GLUT1 proteolysis. They reveal that subunit cooperativity can be lost under circumstances where cohesive structural interactions between transporter subunits are maintained. They also refute the hypothesis [Hebert, D. N., & Carruthers, A. (1992) J. Biol. Chem. 267, 23829-23838] that rapid substrate translocation by the multisubunit erythroid glucose transporter requires cooperative interactions between subunit ligand binding sites.

The entD gene of the Escherichia coli K12 enterobactin gene cluster.

The Escherichia coli entD gene encodes a product necessary for the synthesis of the iron-chelating and transport molecule enterobactin (Ent); cells harbouring entD mutations fail to grow in iron-deficient environments. For unknown reasons, it has not been possible to identify the entD product. The nucleotide sequence of the entD region has now been determined. An open reading frame extending in the same direction as the adjacent fepA gene and capable of encoding an approximately 24 kDa polypeptide was found; it contained a high percentage of rare codons and two possible translational start sites. Complementation data suggested that EntD proteins truncated at the carboxy terminus retain some activity. Two REP sequences were present upstream of entD and an IS186 sequence was observed downstream. RNA dot-blot hybridizations demonstrated that entD is transcribed from the strand predicted by the sequencing results. An entD-lacZ recombinant plasmid was constructed and shown to express low amounts of a fusion protein of the anticipated size (approximately 125 kDa). The evidence suggests a number of possible explanations for difficulties in detecting the entD product. Sequence data indicate that if entD has its own promoter, it is weak; the REP sequences suggest that entD mRNA may be destabilized; and translation may be slow because of the frequency of rare codons and a possible unusual start codon (UUG). The data are also consistent with previous evidence that the entD product is unstable.