RESUMO
The helABC genes are predicted to encode an ATP-binding cassette (ABC) transporter necessary for heme export for ligation in bacterial cytochrome c biogenesis. The recent discoveries of homologs of the helB and helC genes in plant mitochondrial genomes suggest this is a highly conserved transporter in prokaryotes and some eukaryotes with the HelB and HelC proteins comprising the transmembrane components. Molecular genetic analysis in the Gram-negative bacterium Rhodobacter capsulatus was used to show that the helABC and helDX genes are part of an operon linked to the secDF genes. To facilitate analysis of this transporter, strains with non-polar deletions in each gene, epitope and reporter-tagged HelABCD proteins, and antisera specific to the HelA and HelX proteins were generated. We directly demonstrate that this transporter is present in the cytoplasmic membrane as an HelABCD complex. The HelB and HelC but not HelD proteins are necessary for the binding and stability of the HelA protein, the cytoplasmic subunit containing the ATP-binding region. In addition we show that the HelA protein co-immunoprecipitates with either the HelC or HelD proteins. Thus, the HelABCD heme export complex is distinguished by the presence of four membrane-associated subunits and represents a unique subfamily of ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias , Grupo dos Citocromos c/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genética , Epitopos/genética , Deleção de Genes , Genes Bacterianos , Substâncias Macromoleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Óperon , Fenótipo , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/imunologia , Rhodobacter capsulatus/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Two contiguous mcp genes, mcpA and mcpB, transcribed from the same DNA strand and capable of encoding methyl-accepting chemotaxis proteins (Mcp) have been isolated from Rhodobacter capsulatus (Rc), sequences and overexpressed in Escherichia coli (Ec). The deduced proteins (McpA, 69 171 Da; McpB, 81 629 Da) show a structure similar to that of Ec Mcp. The products of mcpA and mcpB, overproduced in Ec, were recognized by anti-Ec Mcp (Trg) antibodies.
Assuntos
Proteínas de Bactérias/genética , Células Quimiorreceptoras , Genes Bacterianos , Proteínas de Membrana/genética , Rhodobacter capsulatus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/imunologia , Sequência de Bases , Escherichia coli/genética , Expressão Gênica , Proteínas de Membrana/biossíntese , Proteínas de Membrana/imunologia , Proteínas Quimiotáticas Aceptoras de Metil , Metilação , Dados de Sequência Molecular , Peso Molecular , Rhodobacter capsulatus/imunologia , Homologia de Sequência , Especificidade da EspécieRESUMO
Shock due to Gram-negative bacterial sepsis is a consequence of acute inflammatory response to lipopolysaccharide (LPS) or endotoxin released from bacteria. LPS is a major constituent of the outer membrane of Gram-negative bacteria, and its terminal disaccharide phospholipid (lipid A) portion contains the key structural features responsible for toxic activity. Based on the proposed structure of nontoxic Rhodobacter capsulatus lipid A, a fully stabilized endotoxin antagonist E5531 has been synthesized. In vitro, E5531 demonstrated potent antagonism of LPS-mediated cellular activation in a variety of systems. In vivo, E5531 protected mice from LPS-induced lethality and, in cooperation with an antibiotic, protected mice from a lethal infection of viable Escherichia coli.
Assuntos
Endotoxinas/antagonistas & inibidores , Lipídeo A/análogos & derivados , Animais , Vacina BCG/imunologia , Citocinas/metabolismo , Desenho de Fármacos , Infecções por Escherichia coli/imunologia , Bactérias Gram-Negativas/imunologia , Humanos , Técnicas In Vitro , Lipídeo A/síntese química , Lipídeo A/química , Lipídeo A/farmacologia , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Moxalactam/farmacologia , Óxido Nítrico/metabolismo , Rhodobacter capsulatus/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The PufQ protein has been detected in vivo for the first time by Western blot (immunoblot) analyses of the chromatophore membranes of Rhodobacter capsulatus. The PufQ protein was not visible in Western blots of membranes of a mutant (delta RC6) lacking the puf operon but appeared in membranes of the same mutant to which the pufQ gene had been added in trans. It was also detected in elevated amounts in a mutant (CB1200) defective in two bch genes and unable, therefore, to make bacteriochlorophyll. The extremely hydrophobic nature of the PufQ protein was also apparent in these studies since it was not extracted from chromatophores by 3% (wt/vol) n-octyl-beta-D-glucopyranoside, a procedure which solubilized the reaction center and light-harvesting complexes. During adaptation of R. capsulatus from aerobic to semiaerobic growth conditions (during which time the synthesis of bacteriochlorophyll was induced), the PufQ protein was observed to increase to the level of detection in the developing chromatophore fraction approximately 3 h after the start of the adaptation. The enzyme, S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase, also increased in amount in the developing chromatophore fraction but was present in a cell membrane fraction at the start of the adaptation as well.
