Bacterial metal detectors

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
Molecular Microbiology (Impact Factor: 4.42). 01/2006; 58(5):1205-9. DOI: 10.1111/j.1365-2958.2005.04904.x
Source: PubMed


Gram negative bacteria can detect environmental iron using outer membrane transporters (OMTs), and then regulate certain transport genes to take advantage of a readily available iron source. This process begins with an iron complex being bound by an OMT, and results in a signal being sent across the outer membrane, the periplasmic space, and the inner membrane, to a sigma factor that interacts with RNA polymerase and initiates transcription of relevant genes. Many of the interactions contributing to signalling have been observed by genetic and biochemical studies, but structural studies, which potentially show these interactions in molecular detail, have been limited. In this issue, Garcia-Herrero and Vogel describe an NMR structure of the periplasmic domain of an OMT, which had not been seen in previous X-ray crystal structures. This domain transmits the 'iron availability' signal to the next protein in the signal transduction cascade, which sits in the inner membrane and extends into the periplasm. The new structure extends our knowledge of transporter architecture and suggests how signalling may occur across the outer membrane.

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    ABSTRACT: Membrane proteins (MPs) are the coveted, yet elusive, targets of structural genomics, structural proteomics, and the pharmaceutical industry. Characterized by amphiphilic surfaces, and lipid stabilized in vivo, MPs require unique combinations of detergent and additives for solubility and stability in vitro. We sought to bypass the exhaustive, and often unsuccessful exploration of detergents and additives for our target hemoglobin binding protein (HgbA): a 105 kDa, 22-stranded ß-barrel outer membrane protein (OMP) from Actinobacillus pleuropneumoniae. To address requirements of structural studies for milligram amounts of soluble target protein, a novel series of fractionating steps was developed to extract and isolate soluble HgbA. Two well established OMP properties were exploited in this pursuit: sarkosyl-insolubility and a robust structural architecture. Total membrane solubilization by sarkosyl detergent was modified for efficiency and fractionated insoluble OMPs from cytoplasmic membrane and soluble cytoplasmic proteins. Liberation of HgbA from the insoluble fraction required treatments more aggressive than variations of detergent and additives. Rigidified with extensive hydrogen bonding, the structural tolerance of ß-barrel proteins was exploited against elevated temperatures. Heat treatment of insoluble OMP fractions at 55 oC preferentially solubilized HgbA yielding 5mg HgbA per litre culture that retained its ability to bind hemoglobin. Protein profiles of these extraction products resolved one major band representing excess amounts of HgbA in the presence of limited quantities of minor species. This extraction protocol produces high quality HgbA that is markedly enriched from fractions that are otherwise inaccessible. Such preparations are advantageous for structural studies as well as promising for application to other ß-barrel proteins. Les protéines de la membrane externe (OMPs) sont les plus en demande pour les études structurales et pharmacologiques, mais sont en même temps les plus difficiles à travailler. Ces protéines, qui sont caractérisées par des surfaces amphiphiles et stabilisées par les lipides in vivo, requièrent des combinaisons uniques de détergents et d'additifs pour les solubiliser et les stabiliser in vitro. Pour des études structurales, nous avons voulu étudier la protéine HgbA (hemoglobin binding protein), une OMP de 105 kDa de la bactérie Actinobacillus pleuropneumoniae, comportant 22 feuillets ß antiparallèles organisés en un tonneau transmembranaire. Comme ces études demandent une grande quantité de protéine soluble, nous avons développé une nouvelle méthode d'extraction et d'isolation de HgbA par fractionnation, qui va au-delà du choix d'un détergent et d'un additif. Nous avons décidé d'exploiter deux propriétés établies des OMPs: leur insolubilité dans le détergent sarkosyl ainsi que leur structure robuste. Les OMPs ne sont pas solubilisées dans le sarkosyl et peuvent ainsi être séparées des protéines du cytoplasme et de la membrane interne. L'isolation de HgbA à partir de la fraction insoluble s'est avérée nécessiter des procédés plus agressifs que la combinaison de détergents et d'additifs. Les protéines à tonneau ß sont stabilisées par de nombreuses liaisons hydrogène, et de ce fait présentent une forte résistance structurale aux hautes températures. L'incubation à 55 oC de fractions OMP insolubles a ainsi permis de solubiliser préférentiellement HgbA, avec un rendement de 5 mg par litre de culture et une préservation de sa capacité de se lier à l'hémoglobine. La résolution de cette fraction solubilisée sur un gel SDS-PAGE montre une bande majeure, correspondant à une grande quantité de HgbA, ainsi qu'un nombre faible d'espèces mineures. Ce protocole d'extraction permet un fort enrichissement en HgbA à partir de fractions jusqu' à présent inaccessibles. De tels procédés seront profitables pour des analyses structurales ainsi que dans l'étude d'autres protéines à tonneau ß.
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    ABSTRACT: Milk mineral (MM) is a type II antioxidant (metal chelator) that can bind iron and prevent iron catalysis of lipid oxidation. Thus, MM might have microbial growth inhibition effects on iron-dependent bacteria. Objective 1 was to evaluate effects of MM on growth of non-pathogenic iron-dependent bacterial strains (Listeria innocua, Eschericia coli, Pseudomonas fluorescens). MM (1.5 % w/v) did not significantly inhibit growth of Listeria and E. coli. However, growth of Pseudomonas fluorescens was consistently and significantly reduced by ~1 log colony forming units per ml (CFU/ml) with all levels of MM (0.5, 0.75, 1.5 % w/v). All levels of MM also had no growth inhibition effects against the mixed microflora of fresh ground beef during storage for up to 10 days at 2°C. In conclusion, MM had little or no effect to inhibit microbial growth. The strong affinity of MM to ionic iron inhibits lipid oxidation, but does not inhibit bacterial growth supported by other forms of iron (heme or amino acid + iron complexes).Several studies report that MM has greater antioxidant effect than sodium tripolyphosphate (STP) in ground meats, especially at longer storage time. Objective 2 was to compare stability of MM and STP in ground beef patties by monitoring the decomposition to soluble orthophosphates (Pi). Patties (control) and patties with 0.75 % MM or 0.5 % STP were stored at 2 or 22°C for 0, 1, or 2 days. CFU/g and Pi were measured. As expected, CFU/g at 22°C was much higher than treatment at 2°C. Pi levels at 2°C were lower (P < 0.05) than at 22°C. At day 0, for both temperatures, patties formulated with MM had the highest Pi levels. However, after 2 days storage, samples with added STP had the highest level of Pi, followed by MM and control. Thus, decomposition as measured by release of Pi was significantly higher for STP than for MM added to beef patties. There was a significant positive correlation (0.77) between CFU/g and Pi during storage of beef patties for 2 days at 22°C. In conclusion, increased Pi during storage of beef patties was at least partially due to bacterial phosphatases. A third experiment was conducted to examine the stability of 0.75 % MM or 0.5 % STP added to growing cultures of Pseudomonas fluorescens at 2°C or 22°C for 0, 1, and 2 days. Neither MM nor STP was stable in autoclaved media (Pi increased significantly). The factors responsible for decomposition of MM or STP in autoclaved media remain to be determined.
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    ABSTRACT: In this critical review we discuss recent advances in understanding the modes of interaction of metal ions with membrane proteins, including channels, pumps, transporters, ATP-binding cassette proteins, G-protein coupled receptors, kinases and respiratory enzymes. Such knowledge provides a basis for elucidating the mechanism of action of some classes of metallodrugs, and a stimulus for the further exploration of the coordination chemistry of metal ions in membranes. Such research offers promise for the discovery of new drugs with unusual modes of action. The article will be of interest to bioinorganic chemists, chemical biologists, biochemists, pharmacologists and medicinal chemists. (247 references).
    Chemical Society Reviews 07/2007; 36(6):968-92. DOI:10.1039/b617040b · 33.38 Impact Factor
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