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Requirements for CuA and Cu-S Center Assembly of Nitrous Oxide Reductase Deduced from Complete Periplasmic Enzyme Maturation in the Nondenitrifier Pseudomonas putida. Patrick Wunsch, 2003.Bacterial nitrous oxide (N2O) reductase is the terminal oxidoreductase of a respiratory process that generates dinitrogen from N2O . To attain its functional state, the enzyme is subjected to a maturation process which involves the protein-driven synthesis of a unique copper-sulfur cluster and metallation of the binuclear CuA site in the periplasm . There are seven putative maturation factors, encoded by nosA, nosD, nosF, nosY, nosL, nosX, and sco . We wanted to determine the indispensable proteins by expressing nos genes from Pseudomonas stutzeri in the nondenitrifying organism Pseudomonas putida . An in silico study of denitrifying bacteria revealed that nosL, nosX (or a homologous gene, apbE), and sco, but not nosA, coexist consistently with the N2O reductase structural gene and other maturation genes . Nevertheless, we found that expression of only three maturation factors (periplasmic protein NosD, cytoplasmic NosF ATPase, and the six-helix integral membrane protein NosY) together with nosRZ in trans was sufficient to produce catalytically active holo-N2O reductase in the nondenitrifying background . We suggest that these obligatory factors are required for Cu-S center assembly . Using a mutational approach with P . stutzeri, we also studied NosA, the Cu-containing outer membrane protein previously thought to have Cu insertase function, and ScoP, a putative membrane-anchored chaperone for CuA metallation . Both of these were found to be dispensable elements for N2O reductase biosynthesis . Our experimental and in silico data were integrated in a model of N2O reductase maturation . Effect of Stress on the Ability of a phlA-Based Quantitative Competitive PCR Assay To Monitor Biocontrol Strain Pseudomonas fluorescens CHA0. Fabio Rezzonico, 2003.A quantitative competitive PCR (QC-PCR) assay targeting the phlA gene of Pseudomonas fluorescens CHA0 was developed and tested in vitro . Statistically significant, positive correlations were found between QC-PCR and both CFU and total cell number when studying cells in log or stationary phase . The correlations disappeared when considering stressed cells . A Rapid Microtiter Plate Method To Measure Carbon Dioxide Evolved from Carbon Substrate Amendments so as To Determine the Physiological Profiles of Soil Microbial Communities by Using Whole Soil. Colin D. Campbell, 2003.Sole-carbon-source tests (Biolog), designed to identify bacteria, have become very popular for metabolically fingerprinting soil microbial communities, despite disadvantages associated with the use of carbon source profiles that primarily select for fast-growing bacteria . In this paper we describe the use of an alternative method that combines the advantages of the Biolog community-level physiological profile (CLPP) method, in which microtiter-based detection plates are used, with the ability to measure carbon dioxide evolution from whole soil . This method facilitates measurement over short periods of time (4 to 6 h) and does not require the extraction and culturing of organisms . Deep-well microtiter plates are used as test wells into which soil is placed . The apparatus to fill the deep-well plates and interface it with a second removable detection plate is described . Two detection systems, a simple colorimetric reaction in absorbent alkali and scintillation counting with radioactive carbon sources, are described . The methods were compared to the Biolog-CLPP system by using soils under different vegetation types and soil treated with wastewater sludge . We aimed to test the hypothesis that using whole soil would have specific advantages over using extracts in that more immediate responses to substrates could be obtained that would reflect activity rather than growth . The whole-soil method was more rapid and gave earlier detection of C source use . Also, the metabolic fingerprints obtained could discriminate between sludge treatments .
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