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Membrane Topology of the ZntB Efflux System of Salmonella enterica Serovar Typhimurium. Andreia M. Caldwell, 2003.The membrane topology of the ZntB Zn2+ transport protein of Salmonella enterica serovar Typhimurium was determined by constructing deletion derivatives of the protein and genetically fusing them to blaM or lacZ cassettes . The enzymatic activities of the hybrid proteins indicate that ZntB is a bitopic integral membrane protein consisting largely of two independent domains . The first 266 amino acids form a large, highly charged domain within the cytoplasm, while the remaining 61 residues form a small membrane domain containing two membrane-spanning segments . The overall orientation towards the cytoplasm is consistent with the ability of ZntB to facilitate zinc efflux . Nitrite-Responsive Activation of the Nitrate Assimilation Operon in Cyanobacteria Plays an Essential Role in Up-Regulation of Nitrate Assimilation Activities under Nitrate-Limited Growth Conditions. Makiko Aichi, 2004.NtcB of the cyanobacterium Synechococcus elongatus strain PCC 7942 is a LysR family protein that enhances expression of the nitrate assimilation operon (nirA operon) in response to the presence of nitrite, an intermediate of assimilatory nitrate reduction . Inactivation of ntcB in this cyanobacterium specifically abolishes the nitrite responsiveness of nirA operon expression, but under nitrate-replete conditions (wherein negative feedback by intracellularly generated ammonium prevails over the positive effect of nitrite) activity levels of the nitrate assimilation enzymes are marginally higher in the wild-type cells than in the mutant cells, raising the issue of whether the nitrite-promoted regulation has physiological importance . On the other hand, the strains carrying ntcB expressed much higher nitrate assimilation enzyme activities under nitrate-limited growth conditions than under nitrate-replete conditions whereas the ntcB-deficient strains showed levels of the enzyme activities lower than those seen under the nitrate-replete conditions . Although the ntcB mutant maintained a constant cell population in a nitrate-limited chemostat when grown as a single culture, it was diluted at a rate expected for nondividing cells when mixed with the wild-type cells and subjected to nitrate limitation in the chemostat culture system . These results demonstrated that the nitrite-promoted activation of the nitrate assimilation operon is essential for up-regulation of the nitrate assimilation activities under the conditions of nitrate limitation and for competitive utilization of nitrate . Automated Enumeration of Groups of Marine Picoplankton after Fluorescence In Situ Hybridization. Jakob Pernthaler, 2003.We describe here an automated system for the counting of multiple samples of double-stained microbial cells on sections of membrane filters . The application integrates an epifluorescence microscope equipped with motorized z-axis drive, shutters, and filter wheels with a scanning stage, a digital camera, and image analysis software . The relative abundances of specific microbial taxa are quantified in samples of marine picoplankton, as detected by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition . Pairs of microscopic images are automatically acquired from numerous positions at two wavelengths, and microbial cells with both general DNA and FISH staining are counted after object edge detection and signal-to-background ratio thresholding . Microscopic fields that are inappropriate for cell counting are automatically excluded prior to measurements . Two nested walk paths guide the device across a series of triangular preparations until a user-defined number of total cells has been analyzed per sample . A backup autofocusing routine at incident light allows automated refocusing between individual samples and can reestablish the focal plane after fatal focusing errors at epifluorescence illumination . The system was calibrated to produce relative abundances of FISH-stained cells in North Sea samples that were comparable to results obtained by manual evaluation . Up to 28 preparations could be analyzed within 4 h without operator interference . The device was subsequently applied for the counting of different microbial populations in incubation series of North Sea waters . Automated digital microscopy greatly facilitates the processing of numerous FISH-stained samples and might thus open new perspectives for bacterioplankton population ecology .
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