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Cellular Stoichiometry of the Components of the Chemotaxis Signaling Complex. Mingshan Li, 2004.The chemotactic sensory system of Escherichia coli comprises membrane-embedded chemoreceptors and six soluble chemotaxis (Che) proteins . These components form signaling complexes that mediate sensory excitation and adaptation . Previous determinations of cellular content of individual components provided differing and apparently conflicting values . We used quantitative immunoblotting to perform comprehensive determinations of cellular amounts of all components in two E . coli strains considered wild type for chemotaxis, grown in rich and minimal media . Cellular amounts varied up to 10-fold, but ratios between proteins varied no more than 30% . Thus, cellular stoichiometries were almost constant as amounts varied substantially . Calculations using those cellular stoichiometries and values for in vivo proportions of core components in complexes yielded an in vivo stoichiometry for core complexes of 3.4 receptor dimers and 1.6 CheW monomers for each CheA dimer and 2.4 CheY, 0.5 CheZ dimers, 0.08 CheB, and 0.05 CheR per complex . The values suggest a core unit of a trimer of chemoreceptor dimers, a dimer (or two monomers) of kinase CheA, and two CheW . These components may interact in extended arrays and, thus, stoichiometries could be nonintegral . In any case, cellular stoichiometries indicate that CheY could be bound to all signaling complexes and this binding would recruit essentially the entire cellular complement of unphosphorylated CheY, and also that phosphatase CheZ, methylesterase CheB, and methyltransferase CheR would be present at 1 per 2, per 14, and per 20 core complexes, respectively . These characteristic ratios will be important in quantitative treatments of chemotaxis, both experimental and theoretical . Inhibition of Adherence and Killing of Candida albicans with a 23-Mer Peptide (Fn/23) with Dual Antifungal Properties. Stephen A. Klotz, 2004.Candida albicans adheres to host tissue and then proliferates in order to establish a commensal as well as a pathogenic state . Specific adherence to proteins is provided by several surface adhesins of Candida . Two well-studied proteins, Als1p and Als5p, do not require energy for adherence to occur (dead as well as living cells adhere) and have a multiplier effect of cell-cell aggregation that mediates the formation of microcolonies of Candida cells . The entire process is spontaneous, reversible, and stable for physiologically relevant chemical and physical forces . This adherence process is inhibited by the addition of free peptide ligands, including a 23-mer derived from fibronectin (Fn/23) that binds to the adhesins through H bond formation . Adherence was measured by determining the number of yeast cells that adhered to 90-µm-diameter polyethylene glycol (PEG) beads with a 7-mer peptide (KLRIPSV) synthesized on the surfaces of the beads . The concentration of the Fn/23 peptide that inhibited the adherence of cells to the peptide-coated beads by 50% was 4 to 5 µM, and the magnitudes of adherence were similar regardless of the presence or absence of physiologic salt concentrations . The minimum fungicidal concentration of Fn/23 was 2 to 4 µM in water, but there was no killing in physiologic salt concentrations . Peptides from the C and N termini or the center sequence of Fn/23 had no effect on inhibition of adherence and little effect on fungal viability . The fungicidal effect was similar to that seen with 23-, 19-, and 18-mer peptides derived from porcine myeloid cells, a Helicobacter pylori ribosomal protein, and a hybrid of cecropin and magainin, respectively . However, these fungicidal peptides did not inhibit C . albicans adherence to the peptide-coated PEG beads . This dual property of Fn/23, i.e., inhibition of adherence and killing of C . albicans, may provide important adjuvant effects in the treatment of disease caused by this fungus . Initial Proteome Analysis of Model Microorganism Haemophilus influenzae Strain Rd KW20. Eugene Kolker, 2003.The proteome of Haemophilus influenzae strain Rd KW20 was analyzed by liquid chromatography (LC) coupled with ion trap tandem mass spectrometry (MS/MS) . This approach does not require a gel electrophoresis step and provides a rapidly developed snapshot of the proteome . In order to gain insight into the central metabolism of H . influenzae, cells were grown microaerobically and anaerobically in a rich medium and soluble and membrane proteins of strain Rd KW20 were proteolyzed with trypsin and directly examined by LC-MS/MS . Several different experimental and computational approaches were utilized to optimize the proteome coverage and to ensure statistically valid protein identification . Approximately 25% of all predicted proteins (open reading frames) of H . influenzae strain Rd KW20 were identified with high confidence, as their component peptides were unambiguously assigned to tandem mass spectra . Approximately 80% of the predicted ribosomal proteins were identified with high confidence, compared to the 33% of the predicted ribosomal proteins detected by previous two-dimensional gel electrophoresis studies . The results obtained in this study are generally consistent with those obtained from computational genome analysis, two-dimensional gel electrophoresis, and whole-genome transposon mutagenesis studies . At least 15 genes originally annotated as conserved hypothetical were found to encode expressed proteins . Two more proteins, previously annotated as predicted coding regions, were detected with high confidence; these proteins also have close homologs in related bacteria . The direct proteomics approach to studying protein expression in vivo reported here is a powerful method that is applicable to proteome analysis of any (micro)organism . Effect of Associated Bacteria on the Growth and Toxicity of Alexandrium catenella. Paulina Uribe, 2003.Saprophytic bacteria in cultures of the marine dinoflagellate Alexandrium catenella were removed to assess their effect on growth and paralytic shellfish poisoning toxin production of this dinoflagellate . The actual axenic status was demonstrated by the lack of observable bacteria both immediately after treatment and following extended incubation in the absence of antibiotics . Bacteria were measured by counting CFU and also by epifluorescence microscopy and PCR amplification of bacterial 16S-23S spacer ribosomal DNA to detect noncultivable bacteria . Removal of bacteria did not have any effect on the growth of the dinoflagellate except for the inhibition of A . catenella disintegration after reaching the stationary phase . Toxicity was determined in dinoflagellate cell extracts by different methods: high-performance liquid chromatography (HPLC); an electrophysiological test called the Electrotest, which measures the inhibition of saxitoxin-sensitive Na+ channels expressed in a cell line; and a mouse bioassay, which measures the toxic effect on the whole mammal neuromuscular system . A lower toxicity of the dinoflagellates in axenic culture was observed by these three methods, though the difference was significant only by the mouse bioassay and HPLC methods . Altogether the results indicate that axenic cultures of A . catenella are able to produce toxin, though the total toxicity is probably diminished to about one-fifth of that in nonaxenic cultures .
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