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Three-Dimensional Electron Microscopic Imaging of Membrane Invaginations in Escherichia coli Overproducing the Chemotaxis Receptor Tsr. Jonathan Lefman, 2004.Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable . Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr . Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures . We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters . Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr . We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E . coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis . Enhanced Production of Farnesol by Candida albicans Treated with Four Azoles. Jacob M. Hornby, 2004.The dimorphic fungus Candida albicans excretes farnesol, which is produced enzymatically from the sterol biosynthetic intermediate farnesyl pyrophosphate . Inhibition of C . albicans by four azole antifungals, fluconazole, ketoconazole, miconazole, and clotrimazole, caused elevated farnesol production (10- to 45-fold) . Furthermore, farnesol production occurs in both laboratory strains and clinical isolates (J . M . Hornby et al., Appl . Environ . Microbiol . 67:2982-2992, 2001) of C . albicans . Anaerobic Nitrogen-Fixing Consortia Consisting of Clostridia Isolated from Gramineous Plants. Kiwamu Minamisawa, 2004.We report here the existence of anaerobic nitrogen-fixing consortia (ANFICOs) consisting of N2-fixing clostridia and diverse nondiazotrophic bacteria in nonleguminous plants; we found these ANFICOs while attempting to overcome a problem with culturing nitrogen-fixing microbes from various gramineous plants . A major feature of ANFICOs is that N2 fixation by the anaerobic clostridia is supported by the elimination of oxygen by the accompanying bacteria in the culture . In a few ANFICOs, nondiazotrophic bacteria specifically induced nitrogen fixation of the clostridia in culture . ANFICOs are widespread in wild rice species and pioneer plants, which are able to grow in unfavorable locations . These results indicate that clostridia are naturally occurring endophytes in gramineous plants and that clostridial N2 fixation arises in association with nondiazotrophic endophytes . Respiration-Dependent Utilization of Sugars in Yeasts: a Determinant Role for Sugar Transporters. Paola Goffrini, 2002.In many yeast species, including Kluyveromyces lactis, growth on certain sugars (such as galactose, raffinose, and maltose) occurs only under respiratory conditions . If respiration is blocked by inhibitors, mutation, or anaerobiosis, growth does not take place . This apparent dependence on respiration for the utilization of certain sugars has often been suspected to be associated with the mechanism of the sugar uptake step . We hypothesized that in many yeast species, the permease activities for these sugars are not sufficient to ensure the high substrate flow that is necessary for fermentative growth . By introducing additional sugar permease genes, we have obtained K . lactis strains that were capable of growing on galactose and raffinose in the absence of respiration . High dosages of both the permease and maltase genes were indeed necessary for K . lactis cells to grow on maltose in the absence of respiration . These results strongly suggest that the sugar uptake step is the major bottleneck in the fermentative assimilation of certain sugars in K . lactis and probably in many other yeasts . The Moraxella catarrhalis Immunoglobulin D-Binding Protein MID Has Conserved Sequences and Is Regulated by a Mechanism Corresponding to Phase Variation. Andrea Möllenkvist, 2003.The prevalence of the Moraxella catarrhalis immunoglobulin D (IgD)-binding outer membrane protein MID and its gene was determined in 91 clinical isolates and in 7 culture collection strains . Eighty-four percent of the clinical Moraxella strains expressed MID-dependent IgD binding . The mid gene was detected in all strains as revealed by homology of the signal peptide sequence and a conserved area in the 3' end of the gene . When MID proteins from five different strains were compared, an identity of 65.3 to 85.0% and a similarity of 71.2 to 89.1% were detected . Gene analyses showed several amino acid repeat motifs in the open reading frames, and MID could be called a putative autotransport protein . Interestingly, homopolymeric {polyguanine [poly(G)]} tracts were detected at the 5' ends within the open reading frames . By flow cytometry, using human IgD and fluorescein isothiocyanate-conjugated anti-IgD polyclonal antibodies, most strains showed two peaks: one high- and one low-intensity peak . All isolates expressing high levels of MID had 1, 2, or 3 triplets of G's in their poly(G) tracts, while strains not expressing MID had 4, 7, 8, or 10 G’s in their poly(G) tracts or point mutations causing a putative preterminated translation . Northern blot analysis revealed that the mid gene was regulated at the transcriptional level . Experiments with nonclumping variants of M . catarrhalis proved that bacteria lost their MID expression by removing a G in their poly(G) tracts . Moraxella strains isolated from the nasopharynx or from blood and sputum specimens expressed MID at approximately the same frequency . In addition, no variation was observed between strains of different geographical origins (Australia, Europe, Japan, or the United States) . MID and the mid gene were found solely in M . catarrhalis, whereas related Neisseria and Moraxella species did not express MID . Taken together, MID appears to be a conserved protein that can be found in essentially all M . catarrhalis strains . Furthermore, MID is governed by poly(G) tracts when bacteria undergo phase variation . Curvularia Haloperoxidase: Antimicrobial Activity and Potential Application as a Surface Disinfectant. Eva H. Hansen, 2003.A presumed antimicrobial enzyme system, the Curvularia haloperoxidase system, was examined with the aim of evaluating its potential as a sanitizing agent . In the presence of hydrogen peroxide, Curvularia haloperoxidase facilitates the oxidation of halides, such as chloride, bromide, and iodide, to antimicrobial compounds . The Curvularia haloperoxidase system caused several-log-unit reductions in counts of bacteria (Pseudomonas spp., Escherichia coli, Serratia marcescens, Aeromonas salmonicida, Shewanella putrefaciens, Staphylococcus epidermidis, and Listeria monocytogenes), yeasts (Candida sp . and Rhodotorula sp.), and filamentous fungi (Aspergillus niger, Aspergillus tubigensis, Aspergillus versicolor, Fusarium oxysporum, Penicillium chrysogenum, and Penicillium paxilli) cultured in suspension . Also, bacteria adhering to the surfaces of contact lenses were killed . The numbers of S . marcescens and S . epidermidis cells adhering to contact lenses were reduced from 4.0 and 4.9 log CFU to 1.2 and 2.7 log CFU, respectively, after treatment with the Curvularia haloperoxidase system . The killing effect of the Curvularia haloperoxidase system was rapid, and 106 CFU of E . coli cells/ml were eliminated within 10 min of treatment . Furthermore, the antimicrobial effect was short lived, causing no antibacterial effect against E . coli 10 min after the system was mixed . Bovine serum albumin (1%) and alginate (1%) inhibited the antimicrobial activity of the Curvularia haloperoxidase system, whereas glucose and Tween 20 did not affect its activity . In conclusion, the Curvularia haloperoxidase system is an effective sanitizing system and has the potential for a vast range of applications, for instance, for disinfection of contact lenses or medical devices .
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