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Identification of the Secretion and Translocation Domain of the Enteropathogenic and Enterohemorrhagic Escherichia coli Effector Cif, Using TEM-1 ß-Lactamase as a New Fluorescence-Based Reporter. Xavier Charpentier, 2004.Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS) . Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton . Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains . In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 ß-lactamase . Translocation was detected directly in living host cells by using the fluorescent ß-lactamase substrate CCF2/AM . We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells . Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner . A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation . In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation . In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells . Comparison of Fluorescence Microscopy and Solid-Phase Cytometry Methods for Counting Bacteria in Water. John T. Lisle, 2004.Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications . Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data . Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 106 cells filter–1 . In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable . Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 105, the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%) . The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%) . The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods . It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter . In-Field Spatial Variability in the Degradation of the Phenyl-Urea Herbicide Isoproturon Is the Result of Interactions between Degradative Sphingomonas spp . and Soil pH. Gary D. Bending, 2003.Substantial spatial variability in the degradation rate of the phenyl-urea herbicide isoproturon (IPU) [3-(4-isopropylphenyl)-1,1-dimethylurea] has been shown to occur within agricultural fields, with implications for the longevity of the compound in the soil, and its movement to ground- and surface water . The microbial mechanisms underlying such spatial variability in degradation rate were investigated at Deep Slade field in Warwickshire, United Kingdom . Most-probable-number analysis showed that rapid degradation of IPU was associated with proliferation of IPU-degrading organisms . Slow degradation of IPU was linked to either a delay in the proliferation of IPU-degrading organisms or apparent cometabolic degradation . Using enrichment techniques, an IPU-degrading bacterial culture (designated strain F35) was isolated from fast-degrading soil, and partial 16S rRNA sequencing placed it within the Sphingomonas group . Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified bacterial community 16S rRNA revealed two bands that increased in intensity in soil during growth-linked metabolism of IPU, and sequencing of the excised bands showed high sequence homology to the Sphingomonas group . However, while F35 was not closely related to either DGGE band, one of the DGGE bands showed 100% partial 16S rRNA sequence homology to an IPU-degrading Sphingomonas sp . (strain SRS2) isolated from Deep Slade field in an earlier study . Experiments with strains SRS2 and F35 in soil and liquid culture showed that the isolates had a narrow pH optimum (7 to 7.5) for metabolism of IPU . The pH requirements of IPU-degrading strains of Sphingomonas spp . could largely account for the spatial variation of IPU degradation rates across the field . Mutants and Intersexual Heterokaryons of Blakeslea trispora for Production of ß-Carotene and Lycopene. Bina J. Mehta, 2003.The industrial production of ß-carotene with the zygomycete Blakeslea trispora involves the joint cultivation of mycelia of opposite sex in the presence of ß-ionone and other chemical activators . We have obtained improved strains by mutation and heterokaryosis . We chose wild strains on the basis of their growth and carotene content in single and mated cultures . Following exposure of their spores to N-methyl-N'-nitro-N-nitrosoguanidine, we obtained high-carotene mutants, which were more productive than their parents but similar to them in having ß-carotene as the main product . Further increases in carotene content were obtained after a new round of mutagenesis in one of the mutants . The production was shifted to lycopene in cultures incubated in the presence of nicotine and in lycopene-rich mutants derived from the wild strains . The highest production levels were achieved in intersexual heterokaryons, which contained mutant nuclei of opposite sex . These contained up to 39 mg of ß-carotene or 15 mg of lycopene per g (dry mass) under standard laboratory conditions in which the original wild strains contained about 0.3 mg of ß-carotene per g (dry mass) . ß-Ionone did not increase the carotene content of these strains . Not all wild strains lent themselves to these improvements, either because they produced few mutants or because they did not increase their carotene production in mated cultures .
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