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Protein Synthesis in Escherichia coli with Mischarged tRNA. Bokkee Min, 2003.Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNAAsp, while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNAAsn, a required intermediate in protein synthesis in many organisms (but not in Escherichia coli) . On the basis of the E . coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNAAsn and its acceptance by elongation factor EF-Tu . While large amounts of Asp-tRNAAsn are detrimental to E . coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase . Inhibitory Activities of Colicins against Escherichia coli Strains Responsible for Postweaning Diarrhea and Edema Disease in Swine. Chad H. Stahl, 2004.The efficacies of colicins E1 and N against Escherichia coli strains responsible for postweaning diarrhea and edema disease, two of the most prevalent disease problems for pigs in the United States, were determined in vitro . These proteins may provide an environmentally sound means for the prevention of these infections in swine . The Type IV Pilus Assembly Complex: Biogenic Interactions among the Bundle-Forming Pilus Proteins of Enteropathogenic Escherichia coli. Sandra W. Ramer, 2002.Production of type IV bundle-forming pili (BFP) by enteropathogenic Escherichia coli (EPEC) requires the protein products of 12 genes of the 14-gene bfp operon . Antisera against each of these proteins were used to demonstrate that in-frame deletion of individual genes within the operon reduces the abundance of other bfp operon-encoded proteins . This result was demonstrated not to be due to downstream polar effects of the mutations but rather was taken as evidence for protein-protein interactions and their role in the stabilization of the BFP assembly complex . These data, combined with the results of cell compartment localization studies, suggest that pilus formation requires the presence of a topographically discrete assembly complex that is composed of BFP proteins in stoichiometric amounts . The assembly complex appears to consist of an inner membrane component containing three processed, pilin-like proteins, BfpI, -J, and -K, that localize with BfpE, -L, and -A (the major pilin subunit); an outer membrane, secretin-like component, BfpB and -G; and a periplasmic component composed of BfpU . Of these, only BfpL consistently localizes with both the inner and outer membranes and thus, together with BfpU, may articulate between the Bfp proteins in the inner membrane and outer membrane compartments . The Complex Flagellar Torque Generator of Pseudomonas aeruginosa. Timothy B. Doyle, 2004.Flagella act as semirigid helical propellers that are powered by reversible rotary motors . Two membrane proteins, MotA and MotB, function as a complex that acts as the stator and generates the torque that drives rotation . The genome sequence of Pseudomonas aeruginosa PAO1 contains dual sets of motA and motB genes, PA1460-PA1461 (motAB) and PA4954-PA4953 (motCD), as well as another gene, motY (PA3526), which is known to be required for motor function in some bacteria . Here, we show that these five genes contribute to motility . Loss of function of either motAB-like locus was dispensable for translocation in aqueous environments . However, swimming could be entirely eliminated by introduction of combinations of mutations in the two motAB-encoding regions . Mutation of both genes encoding the MotA homologs or MotB homologs was sufficient to abolish motility . Mutants carrying double mutations in nonequivalent genes (i.e., motA motD or motB motC) retained motility, indicating that noncognate components can function together . motY appears to be required for motAB function . The combination of motY and motCD mutations rendered the cells nonmotile . Loss of function of motAB, motY, or motAB motY produced similar phenotypes; although the swimming speed was only reduced to  85% of the wild-type speed, translocation in semisolid motility agar and swarming on the surface of solidified agar were severely impeded . Thus, the flagellar motor of P . aeruginosa represents a more complex configuration than the configuration that has been studied in other bacteria, and it enables efficient movement under different circumstances .
Electrophoretic Analysis of Genetic Variability within Cryptosporidium parvum from Imported and Autochthonous Cases of Human Cryptosporidiosis in the United Kingdom. R. B. Gasser, 2003.Cryptosporidium parvum oocyst DNA samples (n = 184) from humans with cryptosporidiosis contracted during foreign travel or during outbreaks in the United Kingdom were characterized genetically and categorized by single-strand conformation polymorphism (SSCP)-based analysis of the small-subunit gene (pSSU) (  300 bp) and second internal transcribed spacer (pITS-2) (230 bp) of nuclear ribosomal DNA . The two recognized genotypes (types 1 and 2) of C . parvum could be readily differentiated by a distinct electrophoretic shift in the pSSU SSCP profile, associated with a nucleotide difference of
1.3 to 1.7% . Of the 102 samples from cases contracted during foreign travel, 88 (86.3%) were identified as C . parvum type 1 and 14 (13.7%) were identified as type 2 . For outbreak samples, unequivocal differentiation between type 1 (n = 20; one child nursery outbreak) and type 2 (n = 62; two waterborne outbreaks) was also achieved . Nucleotide variation in pITS-2 (both within and among samples representing each genotype) was substantially greater (10 to 13 different profiles for each genotype, relating to sequence differences of
1 to 42%) than that in pSSU . SSCP analysis of pITS-2 for all samples revealed that some profiles had a broad geographical distribution whereas others were restricted to particular locations, suggesting a link between some subgenotypes and the geographical origin or source . Comparative denaturing polyacrylamide gel electrophoretic analysis revealed the same genotypic identification and a similar subgenotypic classification of samples as SSCP analysis . The findings of this study, particularly the detection of intragenotypic variation by SSCP, should have significant diagnostic implications for investigating transmission patterns and the monitoring of outbreaks .
Foreshore Sand as a Source of Escherichia coli in Nearshore Water of a Lake Michigan Beach. Richard L. Whitman, 2003.Swimming advisories due to excessive Escherichia coli concentrations are common at 63rd Street Beach, Chicago, Ill . An intensive study was undertaken to characterize the source and fate of E . coli in beach water and sand at the beach . From April through September 2000, water and sand samples were collected daily or twice daily at two depths on three consecutive days per week (water samples, n = 1,747; sand samples, n = 858); hydrometeorological conditions and bird and bather distributions were also recorded . E . coli concentrations in sand and water were significantly correlated, with the highest concentration being found in foreshore sand, followed by those in submerged sediment and water of increasing depth . Gull contributions to E . coli densities in sand and water were most apparent on the day following gull activity in a given area . E . coli recolonized newly placed foreshore sand within 2 weeks . Analysis of variance, correlation, cluster analyses, concentration gradients, temporal-spatial distribution, demographic patterns, and DNA fingerprinting suggest that E . coli may be able to sustain population density in temperate beach sand during summer months without external inputs . This research presents evidence that foreshore beach sand (i) plays a major role in bacterial lake water quality, (ii) is an important non-point source of E . coli to lake water rather than a net sink, (iii) may be environmentally, and perhaps hygienically, problematic, and (iv) is possibly capable of supporting an autochthonous, high density of indicator bacteria for sustained periods, independent of lake, human, or animal input .
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