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Genetic Basis of Erythromycin Resistance in Oral Bacteria. A. Villedieu, 2004.We determined the prevalence of erythromycin-resistant bacteria in the oral cavity and identified mef and erm(B) as the most common resistance determinants . In addition, we demonstrate the genetic linkage, on various Tn1545-like conjugative transposons, between erythromycin and tetracycline resistance in a number of isolates . Novel Forms of Structural Integration between Microbes and a Hydrothermal Vent Gastropod from the Indian Ocean. Shana K. Goffredi, 2004.Here we describe novel forms of structural integration between endo- and episymbiotic microbes and an unusual new species of snail from hydrothermal vents in the Indian Ocean . The snail houses a dense population of  -proteobacteria within the cells of its greatly enlarged esophageal gland . This tissue setting differs from that of all other vent mollusks, which harbor sulfur-oxidizing endosymbionts in their gills . The significantly reduced digestive tract, the isotopic signatures of the snail tissues, and the presence of internal bacteria suggest a dependence on chemoautotrophy for nutrition . Most notably, this snail is unique in having a dense coat of mineralized scales covering the sides of its foot, a feature seen in no other living metazoan . The scales are coated with iron sulfides (pyrite and greigite) and heavily colonized by
 - and
 -proteobacteria, likely participating in mineralization of the sclerites . This novel metazoan-microbial collaboration illustrates the great potential of organismal adaptation in chemically and physically challenging deep-sea environments .
Characterization of comQ and comX, Two Genes Required for Production of ComX Pheromone in Bacillus subtilis. Katherine Bacon Schneider, 2002.Many microbes use secreted peptide-signaling molecules to stimulate changes in gene expression in response to high population density, a process called quorum sensing . ComX pheromone is a modified 10-amino-acid peptide used by Bacillus subtilis to modulate changes in gene expression in response to crowding . comQ and comX are required for production of ComX pheromone . We found that accumulation of ComX pheromone in culture supernatant paralleled cell growth, indicating that there was no autoinduction of production of ComX pheromone . We overexpressed comQ and comX separately and together and found that overexpression of comX alone was sufficient to cause an increase in production of ComX pheromone and early induction of a quorum-responsive promoter . These results indicate that the extracellular concentration of ComX pheromone plays a major role in determining the timing of the quorum response and that expression of comX is limiting for production of ComX pheromone . We made alanine substitutions in the residues that comprise the peptide backbone of ComX pheromone . Analysis of these mutants highlighted the importance of the modification for ComX pheromone function and identified three residues (T50, G54, and D55) that are unlikely to interact with proteins involved in production of or response to ComX pheromone . We have also identified and mutated a putative isoprenoid binding domain of ComQ . Mutations in this domain eliminated production of ComX pheromone, consistent with the hypothesis that ComQ is involved in modifying ComX pheromone and that the modification is likely to be an isoprenoid . Characterization of a Novel Thermostable O-Acetylserine Sulfhydrylase from Aeropyrum pernix K1. Koshiki Mino, 2003.An O-acetylserine sulfhydrylase (OASS) from the hyperthermophilic archaeon Aeropyrum pernix K1, which shares the pyridoxal 5'-phosphate binding motif with both OASS and cystathionine ß-synthase (CBS), was cloned and expressed by using Escherichia coli Rosetta(DE3) . The purified protein was a dimer and contained pyridoxal 5'-phosphate . It was shown to be an enzyme with CBS activity as well as OASS activity in vitro . The enzyme retained 90% of its activity after a 6-h incubation at 100°C . In the O-acetyl-L-serine sulfhydrylation reaction, it had a pH optimum of 6.7, apparent Km values for O-acetyl-L-serine and sulfide of 28 and below 0.2 mM, respectively, and a rate constant of 202 s-1 . In the L-cystathionine synthetic reaction, it showed a broad pH optimum in the range of 8.1 to 8.8, apparent Km values for L-serine and L-homocysteine of 8 and 0.51 mM, respectively, and a rate constant of 0.7 s-1 . A . pernix OASS has a high activity in the L-cysteine desulfurization reaction, which produces sulfide and S-(2,3-hydroxy-4-thiobutyl)-L-cysteine from L-cysteine and dithiothreitol . Improved Quantitative Estimates of Low Environmental Loading and Sporadic Periparturient Shedding of Cryptosporidium parvum in Adult Beef Cattle. E. R. Atwill, 2003.Our primary goal was to generate an accurate estimate of the daily environmental loading rate of Cryptosporidium parvum oocysts for adult beef cattle, using immunomagnetic separation coupled with direct immunofluorescence microscopy for a highly sensitive diagnostic assay . An additional goal was to measure the prevalence and intensity of fecal shedding of C . parvum oocysts in pre- and postparturient cows as an indicator of their potential to infect young calves . This diagnostic method could detect with a  90% probability oocyst concentrations as low as 3.2 oocysts g of feces-1, with a 54% probability of detecting just one oocyst g of feces-1 . Using this diagnostic method, the overall apparent prevalence of adult beef cattle testing positive for C . parvum was 7.1% (17 of 240), with 8.3 and 5.8% of cattle shedding oocysts during the pre- and postcalving periods, respectively . The mean intensity of oocyst shedding for test-positive cattle was 3.38 oocysts g of feces-1 . The estimated environmental loading rate of C . parvum ranged from 3,900 to 9,200 oocysts cow-1 day-1, which is substantially less than a previous estimate of 1.7 x 105 oocysts cow-1 day-1 (range of 7.7 x 104 to 2.3 x 105 oocysts cow-1 day-1) (B . Hoar, E . R . Atwill, and T . B . Farver, Quant . Microbiol . 2:21-36, 2000) . Use of this highly sensitive assay functioned to detect a greater proportion of low-intensity shedders in our population of cattle, which reduced the estimated mean intensity of shedding and thereby reduced the associated environmental loading rate compared to those of previous studies .
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