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Novel Inhalational Murine Model of Invasive Pulmonary Aspergillosis. Donald C. Sheppard, 2004.We developed a novel model of invasive aspergillosis (IA) that recapitulates human disease . Mice were immunosuppressed with cyclophosphamide and cortisone acetate and then infected in an aerosol chamber . This procedure reproducibly delivered 1 x 103 to 3 x 103 conidia to the lungs . Lethal pulmonary IA developed over 2 weeks and was prevented by amphotericin B . Integration of Microbial Ecology and Statistics: a Test To Compare Gene Libraries. Patrick D. Schloss, 2004.Libraries of 16S rRNA genes provide insight into the membership of microbial communities . Statistical methods help to determine whether differences in library composition are artifacts of sampling or are due to underlying differences in the communities from which they are derived . To contribute to a growing statistical framework for comparing 16S rRNA libraries, we present a computer program,  -LIBSHUFF, which calculates the integral form of the Cramér-von Mises statistic . This implementation builds upon the LIBSHUFF program, which uses an approximation of the statistic and makes a number of modifications that improve precision and accuracy . Once
-LIBSHUFF calculates the P values, when pairwise comparisons are tested at the 0.05 level, the probability of falsely identifying a significant P value is 0.098 for a study with two libraries, 0.265 for three libraries, and 0.460 for four libraries . The potential negative effects of making the multiple pairwise comparisons necessitate correcting for the increased likelihood that differences between treatments are due to chance and do not reflect biological differences . Using
-LIBSHUFF, we found that previously published 16S rRNA gene libraries constructed from Scottish and Wisconsin soils contained different bacterial lineages . We also analyzed the published libraries constructed for the zebrafish gut microflora and found statistically significant changes in the community during development of the host . These analyses illustrate the power of
-LIBSHUFF to detect differences between communities, providing the basis for ecological inference about the association of soil productivity or host gene expression and microbial community composition .
Regulatory Circuitry of the CsrA/CsrB and BarA/UvrY Systems of Escherichia coli. Kazushi Suzuki, 2002.The global regulator CsrA (carbon storage regulator) is an RNA binding protein that coordinates central carbon metabolism, activates flagellum biosynthesis and motility, and represses biofilm formation in Escherichia coli . CsrA activity is antagonized by the untranslated RNA CsrB, to which it binds and forms a globular ribonucleoprotein complex . CsrA indirectly activates csrB transcription, in an apparent autoregulatory mechanism . In the present study, we elucidate the intermediate regulatory circuitry of this system . Mutations affecting the BarA/UvrY two-component signal transduction system decreased csrB transcription but did not affect csrA'-'lacZ expression . The uvrY defect was severalfold more severe than that of barA . Both csrA and uvrY were required for optimal barA expression . The latter observation suggests an autoregulatory loop for UvrY . Ectopic expression of uvrY suppressed the csrB-lacZ expression defects caused by uvrY, csrA, or barA mutations; csrA suppressed csrA or barA defects; and barA complemented only the barA mutation . Purified UvrY protein stimulated csrB-lacZ expression approximately sixfold in S-30 transcription-translation reactions, revealing a direct effect of UvrY on csrB transcription . Disruption of sdiA, which encodes a LuxR homologue, decreased the expression of uvrY'-'lacZ and csrB-lacZ fusions but did not affect csrA'-'lacZ . The BarA/UvrY system activated biofilm formation . Ectopic expression of uvrY stimulated biofilm formation by a csrB-null mutant, indicative of a CsrB-independent role for UvrY in biofilm development . Collectively, these results demonstrate that uvrY resides downstream from csrA in a signaling pathway for csrB and that CsrA stimulates UvrY-dependent activation of csrB expression by BarA-dependent and -independent mechanisms . Monitoring Key Reactions in Degradation of Chloroaromatics by In Situ 1H Nuclear Magnetic Resonance: Solution Structures of Metabolites Formed from cis-Dienelactone. Dietmar H. Pieper, 2002.A 1H nuclear magnetic resonance (1H NMR) assay was used to study the enzymatic transformation of cis-dienelactone, a central intermediate in the degradation of chloroaromatics . It was shown that the product of the cis-dienelactone hydrolase reaction is maleylacetate, in which there is no evidence for the formation of 3-hydroxymuconate . Under acidic conditions, the product structure was 4-carboxymethyl-4-hydroxybut-2-en-4-olide . Maleylacetate was transformed by maleylacetate reductase into 3-oxoadipate, a reaction competing with spontaneous decarboxylation into cis-acetylacrylate . One-dimensional 1H NMR in 1H2O could thus be shown to be an excellent noninvasive tool for monitoring enzyme activities and assessing the solution structure of substrates and products . Characterization of a Thermostable D-Stereospecific Alanine Amidase from Brevibacillus borstelensis BCS-1. Dae Heoun Baek, 2003.A gene encoding a new thermostable D-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced . The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits . The purified enzyme exhibited strong amidase activity towards D-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards L-amino acid amides, D-amino acid-containing peptides, and NH2-terminally protected amino acid amides . The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively . The enzyme remained stable within a broad pH range from 7.0 to 10.0 . The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co2+ and Mn2+ . The kcat/Km for D-alaninamide was measured as 544.4 ± 5.5 mM-1 min-1 at 50°C with 1 mM Co2+ .
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