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Novel Murine Model of Pneumococcal Pneumonia: Use of Temperature as a Measure of Disease Severity To Compare the Efficacies of Moxifloxacin and Levofloxacin. Darrin J. Bast, 2004.Surface temperature measured by an infrared temperature-scanning thermometer was used to evaluate disease severity and predict imminent death in a murine model of pneumococcal pneumonia . We showed that a decrease in temperature was associated with increasing severity of disease and concomitant histological changes and also that a temperature of 30°C or less was a predictor of death . Furthermore, viable bacterial counts in the lungs of mice euthanized at a temperature of  30°C were not significantly different from those seen in the lungs of mice allowed to die without intervention . These data support temperature change as a more subtle indicator of outcome than death and demonstrate that this could be used as a reliable end point for euthanasia . To test the utility of our model in a drug trial, we examined the efficacies of moxifloxacin and levofloxacin by using temperature as a measure of disease severity prior to and during treatment . Regardless of the antibiotic used, mice assessed as moderately ill (temperature
 32°C) at the start of treatment had better clinical and bacteriological outcomes than mice assessed as severely ill (temperature < 32°C) . However, moxifloxacin offered better protection and greater bacterial clearance than did levofloxacin in all infected mice independent of disease severity . This model not only allows a more subtle evaluation of drug efficacy but also ensures a better degree of standardization and a more humane approach to drug efficacy studies involving animals .
Natural Resistance to Inhibitors of the Ubiquinol Cytochrome c Oxidoreductase of Rubrivivax gelatinosus: Sequence and Functional Analysis of the Cytochrome bc1 Complex. Soufian Ouchane, 2002.Biochemical analyses of Rubrivivax gelatinosus membranes have revealed that the cytochrome bc1 complex is highly resistant to classical inhibitors including myxothiazol, stigmatellin, and antimycin . This is the first report of a strain exhibiting resistance to inhibitors of both catalytic Q0 and Qi sites . Because the resistance to cytochrome bc1 inhibitors is primarily related to the cytochrome b primary structure, the petABC operon encoding the subunits of the cytochrome bc1 complex of Rubrivivax gelatinosus was sequenced . In addition to homologies to the corresponding proteins from other organisms, the deduced amino acid sequence of the cytochrome b polypeptide shows (i) an E303V substitution in the highly conserved PEWY loop involved in quinol/stigmatellin binding, (ii) other substitutions that could be involved in resistance to cytochrome bc1 inhibitors, and (iii) 14 residues instead of 13 between the histidines in helix IV that likely serve as the second axial ligand to the bH and bL hemes, respectively . These characteristics imply different functional properties of the cytochrome bc1 complex of this bacterium . The consequences of these structural features for the resistance to inhibitors and for the properties of R . gelatinosus cytochrome bc1 are discussed with reference to the structure and function of the cytochrome bc1 complexes from other organisms . Arginine Operator Binding by Heterologous and Chimeric ArgR Repressors from Escherichia coli and Bacillus stearothermophilus. Anahit Ghochikyan, 2002.Bacillus stearothermophilus ArgR binds efficiently to the Escherichia coli carAB operator, whereas the E . coli repressor binds very poorly to the argCo operator of B . stearothermophilus . In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide . Chimeras carrying the E . coli DNA-binding domain and the B . stearothermophilus oligomerization domain showed sequence-nonspecific rather than sequence-specific interactions with arg operators . Chimeras carrying the B . stearothermophilus DNA-binding domain and E . coli oligomerization domain exhibited a high DNA-binding affinity for the B . stearothermophilus argCo and E . coli carAB operators and repressed the reporter-gene transcription from the B . stearothermophilus PargCo control region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity . With the protein array method, we showed that the wild-type B . stearothermophilus ArgR and derivatives of it containing only the exchanged linker from E . coli ArgR or carrying the B . stearothermophilus DNA-binding domain along with the linker and the  4 regions were able to bind argCo containing the single Arg box . This binding was weaker than binding to the two-box operator but was no longer arginine dependent . Several lines of observations indicate that the
4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria .
FimX, a Multidomain Protein Connecting Environmental Signals to Twitching Motility in Pseudomonas aeruginosa. Bixing Huang, 2003.Twitching motility is a form of surface translocation mediated by the extension, tethering, and retraction of type IV pili . Three independent Tn5-B21 mutations of Pseudomonas aeruginosa with reduced twitching motility were identified in a new locus which encodes a predicted protein of unknown function annotated PA4959 in the P. aeruginosa genome sequence . Complementation of these mutants with the wild-type PA4959 gene, which we designated fimX, restored normal twitching motility . fimX mutants were found to express normal levels of pilin and remained sensitive to pilus-specific bacteriophages, but they exhibited very low levels of surface pili, suggesting that normal pilus function was impaired . The fimX gene product has a molecular weight of 76,000 and contains four predicted domains that are commonly found in signal transduction proteins: a putative response regulator (CheY-like) domain, a PAS-PAC domain (commonly involved in environmental sensing), and DUF1 (or GGDEF) and DUF2 (or EAL) domains, which are thought to be involved in cyclic di-GMP metabolism . Red fluorescent protein fusion experiments showed that FimX is located at one pole of the cell via sequences adjacent to its CheY-like domain . Twitching motility in fimX mutants was found to respond relatively normally to a range of environmental factors but could not be stimulated by tryptone and mucin . These data suggest that fimX is involved in the regulation of twitching motility in response to environmental cues . Attachment of Listeria monocytogenes to Radish Tissue Is Dependent upon Temperature and Flagellar Motility. Lisa Gorski, 2003.Outbreaks of listeriosis and febrile gastroenteritis have been linked to produce contamination by Listeria monocytogenes . In order to begin to understand the physiology of the organism in a produce habitat, the ability of L . monocytogenes to attach to freshly cut radish tissue was examined . All strains tested had the capacity to attach sufficiently well such that they could not be removed during washing of the radish slices . A screen was developed to identify Tn917-LTV3 mutants that were defective in attachment to radish tissue, and three were characterized . Two of the three mutations were in genes with unknown functions . Both of the unknown genes mapped to a region predicted to contain genes necessary for flagellar export; however, only one of the two insertions caused a motility defect . The third insertion was found to be in an operon encoding a phosphoenolpyruvate-sugar phosphotransferase system . All three mutants were defective in attachment when tested at 30°C; the motility mutant had the most severe phenotype . However, not all of the mutants were defective when tested at other temperatures . These results indicate that L . monocytogenes may use different attachment factors at different temperatures and that temperature should be considered an important variable in studies of the molecular mechanisms of Listeria fitness in complex environments . Unexpected Thermal Destruction of Dried, Glass Bead-Immobilized Microorganisms as a Function of Water Activity. C. Laroche, 2003.To help us understand the factors and mechanisms implicated in the death of microorganisms or their resistance to temperature in a low water activity environment, microorganisms were dried on the surface of glass beads before being subjected to high temperatures for a short period followed by rapid cooling . Two microorganisms were studied: the yeast Saccharomyces cerevisiae and the bacterium Lactobacillus plantarum . Experiments were carried out at 150, 200, and 250°C, with four durations of heat treatment and seven levels of initial water activity between 0.10 and 0.70 . We observed an unexpected range of water activity, between 0.30 and 0.50, at which microorganisms were more resistant to the various treatments, with maximal viability at 0.35 for L . plantarum and 0.40 for S . cerevisiae.
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