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Sequence Diversity and Molecular Evolution of the Heat-Modifiable Outer Membrane Protein Gene (ompA) of Mannheimia(Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi. Robert L. Davies, 2004.The OmpA (or heat-modifiable) protein is a major structural component of the outer membranes of gram-negative bacteria . The protein contains eight membrane-traversing ß-strands and four surface-exposed loops . The genetic diversity and molecular evolution of OmpA were investigated in 31 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains by comparative nucleotide sequence analysis . The OmpA proteins of M . haemolytica and M . glucosida contain four hypervariable domains located at the distal ends of the surface-exposed loops . The hypervariable domains of OmpA proteins from bovine and ovine M . haemolytica isolates are very different but are highly conserved among strains from each of these two host species . Fourteen different alleles representing four distinct phylogenetic classes, classes I to IV, were identified in M . haemolytica and M . glucosida . Class I, II, and IV alleles were associated with bovine M . haemolytica, ovine M . haemolytica, and M . glucosida strains, respectively, whereas class III alleles were present in certain M . haemolytica and M . glucosida isolates . Class I and II alleles were associated with divergent lineages of bovine and ovine M . haemolytica strains, respectively, indicating a history of horizontal DNA transfer and assortative (entire gene) recombination . Class III alleles have mosaic structures and were derived by horizontal DNA transfer and intragenic recombination . Our findings suggest that OmpA is under strong selective pressure from the host species and that it plays an important role in host adaptation . It is proposed that the OmpA protein of M . haemolytica acts as a ligand and is involved in binding to specific host cell receptor molecules in cattle and sheep. P . trehalosi expresses two OmpA homologs that are encoded by different tandemly arranged ompA genes . The P . trehalosi ompA genes are highly diverged from those of M . haemolytica and M . glucosida, and evidence is presented to suggest that at least one of these genes was acquired by horizontal DNA transfer . Dominant Microbial Composition and Its Vertical Distribution in Saline Meromictic Lake Kaiike (Japan) as Revealed by Quantitative Oligonucleotide Probe Membrane Hybridization. Yoshikazu Koizumi, 2004.Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization . Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed . In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two  -Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions . The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68% . The abundance of the
-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca . 34% . The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment . Any SSU rRNA of Chloroflexi in the water column was under the detection limit . The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations .
In Vivo Characterization of Escherichia coli ftsZ Mutants: Effects on Z-Ring Structure and Function. Jesse Stricker, 2003.We have characterized the in vivo phenotypes of 17 mutations of Escherichia coli ftsZ . In particular, we determined whether these mutations can complement a null ftsZ phenotype, and we demonstrated that two noncomplementing mutations show partial dominant-negative behavior . We performed immunofluorescence microscopy to determine whether these mutants could assemble into normal or abnormal structures in vivo . The mutants separated into four classes—those that complemented the null and formed normal FtsZ rings, those that complemented the null but formed aberrant FtsZ structures, those that formed aberrant FtsZ structures and did not complement, and those that were unable to form any FtsZ structures . We did not find any mutations that produced nonfunctional Z rings of normal appearance . Surprisingly, some mutants that produced extensively spiraled Z-ring structures divided and grew with a normal doubling time . The analysis was carried out using a complementation system based on an ftsZ deletion strain, a temperature-sensitive rescue plasmid, and a complementation vector that placed mutated ftsZ alleles under the control of the pBAD promoter, which offered several advantages over previous systems . Bacteriocin Detection from Whole Bacteria by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry. Thomas Hindré, 2003.Class I bacteriocins (lantibiotics) and class II bacteriocins are antimicrobial peptides secreted by gram-positive bacteria . Using two lantibiotics, lacticin 481 and nisin, and the class II bacteriocin coagulin, we showed that bacteriocins can be detected without any purification from whole producer bacteria grown on plates by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) . When we compared the results of MALDI-TOF-MS performed with samples of whole cells and with samples of crude supernatants of liquid cultures, the former samples led to more efficient bacteriocin detection and required less handling . Nisin and lacticin 481 were both detected from a mixture of their producer strains, but such a mixture can yield additional signals . We used this method to determine the masses of two lacticin 481 variants, which confirmed at the peptide level the effect of mutations in the corresponding structural gene . Reactive Oxygen Species and Induction of Lignin Peroxidase in Phanerochaete chrysosporium. Paula A. Belinky, 2003.We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O2) of Phanerochaete chrysosporium by comparing levels of reactive oxygen species (ROS), cumulative oxidative damage, and antioxidant enzymes with those found in non-LIP-producing cultures (cultures grown with free exchange of atmospheric air [control cultures]) . A significant increase in the intracellular peroxide concentration and the degree of oxidative damage to macromolecules, e.g., DNA, lipids, and proteins, was observed when the fungus was exposed to pure O2 gas . The specific activities of manganese superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase and the consumption of glutathione were all higher in cultures exposed to pure O2 (oxygenated cultures) than in cultures grown with atmospheric air . Significantly higher gene expression of the LIP-H2 isozyme occurred in the oxygenated cultures . A hydroxyl radical scavenger, dimethyl sulfoxide (50 mM), added to the culture every 12 h, completely abolished LIP expression at the mRNA and protein levels . This effect was confirmed by in situ generation of hydroxyl radicals via the Fenton reaction, which significantly enhanced LIP expression . The level of intracellular cyclic AMP (cAMP) was correlated with the starvation conditions regardless of the oxygenation regimen applied, and similar cAMP levels were obtained at high O2 concentrations and in cultures grown with atmospheric air . These results suggest that even though cAMP is a prerequisite for LIP expression, high levels of ROS, preferentially hydroxyl radicals, are required to trigger LIP synthesis . Thus, the induction of LIP expression by O2 is at least partially mediated by the intracellular ROS .
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