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Impact of Nelfinavir Resistance Mutations on In Vitro Phenotype, Fitness, and Replication Capacity of Human Immunodeficiency Virus Type 1 with Subtype B and C Proteases. Luis M. F. Gonzalez, 2004.Human immunodeficiency virus type 1 subtype B and C proteases were manipulated to contain 90M, 88D, or 89L, and their in vitro biological properties were studied . We showed that D30N has significantly more impact in subtype C than in subtype B counterparts, accounting for the reported low prevalence of this mutation in patients failing nelfinavir-based regimens . The Iron-Binding Protein Dps Confers Hydrogen Peroxide Stress Resistance to Campylobacter jejuni. Takahiko Ishikawa, 2003.We identified and characterized the iron-binding protein Dps from Campylobacter jejuni . Electron microscopic analysis of this protein revealed a spherical structure of 8.5 nm in diameter, with an electron-dense core similar to those of other proteins of the Dps (DNA-binding protein from starved cells) family . Cloning and sequencing of the Dps-encoding gene (dps) revealed that a 450-bp open reading frame (ORF) encoded a protein of 150 amino acids with a calculated molecular mass of 17,332 Da . Amino acid sequence comparison indicated a high similarity between C . jejuni Dps and other Dps family proteins . In C . jejuni Dps, there are iron-binding motifs, as reported in other Dps family proteins . C . jejuni Dps bound up to 40 atoms of iron per monomer, whereas it did not appear to bind DNA . An isogenic dps-deficient mutant was more vulnerable to hydrogen peroxide than its parental strain, as judged by growth inhibition tests . The iron chelator Desferal restored the resistance of the Dps-deficient mutant to hydrogen peroxide, suggesting that this iron-binding protein prevented generation of hydroxyl radicals via the Fenton reaction . Dps was constitutively expressed during both exponential and stationary phase, and no induction was observed when the cells were exposed to H2O2 or grown under iron-supplemented or iron-restricted conditions . On the basis of these data, we propose that this iron-binding protein in C . jejuni plays an important role in protection against hydrogen peroxide stress by sequestering intracellular free iron and is expressed constitutively to cope with the harmful effect of hydrogen peroxide stress on this microaerophilic organism without delay . Identification and Characterization of the Conjugal Transfer Region of the pCg1 plasmid from Naphthalene-Degrading Pseudomonas putida Cg1. Woojun Park, 2003.Hybridization and restriction fragment length polymorphism data (K . G . Stuart-Keil, A . M . Hohnstock, K . P . Drees, J . B . Herrick, and E . L . Madsen, Appl . Environ . Microbiol . 64:3633-3640, 1998) have shown that pCg1, a naphthalene catabolic plasmid carried by Pseudomonas putida Cg1, is homologous to the archetypal naphthalene catabolic plasmid, pDTG1, in P . putida NCIB 9816-4 . Sequencing of the latter plasmid allowed PCR primers to be designed for amplifying and sequencing the conjugal transfer region in pCg1 . The mating pair formation (mpf) gene, mpfA encoding the putative precursor of the conjugative pilin subunit from pCg1, was identified along with other trb-like mpf genes . Sequence comparison revealed that the 10 mpf genes in pCg1 and pDTG1 are closely related (61 to 84% identity) in sequence and operon structure to the putative mpf genes of catabolic plasmid pWW0 (TOL plasmid of P . putida) and pM3 (antibiotic resistance plasmid of Pseudomonas. spp) . A polar mutation caused by insertional inactivation in mpfA of pCg1 and reverse transcriptase PCR analysis of mRNA showed that this mpf region was involved in conjugation and was transcribed from a promoter located upstream of an open reading frame adjacent to mpfA . lacZ transcriptional fusions revealed that mpf genes of pCg1 were expressed constitutively both in liquid and on solid media . This expression did not respond to host exposure to naphthalene . Conjugation frequency on semisolid media was consistently 10- to 100-fold higher than that in liquid media . Thus, conjugation of pCg1 in P . putida Cg1 was enhanced by expression of genes in the mpf region and by surfaces where conditions fostering stable, high-density cell-to-cell contact are manifest . RpoS-Dependent Stress Response and Exoenzyme Production in Vibrio vulnificus. A. Hülsmann, 2003.Vibrio vulnificus is an estuarine bacterium capable of causing rapidly fatal infections through both ingestion and wound infection . Like other opportunistic pathogens, V . vulnificus must adapt to potentially stressful environmental changes while living freely in seawater, upon colonization of the oyster gut, and upon infection of such diverse hosts as humans and eels . In order to begin to understand the ability of V . vulnificus to respond to such stresses, we examined the role of the alternate sigma factor RpoS, which is important in stress response and virulence in many pathogens . An rpoS mutant of V . vulnificus strain C7184o was constructed by homologous recombination . The mutant strain exhibited a decreased ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and acidic conditions . The most striking difference was a high sensitivity of the mutant to hydrogen peroxide . Albuminase, caseinase, and elastase activity were detected in the wild type but not in the mutant strain, and an additional two hydrolytic activities (collagenase and gelatinase) were reduced in the mutant strain compared to the wild type . Additionally, the motility of the rpoS mutant was severely diminished . Overall, these studies suggest that rpoS in V . vulnificus is important for adaptation to environmental changes and may have a role in virulence .
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