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Characterization and Functional Complementation of a Nonlethal Deletion in the Chromosome of a ß-Glycosidase Mutant of Sulfolobus solfataricus. Simonetta Bartolucci, 2003.LacS- mutants of Sulfolobus solfataricus defective in ß-glycosidase activity were isolated in order to explore genomic instability and exploit novel strategies for transformation and complementation . One of the mutants showed a stable phenotype with no reversion; analysis of its chromosome revealed the total absence of the ß-glycosidase gene (lacS) . Fine mapping performed in comparison to the genomic sequence of S . solfataricus P2 indicated an extended deletion of  13 kb . The sequence analysis also revealed that this chromosomal rearrangement was a nonconservative transposition event driven by the mobile insertion sequence element ISC1058 . In order to complement the LacS- phenotype, an expression vector was constructed by inserting the lacS coding sequence with its 5' and 3' flanking regions into the pEXSs plasmid . Since no transformant could be recovered by selection on lactose as the sole nutrient, another plasmid construct containing a larger genomic fragment was tested for complementation; this region also comprised the lacTr (lactose transporter) gene encoding a putative membrane protein homologous to the major facilitator superfamily . Cells transformed with both genes were able to form colonies on lactose plates and to be stained with the ß-glycosidase chromogenic substrate X-Gal (5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside) .
High pH during Trisodium Phosphate Treatment Causes Membrane Damage and Destruction of Salmonella enterica Serovar Enteritidis. Balamurugan Sampathkumar, 2003.Trisodium phosphate (TSP) is now widely used during the processing of poultry and red meats, but the mechanism whereby it inactivates gram-negative bacteria such Salmonella spp . remains unclear . Thus, Salmonella enterica serovar Enteritidis (ATCC 4931) cells were treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) and compared with (i) cells treated with the same pH as the TSP treatments (pH 10.0, 10.5, and 11.0, respectively) and (ii) cells treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) adjusted to a pH of 7.0 ± 0.2 (mean ± standard deviation) . Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides, and cell morphology were accordingly examined and quantified under the above treatment conditions . Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or alkaline pH-dependent manner . In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity . A 30-min pretreatment with 1.0 mM EDTA significantly enhanced the loss of membrane integrity only when followed by TSP or alkaline pH treatments . Measuring the absorbance at 260 nm, agarose gel electrophoresis, Bradford assay, and Tricine-sodium dodecyl sulfate gel electrophoresis of filtrates of treated cell suspensions revealed considerable release of DNA, proteins, and lipopolysaccharides compared to controls and pH 7.0 TSP treatments . Electron microscopic examination of TSP- or alkaline pH-treated cells showed disfigured cell surface topology and wrinkled appearance and showed evidence of a TSP concentration- and pH-dependent disruption of the cytoplasmic and outer membranes . These results demonstrate that TSP treatment permeabilizes and disrupts the cytoplasmic and outer membranes of serovar Enteritidis cells because of the alkaline pH, which in turn leads to release of intracellular contents and eventual cell death . Selective Plating Underestimates Abundance and Shows Differential Recovery of Bifidobacterial Species from Human Feces. Juha H. A. Apajalahti, 2003.The aim of the present work was to compare the efficacies and levels of selectivity of different culture-dependent and -independent methods for analyzing bifidobacteria in human stool samples . The three different culture media used here significantly differed from each other, particularly with regard to the recovery of Bifidobacterium adolescentis . Bifidobacterium medium failed to recover B . adolescentis; Beerens medium recovered some B . adolescentis organisms (17% of total bifidobacteria), whereas tomato-Eugon medium recovered mainly B . adolescentis organisms (58% of total bifidobacteria) . A culture-independent method that combines GC fractionation of bacterial community DNA and 16S rRNA sequencing indicated that B . adolescentis organisms accounted for 85% of all bifidobacteria . Methodological biases, such as those described in this paper, should be taken into account in interpreting earlier studies and designing future experiments .
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