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Identification of the Receptor-Binding Protein in 936-Species Lactococcal Bacteriophages. Kitt Dupont, 2004.The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936 . These phages have a high level of DNA identity but different host ranges . Bioinformatic analysis indicated that homologous genes, orf18 in sk1 and orf20 in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition . Consequently, chimeric bIL170 phages carrying orf18 from sk1 were generated . The recombinant phages were able to form plaques on the sk1 host Lactococcus lactis MG1614, and recombination was verified by PCR analysis directly with the plaques . A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail . Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding . The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and  7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively . Interestingly, sk1-like phages bind to and infect a defined group of L . lactis subsp . cremoris strains, while bIL170-like phages bind to and infect a defined group of L . lactis subsp . lactis strains .
Enhanced Mineralization of [U-14C]2,4-Dichlorophenoxyacetic Acid in Soil from the Rhizosphere of Trifolium pratense. Liz J. Shaw, 2004.Enhanced biodegradation in the rhizosphere has been reported for many organic xenobiotic compounds, although the mechanisms are not fully understood . The purpose of this study was to discover whether rhizosphere-enhanced biodegradation is due to selective enrichment of degraders through growth on compounds produced by rhizodeposition . We monitored the mineralization of [U-14C]2,4-dichlorophenoxyacetic acid (2,4-D) in rhizosphere soil with no history of herbicide application collected over a period of 0 to 116 days after sowing of Lolium perenne and Trifolium pratense . The relationships between the mineralization kinetics, the number of 2,4-D degraders, and the diversity of genes encoding 2,4-D/  -ketoglutarate dioxygenase (tfdA) were investigated . The rhizosphere effect on [14C]2,4-D mineralization (50 µg g–1) was shown to be plant species and plant age specific . In comparison with nonplanted soil, there were significant (P < 0.05) reductions in the lag phase and enhancements of the maximum mineralization rate for 25- and 60-day T . pratense soil but not for 116-day T . pratense rhizosphere soil or for L . perenne rhizosphere soil of any age . Numbers of 2,4-D degraders in planted and nonplanted soil were low (most probable number, <100 g–1) and were not related to plant species or age . Single-strand conformational polymorphism analysis showed that plant species had no impact on the diversity of
-Proteobacteria tfdA-like genes, although an impact of 2,4-D application was recorded . Our results indicate that enhanced mineralization in T . pratense rhizosphere soil is not due to enrichment of 2,4-D-degrading microorganisms by rhizodeposits . We suggest an alternative mechanism in which one or more components of the rhizodeposits induce the 2,4-D pathway .
Regulation of mutY and Nature of Mutator Mutations in Escherichia coli Populations under Nutrient Limitation. Lucinda Notley-McRobb, 2002.Previous analysis of aerobic, glucose-limited continuous cultures of Escherichia coli revealed that G:C-to-T:A (G:C  T:A) transversions were the most commonly occurring type of spontaneous mutation . One possible explanation for the preponderance of these mutations was that nutrient limitation repressed MutY-dependent DNA repair, resulting in increased proportions of G:CT:A transversions . The regulation of the mutY-dependent DNA repair system was therefore studied with a transcriptional mutY-lacZ fusion recombined into the chromosome . Expression from the mutY promoter was fourfold higher under aerobic conditions than under anaerobic conditions . But mutY expression was higher in glucose- or ammonia-limited chemostats than in nutrient-excess batch culture, so mutY was not downregulated by nutrient limitation . An alternative explanation for the frequency of G:CT:A transversions was the common appearance of mutY mutator mutations in the chemostat populations . Of 11 chemostat populations screened in detail, six contained mutators, and the mutator mutation in four cultures was located in the region of mutY at 66 min on the chromosome . The spectrum of mutations and rate of mutation in these isolates were fully consistent with a mutY-deficiency in each strain . Based on PCR analysis of the region within and around mutY, isolates from three individual populations contained deletions extending at least 2 kb upstream of mutY and more than 5 kb downstream . In the fourth population, the deletion was even longer, extending at least 5 kb upstream and 5 kb downstream of mutY . The isolation of mutY mutator strains from four independent populations with extensive chromosomal rearrangements suggests that mutY inactivation by deletion is a means of increasing mutation rates under nutrient limitation and explains the observed frequency of G:CT:A mutations in glucose-limited chemostats .
The Escherichia coli BarA-UvrY Two-Component System Is Needed for Efficient Switching between Glycolytic and Gluconeogenic Carbon Sources. Anna-Karin Pernestig, 2003.The Escherichia coli BarA and UvrY proteins were recently demonstrated to constitute a novel two-component system, although its function has remained largely elusive . Here we show that mutations in the sensor kinase gene, barA, or the response regulator gene, uvrY, in uropathogenic E . coli drastically affect survival in long-term competition cultures . Using media with gluconeogenic carbon sources, the mutants have a clear growth advantage when competing with the wild type, but using media with carbon sources feeding into the glycolysis leads to a clear growth advantage for the wild type . Results from competitions with mutants in the carbon storage regulation system, CsrA/B, known to be a master switch between glycolysis and gluconeogenesis, led us to propose that the BarA-UvrY two-component system controls the Csr system . Taking these results together, we propose the BarA-UvrY two-component system is crucial for efficient adaptation between different metabolic pathways, an essential function for adaptation to a new environment . Inactivation of Salmonella enterica Serovar Enteritidis by Ultrasonic Waves under Pressure at Different Water Activities. I. Álvarez, 2003.The inactivation of Salmonella enterica serovar Enteritidis by ultrasonic waves (20 kHz; 117-µm wavelength) under pressure (175 kPa) at nonlethal temperatures (manosonication [MS]) and lethal temperatures (manothermosonication [MTS]) in media of different water activities has been investigated . Heat decimal reduction time values increased 30 times when the water activity was decreased from nearly 1 to 0.96, but the MS resistance was increased only twofold . The inactivation of Salmonella serovar Enteritidis by ultrasound under pressure at low water activities was a phenomenon of the "all-or-nothing" type . A synergistic lethal effect was observed between heat and ultrasound in media with reduced water activity; the lower the water activity, the greater the synergistic effect . This work could be useful for improving sanitation and preservation treatments of foods, especially those which are sensitive to temperature and those in which components protect microorganisms to heat . It also contributes to our knowledge of microbial inactivation mechanisms by MS and MTS treatments . Cold Shock Response and Major Cold Shock Proteins of Vibrio cholerae. Partha Pratim Datta, 2003.When exponentially growing Vibrio cholerae cells were shifted from 37°C to various lower temperatures, it was found that the organism could adapt and grow at temperatures down to 15°C, below which the growth was completely arrested . There was no difference between the patterns of the cold shock responses in toxinogenic and nontoxinogenic strains of V . cholerae . Gel electrophoretic analyses of proteins of cold-exposed cells revealed significant induction of two major cold shock proteins (Csps), whose molecular masses were 7.7 kDa (CspAVC) and 7.5 kDa (CspV), and six other Csps, most of which were much larger . We cloned, sequenced, and analyzed the cspV gene encoding the CspV protein of V . cholerae O139 strain SG24 . Although CspAVC and CspV have similar kinetics of synthesis and down-regulation, the corresponding genes, cspA and cspV, which are located in the small chromosome, are not located in the same operon . A comparative analysis of the kinetics of synthesis revealed that the CspV protein was synthesized de novo only during cold shock . Although both CspAVC and CspV were stable for several hours in the cold, the CspV protein was degraded rapidly when the culture was shifted back to 37°C, suggesting that this protein is probably necessary for adaptation at lower temperatures . Northern blot analysis confirmed that the cspV gene is cold shock inducible and is regulated tightly at the level of transcription . Interestingly, the cspV gene has a cold shock-inducible promoter which is only 12 nucleotides from the translational start site, and therefore, it appears that no unusually long 5' untranslated region is present in its mRNA transcript . Thus, this promoter is an exception compared to other promoters of cold shock-inducible genes of different organisms, including Escherichia coli . Our results suggest that V . cholerae may use an alternative pathway for regulation of gene expression during cold shock .
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