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Systematic Mutagenesis of the Escherichia coli Genome. Yisheng Kang, 2004.A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome . The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame . The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively . Reaction products are then introduced into the genome by homologous recombination via the  Red
proteins . The method has yielded insertion alleles for 1976 genes
during a first pass through the genome including, unexpectedly, a
number of known and putative essential genes . Sce-poson insertions
can be easily replaced by markerless mutations by using the I-SceI
homing endonuclease to select against retention of the transposon as
demonstrated by the substitution of amber and/or in-frame deletions
in six different genes . This allows a Sce-poson-containing gene to be
specifically targeted for either designed or random modifications, as
well as permitting the stepwise engineering of strains with multiple
mutations . The promiscuous nature of Tn5 transposition also
enables a targeted gene to be dissected by using randomly inserted
Sce-posons as shown by a lacZ allelic series . Finally,
assessment of the insertion sites by an iterative weighted matrix
algorithm reveals that these hyperactive Tn5 complexes
generally recognize a highly degenerate asymmetric motif on one end
of the target site helping to explain the randomness of Tn5
transposition .
Regulatory Regions of smeDEF in Stenotrophomonas maltophilia Strains Expressing Different Amounts of the Multidrug Efflux Pump SmeDEF. Patricia Sanchez, 2004.The smeT-smeDEF region and the smeT gene, which encodes the smeDEF repressor, are highly polymorphic . Few changes in smeT might be associated with smeDEF overexpression. The results obtained with cellular extracts suggest that mutant SmeT proteins cannot bind to the operator and that other transcription factors besides SmeT are involved in the regulation of smeDEF expression . Effects of Escherichia coli Physiology on Growth of Phage T7 In Vivo and In Silico. Lingchong You, 2002.Phage development depends not only upon phage functions but also on the physiological state of the host, characterized by levels and activities of host cellular functions . We established Escherichia coli at different physiological states by continuous culture under different dilution rates and then measured its production of phage T7 during a single cycle of infection . We found that the intracellular eclipse time decreased and the rise rate increased as the growth rate of the host increased . To develop mechanistic insight, we extended a computer simulation for the growth of phage T7 to account for the physiology of its host . Literature data were used to establish mathematical correlations between host resources and the host growth rate; host resources included the amount of genomic DNA, pool sizes and elongation rates of RNA polymerases and ribosomes, pool sizes of amino acids and nucleoside triphosphates, and the cell volume . The in silico (simulated) dependence of the phage intracellular rise rate on the host growth rate gave quantitatively good agreement with our in vivo results, increasing fivefold for a 2.4-fold increase in host doublings per hour, and the simulated dependence of eclipse time on growth rate agreed qualitatively, deviating by a fixed delay . When the simulation was used to numerically uncouple host resources from the host growth rate, phage growth was found to be most sensitive to the host translation machinery, specifically, the level and elongation rate of the ribosomes . Finally, the simulation was used to follow how bottlenecks to phage growth shift in response to variations in host or phage functions . Phase Variation of Ag43 Is Independent of the Oxidation State of OxyR. Anu Wallecha, 2003.OxyR is a DNA binding protein that differentially regulates a cell's response to hydrogen peroxide-mediated oxidative stress . We previously reported that the reduced form of OxyR is sufficient for repression of transcription of agn43 from unmethylated template DNA, which is essential for deoxyadenosine methylase (Dam)- and OxyR-dependent phase variation of agn43 . Here we provide evidence that the oxidized form of OxyR [OxyR(ox)] also represses agn43 transcription . In vivo, we found that exogenous addition of hydrogen peroxide, sufficient to oxidize OxyR, did not affect the expression of agn43. OxyR(ox) repressed in vitro transcription but only from an unmethylated agn43 template . The -10 sequence of the promoter and three Dam target sequences were protected in an in vitro DNase I footprint assay by OxyR(ox) . Furthermore, OxyR(ox) bound to the agn43 regulatory region DNA with an affinity similar to that for the regulatory regions of katG and oxyS, which are activated by OxyR(ox), indicating that binding at agn43 can occur at biologically relevant concentrations . OxyR-dependent regulation of Ag43 expression is therefore unusual in firstly that OxyR binding at agn43 is dependent on the methylation state of Dam target sequences in its binding site and secondly that OxyR-dependent repression appears to be independent of hydrogen-peroxide mediated oxidative stress and the oxidation state of OxyR .
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