Isolation and characterization of methyl viologen-sensitive mutants of Escherichia coli K-12.

Escherichia coli mutants sensitive to methyl viologen (MV), an active oxygen propagator, were isolated. Among them, the new genes mvrA and mvrB were mapped at 7 and 28 min on the E. coli linkage map, respectively. MV toxicity was exerted only in the presence of oxygen and was suppressed by the radical scavenger uric acid but not by the hydroxyl radical scavenger mannitol. The mvr mutants were sensitive only to MV and had a normal repair capacity for the MV-damaged DNA. From these results, these mutants were assumed to be related to the elimination of MV-specific toxic species. Gene mvrA was cloned into vector pBR322 and its sequence was determined. The mvrA gene, which was predicted to range in size from 600 to 900 base pairs (bp) by transposon Tn1000 insertion analysis, was identified to be 807 bp, with an approximately 60-bp promoter sequence carrying consensus sequences for the -35 region, the -10 region, and a ribosome-binding site. The MvrA protein deduced from the DNA sequence was 29.7 kilodaltons, which was in good agreement with the 29 kilodaltons of the MvrA protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after a maxicell labeling experiment

A simple and rapid amplification procedure for cDNA cloned in dephosphorylated plasmid.

The preparation of cDNA libraries usually involves multiple steps, such as isolation of poly A+ mRNA, synthesis of cDNA, cloning of cDNA into plasmid, and amplification of cloned plasmid. The last step, the amplification of insert DNA fragment after cloned in dephosphorylated plasmid vector, is done in either of two ways. The recombinant DNA can be extracted after amplification in host cells, or the amplification of insert DNA can be done by the PCR using primers complementary to both ends of the insertion site of the vector. However, the ligation of dephosphorylated nicks or nick translation beyond the primer sites is essential in the latter case prior to the PCR (see step 1 in Figure 1 (B)). Taq DNA polymerase, by virtue of its 5 ' to 3 ' exonuclease activity (Ref 1), may be utilized for nick translation instead of E. coli DNA polymerase 1. This would simplify the amplification of insert cDNA by doing nick translation and PCR steps successively, controlling each reaction condition. We examined the conditions for nick translation by Taq DNA polymerase. The reaction mixture, 20 /tl containing 25 yCi ^P-dNTPs (600 Ci/mmol), 0.5 ng RF-M13mpl8 DNA, 0.02 ng pancreatic DNase I, and 2.5 units of Taq DNA polymerase was incubated at 37, 50 and 65°C and the incorporation of 32P-dNTPs into acid-insoluble fraction was measured. Radioactivities incorporated were 167 k, 323 k, and 286 k DPM, respectively, compared with 82 k DPM by a standard nick translation reaction at 12°C using DNA polymerase I and DNase I. These results indicated that the nick translation reaction by Taq DNA polymerase worked normally at all temperatures examined, although the incorporation was reduced at 65°C. Then we amplified insert DNA by nick translation reactio

One step DNA sequencing of single-stranded DNA with reverse dye-primer.

DNA sequencing can be performed much faster and more easily by an automated DNA sequencer, which can process a large quantity of samples at one time. We use an Applied Biosystems DNA sequencer, Model 373A. Among several methods applicable to the sequencer, the dye-primer method of single-stranded template with Taq DNA polymerase gives the most reliable results. If both dye-primers (forward dye-primer (-21M13) and reverse dye-primer (M13RP1)) are available, the dye-primer method will be able to give at least 400 nucleotide sequences from each end. However, only the forward dye-primer is available for a single-stranded template. For use of a reverse dye-primer, two methods are available. 1) The insert can be recloned into another vector. 2) A double-stranded fragment can be produced by PCR with both primers. In both cases, an additional step is required, providing a large obstacle to extensiv

Antibiotic Susceptibility Profiles of Escherichia coli Strains Lacking Multidrug Efflux Pump Genes

The contribution of seven known and nine predicted genes or operons associated with multidrug resistance to the susceptibility of Escherichia coli W3110 was assessed for 20 different classes of antimicrobial compounds that include antibiotics, antiseptics, detergents, and dyes. Strains were constructed with deletions for genes in the major facilitator superfamily, the resistance nodulation-cell division family, the small multidrug resistance family, the ATP-binding cassette family, and outer membrane factors. The agar dilution MICs of 35 compounds were determined for strains with deletions for multidrug resistance (MDR) pumps. Deletions in acrAB or tolC resulted in increased susceptibilities to the majority of compounds tested. The remaining MDR pump gene deletions resulted in increased susceptibilities to far fewer compounds. The results identify which MDR pumps contribute to intrinsic resistance under the conditions tested and supply practical information useful for designing sensitive assay strains for cell-based screening of antibacterial compounds