Real-time turbidimetry of LAMP reaction for quantifying template DNA.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method that allows the synthesis of large amounts of DNA in a short period of time with high specificity. As the LAMP reaction progresses, the reaction by-product pyrophosphate ions bind to magnesium ions and form a white precipitate of magnesium pyrophosphate. We designed an apparatus capable of measuring the turbidity of multiple samples simultaneously while maintaining constant temperature to conduct real-time measurements of the changes in the turbidity of LAMP reactions. The time (Tt) required for the turbidity of the LAMP reaction solution to exceed a given value was dependent on the quantity of the initial template DNA. That is, a graph with the plot of Tt versus the log of the amount of initial template DNA was linear from 2 x 10(3) copies (0.01 pg/tube) to 2 x 10(9) copies (100 ng/tube) of template DNA. These results indicate that real-time turbidity measurements of the LAMP reaction permit the quantitative analysis of minute amounts of nucleic acids present in a sample, with a high precision over a wide range, using a simple apparatus reported in this study.

A novel assembly process of the multicomponent xenobiotic efflux pump in Pseudomonas aeruginosa.

The nfxC-type cells of Pseudomonas aeruginosa show resistance to a wide range of structurally and functionally diverse antibiotics, which is a phenomenon that is mainly attributable to the expression of the MexEF-OprN xenobiotic transporter. The MexF, MexE and OprN subunits of this transporter are located on the inner membrane, the periplasm and the outer membrane, respectively, and are assumed to function as an energy-dependent transporter, a bridge connecting the inner and outer membranes and outer membrane channel respectively. The nfxC-type cells showed a single protein band of MexF and OprN, whereas MexE appeared as three distinct bands in an SDS-polyacrylamide gel electrophoretogram. The mutant cells lacking MexF produced undetectable OprN and only a full-size of MexE even though the cells had unimpaired oprN and mexE. Expression of the plasmid-borne MexF in this mutant fully restored OprN and three MexE bands. Another class of mutants producing a full amount of MexF yielded undetectable OprN and two MexE bands lacking the smallest protein species suggesting that the presence of the smallest MexE subunit is required for stabilization of OprN. To identify which part of MexE was needed for stabilization and assembly of OprN, the carboxyl-terminal-truncated MexE tagged with polyhistidine was constructed and protein bands were visualized in the presence of MexF with an antibody raised against polyhistidine or MexE. The results revealed that the proteolytic processing of MexE would occur at carboxyl terminal amino acids between 11 and 16, thereby suggesting that the presence of the C-terminal truncated MexE is essential for stabilization and the proper assembly of OprN. Nucleotide sequencing of mutant mexFs, which produce a wild-type level of MexF but are unable to support the production of the smallest MexE, thereby destabilizing OprN, revealed that all the mutations were located within two large periplasmic domains of MexF between transmembrane segments 1-2 and 7-8. Taking these findings together, we concluded that two large periplasmic domains of MexF interact with MexE thereby promoting programmed processing of MexE, and this complex eventually assists the correct assembly and sorting of OprN.