Use of signal-mediated amplification of RNA technology (SMART) to detect marine cyanophage DNA.

Here, we describe the application of an isothermal nucleic acid amplification assay, signal-mediated amplification of RNA technology (SMART), to detect DNA extracted from marine cyanophages known to infect unicellular cyanobacteria from the genus Synechococcus. The SMART assay is based on the target-dependent production of multiple copies of an RNA signal, which is measured by an enzyme-linked oligosorbent assay. SMART was able to detect both synthetic oligonucleotide targets and genomic cyanophage DNA using probes designed against the portal vertex gene (g20). Specific signals were obtained for each cyanophage strain (S-PM2 and S-BnMI). Nonspecific genomic DNA did not produce false signals or inhibit the detection of a specific target. In addition, we found that extensive purification of target DNA may not be required since signals were obtained from crude cyanophage lysates. This is the first report of the SMART assay being used to discriminate between two similar target sequences.

Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure.

The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets.

Conditional mutations in OutE and OutL block exoenzyme secretion across the Erwinia carotovora outer membrane.

The phytopathogen Erwinia carotovora subspecies carotovora secretes pectinases and cellulase via the general secretory pathway, a process requiring at least 13 proteins encoded by the out gene cluster. By exploiting delta::Tn5, a generalised transducing phage (psi KP) and localised mutagenesis of the out gene cluster, we have produced a histidine auxotroph and 19 new secretory mutants, including two (HJN1003 and HJN1004) which were conditional (temperature sensitive) for secretion. All of the mutants accumulated pectinases and cellulase in the periplasm, but in the case of HJN1003 and HJN1004, only at the restrictive temperature. HJN1003 and HJN1004 were complemented by the outE and outL wild-type genes, respectively, and both mutant alleles were cloned and sequenced to reveal single missense substitutions. HJN1003 carries an Arg166 to His alteration in OutE and HJN1004 carries a Pro159 to Leu alteration in OutL. Topology mapping of OutL using a beta-lactamase probe confirmed that OutL is a type II bitopic trans-inner membrane protein and that the mutated Pro159 residue in HJN1004 is located in the cytoplasmic domain of OutL. Hence, the secretion of exoenzymes across the outer membrane is critically dependent on the conformation of secretory components located at the cytoplasmic face of the inner membrane.