Susan Wharam

The chemical mutagen ethylmethanesulphonate (EMS) has been used to generate mutants of Erwinia carotovora subspecies carotovora which are defective in the secretion of pectinases (Pel) and cellulases (Cel) but unaltered for protease (Prt) secretion. Such mutants, called Out-, still synthesize Pel and Cel but these enzymes accumulate within the periplasm. Cosmid clones carrying wild-type E. carotovora ssp. carotovora DNA, identified by their ability to restore the Out+ phenotype when transferred to some Out- mutants, were classified into six complementation groups using cosmids and cosmid derivatives. Analysis of the nucleotide sequence of a 12.7 kb DNA fragment, encompassing complementing cosmid inserts, revealed a coding capacity for 13 potential open reading frames (ORFs), and these were designated outC-outO. Some of the out gene products were visualized using a T7 gene 10 expression system. The predicted Out proteins are highly similar to components of extracellular enzyme secretion systems from a diverse range of eubacteria including Erwinia chrysanthemi, Klebsiella oxytoca, Aeromonas hydrophila, Pseudomonas aeruginosa and Xanthomonas campestris. Lower levels of similarity exist between Ecc Out proteins and components of macromolecular trafficking systems from Bacillus subtilis, Haemophilus influenzae, Agrobacterium tumefaciens, Yersinia pestis and a protein involved in the morphogenesis of filamentous bacteriophages such as M13.

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.

Rapid Na+/Ca2+-based action potentials govern essential cellular functions in eukaryotes, from the motile responses of unicellular protists, such as Paramecium [1Fujiu K. Nakayama Y. Yanagisawa A. Sokabe M. Yoshimura K. Chlamydomonas CAV2 encodes a voltage- dependent calcium channel required for the flagellar waveform conversion.Curr. Biol. 2009; 19: 133-139Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 2Lodh S. Yano J. Valentine M.S. Van Houten J.L. Voltage-gated calcium channels of Paramecium cilia.J. Exp. Biol. 2016; 219: 3028-3038Crossref PubMed Scopus (26) Google Scholar], to complex animal neuromuscular activity [3Hille B. Ion Channels of Excitable Membranes.Third Edition. Sinauer, 2001Google Scholar]. A key innovation underpinning this fundamental signaling process has been the evolution of four-domain voltage-gated Na+/Ca2+ channels (4D-Cavs/Navs). These channels are widely distributed across eukaryote diversity [4Verret F. Wheeler G. Taylor A.R. Farnham G. Brownlee C. Calcium channels in photosynthetic eukaryotes: implications for evolution of calcium-based signalling.New Phytol. 2010; 187: 23-43Crossref PubMed Scopus (109) Google Scholar], albeit several eukaryotes, including land plants and fungi, have lost voltage-sensitive 4D-Cav/Navs [5Liebeskind B.J. Hillis D.M. Zakon H.H. Phylogeny unites animal sodium leak channels with fungal calcium channels in an ancient, voltage-insensitive clade.Mol. Biol. Evol. 2012; 29: 3613-3616Crossref PubMed Scopus (38) Google Scholar, 6Pozdnyakov I. Matantseva O. Skarlato S. Diversity and evolution of four-domain voltage-gated cation channels of eukaryotes and their ancestral functional determinants.Sci. Rep. 2018; 8: 3539Crossref PubMed Scopus (18) Google Scholar, 7Edel K.H. Marchadier E. Brownlee C. Kudla J. Hetherington A.M. The evolution of calcium-based signalling in plants.Curr. Biol. 2017; 27: R667-R679Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar]. Because these lineages appear to lack rapid Na+/Ca2+-based action potentials, 4D-Cav/Navs are generally considered necessary for fast Na+/Ca2+-based signaling [7Edel K.H. Marchadier E. Brownlee C. Kudla J. Hetherington A.M. The evolution of calcium-based signalling in plants.Curr. Biol. 2017; 27: R667-R679Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar]. However, the cellular mechanisms underpinning the membrane physiology of many eukaryotes remain unexamined. Eukaryotic phytoplankton critically influence our climate as major primary producers. Several taxa, including the globally abundant diatoms, exhibit membrane excitability [8Oami K. Naitoh Y. Sibaoka T. Modification of voltage-sensitive inactivation of Na+ current by external Ca2+ in the marine dinoflagellate Noctiluca miliaris.J. Comp. Physiol. A. 1995; 176: 635-640Crossref Google Scholar, 9Taylor A.R. Brownlee C. A novel Cl- inward-rectifying current in the plasma membrane of the calcifying marine phytoplankton Coccolithus pelagicus.Plant Physiol. 2003; 131: 1391-1400Crossref PubMed Scopus (37) Google Scholar, 10Taylor A.R. A fast Na+/Ca2+-based action potential in a marine diatom.PLoS ONE. 2009; 4: e4966Crossref PubMed Scopus (41) Google Scholar]. We previously demonstrated that certain diatom genomes encode 4D-Cav/Navs [4Verret F. Wheeler G. Taylor A.R. Farnham G. Brownlee C. Calcium channels in photosynthetic eukaryotes: implications for evolution of calcium-based signalling.New Phytol. 2010; 187: 23-43Crossref PubMed Scopus (109) Google Scholar] but also proteins of unknown function, resembling prokaryote single-domain, voltage-gated Na+ channels (BacNavs) [4Verret F. Wheeler G. Taylor A.R. Farnham G. Brownlee C. Calcium channels in photosynthetic eukaryotes: implications for evolution of calcium-based signalling.New Phytol. 2010; 187: 23-43Crossref PubMed Scopus (109) Google Scholar]. Here, we show that single-domain channels are actually broadly distributed across major eukaryote phytoplankton lineages and represent three novel classes of single-domain channels, which we refer collectively to as EukCats. Functional characterization of diatom EukCatAs indicates that they are voltage-gated Na+- and Ca2+-permeable channels, with rapid kinetics resembling metazoan 4D-Cavs/Navs. In Phaeodactylum tricornutum, which lacks 4D-Cav/Navs, EukCatAs underpin voltage-activated Ca2+ signaling important for membrane excitability, and mutants exhibit impaired motility. EukCatAs therefore provide alternative mechanisms for rapid Na+/Ca2+ signaling in eukaryotes and may functionally replace 4D-Cavs/Navs in pennate diatoms. Marine phytoplankton thus possess unique signaling mechanisms that may be key to environmental sensing in the oceans.

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.

BACKGROUND: Signal-Mediated Amplification of RNA Technology (SMART) is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression) or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus. RESULTS: Here we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20) was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240-360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis. CONCLUSION: The SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.