Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases

The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation

Discovery of enzymes for toluene synthesis from anoxic microbial communities

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum

Label-free detection of DNA hybridization at a liquid|liquid interface

A novel electrochemical approach for label-free detection of DNA primary sequence has been proposed. The flow of nonelectroactive ions across a liquid|liquid interface was used as an electrochemical probe for detection of DNA hybridization. Disposable graphite screen-printed electrodes shielded with a thin layer of inert polymer plasticized with water-immiscible polar organic solvent were modified by probe oligonucleotide and used as a DNA sensor. The specific DNA coupling has been detected with impedance spectroscopy by decrease of ion-transfer resistance. The detection limit was of 10(-8) M of target oligonucleotide. The reported sensor was suitable for discrimination of a single mismatch oligonucleotide from the full complementary one. The reported DNA sensor was advantageous over known physicochemical approaches, providing the most significant changes in the measured parameters

From geography to genes: evolutionary perspectives on salinity tolerance in the brackish water barnacle Balanus improvisus

How species respond to changes in their environment is a fundamental question in biology. This has become an increasingly important issue as anthropogenic effects of climate change and biological invasions have major impacts on marine ecosystems worldwide. In this thesis I investigated the role of salinity tolerance from an evolutionary perspective, using a wide range of techniques, spanning from population genetics and common-garden experiments to characterizing potential genes involved in osmoregulation in barnacles. I used the acorn barnacle species Balanus (Amphibalanus) improvisus, which displays a remarkably broad salinity tolerance, to investigate how this trait has influenced the species' potential to establish in new environments, and respond to projected near-future salinity reductions in coastal seas. I also examined physiological and molecular mechanisms that may be involved in osmoregulation in B. improvisus. I further analysed population genetic structure using microsatellites and mitochondrial DNA, and related the results to anthropogenic and natural dispersal dynamics on both global and regional (Baltic Sea) scales. I found high genetic diversity in most populations, with many shared haplotypes between distant populations. This supports the hypothesis that maritime shipping is an important vector for the dispersal of the cosmopolitan species B. improvisus. Nonetheless, natural larval dispersal is also important on smaller geographical scales, such as within the Baltic Sea. Marked genetic differentiation between northern and southern Baltic Sea populations raises the question whether there is restricted gene flow within the Baltic Sea, creating potential for local adaptations to evolve. To investigate the extent to which the broad distribution of B. improvisus along the Baltic Sea salinity gradient is explained by local adaptation versus physiological plasticity, I performed a common-garden experiment in which multiple populations were exposed to different salinities and multiple fitness-related phenotypic traits were recorded. The experiment confirmed that phenotypic plasticity, rather than local adaptation, explained the broad distribution of the species along the salinity gradient. Interestingly, all populations of B. improvisus performed best at low and intermediate salinities in many fitness-related traits (survival, growth and reproduction), although other traits (e.g. shell strength an juvenile growth) indicated higher costs associated with low salinity. A candidate gene approach was used to investigate the molecular basis of broad salinity tolerance in B. improvisus by characterizing the Na+/K+ ATPase (NAK) of B. improvisus – an ion transporter commonly involved in active osmoregulation in many species. We identified two main gene variants in B. improvisus (NAK1 and NAK2), and found that NAK1 mRNA existed in two isoforms that were differentially expressed in different life stages and adult tissues, suggesting an active role in osmoregulation. Lastly, I summarise current knowledge about salinity tolerance in barnacles and outline new research directions to further our understanding of the physiological and molecular mechanisms involved in salinity tolerance in barnacles