Novel integrative elements and genomic plasticity in ocean ecosystems.

Horizontal gene transfer accelerates microbial evolution. The marine picocyanobacterium Prochlorococcus exhibits high genomic plasticity, yet the underlying mechanisms are elusive. Here, we report a novel family of DNA transposons-"tycheposons"-some of which are viral satellites while others carry cargo, such as nutrient-acquisition genes, which shape the genetic variability in this globally abundant genus. Tycheposons share distinctive mobile-lifecycle-linked hallmark genes, including a deep-branching site-specific tyrosine recombinase. Their excision and integration at tRNA genes appear to drive the remodeling of genomic islands-key reservoirs for flexible genes in bacteria. In a selection experiment, tycheposons harboring a nitrate assimilation cassette were dynamically gained and lost, thereby promoting chromosomal rearrangements and host adaptation. Vesicles and phage particles harvested from seawater are enriched in tycheposons, providing a means for their dispersal in the wild. Similar elements are found in microbes co-occurring with Prochlorococcus, suggesting a common mechanism for microbial diversification in the vast oligotrophic oceans.

Coping with darkness: The adaptive response of marine picocyanobacteria to repeated light energy deprivation.

The picocyanobacteria Prochlorococcus and Synechococcus are found throughout the ocean's euphotic zone, where the daily light:dark cycle drives their physiology. Periodic deep mixing events can, however, move cells below this region, depriving them of light for extended periods of time. Here, we demonstrate that members of these genera can adapt to tolerate repeated periods of light energy deprivation. Strains kept in the dark for 3 d and then returned to the light initially required 18-26 d to resume growth, but after multiple rounds of dark exposure they began to regrow after only 1-2 d. This dark-tolerant phenotype was stable and heritable; some cultures retained the trait for over 132 generations even when grown in a standard 13:11 light:dark cycle. We found no genetic differences between the dark-tolerant and parental strains of Prochlorococcus NATL2A, indicating that an epigenetic change is likely responsible for the adaptation. To begin to explore this possibility, we asked whether DNA methylation-one potential mechanism mediating epigenetic inheritance in bacteria-occurs in Prochlorococcus. LC-MS/MS analysis showed that while DNA methylations, including 6 mA and 5 mC, are found in some other Prochlorococcus strains, there were no methylations detected in either the parental or dark-tolerant NATL2A strains. These findings suggest that Prochlorococcus utilizes a yet-to-be-determined epigenetic mechanism to adapt to the stress of extended light energy deprivation, and highlights phenotypic heterogeneity as an additional dimension of Prochlorococcus diversity.

Reversible Switching of the Dirac Point in Graphene Field-Effect Transistors Functionalized with Responsive Polymer Brushes.

The reversible control of the graphene Dirac point using external chemical stimuli is of major interest in the development of advanced electronic devices such as sensors and smart logic gates. Here, we report the coupling of chemoresponsive polymer brushes to reduced graphene oxide (rGO)-based field-effect transistors to modulate the graphene Dirac point in the presence of specific divalent cations. Poly[2-(methacryloyloxy)ethyl] phosphate (PMEP) brushes were grown on the transistor channel by atom transfer radical polymerization initiated from amine-pyrene linkers noncovalently attached to rGO surfaces. Our results show an increase in the Dirac point voltage due to electrostatic gating effects upon the specific binding of Ca2+ and Mg2+ to the PMEP brushes. We demonstrate that the electrostatic gating is reversibly controlled by the charge density of the polymer brushes, which depends on the divalent cation concentration. Moreover, a theoretical formalism based on the Grahame equation and a Langmuir-type binding isotherm is presented to obtain the PMEP-cation association constant from the experimental data.

In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis.

Liquid-phase electrophoresis either in the classical capillary format or miniaturized (chip CE) is a valuable tool for quality control of virus preparations and for targeting questions related to conformational changes of viruses during infection. We present an in vitro assay to follow the release of the RNA genome from a human rhinovirus (common cold virus) by using a molecular beacon (MB) and chip CE. The MB, a probe that becomes fluorescent upon hybridization to a complementary sequence, was designed to bind close to the 3' end of the viral genome. Addition of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a well-known additive for reduction of bleaching and blinking of fluorophores in fluorescence microscopy, to the background electrolyte increased the sensitivity of our chip CE set-up. Hence, a fast, sensitive and straightforward method for the detection of viral RNA is introduced. Additionally, challenges of our assay will be discussed. In particular, we found that (i) desalting of virus preparations prior to analysis increased the recorded signal and (ii) the MB-RNA complex signal decreased with the time of virus storage at -70 °C. This suggests that 3'-proximal sequences of the viral RNA, if not the whole genome, underwent degradation during storage and/or freezing and thawing. In summary, we demonstrate, for two independent virus batches, that chip electrophoresis can be used to monitor MB hybridization to RNA released upon incubation of the native virus at 56 °C. Graphical Abstract Schematic of the study strategy: RNA released from HRV-A2 is detected by chip electrophoresis through the increase in fluorescence after genom complexation to a cognate molecular beacon.

Toward a genetic system in the marine cyanobacterium Prochlorococcus.

As the smallest and most abundant primary producer in the oceans, the cyanobacterium Prochlorococcus is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically. Multiple attempts have been made to develop an efficient genetic transformation method for Prochlorococcus over the years; all have been unsuccessful to date, despite some success with their close relative, Synechococcus . To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into Prochlorococcus cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.

Enzyme Multilayers on Graphene-Based FETs for Biosensing Applications.

Electrochemical sensors represent a powerful tool for real-time measurement of a variety of analytes of much significance to different areas, ranging from clinical diagnostics to food technology. Point-of-care devices which can be used at patient bedside or for online monitoring of critical parameters are of great importance in clinical daily routine. In this work, portable, low-cost electrochemical sensors for a fast and reliable detection of the clinically relevant analyte urea have been developed. The intrinsic pH sensitivity of reduced graphene oxide (rGO)-based field-effect transistors (FETs) was exploited to monitor the enzymatic hydrolysis of urea. The functionalization of the sensor platform using the layer-by-layer technique is especially advantageous for the immobilization of the biorecognition element provided that this approach preserves the enzyme integrity as well as the rGO surface. The great selectivity of the enzyme (urease) combined with the high sensitivity of rGO-based FETs result in the construction of urea biosensors with a limit of detection (LOD) of 1µM and a linear range up to 1mM. Quantification of Cu2+ with a LOD down to 10nM was performed by taking advantage of the specific inhibition of urease in the presence of heavy metals. These versatile biosensors offer great possibilities for further development of highly sensitive enzyme-based FETs for real-time, label-free detection of a wide variety of clinically relevant analytes.

Cascading reaction of arginase and urease on a graphene-based FET for ultrasensitive, real-time detection of arginine.

Herein, a biosensor based on a reduced graphene oxide field effect transistor (rGO-FET) functionalized with the cascading enzymes arginase and urease was developed for the detection of L-arginine. Arginase and urease were immobilized on the rGO-FET sensing surface via electrostatic layer-by-layer assembly using polyethylenimine (PEI) as cationic building block. The signal transduction mechanism is based on the ability of the cascading enzymes to selectively perform chemical transformations and prompt local pH changes, that are sensitively detected by the rGO-FET. In the presence of L-arginine, the transistors modified with (PEI/urease(arginase)) multilayers showed a shift in the Dirac point due to the change in the local pH close to the graphene surface, produced by the catalyzed urea hydrolysis. The transistors were able to monitor L-arginine in the 10-1000 µM linear range with a LOD of 10 µM, displaying a fast response and a good long-term stability. The sensor showed stereospecificity and high selectivity in the presence of non-target amino acids. Taking into account the label-free, real-time measurement capabilities and the easily quantifiable, electronic output signal, this biosensor offers advantages over state-of-the-art L-arginine detection methods.

Monolithic anion-exchange chromatography yields rhinovirus of high purity.

For vaccine development, 3D-structure determination, direct fluorescent labelling, and numerous other studies, homogeneous virus preparations of high purity are essential. Working with human rhinoviruses (RVs), members of the picornavirus family and the main cause of generally mild respiratory infections, we noticed that our routine preparations appeared highly pure on analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), exclusively showing the four viral capsid proteins (VPs). However, the preparations turned out to contain substantial amounts of contaminating material when analyzed by orthogonal analytical methods including capillary zone electrophoresis, nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA), and negative stain transmission electron microscopy (TEM). Because these latter analyses are not routine to many laboratories, the above contaminations might remain unnoticed and skew experimental results. By using human rhinovirus serotype A2 (RV-A2) as example we report monolithic anion-exchange chromatography (AEX) as a last polishing step in the purification and demonstrate that it yields infective, highly pure, virus (RV-A2 in the respective fractions was confirmed by peptide mass fingerprinting) devoid of foreign material as judged by the above criteria.

Proteo-lipobeads to encapsulate cytochrome c oxidase from Paracoccus denitrificans.

Proteo-lipobeads (PLBs) are investigated as cell-free model systems to encapsulate membrane proteins such as ion channels and transporters. PLBs are based on nickel nitrile tri-acetic acid (Ni-NTA)-functionalized agarose beads, onto which membrane proteins (MP) are bound via histidine(his)-tag. Composite beads thus obtained (subsequently called proteobeads) are dialyzed in the presence of lipid micelles to form PLBs. As an example we employed cytochrome c oxidase from P. denitrificans with a his-tag fused to the C-terminus of subunitI. In this orientation the P side of CcO faces the outside of the PLB and hence protons are released to the outer aqueous phase, when electron transfer is initiated by light excitation of Ru complexes. Proton release kinetics was probed by fluorescence microscopy using the pH-sensitive sensor molecule fluorescein DHPE inserted into the lipid layer. In order to monitor the generation of membrane potentials we performed a FLIPR assay on the CcO embedded in PLBs using the FRET pair CC2-DMPE/DiSBAC2(3). The combined results show that PLBs can be used as a model system designed to quantify the kinetic parameters of membrane proteins. In addition, the FLIPR assay demonstrates the feasibility of PLBs for high throughput screening applications.

Enzyme-polyelectrolyte multilayer assemblies on reduced graphene oxide field-effect transistors for biosensing applications.

We present the construction of layer-by-layer (LbL) assemblies of polyethylenimine and urease onto reduced-graphene-oxide based field-effect transistors (rGO FETs) for the detection of urea. This versatile biosensor platform simultaneously exploits the pH dependency of liquid-gated graphene-based transistors and the change in the local pH produced by the catalyzed hydrolysis of urea. The use of an interdigitated microchannel resulted in transistors displaying low noise, high pH sensitivity (20.3µA/pH) and transconductance values up to 800 µS. The modification of rGO FETs with a weak polyelectrolyte improved the pH response because of its transducing properties by electrostatic gating effects. In the presence of urea, the urease-modified rGO FETs showed a shift in the Dirac point due to the change in the local pH close to the graphene surface. Markedly, these devices operated at very low voltages (less than 500mV) and were able to monitor urea in the range of 1-1000µm, with a limit of detection (LOD) down to 1µm, fast response and good long-term stability. The urea-response of the transistors was enhanced by increasing the number of bilayers due to the increment of the enzyme surface coverage onto the channel. Moreover, quantification of the heavy metal Cu2+(with a LOD down to 10nM) was performed in aqueous solution by taking advantage of the urease specific inhibition.

pH and Potential Transients of the bc1 Complex Co-Reconstituted in Proteo-Lipobeads with the Reaction Center from Rb. sphaeroides.

His-tag technology is employed to bind membrane proteins, such as the bc1 complex and the reaction center (RC) from Rhodobacter sphaeroides, to spherical as well as planar surfaces in a strict orientation. Subsequently, the spherical and planar surfaces are subjected to in situ dialysis to form proteo-lipobeads (PLBs) and protein-tethered bilayer membranes, respectively. PLBs based on Ni-nitrileotriacetic acid-functionalized agarose beads that have diameters ranging from 50 to 150 µm are used to assess proton release and membrane potential parameters by confocal laser-scanning microscopy. The pH and potential transients are thus obtained from bc1 activated by the RC. To assess the turnover of bc1 excited by the RC in a similar setting, we used the planar surface of an attenuated total reflection crystal modified with a thin gold layer to carry out time-resolved surface-enhanced IR absorption spectroscopy triggered by flash lamp excitation. The experiments suggest that both proteins interact in a cyclic manner in both environments. The activity of the proteins seems to be preserved in the same manner as that in chromatophores or reconstituted in liposomes.