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A key goal of synthetic biology is to engineer organisms that can use solar energy to convert CO

Significance We constructed simple cells (SimCells) whose native chromosomes were removed and replaced by synthetic genetic circuits. The chromosome-free SimCells can process designed DNA and express target genes for an extended period of time. The strategy of SimCell generation is applicable to most bacteria, creating a universal platform for reprogramming bacteria. We demonstrated that SimCells can be designed as safe agents for bacterial therapy through synthesis and delivery of a potent anticancer drug against a variety of cancer cell lines. This showed that the nonreplicating and programmable property of SimCells is advantageous for applications in sensitive environments. The results of this work will both improve our understanding of natural living systems and simultaneously lay the foundations for future advances in synthetic biology., A type of chromosome-free cell called SimCells (simple cells) has been generated from Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. The removal of the native chromosomes of these bacteria was achieved by double-stranded breaks made by heterologous I-CeuI endonuclease and the degradation activity of endogenous nucleases. We have shown that the cellular machinery remained functional in these chromosome-free SimCells and was able to process various genetic circuits. This includes the glycolysis pathway (composed of 10 genes) and inducible genetic circuits. It was found that the glycolysis pathway significantly extended longevity of SimCells due to its ability to regenerate ATP and NADH/NADPH. The SimCells were able to continuously express synthetic genetic circuits for 10 d after chromosome removal. As a proof of principle, we demonstrated that SimCells can be used as a safe agent (as they cannot replicate) for bacterial therapy. SimCells were used to synthesize catechol (a potent anticancer drug) from salicylic acid to inhibit lung, brain, and soft-tissue cancer cells. SimCells represent a simplified synthetic biology chassis that can be programmed to manufacture and deliver products safely without interference from the host genome.

Common reed (Phragmites) interacts with a large suite of other organisms including cryptogams attached to live or dead culm bases. We report an unusual observation of at least ten taxa of lichens and hepatics attached to persistent reed stubble on the swampy bank of freshwater Cypress Creek north of Tampa, Hillsborough County, Florida. This bryoid material included the minute, leafy hepatics Microlejeunea globosa, Microlejeunea cf. ulicina, Myriocoleopsis minutissima, and Frullania cf. inflata; and the lichens Arthonia subdiffusa, Chrysothrix xanthina, Opegrapha viridis, Phaeographis sp., Physcia sp., and an unidentified species of Parmeliaceae. These generally corticolous or epiphyllous taxa have not previously been reported from common reed, and suggest unrecognized complexity in both cryptogamic microhabitats and reed epiphytes.

In 1998, Gold and Heffner authored a landmark review in Clinical Psychology Review on the topic of sexual addiction that concluded that sexual addiction, though increasingly popular in mental health settings, was largely based on speculation, with virtually no empirical basis. In the more than two decades since that review, empirical research around compulsive sexual behaviors (which subsumes prior research about sexual addiction) has flourished, ultimately culminating in the inclusion of a novel diagnosis of Compulsive Sexual Behavior Disorder in the eleventh edition of the World Health Organization’s International Classification of Diseases. The present work details a systematic review of empirical research published between January 1st, 1995 and August 1st, 2020 related to compulsive sexual behaviors, with a specific focus on evaluating the methodologies of that literature. This review yielded 371 papers detailing 415 individual studies. In general, the present review finds that, although research related to compulsive sexual behaviors has proliferated, much of this work is characterized by simplistic methodological designs, a lack of theoretical integration, and an absence of quality measurement. Moreover, the present review finds a virtual absence of high-quality treatment-related research published within this time frame. Implications of these findings for both clinical practice and future research are discussed.

The insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX, and the rearrangement of several loops envelops this substrate, forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for the delivery of magnesium or abstraction of protons.

A toxicity biosensor Acinetobacter baylyi Tox2 was constructed with the host strain A. baylyi ADP1 harboring a new and medium-copy-number plasmid pWH1274_lux, and was applied to detect the cytotoxicity of heavy metal contaminated seawater. The gene cassette luxCDABE was controlled by constitutively expressed promoter Ptet on pWH1274_lux and the bioluminescence intensity of the biosensor reduces in proportional to the concentrations of toxic compounds. A. baylyi Tox2 exhibits tolerance to salinity, hence it is applicable to seawater samples. A. baylyi Tox2 and Mugilogobius chulae were exposed to different concentrations of heavy metals (Hg2+, Zn2+, Cu2+, and Cd2+) in artificial seawater for performance comparison and Pearson correlation analysis showed a significant correlation (p

The genus Frullania (Frullaniaceae, Porellales, Jungermanniopsida) comprises 21 species in Southeastern North America. Screening about 850 independent collections of common species of Frullania taken from the hark of trees in Alabama, Georgia, Mississippi, North Carolina, Tennessee, and Virginia revealed a surprisingly high number of parasitic fruit-body forming ascomycetes. Particularly well represented are the Hypocreales (Sordariomycetes), of which 14 species (out of 17 occurring on Frullania worldwide) in five genera on eight host species are documented here based on 235 specimens. The genera Bryotria, Laniatria, and Periantria are proposed as new as well as the following ten species: Bryocentria biannulata, B. chlysothrix, B. lusor, B. navicula. B. pentarnera. Bryonectria anisopoda, Bryotria adelpha, B. urophora, Laniatria myxostoma, and Periantria bellacaptiva. Three new combinations are proposed. Calonectria frullaniae is transferred to Periantria. Nectria foertheri (on Porella sp.) and N. lagodes to Bryotria. Bryocentria brongniartii, Bryonectria callicarpa. Bryotria lagodes, and Periantria frullaniae are new records for North America. The latter species which destroys developing sporophytes within the perianths was found to be by far the most frequent. The hypocrealean fungi on Frullania in Southeastern North America are much more diverse than those of Central Europe. Biotrophic and necrotrophic species occur in both regions. The main distinguishing characters are size and colour of perithecium, structure of the perithecial wall, and size, shape and septation of the ascospores. Perithecia sometimes occur in high densities (up to eight per square mm) and, in spite of their small size, are relatively conspicuous due to their vivid colouration. Nevertheless, beyond the present study, knowledge of the Frullania inhabiting ascomycetes in North America is almost non-existent.

Proteorhodopsin (PR) is part of a diverse, abundant, and widespread superfamily of photoreactive proteins, the microbial rhodopsins. PR, a light-driven proton pump, enhances the ability of the marine bacterium Vibrio strain AND4 to survive and recover from periods of starvation, and heterologously produced PR extends the viability of nutrient-limited Shewanella oneidensis. We show that heterologously produced PR enhances the viability of E. coli cultures over long periods of several weeks and use single-cell Raman spectroscopy (SCRS) to detect PR in 9-month-old cells. We identify a densely packed and consequently stabilized cell membrane as the likely basis for extended viability. Similar considerations are suggested to apply to marine bacteria, for which high PR levels represent a significant investment in scarce metabolic resources. PR-stabilized cell membranes in marine bacteria are proposed to keep a population viable during extended periods of light or nutrient limitation, until conditions improve., Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli. Quantitative mass spectrometry shows that E. coli, exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize ∼148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans. IMPORTANCE Proteorhodopsin (PR) is part of a diverse, abundant, and widespread superfamily of photoreactive proteins, the microbial rhodopsins. PR, a light-driven proton pump, enhances the ability of the marine bacterium Vibrio strain AND4 to survive and recover from periods of starvation, and heterologously produced PR extends the viability of nutrient-limited Shewanella oneidensis. We show that heterologously produced PR enhances the viability of E. coli cultures over long periods of several weeks and use single-cell Raman spectroscopy (SCRS) to detect PR in 9-month-old cells. We identify a densely packed and consequently stabilized cell membrane as the likely basis for extended viability. Similar considerations are suggested to apply to marine bacteria, for which high PR levels represent a significant investment in scarce metabolic resources. PR-stabilized cell membranes in marine bacteria are proposed to keep a population viable during extended periods of light or nutrient limitation, until conditions improve.

Saxifraga tridactylites (Saxifragaceae), an annual herb native to northwest Africa, southwestern Asia, Europe, northeastern Iran, and western Russia, has rapidly naturalized in two geographically distinct areas of the United States: the Southeast and the Northwest. In the Southeast, the spread has been exceedingly fast and poses a potential threat to xeric limestone habitats of the Interior Low Plateau and Ridge and Valley physiographic provinces. Prior to our work, S. tridactylites appeared to be an insignificant introduction, only documented in a few North American locations in British Columbia and Oregon. Here, we show that the North American distribution is much greater than previously reported, with records from four counties in the Northwest and 53 counties in the Southeast: northern Alabama (14 counties), northwest Georgia (two counties) northern Mississippi (five counties), and southern Tennessee (32 counties). To our knowledge, it has not yet dispersed into Arkansas, Kentucky, or North Carolina.

The cyclase step in chlorophyll (Chl) biosynthesis has not been characterized biochemically, although there are some plausible candidates for cyclase subunits. Two of these, Sll1214 and Sll1874 from the cyanobacterium Synechocystis 6803, were FLAG-tagged in vivo and used as bait in separate pulldown experiments. Mass spectrometry identified Ycf54 as an interaction partner in each case, and this interaction was confirmed by a reciprocal pulldown using FLAG-tagged Ycf54 as bait. Inactivation of the ycf54 gene (slr1780) in Synechocystis 6803 resulted in a strain that exhibited significantly reduced Chl levels. A detailed analysis of Chl precursors in the ycf54 mutant revealed accumulation of very high levels of Mg-protoporphyrin IX methyl ester and only traces of protochlorophyllide, the product of the cyclase, were detected. Western blotting demonstrated that levels of the cyclase component Sll1214 and the Chl biosynthesis enzymes Mg-protoporphyrin IX methyltransferase and protochlorophyllide reductase are significantly impaired in the ycf54 mutant. Ycf54 is, therefore, essential for the activity and stability of the oxidative cyclase. We discuss a possible role of Ycf54 as an auxiliary factor essential for the assembly of a cyclase complex or even a large multienzyme catalytic center.

Single cell Raman spectroscopy measures a spectral fingerprint of the biochemistry of cells, and provides a powerful method for label-free detection of living cells without the involvement of a chemical labelling strategy. However, as the intrinsic Raman signals of cells are inherently weak, there is a significant challenge in discriminating and isolating cells in a flowing stream. Here we report an integrated Raman-microfluidic system for continuous sorting of a stream of cyanobacteria, Synechocystis sp. PCC6803. These carotenoid-containing microorganisms provide an elegant model system enabling us to determine the sorting accuracy using the subtly different resonance Raman spectra of microorganism cultured in a (12)C or (13)C carbon source. Central to the implementation of continuous flow sorting is the use of "pressure dividers" that eliminate fluctuations in flow in the detection region. This has enabled us to stabilise the flow profile sufficiently to allow automated operation with synchronisation of Raman acquisition, real-time classification and sorting at flow rates of ca.

Two bryophilous species of the order Hypocreales (Ascomycetes) are introduced as new. Bryonectria aphanes (Bionectriaceae) has hyaline ascomata, four-spored asci, three-celled ascospores and numerous phialids and appressoria on the hyphae. It is recorded on the moss Platygyrium repens (Pylaisiadelphaceae, Hypnales) from Maine, U.S.A. Trichosphaerella goniospora (Niessliaceae) forms dark ascomata beset with setae deeply rooted in the peridial wall and eight two-celled ascospores. They disarticulate very early during sporogenesis resulting in 16 irregularly cubic part-spores. The species was collected on the liverwort Ptilidium pulcherrimum (Ptilidiaceae, Jungermanniales) in New Brunswick, Canada. The Niessliaceae belong to a hitherto unknown line within bryophilous fungi. On the other hand, bryophytes extend the spectrum of substrates for black hypocrealean fungi.

Exercise and heat stress lead to systemic improvements in arterial endothelial function, vascular stiffness, and cardiopulmonary capacity. The improvements in endothelial function may be primarily mediated via increases in shear stress. This study examined whether improvements in arterial function may be achieved in the absence of systemic vascular adaptations. Specifically, we hypothesized that repeated bouts of brief occlusion would improve arterial endothelial function via shear stress-dependent mechanisms.Eleven healthy males underwent a shear stress intervention (5 s brachial occlusion, 10 s rest) for 30 min, five times weekly for 6 weeks on one arm while the other acted as an untreated control. Ultrasound was used to assess brachial arterial forearm blood flow (FBF) and vascular conductance (FVC), diameter, and shear rate (SR), while endothelial function was assessed by flow-mediated dilatation (FMD). Post-occlusive reactive hyperaemia and pulse wave velocity (PWV) were also measured.There were no changes in any of the measures in the control arm (all d 0.2, p 0.05). After 3 weeks of the intervention, FMD was increased from baseline (7.6 ± 0.6 vs. 5.9 ± 0.9%; d = 1.3, p = 0.038) and further increased after 6 weeks to 9.5 ± 2.6% (d = 1.7, p 0.001). SR was also increased following the 6-week intervention (all d ≥ 0.6, p 0.001). Resting and peak FBF and FVC were also increased in response to the intervention (all d ≥ 0.6, p 0.001) and PWV was reduced.These data demonstrate that episodic increases in shear stress elicit marked increases in arterial endothelial function and vascular reactivity.

Previous studies suggest that exercise and heat stress improve cutaneous endothelial function, caused by increases in shear stress. However, as vasodilatation in the skin is primarily a thermogenic phenomenon, we investigated if shear stress alone without increases in skin temperature that occur with exercise and heat stress increases endothelial function. We examined the hypothesis that repeated bouts of brief occlusion would improve cutaneous endothelial function via shear stress-dependent mechanisms. Eleven males underwent a shear stress intervention (forearm occlusion 5 s rest 10 s) for 30 min, five times·week−1 for 6 weeks on one arm, the other was an untreated control. Skin blood flow was measured using laser-Doppler flowmetry, and endothelial function was assessed with and without NOS-inhibition with L-NAME in response to three levels of local heating (39, 42, and 44 °C), ACh administration, and reactive hyperaemia. Data are cutaneous vascular conductance (CVC, laser-Doppler/blood pressure). There were no changes in the control arm (all d ≤ 0.2, p > 0.05). In the experimental arm, CVC to 39 °C was increased after 3 and 6 weeks (d = 0.6; p ≤ 0.01). Nitric oxide contribution was increased after 6 weeks compared to baseline (d = 0.85, p 0.05). For both 42 and 44 °C, nitric oxide contribution was increased after weeks 3 and 6 (d ≥ 0.4, p

Magnesium chelatase (MgCH) initiates chlorophyll biosynthesis by catalysing the ATP-dependent insertion of Mg2+ into protoporphyrin. This large enzyme complex comprises ChlH, I and D subunits, with I and D involved in ATP hydrolysis, and H the protein that handles the substrate and product. The 148 kDa ChlH subunit has a globular N-terminal domain attached by a narrow linker to a hollow cage-like structure. Following deletion of this ~18 kDa domain from the Thermosynechoccus elongatus ChlH, we used single particle reconstruction to show that the apo- and porphyrin-bound forms of the mutant subunit consist of a hollow globular protein with three connected lobes; superposition of the mutant and native ChlH structures shows that, despite the clear absence of the N-terminal ‘head’ region, the rest of the protein appears to be correctly folded. Analyses of dissociation constants shows that the ΔN159ChlH mutant retains the ability to bind protoporphyrin and the Gun4 enhancer protein, although the addition of I and D subunits yields an extremely impaired active enzyme complex. Addition of the Gun4 enhancer protein, which stimulates MgCH activity significantly especially at low Mg2+ concentrations, partially reactivates the ΔN159ChlH–I–D mutant enzyme complex, suggesting that the binding site or sites for Gun4 on H do not wholly depend on the N-terminal domain., The N-terminal domain of the 148 kDa ChlH is essential for normal activity of the ChlH–I–D magnesium chelatase complex. Deleting this 18 kDa domain retains the hollow cage-like structure and porphyrin binding. Chelatase activity is partially restored by the Gun4 protein.

Penetrating thermal tissue damage/spread is an important aspect of many electrosurgical devices and correlates with effective tissue cutting, hemostasis, preservation of adjacent critical structures and tissue healing. This study compared the thermal damage/spread associated with the PhotonBlade, Valleylab Pencil, Valleylab EDGE Coated Pencil, PlasmaBlade 3.0S and PlasmaBlade 4.0, when performing a single pass dynamic tissue cut in fresh extirpated porcine longissimus muscle. These devices were used in a fashion that emulated their use in the clinical setting. Each device’s thermal damage/spread, at Minimum, Median and Maximum power input settings, was assessed with nitroblue tetrazolium viability staining in the WVU Pathology Laboratory for Translational Medicine. The thermal damage/spread associated with the PhotonBlade was compared with the other devices tested based on the individual treatment results (n=179 cuts combined). In summary, the PhotonBlade overall demonstrated the least penetrating thermal tissue damage/spread, followed by the PlasmaBlade 4.0, then Valleylab Pencil and PlasmaBlade 3.0S and then Valleylab EDGE Coated Pencil in order of increasing thermal damage/spread depths.

Summary Cell sorting coupled with single‐cell genomics is a powerful tool to circumvent cultivation of microorganisms and reveal microbial ‘dark matter’. Single‐cell Raman spectra (SCRSs) are label‐free biochemical ‘fingerprints’ of individual cells, which can link the sorted cells to their phenotypic information and ecological functions. We employed a novel Raman‐activated cell ejection (RACE) approach to sort single bacterial cells from a water sample in the Red Sea based on SCRS. Carotenoids are highly diverse pigments and play an important role in phototrophic bacteria, giving strong and distinctive Raman spectra. Here, we showed that individual carotenoid‐containing cells from a Red Sea sample were isolated based on the characteristic SCRS. RACE‐based single‐cell genomics revealed putative novel functional genes related to carotenoid and isoprenoid biosynthesis, as well as previously unknown phototrophic microorganisms including an unculturable Cyanobacteria spp. The potential of Raman sorting coupled to single‐cell genomics has been demonstrated.

The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg(2+) into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ∼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ∼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.

Photosynthetic microorganisms play crucial roles in aquatic ecosystems and are the major primary producers in global marine ecosystems. The discovery of new bacteria and microalgae that play key roles in CO(2) fixation is hampered by the lack of methods to identify hitherto-unculturable microorganisms. To overcome this problem we studied single microbial cells using stable-isotope probing (SIP) together with resonance Raman (RR) microspectroscopy of carotenoids, the light-absorbing pigments present in most photosynthetic microorganisms. We show that fixation of (13)CO(2) into carotenoids produces a red shift in single-cell RR (SCRR) spectra and that this SCRR-SIP technique is sufficiently sensitive to detect as little as 10% of (13)C incorporation. Mass spectrometry (MS) analysis of labelled cellular proteins verifies that the red shift in carotenoid SCRR spectra acts as a reporter of the (13)C content of single cells. Millisecond Raman imaging of cells in mixed cultures and natural seawater samples was used to identify cells actively fixing CO(2), demonstrating that the SCRR-SIP is a noninvasive method for the rapid and quantitative detection of CO(2) fixation at the single cell level in a microbial community. The SCRR-SIP technique may provide a direct method for screening environmental samples, and could help to reveal the ecophysiology of hitherto-unculturable microorganisms, linking microbial species to their ecological function in the natural environment.

Epiphyllous bryophytes are poorly documented for temperate eastern North America. Ten liverwort taxa and one moss species are reported as epiphylls on Rhododendron maximum from the Appalachian Plateau (Fentress County, Tennessee, and Menifee County, Kentucky). The Kentucky localities represent the northernmost documented sites for bryophyte epiphylls in eastern North America. Three North American liverwort endemics, Lejeunea ruthii, Leucolejeunea clypeata, and Radula obconica, and the moss, Platygyrium repens, are newly reported as epiphylls. Colony size ranged from

Bryoplana xerophila, a new genus and species of limnoterrestrial protoplanelline platyhelminth, was found in moss and soil covering a concrete wall in northern Alabama, USA. Bryoplana xerophila is the first taxon of limnoterrestrial Protoplanellinae recorded from North America and is one of the few rhabdocoels known from dry habitats. It is unique within Protoplanellinae in lacking rhabdites, having a pharynx rosulatus in the frontal half of the body, and lacking sclerotized parts in the male system. Notes on encystment, reproduction and feeding behavior are given. An updated identification key to all known genera of Protoplanellinae is presented.

The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes an essential step in the synthesis of the most abundant pigment on Earth, chlorophyll. This unique reaction involves the sequential addition of a hydride and proton across the C17=C18 double bond of protochlorophyllide (Pchlide) by dynamically coupled quantum tunneling and is an important model system for studying the mechanism of hydrogen transfer reactions. In the present work, we have combined site-directed mutagenesis studies with a variety of sensitive spectroscopic and kinetic measurements to provide new insights into the mechanistic role of three universally conserved Cys residues in POR. We show that mutation of Cys-226 dramatically alters the catalytic mechanism of the enzyme. In contrast to wild-type POR, the characteristic charge-transfer intermediate, formed upon hydride transfer from NADPH to the C17 position of Pchlide, is absent in C226S variant enzymes. This suggests a concerted hydrogen transfer mechanism where proton transfer only is rate-limiting. Moreover, Pchlide reduction does not require the network of solvent-coupled conformational changes that play a key role in the proton transfer step of wild-type POR. We conclude that this globally important enzyme is finely tuned to facilitate efficient photochemistry, and the removal of a key interaction with Pchlide in the C226S variants significantly affects the local active site structure in POR, resulting in a shorter donor-acceptor distance for proton transfer.

Seed plant genera often exhibit intercontinental disjunctions where different species are found on different continents. Many morphologically circumscribed bryophyte species exhibit similar disjunctions. We used nucleotide sequences from the plastid and nuclear genomes to test hypotheses of phylogeography within representatives of the genus Metzgeria: Metzgeria furcata, Metzgeria conjugata, and Metzgeria myriopoda. The first two species have sexual and asexual populations, exhibit disjunctions between North America and Europe, and have been split into separate species, numerous subspecies or varieties. The third species occurs in eastern North America but is not reported from Europe. Phylogenetic analyses resolved three distinct lineages within the morphologically defined species, M. furcata: one in North America, and two in Europe. Similarly, three morphologically cryptic clades of M. conjugata were resolved by the molecular data: northern North America, Europe, and south-eastern North America. For both species, molecular divergence among taxa occurred in the absence of morphological change. In the case of M. myriopoda, all plants from eastern North America were both morphologically uniform and genetically homogeneous (although not identical). The present study provides significant insight into a plant group with complex taxonomy, and indicates that these liverwort taxa with wide distributions, extreme sex ratios, and continental disjunctions harbor cryptic lineages. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 745–756.

Identification of diffuse sediment pollution ‘hotspots’ requiring improved management in catchments supporting salmonids can be achieved using computer models. Accordingly, the PSYCHIC (Ph...

PSYCHIC is a process-based model of phosphorus (P) and suspended sediment (SS) mobilisation in land runoff and subsequent delivery to watercourses. Modelled transfer pathways include release of desorbable soil P, detachment of SS and associated particulate P, incidental losses from manure and fertiliser applications, losses from hard standings, the transport of all the above to watercourses in underdrainage (where present) and via surface pathways, and losses of dissolved P from point sources. The model can operate at two spatial scales, although the scientific core is the same in both cases. At catchment scale, the model uses easily available national scale datasets to infer all necessary input data whilst at field scale, the user is required to supply all necessary data. The model is sensitive to a number of crop and animal husbandry decisions, as well as to environmental factors such as soil type and field slope angle. It is envisaged that the catchment-scale model would provide the first tier of a catchment characterisation study, and would be used as a screening tool to identify areas within the catchment which may be at elevated risk of P loss. This would enable targeted data collection, involving farm visits and stakeholder discussion, which would then be followed up with detailed field-scale modelling. Both tiers allow the effects of possible mitigation options at catchment scale (Tier 1) and field scale (Tier 2) to be explored. The PSYCHIC model framework therefore provides a methodology for identifying critical source areas of sediment and P transfer in catchments and assessing what management changes are required to achieve environmental goals.

Arabidopsis plants overexpressing β-carotene hydroxylase 1 accumulate over double the amount of zeaxanthin present in wild-type plants. The final amplitude of non-photochemical quenching (NPQ) was found to be the same in these plants, but the kinetics were different. The formation and relaxation of NPQ consistently correlated with the de-epoxidation state of the xanthophyll cycle pool and not the amount of zeaxanthin. These data indicate that zeaxanthin and violaxanthin antagonistically regulate the switch between the light harvesting and photoprotective modes of the light harvesting system and show that control of the xanthophyll cycle pool size is necessary to optimize the kinetics of NPQ.

Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis. This complex enzyme has at least three substrates and couples ATP hydrolysis to the insertion of Mg2+ into protoporphyrin IX. We directly observed metal-ion chelation fluorometrically, providing the first data describing the on-enzyme reaction. We describe the transient-state kinetics of magnesium chelatase with direct observation of the evolution of an enzyme-product complex EMgDIX. We demonstrate that MgATP2- binding occurs after the rate-determining step. As nucleotide hydrolysis is essential for the overall reaction this must also occur after the rate-determining step. This provides the first evidence for the synchronization of the ATPase and chelatase pathways and suggests a mechanism where nucleotide binding acts to clamp the chelatase in a product complex. Comparison of rate constants for the slow step in the reaction with further transient kinetics under conditions where multiple turnovers can occur reveals that an additional activation step is required to explain the behavior of magnesium chelatase. These data provide a new view of the sequence of events occurring in the reaction catalyzed by magnesium chelatase.

In oxygenic phototrophs, chlorophylls, hemes and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated and an important regulatory role is attributed to Mgchelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the Mg-chelatase activity but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for Mgprotoporphyrin IX. It revealed that the orientation of 6/7 loop is critical for the binding and the magnesium ion held within the porphyrin is coordinated by Asn211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in Mg-chelatase assay showing that tight porphyrin-binding in Gun4 facilitates its interaction with the Mg-chelatase ChlH subunit. Finally, we introduced the W192A mutation into Synechocystis 6803 cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway. This work was supported by project P501/12/G055 of the Czech Science Foundation, and by the National Programme of Sustainability I (LO1416) and by ERC 2009-Adg25027-PELE (to V.G). J.K. was supported by project Algain (EE2.3.30.0059). N.B.P.A., P.A.D., A.A.B. and C.N.H. thank the Biotechnology and Biological Sciences Research Council (BBSRC) U.K. for funding, under award numbers BB/G021546/1 and BB/M000265/1. CNH was also supported by an Advanced Award 338895 from the European Research Council.

Although uncultured microorganisms have important roles in ecosystems, their ecophysiology in situ remains elusive owing to the difficulty of obtaining live cells from their natural habitats. In this study, we employed a novel magnetic nanoparticle-mediated isolation (MMI) method to recover metabolically active cells of a group of previously uncultured phenol degraders, Burkholderiales spp., from coking plant wastewater biosludge; five other culturable phenol degraders-Rhodococcus sp., Chryseobacterium sp. and three different Pseudomonas spp.-were also isolated from the same biosludge using traditional methods. The kinetics of phenol degradation by MMI-recovered cells (MRCs) was similar to that of the original sludge. Stable isotope probing (SIP) and pyrosequencing of the 16S rRNA from the 'heavy' DNA ((13)C-DNA) fractions indicated that Burkholderiales spp. were the key phenol degraders in situ in the biosludge, consistent with the results of MRCs. Single-cell Raman micro-spectroscopy was applied to probe individual bacteria in the MRCs obtained from the SIP experiment and showed that 79% of them were fully (13)C-labelled. Biolog assays on the MRCs revealed the impact of various carbon and nitrogen substrates on the efficiency of phenol degradation in the wastewater treatment plant biosludge. Specifically, hydroxylamine, a metabolite of ammonia oxidisation, but not nitrite, nitrate or ammonia, inhibited phenol degradation in the biosludge. Our results provided a novel insight into the occasional abrupt failure events that occur in the wastewater treatment plant. This study demonstrated that MMI is a powerful tool to recover live and functional cells in situ from a complex microbial community to enable further characterisation of their physiology.

Plagiochila (sect. Arrectae) punctata, previously known from the Neotropics, Africa and Europe, has been collected on a sandstone cliff in a rockhouse type environment in Tennessee, new to North America. Maximum likelihood analyses based on nrITS1–5.8S-ITS2 sequences of several species of Plagiochila sect. Arrectae resolve the Tennessee specimen in a robust P. punctata subclade with accessions from Ecuador and the Democratic Republic of the Congo.

Gun4 has been implicated in a developmental signaling pathway between the chloroplast and the nucleus involving magnesium protoporphyrin IX (MgP(IX)), the first dedicated intermediate in the chlorophyll biosynthetic pathway. Here we present the crystal structure of Thermosynechococcus elongatus Gun4 at 1.5 A, describe the binding affinities of Gun4 for substrate and product porphyrin molecules, and identify a likely (Mg)P(IX) binding site on the protein. Kinetic analyses show that Gun4 dramatically increases the efficiency of transformation of porphyrin substrate to metalloporphyrin product and that it also reduces the threshold Mg2+ concentration required for activity at low porphyrin concentration. Gun4 therefore controls magnesium chelatase at physiologically significant Mg2+ concentrations and likely acts as a molecular switch in vivo so that in its absence magnesium chelatase is inactive. This mechanism could allow Gun4 to mediate magnesium protoporphyrin levels both for chlorophyll biosynthesis and for signaling to the nucleus.

The typically lithophilic Polypodium appalachianum was discovered as a canopy epiphyte 35 to 40 m above ground on a horizontal branch of a champion-size Liriodendron tulipifera in the Great Smoky Mountains National Park. Occurring along with this first documentation of P. appalachianum from the tree canopy was an assemblage of normally terrestrial mosses, an unusual assortment of collembola (springtails), and a flightless proturan insect species previously known only from soil and litter. The distinctive features of this canopy habitat may duplicate some ecological conditions usually found only at ground level, establishing the opportunity for translocating an entire community and providing biologists with new insights on the origin of some epiphytes.

Surface-based observations were taken of cloud and precipitation particles on the Avery Plateau (66°50.34′S 65°29.58′W), Antarctic Peninsula from 25 November to 13 December 1995. This paper considers cloud parameters on three days during this period when the cloud base reached ground level and snow was falling. It was found that on all three days more ice crystals were present in the cloud than would be expected from simple theoretical considerations. The rate of snowfall decreased as the number of ice crystals increased, the large number of ice crystals present effectively suppressing the formation of large precipitation-sized crystals. The source of the ice nuclei that allowed the formation of the large number of crystals is not known for certain but is thought to be the snow surface, possibly in the form of very fine ice crystals blown from the surface during blowing/drifting snow episodes.

Frullania bolanderi is reported new for Cumberland County, Maine, in an area with attributes of an old-growth forest. This report supports the previously published hypothesis that F. bolanderi may be an indicator of old growth forest in eastern North America.

Popular identification guides to bryophytes are not at all common, although their numbers are increasing given the ease of capturing decent images with digital cameras. This book is the first of its kind for North America in that it includes many color photographs and treats all species of liverworts found in a multistate region. I do not know Mary S. G. Lincoln, but I suspect that as an amateur bryologist herself, this book was born out of a labor of love. As stated in the introduction, the book is ‘‘a nontechnical introduction to the liverworts and hornworts of New England.’’ The descriptions of species ‘‘emphasize characters that can be seen with a strong hand lens or a dissecting microscope.’’ Though aimed squarely at the beginner, all species of liverworts and hornworts currently or historically known for New England (Connecticut, Maine, Massachusetts, New Hampshire, Rhode Island and Vermont) are included. I noted only the omission of Pedinophyllum interruptum known from Connecticut and Massachusetts. The hardcover is richly illustrated with attractive photographs of liverworts. While not specifically designed as a field guide, the duplication of the main ‘‘illustrated key to hornworts and liverworts’’ inside the front cover is convenient for field use. Sections preceding the main treatments provide a brief history of regional hepaticology, describe the New England landscape, give an overview of the distributions compiled and explain basic morphology and life cycle characteristics. Given the growing interest in bryophyte conservation, the paragraph on rare and endangered species of liverworts is misleading. It includes the following statement (p. 7): ‘‘Because liverworts are so infrequently studied, no one can really say that a particular species is rare or endangered, either worldwide or in a particular area.’’ The statement is later contradicted in the treatment of species where some are described as ‘‘rare.’’ There are three appendices: a traditional dichotomous key to 24 ‘‘common and easily identified’’ species; a list of ‘‘leafy liverwort genera arranged by size’’ and a list of ‘‘liverworts characteristic of specialized habitats.’’ A glossary of 90 terms, a bibliography and an index to scientific names are included. The main body of the book (pp. 12–142) consists of 15 sections (A–O), each a logical group identified by the two-paged ‘‘illustrated key.’’ For example, section A presents ‘‘complex thalloid liverworts with elevated receptacles,’’ and section I ‘‘leafy liverworts with lobed leaves and succubous insertion.’’ Each section begins with a key diagram to

Bacterial whole-cell biosensors (BWBs) have unique advantages over conventional environmental monitoring techniques on the detection of toxicity and bioavailability of contaminants in water and soils. BWBs can also be rapid, sensitive, semiquantitative, cost-effective, and easy to use. In this study, a standard method is described for the detection of contaminants and toxicity in real water and soil samples using Acinetobacter baylyi ADP1-based biosensors.

Bacterial whole-cell biosensors (BWBs) have unique advantages over conventional environmental monitoring techniques on the detection of toxicity and bioavailability of contaminants in water and soils. BWBs can also be rapid, sensitive, semiquantitative, cost-effective, and easy to use. In this study, a standard method is described for the detection of contaminants and toxicity in real water and soil samples using Acinetobacter baylyi ADP1-based biosensors.

Synthetic biology involves reprogramming and engineering of regulatory genes in innovative ways for the implementation of novel tasks. Transcriptional gene regulation systems induced by small molecules in prokaryotes provide a rich source for logic gates. Cross-regulation, whereby a promoter is activated by different molecules or different promoters are activated by one molecule, can be used to design an OR-gate and achieve cross-talk between gene networks in cells. Acinetobacter baylyi ADP1 is naturally transformable, readily editing its chromosomal DNA, which makes it a convenient chassis for synthetic biology. The catabolic genes for salicylate, benzoate, and catechol metabolism are located within a supraoperonic cluster (-sal-are-ben-cat-) in the chromosome of A. baylyi ADP1, which are separately regulated by LysR-type transcriptional regulators (LTTRs). ADP1-based biosensors were constructed in which salA, benA, and catB were fused with a reporter gene cassette luxCDABE under the separate control of SalR, BenM, and CatM regulators. Salicylate, benzoate, catechol, and associated metabolites were found to mediate cross-regulation among sal, ben, and cat operons. A new mathematical model was developed by considering regulator-inducer binding and promoter activation as two separate steps. This model fits the experimental data well and is shown to predict cross-regulation performance.

• Premise of the study: The Frullania tamarisci complex includes eight Holarctic liverwort species. One of these, F. asagrayana, is distributed broadly throughout eastern North America from Canada to the Gulf Coast. Preliminary genetic data suggested that the species includes two groups of populations. This study was designed to test whether the two groups are reproductively isolated biological species. • Methods: Eighty-eight samples from across the range of F. asagrayana, plus 73 samples from one population, were genotyped for 13 microsatellite loci. Sequences for two plastid loci and nrITS were obtained from 13 accessions. Genetic data were analyzed using coalescent models and Bayesian inference. • Key results: Frullania asagrayana is sequence-invariant at the two plastid loci and ITS2, but two clear groups were resolved by microsatellites. The two groups are largely reproductively isolated, but there is a low level of gene flow from the southern to the northern group. No gene flow was detected in the other direction. A local population was heterogeneous but displayed strong genetic structure. • Conclusions: The genetic structure of F. asagrayana in eastern North America reflects morphologically cryptic differentiation between reproductively isolated groups of populations, near-panmixis within groups, and clonal propagation at local scales. Reproductive isolation between groups that are invariant at the level of nucleotide sequences shows that caution must be exercised in making taxonomic and evolutionary inferences from reciprocal monophyly (or lack thereof) between putative species. peerReviewed

The chlorophyll-deficient gun5-1 and cch Arabidopsis mutants carry single point mutations in the CHLH subunit of the magnesium chelatase enzyme, which catalyses the first committed step of chlorophyll biosynthesis. Recombinant Synechocystis ChlH subunits carrying the gun5-1 or cch mutations are inactive in Mg-chelatase assays, despite being able to bind both substrate and product, and retaining a capacity to form a ChlH–ChlI–ChlD Mg-chelatase complex. These mutant subunits act as inhibitors of ChlH, showing that the ChlH-porphyrin complex associates reversibly with the ChlI and D subunits during the catalytic cycle. This inhibition is reversed upon addition of Gun4.

A simple method is described for the site-specific attachment of yellow fluorescent protein (YFP) to glass surfaces on length scales ranging from tens of micrometers to ca. 200 nm. 3-Mercaptopropyl(triethoxy silane) is adsorbed onto a glass substrate and subsequently derivatized using a maleimide-functionalized oligomer of ethylene glycol. The resulting protein-resistant surface is patterned by exposure to UV light, causing photochemical degradation of the oligo(ethylene glycol) units to yield aldehyde groups in exposed regions. These are covalently bound to N-(5-amino-1-carboxypentyl)iminoacetic acid, yielding a nitrilotriacetic acid (NTA)-functionalized surface, which following complexation with Ni(2+), is coupled to His-tagged YFP. Using scanning near-field photolithography, in which a UV laser coupled to a scanning near-field optical microscope is utilized as the light source for photolithography, it is possible to fabricate lines of protein smaller than 200 nm, in which the biomolecules remain strongly optically active, facilitating the acquisition of diffraction-limited fluorescence images by confocal microscopy.