Revealing CO-Fixing SAR11 Bacteria in the Ocean by Raman-Based Single-Cell Metabolic Profiling and Genomics.

The majority of marine microbes remain uncultured, which hinders the identification and mining of CO-fixing genes, pathways, and chassis from the oceans. Here, we investigated CO-fixing microbes in seawater from the euphotic zone of the Yellow Sea of China by detecting and tracking their C-bicarbonate (C-HCO) intake via single-cell Raman spectra (SCRS) analysis. The target cells were then isolated by Raman-activated Gravity-driven Encapsulation (RAGE), and their genomes were amplified and sequenced at one-cell resolution.

Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO Fixation.

A key goal of synthetic biology is to engineer organisms that can use solar energy to convert CO to biomass, chemicals, and fuels. We engineered a light-dependent electron transfer chain by integrating rhodopsin and an electron donor to form a closed redox loop, which drives rhodopsin-dependent CO fixation. A light-driven proton pump comprising rhodopsin (GR) and its cofactor retinal have been assembled in () H16.

The active site of magnesium chelatase.

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.

Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology.

A type of chromosome-free cell called SimCells (simple cells) has been generated from , , and 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.

Proteorhodopsin Overproduction Enhances the Long-Term Viability of Escherichia coli.

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 Quantitative mass spectrometry shows that , 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.

Application of a bacterial whole cell biosensor for the rapid detection of cytotoxicity in heavy metal contaminated seawater.

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 P on pWH1274_lux and the bioluminescence intensity of the biosensor reduces in proportional to the concentrations of toxic compounds.

Development of SimCells as a novel chassis for functional biosensors.

This work serves as a proof-of-concept for bacterially derived SimCells (Simple Cells), which contain the cell machinery from bacteria and designed DNA (or potentially a simplified genome) to instruct the cell to carry out novel, specific tasks. SimCells represent a reprogrammable chassis without a native chromosome, which can host designed DNA to perform defined functions. In this paper, the use of Escherichia coli MC1000 ∆minD minicells as a non-reproducing chassis for SimCells was explored, as demonstrated by their ability to act as sensitive biosensors for small molecules.

Single-cell genomics based on Raman sorting reveals novel carotenoid-containing bacteria in the Red Sea.

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.

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway.

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 magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown.

Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus.

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.

Magnetic nanoparticle-mediated isolation of functional bacteria in a complex microbial community.

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.

Bacterial whole-cell biosensors for the detection of contaminants in water and soils.

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.

Characterization and modeling of transcriptional cross-regulation in Acinetobacter baylyi ADP1.

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.

Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803.

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.

Structure of the cyanobacterial Magnesium Chelatase H subunit determined by single particle reconstruction and small-angle X-ray scattering.

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.

Rapid resonance Raman microspectroscopy to probe carbon dioxide fixation by single cells in microbial communities.

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.

Abolition of magnesium chelatase activity by the gun5 mutation and reversal by Gun4.

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.

Mutagenesis alters the catalytic mechanism of the light-driven enzyme protochlorophyllide oxidoreductase.

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.

Site-specific immobilization and micrometer and nanometer scale photopatterning of yellow fluorescent protein on glass surfaces.

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 xanthophyll cycle pool size controls the kinetics of non-photochemical quenching in Arabidopsis thaliana.

Arabidopsis plants overexpressing beta-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.

Direct measurement of metal-ion chelation in the active site of the AAA+ ATPase magnesium chelatase.

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.

Elevated zeaxanthin bound to oligomeric LHCII enhances the resistance of Arabidopsis to photooxidative stress by a lipid-protective, antioxidant mechanism.

The xanthophyll cycle has a major role in protecting plants from photooxidative stress, although the mechanism of its action is unclear. Here, we have investigated Arabidopsis plants overexpressing a gene encoding beta-carotene hydroxylase, containing nearly three times the amount of xanthophyll cycle carotenoids present in the wild-type. In high light at low temperature wild-type plants exhibited symptoms of severe oxidative stress: lipid peroxidation, chlorophyll bleaching, and photoinhibition.

Structural and biochemical characterization of Gun4 suggests a mechanism for its role in chlorophyll biosynthesis.

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.