Computer‐aided biochemical programming of synthetic microreactors as diagnostic devices

Biological systems have evolved efficient sensing and decision‐making mechanisms to maximize fitness in changing molecular environments. Synthetic biologists have exploited these capabilities to engineer control on information and energy processing in living cells. While engineered organisms pose important technological and ethical challenges, de novo assembly of non‐living biomolecular devices could offer promising avenues toward various real‐world applications. However, assembling biochemical parts into functional information processing systems has remained challenging due to extensive multidimensional parameter spaces that must be sampled comprehensively in order to identify robust, specification compliant molecular implementations. We introduce a systematic methodology based on automated computational design and microfluidics enabling the programming of synthetic cell‐like microreactors embedding biochemical logic circuits, or protosensors, to perform accurate biosensing and biocomputing operations in vitro according to temporal logic specifications. We show that proof‐of‐concept protosensors integrating diagnostic algorithms detect specific patterns of biomarkers in human clinical samples. Protosensors may enable novel approaches to medicine and represent a step toward autonomous micromachines capable of precise interfacing of human physiology or other complex biological environments, ecosystems, or industrial bioprocesses.     

Characterization of Plant Carotenoid Cyclases as Members of the Flavoprotein Family Functioning with No Net Redox Change

The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene β-cyclase (LCY-B), which converts lycopene into β-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of κ-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to β-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of β-carotene and κ-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of β-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.Later steps of carotenoid biosynthesis involve the formation of diverse cyclic carotenoids. For example, β-carotene, the vitamin A precursor, is synthesized de novo by photosynthetic organisms, limited nonphototrophic bacteria and fungi, and also by aphids (Moran and Jarvik, 2010) according to a multistep pathway that ends with the cyclization of lycopene by lycopene β-cyclase (LCY-B). Similarly, in pepper (Capsicum annuum) chromoplasts, antheraxanthin and violaxanthin are converted into the κ-cyclic carotenoids capsanthin and capsorubin, respectively, by capsanthin-capsorubin synthase (CCS). In both cases, the proposed mechanism involves a concerted protic attack and stabilization of a transient carbocation without any net redox change (Camara, 1980; Bouvier et al., 1994; Britton, 1998). Several cDNAs for LCY-B have been cloned from bacteria (Misawa et al., 1990; Cunningham et al., 1994; Armstrong, 1997; Cunningham and Gantt, 2001), fungi (Verdoes et al., 1999; Velayos et al., 2000; Arrach et al., 2001), and plants (Hugueney et al., 1995; Ronen et al., 2000) using functional complementation. Information available from primary structures suggest that the cyclization of lycopene is catalyzed by holomeric proteins in photosynthetic organisms (Cunningham et al., 1994; Maresca et al., 2007), by holomeric (Misawa et al., 1990) or heteromeric (Krubasik and Sandmann, 2000; Viveiros et al., 2000) proteins in nonphotosynthetic bacteria, and by holomeric, bifunctional proteins in fungi that combine the activities of phytoene synthase and lycopene cyclase (Verdoes et al., 1999; Velayos et al., 2000; Arrach et al., 2001). This structural diversity of LCY-Bs coupled to a lack of significant amino acid sequence identity between the lycopene cyclases from bacteria, fungi, and plants hinder our understanding of the catalytic mechanism of LCY-Bs and CCS. In addition, the N terminus of plant LCY-B and CCS contains an amino sequence motif characteristic of a polypeptide predicted to adopt a Rossmann fold (Rossmann et al., 1974) and suggests the binding of an as yet unknown dinucleotide prosthetic ligand. It has been shown using recombinant bacterial enzyme that the cyclization of lycopene into β-carotene strictly requires NADPH but proceeds without any net redox change (Schnurr et al., 1996; Hornero-Mendez and Britton, 2002). Under the same conditions, FAD alone could not sustain bacterial LCY-B activity (Schnurr et al., 1996). Much less is known about the dinucleotide requirements of plant carotenoid cyclases, which are highly conserved within plants but are extremely divergent in nonplant organisms. Previously, a crucial acidic domain for lycopene cyclase activity was identified using an affinity-labeling strategy followed by site-directed mutagenesis (Bouvier et al., 1997) in the absence of any crystal structures. This so-called 293-FLEET-297 motif of LCY-B and CCS contained two tandem Glu-295-Glu-296 residues that were essential for LCY-B- and κ-cyclase activities (Bouvier et al., 1997). However, it still remains unclear how the protic mechanism is compatible with the requirement of dinucleotide cofactors.To further explore the mechanism of plant carotenoid cyclases, we first choose pepper CCS as a prototypic enzyme because it displays a strong identity (52%) to pepper LCY-B, and we have shown previously that CCS could also catalyze the cyclization of lycopene into β-carotene (up to 25% of activity compared with LCY-B; Hugueney et al., 1995). Herein, we have shown that monomeric CCS purified to homogeneity from plant chromoplasts or recombinant CCS purified from Escherichia coli-transformed cells are typical flavoproteins containing one noncovalently bound FAD. We also observed that CCS-bound FAD is required for enzyme activity in the presence of NADPH, which functions as a reductant of FAD. During this process, no hydrogen is transferred to β-carotene or κ-cyclic carotenoids. In addition to this cofactor requirement, we also show from extensive site-directed mutagenesis using pepper CCS and LCY-B and Erwinia herbicola LCY-B (Mialoundama, 2009) that Glu-295 of pepper CCS and LCY-B plays a key role in the formation of β-carotene and κ-cyclic carotenoids, and we demonstrate that a similar role is played in structurally divergent bacterial LCY-Bs by Glu-196. These characteristics suggest that plant CCS and LCY-Bs are mechanistically similar to non-metal-assisted terpene cyclases, such as squalene:hopene cyclase and oxidosqualene cyclase, and additionally represent a new subfamily of flavoproteins like isopentenyl diphosphate isomerase type II, which catalyze carotenoid cyclization without any net redox modification of the substrate.     

Effects of the entomopathogenic fungus Neozygites tanajoae and the predatory mite Typhlodromalus aripo on cassava green mite densities: screenhouse experiments

To improve biocontrol of the cassava green mite, Mononychellus tanajoa, a series of screenhouse experiments were conducted on cassava to determine the effects of single and combined releases of the predatory mite Typhlodromalus aripo and the entomopathogenic fungus Neozygites tanajoae on the suppression of M. tanajoa. We showed that, separately or in combination, T. aripo and N. tanajoae significantly reduced M. tanajoa populations. Moreover, combined release of T. aripo and N. tanajoae on the same cassava plants improved M. tanajoa control. However, our data suggest between T. aripo and N. tanajoae an asymmetric competition that significantly affected N. tanajoae only. This interference is indirect, probably mediated by their common prey or host mite, M. tanajoa since N. tanajoae is not pathogenic to T. aripo. We conclude that adding N. tanajoae to T. aripo populations in Africa would not negatively affect T. aripo and would further promote biocontrol of M. tanajoa.     

Unraveling the genomic mosaic of a ubiquitous genus of marine cyanobacteria

Background

The picocyanobacterial genus Synechococcus occurs over wide oceanic expanses, having colonized most available niches in the photic zone. Large scale distribution patterns of the different Synechococcus clades (based on 16S rRNA gene markers) suggest the occurrence of two major lifestyles ('opportunists'/'specialists'), corresponding to two distinct broad habitats ('coastal'/'open ocean'). Yet, the genetic basis of niche partitioning is still poorly understood in this ecologically important group.

Results

Here, we compare the genomes of 11 marine Synechococcus isolates, representing 10 distinct lineages. Phylogenies inferred from the core genome allowed us to refine the taxonomic relationships between clades by revealing a clear dichotomy within the main subcluster, reminiscent of the two aforementioned lifestyles. Genome size is strongly correlated with the cumulative lengths of hypervariable regions (or 'islands'). One of these, encompassing most genes encoding the light-harvesting phycobilisome rod complexes, is involved in adaptation to changes in light quality and has clearly been transferred between members of different Synechococcus lineages. Furthermore, we observed that two strains (RS9917 and WH5701) that have similar pigmentation and physiology have an unusually high number of genes in common, given their phylogenetic distance.

Conclusion

We propose that while members of a given marine Synechococcus lineage may have the same broad geographical distribution, local niche occupancy is facilitated by lateral gene transfers, a process in which genomic islands play a key role as a repository for transferred genes. Our work also highlights the need for developing picocyanobacterial systematics based on genome-derived parameters combined with ecological and physiological data.     

Arabidopsis ERG28 Tethers the Sterol C4-Demethylation Complex to Prevent Accumulation of a Biosynthetic Intermediate That Interferes with Polar Auxin Transport

Sterols are vital for cellular functions and eukaryotic development because of their essential role as membrane constituents. Sterol biosynthetic intermediates (SBIs) represent a potential reservoir of signaling molecules in mammals and fungi, but little is known about their functions in plants. SBIs are derived from the sterol C4-demethylation enzyme complex that is tethered to the membrane by Ergosterol biosynthetic protein28 (ERG28). Here, using nonlethal loss-of-function strategies focused on Arabidopsis thaliana ERG28, we found that the previously undetected SBI 4-carboxy-4-methyl-24-methylenecycloartanol (CMMC) inhibits polar auxin transport (PAT), a key mechanism by which the phytohormone auxin regulates several aspects of plant growth, including development and responses to environmental factors. The induced accumulation of CMMC in Arabidopsis erg28 plants was associated with diagnostic hallmarks of altered PAT, including the differentiation of pin-like inflorescence, loss of apical dominance, leaf fusion, and reduced root growth. PAT inhibition by CMMC occurs in a brassinosteroid-independent manner. The data presented show that ERG28 is required for PAT in plants. Furthermore, it is accumulation of an atypical SBI that may act to negatively regulate PAT in plants. Hence, the sterol pathway offers further prospects for mining new target molecules that could regulate plant development.