Berkeley Synthetic Biology Institute

Berkeley, CA, United States

Berkeley Synthetic Biology Institute

Berkeley, CA, United States
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Sutter M.,U.S. Department of Energy | Wilson A.,CEA Saclay Nuclear Research Center | Wilson A.,French National Center for Scientific Research | Leverenz R.L.,University of California at Berkeley | And 10 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2013

Photosynthetic reaction centers are sensitive to high light conditions, which can cause damage because of the formation of reactive oxygen species. To prevent high-light induced damage, cyanobacteria have developed photoprotective mechanisms. One involves a photoactive carotenoid protein that decreases the transfer of excess energy to the reaction centers. This protein, the orange carotenoid protein (OCP), is present in most cyanobacterial strains; it is activated by high light conditions and able to dissipate excess energy at the site of the light-harvesting antennae, the phycobilisomes. Restoration of normal antenna capacity involves the fluorescence recovery protein (FRP). The FRP acts to dissociate the OCP from the phycobilisomes by accelerating the conversion of the active red OCP to the inactive orange form. We have determined the 3D crystal structure of the FRP at 2.5 Å resolution. Remarkably, the FRP is found in two very different conformational and oligomeric states in the same crystal. Based on amino acid conservation analysis, activity assays of FRP mutants, FRP:OCP docking simulations, and coimmunoprecipitation experiments, we conclude that the dimer is the active form. The second form, a tetramer, may be an inactive form of FRP. In addition, we have identified a surface patch of highly conserved residues and shown that those residues are essential to FRP activity.

Stanley D.N.,D1epartment of Energy Joint Genome Institute | Stanley D.N.,University of California at Berkeley | Raines C.A.,University of Essex | Kerfeld C.A.,D1epartment of Energy Joint Genome Institute | And 2 more authors.
Plant Physiology | Year: 2013

CP12 is found almost universally among photosynthetic organisms, where it plays a key role in regulation of the Calvin cycle by forming a ternary complex with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase. Newly available genomic sequence data for the phylum Cyanobacteria reveals a heretofore unobserved diversity in cyanobacterial CP12 proteins. Cyanobacterial CP12 proteins can be classified into eight different types based on primary structure features. Among these are CP12-CBS (for cystathionine-β-synthase) domain fusions. CBS domains are regulatory modules for a wide range of cellular activities; many of these bind adenine nucleotides through a conserved motif that is also present in the CBS domains fused to CP12. In addition, a survey of expression data sets shows that the CP12 paralogs are differentially regulated. Furthermore, modeling of the cyanobacterial CP12 protein variants based on the recently available three-dimensional structure of the canonical cyanobacterial CP12 in complex with GAPDH suggests that some of the newly identified cyanobacterial CP12 types are unlikely to bind to GAPDH. Collectively these data show that, as is becoming increasingly apparent for plant CP12 proteins, the role of CP12 in cyanobacteria is likely more complex than previously appreciated, possibly involving other signals in addition to light.Moreover, our findings substantiate the proposal that this small protein may have multiple roles in photosynthetic organisms.

Sutter M.,Joint Genome Institute | Wilson S.C.,University of California at Berkeley | Deutsch S.,Joint Genome Institute | Kerfeld C.A.,Joint Genome Institute | And 2 more authors.
Photosynthesis Research | Year: 2013

Cyanobacteria have evolved a unique carbon fixation organelle known as the carboxysome that compartmentalizes the enzymes RuBisCO and carbonic anhydrase. This effectively increases the local CO2 concentration at the active site of RuBisCO and decreases its relatively unproductive side reaction with oxygen. Carboxysomes consist of a protein shell composed of hexameric and pentameric proteins arranged in icosahedral symmetry. Facets composed of hexameric proteins are connected at the vertices by pentameric proteins. Structurally homologous pentamers and hexamers are also found in heterotrophic bacteria where they form architecturally related microcompartments such as the Eut and Pdu organelles for the metabolism of ethanolamine and propanediol, respectively. Here we describe two new high-resolution structures of the pentameric shell protein CcmL from the cyanobacteria Thermosynechococcus elongatus and Gloeobacter violaceus and provide detailed analysis of their characteristics and comparison with related shell proteins. © 2013 Springer Science+Business Media Dordrecht.

Kirilovsky D.,CEA Saclay Nuclear Research Center | Kirilovsky D.,French National Center for Scientific Research | Kerfeld C.A.,Joint Genome Institute | Kerfeld C.A.,University of California at Berkeley | Kerfeld C.A.,Berkeley Synthetic Biology Institute
Photochemical and Photobiological Sciences | Year: 2013

This review focuses on the Orange Carotenoid Protein (OCP) which is the first photoactive protein identified containing a carotenoid as the photoresponsive chromophore. This protein is essential for the triggering of a photoprotective mechanism in cyanobacteria which decreases the excess absorbed energy arriving at the photosynthetic reaction centers by increasing thermal dissipation at the level of the phycobilisomes, the cyanobacterial antenna. Blue-green light causes structural changes within the carotenoid and the protein, converting the orange inactive form into a red active form. The activated red form interacts with the phycobilisome and induces the decrease of phycobilisome fluorescence emission and of the energy arriving to the photosynthetic reaction centers. The OCP is the light sensor, the signal propagator and the energy quencher. A second protein, the Fluorescence Recovery Protein (FRP), is needed to detach the red OCP from the phycobilisome and its reversion to the inactive orange form. In the last decade, in vivo and in vitro mechanistic studies combined with structural and genomic data resulted in both the discovery and a detailed picture of the function of the OCP and OCP-mediated photoprotection. Recent structural and functional results are emphasized and important previous results will be reviewed. Similarities to other blue-light responsive proteins will be discussed. This journal is © The Royal Society of Chemistry and Owner Societies.

Aussignargues C.,Michigan State University | Paasch B.C.,Michigan State University | Gonzalez-Esquer R.,Michigan State University | Erbilgin O.,University of California at Berkeley | And 4 more authors.
Communicative and Integrative Biology | Year: 2015

Bacterial microcompartments (BMCs) are proteinaceous organelles used by a broad range of bacteria to segregate and optimize metabolic reactions. Their functions are diverse, and can be divided into anabolic (carboxysome) and catabolic (metabolosomes) processes, depending on their cargo enzymes. The assembly pathway for the β-carboxysome has been characterized, revealing that biogenesis proceeds from the inside out. The enzymes coalesce into a procarboxysome, followed by encapsulation in a protein shell that is recruited to the procarboxysome by a short (∼17 amino acids) extension on the C-terminus of one of the encapsulated proteins. A similar extension is also found on the N- or C-termini of a subset of metabolosome core enzymes. These encapsulation peptides (EPs) are characterized by a primary structure predicted to form an amphipathic α-helix that interacts with shell proteins. Here, we review the features, function and widespread occurrence of EPs among metabolosomes, and propose an expanded role for EPs in the assembly of diverse BMCs. © Cléement Aussignargues, Bradley C Paasch, Raul Gonzalez-Esquer, Onur Erbilgin, and Cheryl A Kerfeld.

Kerfeld C.A.,Michigan State University | Kerfeld C.A.,University of California at Berkeley | Kerfeld C.A.,Lawrence Berkeley National Laboratory | Kerfeld C.A.,Berkeley Synthetic Biology Institute | Erbilgin O.,University of California at Berkeley
Trends in Microbiology | Year: 2015

Bacterial microcompartments (BMCs) are protein-bound organelles predicted to be present across 23 bacterial phyla. BMCs facilitate carbon fixation as well as the aerobic and anaerobic catabolism of a variety of organic compounds. These functions have been linked to ecological nutrient cycling, symbiosis, pathogenesis, and cardiovascular disease. Within bacterial cells, BMCs are metabolic modules that can be further dissociated into their constituent structural and functional protein domains. Viewing BMCs as genetic, structural, functional, and evolutionary modules provides a framework for understanding both BMC-mediated metabolism and for adapting their architectures for applications in synthetic biology. © 2014 Elsevier Ltd.

Lassila J.K.,University of California at Berkeley | Lassila J.K.,Genencor | Bernstein S.L.,Lawrence Berkeley National Laboratory | Kinney J.N.,Lawrence Berkeley National Laboratory | And 5 more authors.
Journal of Molecular Biology | Year: 2014

Bacterial microcompartments (BMCs) sequester enzymes from the cytoplasmic environment by encapsulation inside a selectively permeable protein shell. Bioinformatic analyses indicate that many bacteria encode BMC clusters of unknown function and with diverse combinations of shell proteins. The genome of the halophilic myxobacterium Haliangium ochraceum encodes one of the most atypical sets of shell proteins in terms of composition and primary structure. We found that microcompartment shells could be purified in high yield when all seven H. ochraceum BMC shell genes were expressed from a synthetic operon in Escherichia coli. These shells differ substantially from previously isolated shell systems in that they are considerably smaller and more homogeneous, with measured diameters of 39 ± 2 nm. The size and nearly uniform geometry allowed the development of a structural model for the shells composed of 260 hexagonal units and 13 hexagons per icosahedral face. We found that new proteins could be recruited to the shells by fusion to a predicted targeting peptide sequence, setting the stage for the use of these remarkably homogeneous shells for applications such as three-dimensional scaffolding and the construction of synthetic BMCs. Our results demonstrate the value of selecting from the diversity of BMC shell building blocks found in genomic sequence data for the construction of novel compartments. © 2014 Elsevier Ltd.

Axen S.D.,U.S. Department of Energy | Erbilgin O.,University of California at Berkeley | Kerfeld C.A.,University of California at Berkeley | Kerfeld C.A.,Michigan State University | And 2 more authors.
PLoS Computational Biology | Year: 2014

Bacterial microcompartments (BMCs) are proteinaceous organelles involved in both autotrophic and heterotrophic metabolism. All BMCs share homologous shell proteins but differ in their complement of enzymes; these are typically encoded adjacent to shell protein genes in genetic loci, or operons. To enable the identification and prediction of functional (sub)types of BMCs, we developed LoClass, an algorithm that finds putative BMC loci and inventories, weights, and compares their constituent pfam domains to construct a locus similarity network and predict locus (sub)types. In addition to using LoClass to analyze sequences in the Non-redundant Protein Database, we compared predicted BMC loci found in seven candidate bacterial phyla (six from single-cell genomic studies) to the LoClass taxonomy. Together, these analyses resulted in the identification of 23 different types of BMCs encoded in 30 distinct locus (sub)types found in 23 bacterial phyla. These include the two carboxysome types and a divergent set of metabolosomes, BMCs that share a common catalytic core and process distinct substrates via specific signature enzymes. Furthermore, many Candidate BMCs were found that lack one or more core metabolosome components, including one that is predicted to represent an entirely new paradigm for BMC-associated metabolism, joining the carboxysome and metabolosome. By placing these results in a phylogenetic context, we provide a framework for understanding the horizontal transfer of these loci, a starting point for studies aimed at understanding the evolution of BMCs. This comprehensive taxonomy of BMC loci, based on their constituent protein domains, foregrounds the functional diversity of BMCs and provides a reference for interpreting the role of BMC gene clusters encoded in isolate, single cell, and metagenomic data. Many loci encode ancillary functions such as transporters or genes for cofactor assembly; this expanded vocabulary of BMC-related functions should be useful for design of genetic modules for introducing BMCs in bioengineering applications. © 2014 Axen et al.

Cai F.,U.S. Department of Energy | Cai F.,University of California at Berkeley | Axen S.D.,U.S. Department of Energy | Kerfeld C.A.,U.S. Department of Energy | And 2 more authors.
RNA Biology | Year: 2013

Members of the phylum Cyanobacteria inhabit ecologically diverse environments. However, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR associated genes), an extremely adaptable defense system, has not been surveyed in this phylum. We analyzed 126 cyanobacterial genomes and, surprisingly, found CRISPR-Cas in the majority except the marine subclade (Synechococcus and Prochlorococcus), in which cyanophages are a known force shaping their evolution. Multiple observations of CRISPR loci in the absence of cas1/cas2 genes may represent an early stage of losing a CRISPR-Cas locus. Our findings reveal the widespread distribution of CRISPR-Cas systems in the phylum Cyanobacteria and provide a first step to systematically understanding their role in cyanobacteria. © 2013 Landes Bioscience.

Sutter M.,Michigan State University | Sutter M.,Lawrence Berkeley National Laboratory | Faulkner M.,University of Liverpool | Aussignargues C.,Michigan State University | And 7 more authors.
Nano Letters | Year: 2016

Bacterial microcompartments (BMCs) are proteinaceous organelles widespread among bacterial phyla. They compartmentalize enzymes within a selectively permeable shell and play important roles in CO2 fixation, pathogenesis, and microbial ecology. Here, we combine X-ray crystallography and high-speed atomic force microscopy to characterize, at molecular resolution, the structure and dynamics of BMC shell facet assembly. Our results show that preformed hexamers assemble into uniformly oriented shell layers, a single hexamer thick. We also observe the dynamic process of shell facet assembly. Shell hexamers can dissociate from and incorporate into assembled sheets, indicating a flexible intermolecular interaction. Furthermore, we demonstrate that the self-assembly and dynamics of shell proteins are governed by specific contacts at the interfaces of shell proteins. Our study provides novel insights into the formation, interactions, and dynamics of BMC shell facets, which are essential for the design and engineering of self-assembled biological nanoreactors and scaffolds based on BMC architectures. © 2015 American Chemical Society.

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