Synechococcus sp. PCC 7002 is an ideal model cyanobacterium for functional genomics and biotechnological applications through metabolic engineering. A gene expression system that takes advantage of its multiple, endogenous plasmids has been constructed in this cyanobacterium. The method involves the integration of foreign DNA cassettes with selectable markers into neutral sites that can be located on any of the several endogenous plasmids of this organism. We have exploited the natural transformability and powerful homologous recombination capacity of this organism by using linear DNA fragments for transformation. This approach overcomes barriers that have made the introduction and expression of foreign genes problematic in the past. Foremost among these is the natural restriction endonuclease barrier that can cleave transforming circular plasmid DNAs before they can be replicated in the cell. We describe herein the general methodology for expressing foreign and homologous genes in Synechococcus sp. PCC 7002, a comparison of several commonly used promoters, and provide examples of how this approach has successfully been used in complementation analyses and overproduction of proteins with affinity tags.

A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL-or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA-and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL-and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria.carotenogenesis ͉ carotenoids ͉ cyanobacteria ͉ green sulfur bacteria ͉ photosynthesis C olored carotenoids are found in nearly all photosynthetic species and in a variety of nonphotosynthetic organisms and play roles in light-harvesting, photoprotection, structural maintenance of pigment-protein complexes (1), and membrane structure and fluidity (2). The cyclization of the linear compound lycopene to produce ␣-, ␤-, ␥-, or -carotene is a branch point in carotenoid biosynthetic pathways in many species of bacteria, plants, and fungi (3, 4). The monocyclic ␥-carotene is a precursor to myxoxanthophylls in cyanobacteria (5, 6), is both an intermediate and an end product of carotenogenesis in orange and yellow flowers and fruits (7-10), and is an intermediate in chlorobactene biosynthesis in green sulfur bacteria (GSB) (11-13). Dicyclic ␤-carotene is a major component of photosystems (PS) I and II in cyanobacteria and plants (14,15) and is modified to isorenieratene in brown-colored GSB and some actinomycetes (16,17).Three classes of lycopene cyclases have previously been identified in bacteria: the CrtY-type ␤-cyclases that are found in many carotenogenic proteobacteria (e.g., refs. 18 and 19), Streptomyces spp. (17), and the Chloroflexi; the CrtL family, which includes the ␤-and -cyclases in some cyanobacteria and plants (20,21); and the heterodimeric cyclases of some Gram-positive bacteria (22, 23), which are related to the lycopene cyclases of archaea (24, 25) and halophilic bacteria (26). These classes are distantly related to each other and share only a few conserved motifs, including an N-terminal flavin-binding domain that is found in the first two groups but appears to be missing in the third (27). Carotenoid monocyclases are found in both the CrtY and CrtL families (28-30), and there are no obvious sequence differences between mono-and dicyclases (28).The cyclization of lycopene to ␥-or ␤-carotene is an isomerization reaction, which produces no net change in mass or redox state o...

In order to enrich the phylogenetic diversity represented in the available sequenced bacterial genomes and as part of an “Assembling the Tree of Life” project, we determined the genome sequence of Thermomicrobium roseum DSM 5159. T. roseum DSM 5159 is a red-pigmented, rod-shaped, Gram-negative extreme thermophile isolated from a hot spring that possesses both an atypical cell wall composition and an unusual cell membrane that is composed entirely of long-chain 1,2-diols. Its genome is composed of two circular DNA elements, one of 2,006,217 bp (referred to as the chromosome) and one of 919,596 bp (referred to as the megaplasmid). Strikingly, though few standard housekeeping genes are found on the megaplasmid, it does encode a complete system for chemotaxis including both chemosensory components and an entire flagellar apparatus. This is the first known example of a complete flagellar system being encoded on a plasmid and suggests a straightforward means for lateral transfer of flagellum-based motility. Phylogenomic analyses support the recent rRNA-based analyses that led to T. roseum being removed from the phylum Thermomicrobia and assigned to the phylum Chloroflexi. Because T. roseum is a deep-branching member of this phylum, analysis of its genome provides insights into the evolution of the Chloroflexi. In addition, even though this species is not photosynthetic, analysis of the genome provides some insight into the origins of photosynthesis in the Chloroflexi. Metabolic pathway reconstructions and experimental studies revealed new aspects of the biology of this species. For example, we present evidence that T. roseum oxidizes CO aerobically, making it the first thermophile known to do so. In addition, we propose that glycosylation of its carotenoids plays a crucial role in the adaptation of the cell membrane to this bacterium's thermophilic lifestyle. Analyses of published metagenomic sequences from two hot springs similar to the one from which this strain was isolated, show that close relatives of T. roseum DSM 5159 are present but have some key differences from the strain sequenced.

Ongoing work has led to the identification of most of the biochemical steps in carotenoid biosynthesis in chlorophototrophic bacteria. In carotenogenesis, a relatively small number of modifications leads to a great diversity of carotenoid structures. This review examines the individual steps in the pathway, discusses how each contributes to structural diversity among carotenoids, and summarizes recent progress in elucidating the biosynthetic pathways for carotenoids in chlorophototrophs.

Despite extensive studies on microbial and enzymatic lignocellulose degradation, relatively few Archaea are known to deconstruct crystalline cellulose. Here we describe a consortium of three hyperthermophilic archaea enriched from a continental geothermal source by growth at 90 °C on crystalline cellulose, representing the first instance of Archaea able to deconstruct lignocellulose optimally above 90 °C. Following metagenomic studies on the consortium, a 90 kDa, multidomain cellulase, annotated as a member of the TIm barrel glycosyl hydrolase superfamily, was characterized. The multidomain architecture of this protein is uncommon for hyperthermophilic endoglucanases, and two of the four domains of the enzyme have no characterized homologues. The recombinant enzyme has optimal activity at 109 °C, a halflife of 5 h at 100 °C, and resists denaturation in strong detergents, high-salt concentrations, and ionic liquids. Cellulases active above 100 °C may assist in biofuel production from lignocellulosic feedstocks by hydrolysing cellulose under conditions typically employed in biomass pretreatment.