Identification and characterization of a multidomain hyperthermophilic cellulase from an archaeal enrichment.

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 half-life 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.

Expression of genes in cyanobacteria: adaptation of endogenous plasmids as platforms for high-level gene expression in Synechococcus sp. PCC 7002.

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.

Polyphasic characterization of a thermotolerant siderophilic filamentous cyanobacterium that produces intracellular iron deposits.

Despite the high potential for oxidative stress stimulated by reduced iron, contemporary iron-depositing hot springs with circum-neutral pH are intensively populated with cyanobacteria. Therefore, studies of the physiology, diversity, and phylogeny of cyanobacteria inhabiting iron-depositing hot springs may provide insights into the contribution of cyanobacteria to iron redox cycling in these environments and new mechanisms of oxidative stress mitigation. In this study the morphology, ultrastructure, physiology, and phylogeny of a novel cyanobacterial taxon, JSC-1, isolated from an iron-depositing hot spring, were determined. The JSC-1 strain has been deposited in ATCC under the name Marsacia ferruginose, accession number BAA-2121. Strain JSC-1 represents a new operational taxonomical unit (OTU) within Leptolyngbya sensu lato. Strain JSC-1 exhibited an unusually high ratio between photosystem (PS) I and PS II, was capable of complementary chromatic adaptation, and is apparently capable of nitrogen fixation. Furthermore, it synthesized a unique set of carotenoids, but only chlorophyll a. Strain JSC-1 not only required high levels of Fe for growth (≥40 µM), but it also accumulated large amounts of extracellular iron in the form of ferrihydrite and intracellular iron in the form of ferric phosphates. Collectively, these observations provide insights into the physiological strategies that might have allowed cyanobacteria to develop and proliferate in Fe-rich, circum-neutral environments.

Roles of xanthophyll carotenoids in protection against photoinhibition and oxidative stress in the cyanobacterium Synechococcus sp. strain PCC 7002.

Synechococcus sp. strain PCC 7002 is a robust, genetically tractable cyanobacterium that produces six different xanthophyll carotenoids (zeaxanthin, cryptoxanthin, myxoxanthophyll (myxol-2'-fucoside), echinenone, 3'-hydroxyechinenone, and synechoxanthin) and tolerates many environmental stresses, including high light intensities. Targeted mutations were introduced to block the branches of the carotenoid biosynthetic pathway leading to specific xanthophylls, and a mutant lacking all xanthophylls was constructed. Some of the mutants showed severe growth defects at high light intensities, and multi-locus mutants had somewhat lower chlorophyll contents and lower photosystem I levels. The results suggested that xanthophylls, particularly zeaxanthin and echinenone, might play regulatory roles in thylakoid biogenesis. Measurements of reactive oxygen (ROS) and nitrogen (RNS) species in the mutants showed that all xanthophylls participate in preventing ROS/RNS accumulation and that a mutant lacking all xanthophylls accumulated very high levels of ROS/RNS. Results from transcription profiling showed that mRNA levels for most genes encoding the enzymes of carotenogenesis are significantly more abundant after exposure to high light. These studies indicated that all xanthophylls contribute to protection against photo-oxidative stress.

The biosynthetic pathway for myxol-2' fucoside (myxoxanthophyll) in the cyanobacterium Synechococcus sp. strain PCC 7002.

Synechococcus sp. strain PCC 7002 produces a variety of carotenoids, which comprise predominantly dicylic beta-carotene and two dicyclic xanthophylls, zeaxanthin and synechoxanthin. However, this cyanobacterium also produces a monocyclic myxoxanthophyll, which was identified as myxol-2' fucoside. Compared to the carotenoid glycosides produced by diverse microorganisms, cyanobacterial myxoxanthophyll and closely related compounds are unusual because they are glycosylated on the 2'-OH rather than on the 1'-OH position of the psi end of the molecule. In this study, the genes encoding two enzymes that modify the psi end of myxoxanthophyll in Synechococcus sp. strain PCC 7002 were identified. Mutational and biochemical studies showed that open reading frame SynPCC7002_A2032, renamed cruF, encodes a 1',2'-hydroxylase [corrected] and that open reading frame SynPCC7002_A2031, renamed cruG, encodes a 2'-O-glycosyltransferase. The enzymatic activity of CruF was verified by chemical characterization of the carotenoid products synthesized when cruF was expressed in a lycopene-producing strain of Escherichia coli. Database searches showed that homologs of cruF and cruG occur in the genomes of all sequenced cyanobacterial strains that are known to produce myxol or the acylic xanthophyll oscillaxanthin. The genomes of many other bacteria that produce hydroxylated carotenoids but do not contain crtC homologs also contain cruF orthologs. Based upon observable intermediates, a complete biosynthetic pathway for myxoxanthophyll is proposed. This study expands the suite of enzymes available for metabolic engineering of carotenoid biosynthetic pathways for biotechnological applications.

Complete genome sequence of the aerobic CO-oxidizing thermophile Thermomicrobium roseum.

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.

The Biosynthetic pathway for synechoxanthin, an aromatic carotenoid synthesized by the euryhaline, unicellular cyanobacterium Synechococcus sp. strain PCC 7002.

The euryhaline, unicellular cyanobacterium Synechococcus sp. strain PCC 7002 produces the dicyclic aromatic carotenoid synechoxanthin (chi,chi-caroten-18,18'-dioic acid) as a major pigment (>15% of total carotenoid) and when grown to stationary phase also accumulates small amounts of renierapurpurin (chi,chi-carotene) (J. E. Graham, J. T. J. Lecomte, and D. A. Bryant, J. Nat. Prod. 71:1647-1650, 2008). Two genes that were predicted to encode enzymes involved in the biosynthesis of synechoxanthin were identified by comparative genomics, and these genes were insertionally inactivated in Synechococcus sp. strain PCC 7002 to verify their function. The cruE gene (SYNPCC7002_A1248) encodes beta-carotene desaturase/methyltransferase, which converts beta-carotene to renierapurpurin. The cruH gene (SYNPCC7002_A2246) encodes an enzyme that is minimally responsible for the hydroxylation/oxidation of the C-18 and C-18' methyl groups of renierapurpurin. Based on observed and biochemically characterized intermediates, a complete pathway for synechoxanthin biosynthesis is proposed.

Synechoxanthin, an aromatic C40 xanthophyll that is a major carotenoid in the cyanobacterium Synechococcus sp. PCC 7002.

A major aromatic, dicarboxylate carotenoid (>15% of total) was isolated from the euryhaline cyanobacterium Synechococcus sp. PCC 7002. This compound, which was given the common name synechoxanthin (1), has been assigned the structure (all-E) chi,chi-caroten-18,18'-dioic acid by a combination of spectroscopic (UV-vis, FT-IR, (1)H and (13)C NMR, LC-MS) and chemical methods. This discovery conclusively establishes that some cyanobacteria are capable of synthesizing aromatic carotenoids.

The biochemical basis for structural diversity in the carotenoids of chlorophototrophic bacteria.

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.

Identification of a fourth family of lycopene cyclases in photosynthetic bacteria.

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.