Niels-Ulrik Frigaard

Proteorhodopsin lateral gene transfer between marine planktonic Bacteria and Archaea

Planktonic Bacteria, Archaea and Eukarya reside and compete in the oceans photic zone under the pervasive influence of light. Bacteria in this environment were recently shown to contain photoproteins called proteorhodopsins, thought to contribute to cellular energy metabolism by catalysing light-driven proton translocation across the cell membrane. So far, proteorhodopsin genes have been well documented only in proteobacteria and a few other bacterial groups. Here we report the presence and distribution of proteorhodopsin genes in Archaea affiliated with the order Thermoplasmatales, in the oceans upper water column. The genomic context and phylogenetic relationships of the archaeal and proteobacterial proteorhodopsins indicate its probable lateral transfer between planktonic Bacteria and Archaea. About 10% of the euryarchaeotes in the photic zone contained the proteorhodopsin gene adjacent to their small-subunit ribosomal RNA. The archaeal proteorhodopsins were also found in other genomic regions, in the same or in different microbial lineages. Although euryarchaeotes were distributed throughout the water column, their proteorhodopsins were found only in the photic zone. The cosmopolitan phylogenetic distribution of proteorhodopsins reflects their significant light-dependent fitness contributions, which drive the photoproteins lateral acquisition and retention, but constrain its dispersal to the photic zone.

Sulfur Metabolism in Phototrophic Sulfur Bacteria

Phototrophic sulfur bacteria are characterized by oxidizing various inorganic sulfur compounds for use as electron donors in carbon dioxide fixation during anoxygenic photosynthetic growth. These bacteria are divided into the purple sulfur bacteria (PSB) and the green sulfur bacteria (GSB). They utilize various combinations of sulfide, elemental sulfur, and thiosulfate and sometimes also ferrous iron and hydrogen as electron donors. This review focuses on the dissimilatory and assimilatory metabolism of inorganic sulfur compounds in these bacteria and also briefly discusses these metabolisms in other types of anoxygenic phototrophic bacteria. The biochemistry and genetics of sulfur compound oxidation in PSB and GSB are described in detail. A variety of enzymes catalyzing sulfur oxidation reactions have been isolated from GSB and PSB (especially Allochromatium vinosum, a representative of the Chromatiaceae), and many are well characterized also on a molecular genetic level. Complete genome sequence data are currently available for 10 strains of GSB and for one strain of PSB. We present here a genome-based survey of the distribution and phylogenies of genes involved in oxidation of sulfur compounds in these strains. It is evident from biochemical and genetic analyses that the dissimilatory sulfur metabolism of these organisms is very complex and incompletely understood. This metabolism is modular in the sense that individual steps in the metabolism may be performed by different enzymes in different organisms. Despite the distant evolutionary relationship between GSB and PSB, their photosynthetic nature and their dependency on oxidation of sulfur compounds resulted in similar ecological roles in the sulfur cycle as important anaerobic oxidizers of sulfur compounds.

Chlorobium tepidum: insights into the structure, physiology, and metabolism of a green sulfur bacterium derived from the complete genome sequence

Green sulfur bacteria are obligate, anaerobic photolithoautotrophs that synthesize unique bacteriochlorophylls (BChls) and a unique light-harvesting antenna structure, the chlorosome. One organism, Chlorobium tepidum, has emerged as a model for this group of bacteria primarily due to its relative ease of cultivation and natural transformability. This review focuses on insights into the physiology and biochemistry of the green sulfur bacteria that have been derived from the recently completed analysis of the 2.15-Mb genome of Chl. tepidum. About 40 mutants of Chl. tepidum have been generated within the last 3 years, most of which have been made based on analyses of the genome. This has allowed a nearly complete elucidation of the biosynthetic pathways for the carotenoids and BChls in Chl. tepidum, which include several novel enzymes specific for BChl c biosynthesis. Facilitating these analyses, both BChl c and carotenoid biosynthesis can be completely eliminated in Chl. tepidum. Based particularly on analyses of mutants lacking chlorosome proteins and BChl c, progress has also been made in understanding the structure and biogenesis of chlorosomes. In silico analyses of the presence and absence of genes encoding components involved in electron transfer reactions and carbon assimilation have additionally revealed some of the potential physiological capabilities, limitations, and peculiarities of Chl. tepidum. Surprisingly, some structural components and biosynthetic pathways associated with photosynthesis and energy metabolism in Chl. tepidum are more similar to those in cyanobacteria and plants than to those in other groups of photosynthetic bacteria.

Mechanisms and evolution of oxidative sulfur metabolism in green sulfur bacteria

Green sulfur bacteria (GSB) constitute a closely related group of photoautotrophic and thiotrophic bacteria with limited phenotypic variation. They typically oxidize sulfide and thiosulfate to sulfate with sulfur globules as an intermediate. Based on genome sequence information from 15 strains, the distribution and phylogeny of enzymes involved in their oxidative sulfur metabolism was investigated. At least one homolog of sulfide:quinone oxidoreductase (SQR) is present in all strains. In all sulfur-oxidizing GSB strains except the earliest diverging Chloroherpeton thalassium, the sulfide oxidation product is further oxidized to sulfite by the dissimilatory sulfite reductase (DSR) system. This system consists of components horizontally acquired partly from sulfide-oxidizing and partly from sulfate-reducing bacteria. Depending on the strain, the sulfite is probably oxidized to sulfate by one of two different mechanisms that have different evolutionary origins: adenosine-5′-phosphosulfate reductase or polysulfide reductase-like complex 3. Thiosulfate utilization by the SOX system in GSB has apparently been acquired horizontally from Proteobacteria. SoxCD does not occur in GSB, and its function in sulfate formation in other bacteria has been replaced by the DSR system in GSB. Sequence analyses suggested that the conserved soxJXYZAKBW gene cluster was horizontally acquired by Chlorobium phaeovibrioides DSM 265 from the Chlorobaculum lineage and that this acquisition was mediated by a mobile genetic element. Thus, the last common ancestor of currently known GSB was probably photoautotrophic, hydrogenotrophic, and contained SQR but not DSR or SOX. In addition, the predominance of the Chlorobium–Chlorobaculum–Prosthecochloris lineage among cultured GSB could be due to the horizontally acquired DSR and SOX systems. Finally, based upon structural, biochemical, and phylogenetic analyses, a uniform nomenclature is suggested for sqr genes in prokaryotes.

Quinones in chlorosomes of green sulfur bacteria and their role in the redox-dependent fluorescence studied in chlorosome-like bacteriochlorophyll c aggregates

Abstract The light-harvesting chlorosome antennae of anaerobic, photosynthetic green sulfur bacteria exhibit a highly redox-dependent fluorescence such that the fluorescence intensity decreases under oxidizing conditions. We found that chlorosomes from Chlorobium tepidum contain three isoprenoid quinone species (chlorobiumquinone, menaquinone-7, and an unidentified quinone that probably is a chlorobiumquinone derivative) at a total concentration of approximately 0.1 mol per mol bacteriochlorophyll c. Most of the cellular chlorobiumquinone was found in the chlorosomes and constituted about 70% of the total chlorosome quinone pool. When the quinones were added to artificial, chlorosome-like bacteriochlorophyll c aggregates in an aqueous solution, a high redox dependency of the fluorescence was observed. Chlorobiumquinones were most effective in this respect. A lesser redox dependency of the fluorescence was still observed in the absence of quinones, probably due to another unidentified redox-active component. These results suggest that quinones play a significant, but not exclusive role in controlling the fluorescence and in inhibiting energy transfer in chlorosomes under oxic conditions. Chlorosomes from Chloroflexus aurantiacus contained menaquinone in an amount similar to that of total quinone in Chlorobium tepdium chlorosomes, but did not contain chlorobiumquinones. This may explain the much lower redox-dependent fluorescence observed in Chloroflexus chlorosomes.

Bioengineering of carbon fixation, biofuels, and biochemicals in cyanobacteria and plants

Development of sustainable energy is a pivotal step towards solutions for todays global challenges, including mitigating the progression of climate change and reducing dependence on fossil fuels. Biofuels derived from agricultural crops have already been commercialized. However the impacts on environmental sustainability and food supply have raised ethical questions about the current practices. Cyanobacteria have attracted interest as an alternative means for sustainable energy productions. Being aquatic photoautotrophs they can be cultivated in non-arable lands and do not compete for land for food production. Their rich genetic resources offer means to engineer metabolic pathways for synthesis of valuable bio-based products. Currently the major obstacle in industrial-scale exploitation of cyanobacteria as the economically sustainable production hosts is low yields. Much effort has been made to improve the carbon fixation and manipulating the carbon allocation in cyanobacteria and their evolutionary photosynthetic relatives, algae and plants. This review aims at providing an overview of the recent progress in the bioengineering of carbon fixation and allocation in cyanobacteria; wherever relevant, the progress made in plants and algae is also discussed as an inspiration for future application in cyanobacteria.

Seeing green bacteria in a new light: genomics-enabled studies of the photosynthetic apparatus in green sulfur bacteria and filamentous anoxygenic phototrophic bacteria

Based upon their photosynthetic nature and the presence of a unique light-harvesting antenna structure, the chlorosome, the photosynthetic green bacteria are defined as a distinctive group in the Bacteria. However, members of the two taxa that comprise this group, the green sulfur bacteria (Chlorobi) and the filamentous anoxygenic phototrophic bacteria (“Chloroflexales”), are otherwise quite different, both physiologically and phylogenetically. This review summarizes how genome sequence information facilitated studies of the biosynthesis and function of the photosynthetic apparatus and the oxidation of inorganic sulfur compounds in two model organisms that represent these taxa, Chlorobium tepidum and Chloroflexus aurantiacus. The genes involved in bacteriochlorophyll (BChl) c and carotenoid biosynthesis in these two organisms were identified by sequence homology with known BChl a and carotenoid biosynthesis enzymes, gene cluster analysis in Cfx. aurantiacus, and gene inactivation studies in Chl. tepidum. Based on these results, BChl a and BChl c biosynthesis is similar in the two organisms, whereas carotenoid biosynthesis differs significantly. In agreement with its facultative anaerobic nature, Cfx. aurantiacus in some cases apparently produces structurally different enzymes for heme and BChl biosynthesis, in which one enzyme functions under anoxic conditions and the other performs the same reaction under oxic conditions. The Chl. tepidum mutants produced with modified BChl c and carotenoid species also allow the functions of these pigments to be studied in vivo.

Gene Inactivation in the Cyanobacterium Synechococcus sp. PCC 7002 and the Green Sulfur Bacterium Chlorobium tepidum Using In Vitro-Made DNA Constructs and Natural Transformation

Inactivation of a chromosomal gene is a useful approach to study the function of the gene in question and can be used to produce a desired phenotype in the organism. This chapter describes how to generate such mutants of the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum by natural transformation with synthetic DNA constructs. Two alternative methods to generate the DNA constructs, both performed entirely in vitro and based on the polymerase chain reaction (PCR), are also presented. These methods are ligation of DNA fragments with T4 DNA ligase, and megaprimer PCR.

Comparative and Functional Genomics of Anoxygenic Green Bacteria from the Taxa Chlorobi, Chloroflexi, and Acidobacteria

Green bacteria are a diverse group of chlorophototrophic organisms belonging to three major taxa within the domain Bacteria: Chlorobi, Chloroflexi, and Acidobacteria. Most, although not all, of these organisms synthesize bacteriochlorophylls c, d or e and utilize chlorosomes for light harvesting. The pace of discoveries concerning the metabolism and physiology of these bacteria has accelerated rapidly since completion of the sequencing of the genomes of the green sulfur bacterium Chlorobaculum tepidum and the filamentous anoxygenic phototroph, Chloroflexus aurantiacus. This chapter summarizes insights gained from the extensive genome sequence data for members of these three taxa. The discovery of the first chlorophototrophic member of the phylum Acidobacteria, Candidatus Chloracidobacterium thermophilum, is also described, and recent insights into the physiology and metabolism of this unique, aerobic photoheterotroph are presented. Based upon phylogenetic inferences derived from analyses of sequences for reaction centers and enzymes of (bacterio)chlorophyll biosynthesis, some implications concerning the evolutionary origins of photosynthesis are discussed.

Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum by Natural Transformation

ABSTRACT Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40°C on agar plates could be completely inhibited by 100 μg of gentamicin ml−1, 2 μg of erythromycin ml−1, 30 μg of chloramphenicol ml−1, or 1 μg of tetracycline ml−1 or a combination of 300 μg of streptomycin ml−1 and 150 μg of spectinomycin ml−1. Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10−7 were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10−3 were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.