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Item Growth of Chlamydomonas reinhardtii in acetate-free medium when co-cultured with alginate-encapsulated, acetate-producing strains ofSynechococcus sp. PCC 7002(2014-10) Therien, Jesse B.; Zadvornyy, Oleg A.; Posewitz, Matthew C.; Bryant, Donald A.; Peters, John W.Background: The model alga Chlamydomonas reinhardtii requires acetate as a co-substrate for optimal production of lipids, and the addition of acetate to culture media has practical and economic implications for algal biofuel production. Here we demonstrate the growth of C. reinhardtii on acetate provided by mutant strains of the cyanobacterium Synechococcus sp. PCC 7002. Results: Optimal growth conditions for co-cultivation of C. reinhardtii with wild-type and mutant strains of Synechococcus sp. 7002 were established. In co-culture, acetate produced by a glycogen synthase knockout mutant of Synechococcus sp. PCC 7002 was able to support the growth of a lipid-accumulating mutant strain of C. reinhardtii defective in starch production. Encapsulation of Synechococcus sp. PCC 7002 using an alginate matrix was successfully employed in co-cultures to limit growth and maintain the stability. Conclusions: The ability of immobilized strains of the cyanobacterium Synechococcus sp. PCC 7002 to produce acetate at a level adequate to support the growth of lipid-accumulating strains of C. reinhartdii offers a potentially practical, photosynthetic alternative to providing exogenous acetate into growth media.Item Occurrence of Far-Red Light Photoacclimation (FaRLiP) in Diverse Cyanobacteria(2014-12) Gan, Fei; Shen, Gaozhong; Bryant, Donald A.Cyanobacteria have evolved a number of acclimation strategies to sense and respond to changing nutrient and light conditions. Leptolyngbya sp. JSC-1 was recently shown to photoacclimate to far-red light by extensively remodeling its photosystem (PS) I, PS II and phycobilisome complexes, thereby gaining the ability to grow in far-red light. A 21-gene photosynthetic gene cluster (rfpA/B/C, apcA2/B2/D2/E2/D3, psbA3/D3/C2/B2/ H2/A4, psaA2/B2/L2/I2/F2/J2) that is specifically expressed in far-red light encodes the core subunits of the three major photosynthetic complexes. The growth responses to far-red light were studied here for five additional cyanobacterial strains, each of which has a gene cluster similar to that in Leptolyngbya sp. JSC-1. After acclimation all five strains could grow continuously in far-red light. Under these growth conditions each strain synthesizes chlorophylls d, f and a after photoacclimation, and each strain produces modified forms of PS I, PS II (and phycobiliproteins) that absorb light between 700 and 800 nm. We conclude that these photosynthetic gene clusters are diagnostic of the capacity to photoacclimate to and grow in far-red light. Given the diversity of terrestrial environments from which these cyanobacteria were isolated, it is likely that FaRLiP plays an important role in optimizing photosynthesis in terrestrial environments.Item Complete genome of Ignavibacterium album, a metabolically versatile, flagellated, facultative anaerobe from the phylum Chlorobi(2012-05) Liu, Zhenhua; Frigaard, N. U.; Vogl, K.; Iino, T.; Ohkuma, M.; Overmann, J.; Bryant, Donald A.Prior to the recent discovery of Ignavibacterium album (I. album), anaerobic photoautotrophic green sulfur bacteria (GSB) were the only members of the bacterial phylum Chlorobi that had been grown axenically. In contrast to GSB, sequence analysis of the 3.7-Mbp genome of I. album shows that this recently described member of the phylum Chlorobi is a chemoheterotroph with a versatile metabolism. I. album lacks genes for photosynthesis and sulfur oxidation but has a full set of genes for flagella and chemotaxis. The occurrence of genes for multiple electron transfer complexes suggests that I. album is capable of organoheterotrophy under both oxic and anoxic conditions. The occurrence of genes encoding enzymes for CO2 fixation as well as other enzymes of the reductive TCA cycle suggests that mixotrophy may be possible under certain growth conditions. However, known biosynthetic pathways for several amino acids are incomplete; this suggests that I. album is dependent upon on exogenous sources of these metabolites or employs novel biosynthetic pathways. Comparisons of I. album and other members of the phylum Chlorobi suggest that the physiology of the ancestors of this phylum might have been quite different from that of modern GSB.Item Genomic analysis reveals key aspects of prokaryotic symbiosis in the phototrophic consortium “Chlorochromatium aggregatum.”(2013-11) Liu, Zhenhua; Müller, J.; Li, T.; Alvey, R. M.; Vogl, K.; Frigaard, N. U.; Rockwell, Nathan C.; Tomsho, Lynn P.; Schuster, Stephan C.; Henke, P.; Rohde, M.; Overmann, J.; Bryant, Donald A.Background: ‘Chlorochromatium aggregatum’ is a phototrophic consortium, a symbiosis that may represent the highest degree of mutual interdependence between two unrelated bacteria not associated with a eukaryotic host. ‘Chlorochromatium aggregatum’ is a motile, barrel-shaped aggregate formed from a single cell of ‘Candidatus Symbiobacter mobilis”, a polarly flagellated, non-pigmented, heterotrophic bacterium, which is surrounded by approximately 15 epibiont cells of Chlorobium chlorochromatii, a non-motile photolithoautotrophic green sulfur bacterium. Results: We analyzed the complete genome sequences of both organisms to understand the basis for this symbiosis. Chl. chlorochromatii has acquired relatively few symbiosis-specific genes; most acquired genes are predicted to modify the cell wall or function in cell-cell adhesion. In striking contrast, ‘Ca. S. mobilis’ appears to have undergone massive gene loss, is probably no longer capable of independent growth, and thus may only reproduce when consortia divide. A detailed model for the energetic and metabolic bases of the dependency of ‘Ca. S. mobilis’ on Chl. chlorochromatii is described. Conclusions: Genomic analyses suggest that three types of interactions lead to a highly sophisticated relationship between these two organisms. Firstly, extensive metabolic exchange, involving carbon, nitrogen, and sulfur sources as well as vitamins, occurs from the epibiont to the central bacterium. Secondly, ‘Ca. S. mobilis’ can sense and move towards light and sulfide, resources that only directly benefit the epibiont. Thirdly, electron cycling mechanisms, particularly those mediated by quinones and potentially involving shared protonmotive force, could provide an important basis for energy exchange in this and other symbiotic relationships.Item Synechococcus sp. strain PCC 7002 transcriptome: acclimation to temperature, salinity, oxidative stress, and mixotrophic growth conditions(2012-10) Ludwig, M.; Bryant, Donald A.Synechococcus sp. strain PCC 7002 is a unicellular, euryhaline cyanobacterium. It is a model organism for studies of cyanobacterial metabolism and has great potential for biotechnological applications. It exhibits an exceptional tolerance of high-light irradiation and shows very rapid growth. The habitats from which this and closely related strains were isolated are subject to changes in several environmental factors, including light, nutrient supply, temperature, and salinity. In this study global transcriptome profiling via RNAseq has been used to perform a comparative and integrated study of global changes in cells grown at different temperatures, at different salinities, and under mixotrophic conditions, when a metabolizable organic carbon source was present. Furthermore, the transcriptomes were investigated for cells that were subjected to a heat shock and that were exposed to oxidative stress. Lower growth temperatures caused relatively minor changes of the transcriptome; the most prominent changes affected fatty acid desaturases. A heat shock caused severe changes of the transcriptome pattern; transcripts for genes associated with major metabolic pathways declined and those for different chaperones increased dramatically. Oxidative stress, however, left the transcript pattern almost unaffected. When grown at high salinity, Synechococcus sp. PCC 7002 had increased expression of genes involved in compatible solute biosynthesis and showed increased mRNA levels for several genes involved in electron transport. Transcripts of two adjacent genes dramatically increased upon growth at high salinity; the respective proteins are putatively involved in coping with oxidative stress and in triggering ion channels. Only minor changes were observed when cells were grown at low salinity or when the growth medium was supplemented with glycerol. However, the transcriptome data suggest that cells must acclimate to excess reducing equivalents when a reduced C-source is present.Item Bacteriochlorophyll f: properties of chlorosomes containing the “forbidden chlorophyll.”(2012-08) Vogl, K.; Tank, M.; Orf, G. S.; Blankenship, R. E.; Bryant, Donald A.The chlorosomes of green sulfur bacteria are mainly assembled from one of three types of bacteriochlorophylls, BChls c, d, and e. By analogy to the relationship between BChl c and BChl d (20-desmethyl-BChl c), a fourth type of BChl, BChl f (20-desmethyl-BChl e), should exist but has not yet been observed in nature. The bchU gene (bacteriochlorophyllide C-20 methyltransferase) of the brown-colored green sulfur bacterium Chlorobaculum limnaeum was inactivated by conjugative transfer from Eshcerichia coli and homologous recombination of a suicide plasmid carrying a portion of the bchU. The resulting bchU mutant was greenish brown in color and synthesized BChl fF. The chlorosomes of the bchU mutant had similar size and polypeptide composition as those of the wild type (WT), but the Qy absorption band of the BChl f aggregates was blue-shifted 16 nm (705 nm vs. 721 nm for the WT). Fluorescence spectroscopy showed that energy transfer to the baseplate was much less efficient in chlorosomes containing BChl f than in WT chlorosomes containing BChl e. When cells were grown at high irradiance with tungsten or fluorescent light, the WT and bchU mutant had identical growth rates. However, the WT grew about 50% faster than the bchU mutant at low irradiance (10 µmol photons m-2 s-1). Less efficient energy transfer from BChl f aggregates to BChl a in the baseplate, the much slower growth of the strain producing BChl f relative to the WT, and competition from other phototrophs, may explain why BChl f is not observed naturally.Item Acclimation of the global transcriptome of the cyanobacterium Synechococcus sp. strain PCC 7002 to nutrient limitations and alternative nitrogen sources(2012-04) Ludwig, M.; Bryant, Donald A.The unicellular, euryhaline cyanobacterium Synechococcus sp. strain PCC 7002 is a model organism for laboratory-based studies of cyanobacterial metabolism and is a potential platform for biotechnological applications. Two of its most notable properties are its exceptional tolerance of high-light intensity and very rapid growth under optimal conditions. In this study, transcription profiling by RNAseq has been used to perform an integrated study of global changes in transcript levels in cells subjected to limitation for the major nutrients CO2, nitrogen, sulfate, phosphate, and iron. Transcriptional patterns for cells grown on nitrate, ammonia, and urea were also studied. Nutrient limitation caused strong decreases of transcript levels of the genes encoding major metabolic pathways, especially for components of the photosynthetic apparatus, CO2 fixation, and protein biosynthesis. Uptake mechanisms for the respective nutrients were strongly up-regulated. The transcription data further suggest that major changes in the composition of the NADH dehydrogenase complex occur upon nutrient limitation. Transcripts for flavoproteins increased strongly when CO2 was limiting. Genes involved in protection from oxidative stress generally showed high, constitutive transcript levels, which possibly explains the high-light tolerance of this organism. The transcriptomes of cells grown with ammonia or urea as nitrogen source showed increased transcript levels for components of the CO2 fixation machinery compared to cells grown with nitrate, but in general transcription differences in cells grown on different N-sources exhibited surprisingly minor differences.Item Components and evolution of oxidative sulfur metabolism in green sulfur bacteria(2011-05) Gregersen, L. H.; Bryant, Donald A.; Frigaard, N. U.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.Item Transcription profiling of the cyanobacterium Synechococcus sp. PCC 7002 using high-throughput cDNA sequencing(2011-03) Ludwig, M.; Bryant, Donald A.The genome of the unicellular, euryhaline cyanobacterium Synechococcus sp. PCC 7002 encodes about 3200 proteins. Transcripts were detected for nearly all annotated open reading frames by a global transcriptomic analysis by Next-Generation (SOLiD™) sequencing of cDNA. In the cDNA samples sequenced, ∼90% of the mapped sequences were derived from the 16S and 23S ribosomal RNAs and ∼10% of the sequences were derived from mRNAs. In cells grown photoautotrophically under standard conditions [38°C, 1% (v/v) CO2 in air, 250 μmol photons m−2 s−1], the highest transcript levels (up to 2% of the total mRNA for the most abundantly transcribed genes; e.g., cpcAB, psbA, psaA) were generally derived from genes encoding structural components of the photosynthetic apparatus. High-light exposure for 1 h caused changes in transcript levels for genes encoding proteins of the photosynthetic apparatus, Type-1 NADH dehydrogenase complex and ATP synthase, whereas dark incubation for 1 h resulted in a global decrease in transcript levels for photosynthesis-related genes and an increase in transcript levels for genes involved in carbohydrate degradation. Transcript levels for pyruvate kinase and the pyruvate dehydrogenase complex decreased sharply in cells incubated in the dark. Under dark anoxic (fermentative) conditions, transcript changes indicated a global decrease in transcripts for respiratory proteins and suggested that cells employ an alternative phosphoenolpyruvate degradation pathway via phosphoenolpyruvate synthase (ppsA) and the pyruvate:ferredoxin oxidoreductase (nifJ). Finally, the data suggested that an apparent operon involved in tetrapyrrole biosynthesis and fatty acid desaturation, acsF2–ho2–hemN2–desF, may be regulated by oxygen concentration.Item Effect of mono-and dichromatic light quality on growth rates and photosynthetic performance of Synechococcus sp. PCC 7002(Frontiers Research Foundation (OA), 2014) Bernstein, Hans C.; Konopka, Allan; Melnicki, Matthew R.; Hill, Eric A.; Kucek, Leo A.; Zhang, Shuyi; Shen, Gaozhong; Bryant, Donald A.; Beliaev, Alexander S.Synechococcus sp. PCC 7002 was grown to steady state in optically thin turbidostat cultures under conditions for which light quantity and quality was systematically varied by modulating the output of narrow-band LEDs. Cells were provided photons absorbed primarily by chlorophyll (680 nm) or phycocyanin (630 nm) as the organism was subjected to four distinct mono- and dichromatic regimes. During cultivation with dichromatic light, growth rates were generally proportional to the total incident irradiance at values <275 2 μmol photons m− · s−1 and were not affected by the ratio of 630:680 nm wavelengths. Notably, under monochromatic light conditions, cultures exhibited similar growth rates only when they were irradiated with 630 nm light; cultures irradiated with only 680 nm light grew at rates that were 60–70% of those under other light quality regimes at equivalent irradiances. The functionality of photosystem II and associated processes such as maximum rate of photosynthetic electron transport, rate of cyclic electron flow, and rate of dark respiration generally increased as a function of growth rate. Nonetheless, some of the photophysiological parameters measured here displayed distinct patterns with respect to growth rate of cultures adapted to a single wavelength including phycobiliprotein content, which increased under severely light-limited growth conditions. Additionally, the ratio of photosystem II to photosystem I increased ∼40% over the range of growth rates, although cells grown with 680 nm light only had the highest ratios. These results suggest the presence of effective mechanisms which allow acclimation of Synechococcus sp. PCC 7002 acclimation to different irradiance conditions.