College of Agriculture

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As the foundation of the land grant mission at Montana State University, the College of Agriculture and the Montana Agricultural Experiment Station provide instruction in traditional and innovative degree programs and conduct research on old and new challenges for Montana’s agricultural community. This integration creates opportunities for students and faculty to excel through hands-on learning, to serve through campus and community engagement, to explore unique solutions to distinct and interesting questions and to connect Montanans with the global community through research discoveries and outreach.

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Harvesting far-red light: Functional integration of chlorophyll f into Photosystem I complexes of Synechococcus sp. PCC 7002
(2020-08) Tros, Martijn; Bersanini, Luca; Shen, Gaozhong; Ho, Ming-Yang; van Stokkum, Ivo H. M.; Bryant, Donald A.; Croce, Roberta
The heterologous expression of the far-red absorbing chlorophyll (Chl) f in organisms that do not synthesize this pigment has been suggested as a viable solution to expand the solar spectrum that drives oxygenic photosynthesis. In this study, we investigate the functional binding of Chl f to the Photosystem I (PSI) of the cyanobacterium Synechococcus 7002, which has been engineered to express the Chl f synthase gene. By optimizing growth light conditions, one-to-four Chl f pigments were found in the complexes. By using a range of spectroscopic techniques, isolated PSI trimeric complexes were investigated to determine how the insertion of Chl f affects excitation energy transfer and trapping efficiency. The results show that the Chls f are functionally connected to the reaction center of the PSI complex and their presence does not change the overall pigment organization of the complex. Chl f substitutes Chl a (but not the Chl a red forms) while maintaining efficient energy transfer within the PSI complex. At the same time, the introduction of Chl f extends the photosynthetically active radiation of the new hybrid PSI complexes up to 750 nm, which is advantageous in far-red light enriched environments. These conclusions provide insights to engineer the photosynthetic machinery of crops to include Chl f and therefore increase the light-harvesting capability of photosynthesis.
Extensive remodeling of the photosynthetic apparatus alters energy transfer among photosynthetic complexes when cyanobacteria acclimate to far-red light
(2020-04) Ho, Ming-Yang; Niedzwiedzki, Dariusz M.; MacGregor-Chatwin, Craig; Gerstenecker, Gary; Hunter, C. Neil; Blankenship, Robert E.; Bryant, Donald A.
Some cyanobacteria remodel their photosynthetic apparatus by a process known as Far-Red Light Photoacclimation (FaRLiP). Specific subunits of the phycobilisome (PBS), photosystem I (PSI), and photosystem II (PSII) complexes produced in visible light are replaced by paralogous subunits encoded within a conserved FaRLiP gene cluster when cells are grown in far-red light (FRL; λ = 700–800 nm). FRL-PSII complexes from the FaRLiP cyanobacterium, Synechococcus sp. PCC 7335, were purified and shown to contain Chl a, Chl d, Chl f, and pheophytin a, while FRL-PSI complexes contained only Chl a and Chl f. The spectroscopic properties of purified photosynthetic complexes from Synechococcus sp. PCC 7335 were determined individually, and energy transfer kinetics among PBS, PSII, and PSI were analyzed by time-resolved fluorescence (TRF) spectroscopy. Direct energy transfer from PSII to PSI was observed in cells (and thylakoids) grown in red light (RL), and possible routes of energy transfer in both RL- and FRL-grown cells were inferred. Three structural arrangements for RL-PSI were observed by atomic force microscopy of thylakoid membranes, but only arrays of trimeric FRL-PSI were observed in thylakoids from FRL-grown cells. Cells grown in FRL synthesized the FRL-specific complexes but also continued to synthesize some PBS and PSII complexes identical to those produced in RL. Although the light-harvesting efficiency of photosynthetic complexes produced in FRL might be lower in white light than the complexes produced in cells acclimated to white light, the FRL-complexes provide cells with the flexibility to utilize both visible and FRL to support oxygenic photosynthesis.
Recombination Does Not Hinder Formation or Detection of Ecological Species of Synechococcus Inhabiting a Hot Spring Cyanobacterial Mat
(Frontiers Media, 2016-01) Melendrez, Melanie C.; Becraft, Eric D.; Wood, Jason M.; Olsen, MIllie T.; Bryant, Donald A.; Heidelberg, John F.; Rusch, Douglas B.; Cohan, Frederick M.; Ward, David M.
Recent studies of bacterial speciation have claimed to support the biological species concept—that reduced recombination is required for bacterial populations to diverge into species. This conclusion has been reached from the discovery that ecologically distinct clades show lower rates of recombination than that which occurs among closest relatives. However, these previous studies did not attempt to determine whether the more-rapidly recombining close relatives within the clades studied may also have diversified ecologically, without benefit of sexual isolation. Here we have measured the impact of recombination on ecological diversification within and between two ecologically distinct clades (A and B') of Synechococcus in a hot spring microbial mat in Yellowstone National Park, using a cultivation-free, multi-locus approach. Bacterial artificial chromosome (BAC) libraries were constructed from mat samples collected at 60°C and 65°C. Analysis of multiple linked loci near Synechococcus 16S rRNA genes showed little evidence of recombination between the A and B' lineages, but a record of recombination was apparent within each lineage. Recombination and mutation rates within each lineage were of similar magnitude, but recombination had a somewhat greater impact on sequence diversity than mutation, as also seen in many other bacteria and archaea. Despite recombination within the A and B' lineages, there was evidence of ecological diversification within each lineage. The algorithm Ecotype Simulation identified sequence clusters consistent with ecologically distinct populations (ecotypes), and several hypothesized ecotypes were distinct in their habitat associations and in their adaptations to different microenvironments. We conclude that sexual isolation is more likely to follow ecological divergence than to precede it. Thus, an ecology-based model of speciation appears more appropriate than the biological species concept for bacterial and archaeal diversification.
Inference of interactions in cyanobacterial-heterotrophic co-cultures via transcriptome sequencing
(2014-04) Beliaev, Alexander S.; Romine, Margaret F.; Serres, Margrethe; Bernstein, Hans C.; Linggi, Bryan E.; Markillie, Lye M.; Isern, Nancy G.; Chrisler, William B.; Kucek, Leo A.; Hill, Eric A.; Pinchuk, Grigoriy E.; Bryant, Donald A.; Wiley, H. Steven; Fredrickson, Jim K.; Konopka, Allan
We used deep sequencing technology to identify transcriptional adaptation of the euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 and the marine facultative aerobe Shewanella putrefaciens W3-18-1 to growth in a co-culture and infer the effect of carbon flux distributions on photoautotroph–heterotroph interactions. The overall transcriptome response of both organisms to co-cultivation was shaped by their respective physiologies and growth constraints. Carbon limitation resulted in the expansion of metabolic capacities, which was manifested through the transcriptional upregulation of transport and catabolic pathways. Although growth coupling occurred via lactate oxidation or secretion of photosynthetically fixed carbon, there was evidence of specific metabolic interactions between the two organisms. These hypothesized interactions were inferred from the excretion of specific amino acids (for example, alanine and methionine) by the cyanobacterium, which correlated with the downregulation of the corresponding biosynthetic machinery in Shewanella W3-18-1. In addition, the broad and consistent decrease of mRNA levels for many Fe-regulated Synechococcus 7002 genes during co-cultivation may indicate increased Fe availability as well as more facile and energy-efficient mechanisms for Fe acquisition by the cyanobacterium. Furthermore, evidence pointed at potentially novel interactions between oxygenic photoautotrophs and heterotrophs related to the oxidative stress response as transcriptional patterns suggested that Synechococcus 7002 rather than Shewanella W3-18-1 provided scavenging functions for reactive oxygen species under co-culture conditions. This study provides an initial insight into the complexity of photoautotrophic–heterotrophic interactions and brings new perspectives of their role in the robustness and stability of the association.
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.
Draft Genome Sequence of the Moderately Thermophilic Bacterium Schleiferia thermophila Strain Yellowstone (Bacteroidetes)
(2014-07) Thiel, Vera; Tomsho, Lynn P.; Burhans, Richard; Gay, Scott E.; Ramaley, R. F.; Schuster, Stephan C.; Steinke, L.; Bryant, Donald A.
The draft genome sequence of the moderately thermophilic bacterium Schleiferia thermophila strain Yellowstone (Bacteroidetes), isolated from Octopus Spring (Yellowstone National Park, WY, USA) was sequenced and comprises 2,617,694 bp in 35 contigs. The draft genome is predicted to encode 2,457 protein coding genes and 37 tRNA encoding genes and two rRNA operons.
Draft Genome Sequence of a Sulfide-Oxidizing, Autotrophic Filamentous Anoxygenic Phototrophic Bacterium, Chloroflexus sp. Strain MS-G (Chloroflexi)
(2014-09) Thiel, Vera; Tomsho, Lynn P.; Burhans, Richard; Schuster, Stephan C.; Bryant, Donald A.; Ward, David M.
The draft genome sequence of the thermophilic filamentous anoxygenic phototrophic bacterium Chloroflexus sp. strain MS-G (Chloroflexi), isolated from Mushroom Spring (Yellowstone National Park, WY, USA) was sequenced and comprises 4,784,183 bp in 251 contigs. The draft genome is predicted to encode 4,059 protein coding genes, 49 tRNA encoding genes, and 3 rRNA operons.
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.
Comparative genomics and functional analysis of rhamnose catabolic pathways and regulons in Bacteria
(2013-12) Rodionova, I. A.; Li, X.; Thiel, Vera; Stolyar, S.; Stanton, K.; Frederickson, J. K.; Bryant, Donald A.; Osterman, A. L.; Best, A. A.; Rodionov, D. A.
L-rhamnose (L-Rha) is a deoxy-hexose sugar commonly found in nature. L-Rha catabolic pathways were previously characterized in various bacteria including Escherichia coli. Nevertheless, homology searches failed to recognize all the genes for the complete L-Rha utilization pathways in diverse microbial species involved in biomass decomposition. Moreover, the regulatory mechanisms of L-Rha catabolism have remained unclear in most species. A comparative genomics approach was used to reconstruct the L-Rha catabolic pathways and transcriptional regulons in the phyla Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria, and Thermotogae. The reconstructed pathways include multiple novel enzymes and transporters involved in the utilization of L-Rha and L-Rha-containing polymers. Large-scale regulon inference using bioinformatics revealed remarkable variations in transcriptional regulators for L-Rha utilization genes among bacteria. A novel bifunctional enzyme, L-rhamnulose-phosphate aldolase (RhaE) fused to L-lactaldehyde dehydrogenase (RhaW), which is not homologous to previously characterized L-Rha catabolic enzymes, was identified in diverse bacteria including Chloroflexi, Bacilli, and Alphaproteobacteria. By using in vitro biochemical assays we validated both enzymatic activities of the purified recombinant RhaEW proteins from Chloroflexus aurantiacus and Bacillus subtilis. Another novel enzyme of the L-Rha catabolism, L-lactaldehyde reductase (RhaZ), was identified in Gammaproteobacteria and experimentally validated by in vitro enzymatic assays using the recombinant protein from Salmonella typhimurium. C. aurantiacus induced transcription of the predicted L-Rha utilization genes when L-Rha was present in the growth medium and consumed L-Rha from the medium. This study provided comprehensive insights to L-Rha catabolism and its regulation in diverse Bacteria.
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.

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