Life Sciences Commons^™

Cpet Is The Phycoerythrobilin Lyase For Cys-165 On Beta-Phycoerythrin From Fremyella Diplosiphon And The Chaperone-Like Protein Cpez Greatly Improves Its Activity., Wendy M. Schluchter, A. A. Nguyen, K. L. Joseph, A. N. Bussell, S. Pokhrel, A. J. Karty, M. C. Kronfel, D. M. Kehoe

Biological Sciences Faculty Publications

Bilin lyases are enzymes which ligate linear tetrapyrrole chromophores to specific cysteine residues on light harvesting proteins present in cyanobacteria and red algae. The lyases responsible for chromophorylating the light harvesting protein phycoerythrin (PE) have not been fully characterized. In this study, we explore the role of CpeT, a putative bilin lyase, in the biosynthesis of PE in the cyanobacterium Fremyella diplosiphon. Recombinant protein studies show that CpeT alone can bind phycoerythrobilin (PEB), but CpeZ, a chaperone-like protein, is needed in order to correctly and efficiently attach PEB to the beta-subunit of PE. MS analyses of the recombinant beta-subunit of …

Cpef Is The Bilin Lyase That Ligates The Doubly Linked Phycoerythrobilin On Phycoerythrin In The Cyanobacterium Fremyella Diplosiphon, Wendy M. Schluchter, R. B. Cole, D. M. Kehoe, M. N. Boutaghou, J. A. Karty, A. Gutu, L. S. Hernandez, J. P. Frick, C. V. Hernandez, C. M. Kronfel

Biological Sciences Faculty Publications

Phycoerythrin (PE) is a green light-absorbing protein present in the light-harvesting complex of cyanobacteria and red algae. The spectral characteristics of PE are due to its prosthetic groups, or phycoerythrobilins (PEBs), that are covalently attached to the protein chain by specific bilin lyases. Only two PE lyases have been identified and characterized so far, and the other bilin lyases are unknown. Here, using in silico analyses, markerless deletion, biochemical assays with purified and recombinant proteins, and site-directed mutagenesis, we examined the role of a putative lyase-encoding gene, cpeF, in the cyanobacterium Fremyella diplosiphon. Analyzing the phenotype of the cpeF deletion, …

The Roles Of The Chaperone-Like Protein Cpez And The Phycoerythrobilin Lyase Cpey In Phycoerythrin Biogenesis, Wendy M. Schluchter, D. M. Kehoe, J. A. Karty, T. Blensdorf, A. Gutu, J. P. Frick, A. Biswas, C. M. Kronfel

Biological Sciences Faculty Publications

Phycoerythrin (PE) present in the distal ends of light-harvesting phycobilisome rods in Fremyella diplosiphon (Tolypothrix sp. PCC 7601) contains five phycoerythrobilin (PEB) chromophores attached to six cysteine residues for efficient green light capture for photosynthesis. Chromophore ligation on PE subunits occurs through bilin lyase catalyzed reactions, but the characterization of the roles of all bilin lyases for phycoerythrin is not yet complete. To gain a more complete understanding about the individual functions of CpeZ and CpeY in PE biogenesis in cyanobacteria, we examined PE and phycobilisomes purified from wild type F. diplosiphon, cpeZ and cpeY knockout mutants. We find that …

Interplay Between Differentially Expressed Enzymes Contributes To Light Color Acclimation In Marine Synechococcus, Wendy M. Schluchter, D. M. Kehoe, F. Partensky, J. A. Strnat, S. Pokhrel, J. A. A. Karty, L. Garczarek, A. A. Nguyen, J. E. Sanfilippo

Biological Sciences Faculty Publications

Marine Synechococcus, a globally important group of cyanobacteria, thrives in various light niches in part due to its varied photosynthetic light-harvesting pigments. Many Synechococcus strains use a process known as chromatic acclimation to optimize the ratio of two chromophores, green-light-absorbing phycoerythrobilin (PEB) and blue-light-absorbing phycourobilin (PUB), within their light-harvesting complexes. A full mechanistic understanding of how Synechococcus cells tune their PEB to PUB ratio during chromatic acclimation has not yet been obtained. Here, we show that interplay between two enzymes named MpeY and MpeZ controls differential PEB and PUB covalent attachment to the same cysteine residue. MpeY attaches PEB to …

Adaptation To Blue Light In Marine Synechococcus Requires Mpeu, An Enzyme With Similarity To Phycoerythrobilin Lyase Isomerases, Wendy M. Schluchter, Rania Mohamed Mahmoud, Joseph Sanfilippo, Adam A. Nguyen, Johann A. Strnat, Frédéric Partensky, Laurence Garczarek, Nabil Kassem, David M. Kehoe

Biological Sciences Faculty Publications

Marine Synechococcus has successfully adapted to environments with different light colors, which likely contributes to this genus being the second most abundant group of microorganisms worldwide. Populations of Synechococcus that grow in deep, blue ocean waters contain large amounts of the blue-light absorbing chromophore phycourobilin (PUB) in their light harvesting complexes (phycobilisomes). Here, we show that all Synechococcus strains adapted to blue light possess a gene called mpeU. MpeU is structurally similar to phycobilin lyases, enzymes that ligate chromophores to phycobiliproteins. Interruption of mpeU caused a reduction in PUB content, impaired phycobilisome assembly and reduced growth rate more strongly in …

The Transcription Factors Adr1 Or Cat8 Are Required For Rtg Pathway Activation And Evasion From Yeast Acetic Acid-Induced Programmed Cell Death In Raffinose, Zhengchang Liu, Luna Laera, Nicoletta Guaragnella, Maša Ždralević, Domenico Marzulli, Sergio Giannattasio

Biological Sciences Faculty Publications

Yeast Saccharomyces cerevisiae grown on glucose undergoes programmed cell death (PCD) induced by acetic acid (AA-PCD), but evades PCD when grown in raffinose. This is due to concomitant relief of carbon catabolite repression (CCR) and activation of mitochondrial retrograde signaling, a mitochondria-to-nucleus communication pathway causing up-regulation of various nuclear target genes, such as CIT2, encoding peroxisomal citrate synthase, dependent on the positive regulator RTG2 in response to mitochondrial dysfunction. CCR down-regulates genes mainly involved in mitochondrial respiratory metabolism. In this work, we investigated the relationships between the RTG and CCR pathways in the modulation of AA-PCD sensitivity under glucose repression …

Adenosine Triphosphate (Atp) Is A Candidate Signaling Molecule In The Mitochondria-To-Nucleus Retrograde Response Pathway, Zhengchang Liu, Feng Zhang, Janet Thornton, Tammy Pracheil

Biological Sciences Faculty Publications

Intracellular communication from the mitochondria to the nucleus is achieved via the retrograde response. In budding yeast, the retrograde response, also known as the RTG pathway, is regulated positively by Rtg1, Rtg2, Rtg3 and Grr1 and negatively by Mks1, Lst8 and two 14-3-3 proteins, Bmh1/2. Activation of retrograde signaling leads to activation of Rtg1/3, two basic helix-loop-helix leucine zipper transcription factors. Rtg1/3 activation requires Rtg2, a cytoplasmic protein with an N-terminal adenosine triphosphate (ATP) binding domain belonging to the actin/Hsp70/sugar kinase superfamily. The critical regulatory step of the retrograde response is the interaction between Rtg2 and Mks1. Rtg2 binds to …

Adenosine Triphosphate (Atp) Is A Candidate Signaling Molecule In The Mitochondria-To-Nucleus Retrograde Response Pathway, Feng Zhang, Tammy Pracheil, Janet Thornton, Zhengchang Liu

Biological Sciences Faculty Publications

Intracellular communication from the mitochondria to the nucleus is achieved via the retrograde response. In budding yeast, the retrograde response, also known as the RTG pathway, is regulated positively by Rtg1, Rtg2, Rtg3 and Grr1 and negatively by Mks1, Lst8 and two 14-3-3 proteins, Bmh1/2. Activation of retrograde signaling leads to activation of Rtg1/3, two basic helix-loop-helix leucine zipper transcription factors. Rtg1/3 activation requires Rtg2, a cytoplasmic protein with an N-terminal adenosine triphosphate (ATP) binding domain belonging to the actin/Hsp70/sugar kinase superfamily. The critical regulatory step of the retrograde response is the interaction between Rtg2 and Mks1. Rtg2 binds to …

Mitochondrial Dna Instability In Cells Lacking Aconitase Correlates With Iron Citrate Toxicity, Muhammad A. Farooq, Tammy M. Pracheil, Zhejun Dong, Fei Xiao, Zhengchang Liu

Biological Sciences Faculty Publications

Aconitase, the second enzyme of the tricarboxylic acid cycle encoded by ACO1 in the budding yeast Saccharomyces cerevisiae, catalyzes the conversion of citrate to isocitrate. aco1 Delta results in mitochondrial DNA (mtDNA) instability. It has been proposed that Aco1 binds to mtDNA and mediates its maintenance. Here we propose an alternative mechanism to account for mtDNA loss in aco1 Delta mutant cells. We found that aco1 Delta activated the RTG pathway, resulting in increased expression of genes encoding citrate synthase. By deleting RTG1, RTG3, or genes encoding citrate synthase, mtDNA instability was prevented in aco1 Delta mutant …

Mitochondrial Dna Instability In Cells Lacking Aconitase Correlates With Iron Citrate Toxicity, Zhengchang Liu, Fei Xiao, Zhejun Dong, Tammy Pracheil, Muhammad Farooq

Biological Sciences Faculty Publications

Aconitase, the second enzyme of the tricarboxylic acid cycle encoded by ACO1 in the budding yeast Saccharomyces cerevisiae, catalyzes the conversion of citrate to isocitrate. aco1Δ results in mitochondrial DNA (mtDNA) instability. It has been proposed that Aco1 binds to mtDNA and mediates its maintenance. Here we propose an alternative mechanism to account for mtDNA loss in aco1Δ mutant cells. We found that aco1Δ activated the RTG pathway, resulting in increased expression of genes encoding citrate synthase. By deleting RTG1, RTG3, or genes encoding citrate synthase, mtDNA instability was prevented in aco1Δ mutant cells. Increased activity of citrate synthase leads …

Cyanolyase: A Database Of Phycobilin Lyase Sequences, Motifs And Functions, Wendy M. Schluchter, Anthony Bretaudeau, Francois Coste, Florian Humily, Laurence Garczarek, Gildas Le Corguille, Christophe Six, Morgane Ratin, Olivier Collin, Frederic Partensky

Biological Sciences Faculty Publications

CyanoLyase (http://cyanolyase.genouest.org/) is a manually curated sequence and motif database of phycobilin lyases and related proteins. These enzymes catalyze the covalent ligation of chromophores (phycobilins) to specific binding sites of phycobiliproteins (PBPs). The latter constitute the building bricks of phycobilisomes, the major light-harvesting systems of cyanobacteria and red algae. Phycobilin lyases sequences are poorly annotated in public databases. Sequences included in CyanoLyase were retrieved from all available genomes of these organisms and a few others by similarity searches using biochemically characterized enzyme sequences and then classified into 3 clans and 32 families. Amino acid motifs were computed for each family …

Phycoerythrin-Specific Bilin Lyase-Isomerase Controls Blue-Green Chromatic Acclimation In Marine Synechococcus, Wendy M. Schluchter, Avijit Biswas, Frédéric Partensky, J. A. Karty, Laurence Garczarek, A. Gutu, David M. Kehoe, A. Shukla, N. Blot, L. A. Hammad

Biological Sciences Faculty Publications

The marine cyanobacterium Synechococcus is the second most abundant phytoplanktonic organism in the world's oceans. The ubiquity of this genus is in large part due to its use of a diverse set of photosynthetic light-harvesting pigments called phycobiliproteins, which allow it to efficiently exploit a wide range of light colors. Here we uncover a pivotal molecular mechanism underpinning a widespread response among marine Synechococcus cells known as type IV chromatic acclimation (CA4). During this process, the pigmentation of the two main phycobiliproteins of this organism, phycoerythrins I and II, is reversibly modified to match changes in the ambient light color …

Population Proteomics: Quantitative Variation Within And Among Populations In Cardiac Protein Expression, Bernard Rees

Biological Sciences Faculty Publications

Population analysis of gene expression is typically achieved by quantifying levels of mRNA; however, gene expression is also a function of protein translation and turnover. Therefore, a complete understanding of population variation in gene expression requires quantitative knowledge of protein expression within and among natural populations. We used two dimensional fluorescence difference gel electrophoresis (2D-DIGE) to quantitatively compare expression of heart ventricle proteins among 18 individuals in three populations of the teleost fish Fundulus. Among populations, expressions between orthologous proteins and mRNAs were generally positively correlated. Additionally, similar to the pattern of cardiac mRNA expression for the same populations, we …

Oxygen Limitation And Tissue Metabolic Potential Of The African Fish Barbus Neumayeri: Roles Of Native Habitat And Acclimatization, Bernard Rees

Biological Sciences Faculty Publications

Oxygen availability in aquatic habitats is a major environmental factor influencing the ecology, behaviour, and physiology of fishes. This study evaluates the contribution of source population and hypoxic acclimatization of the African fish, Barbus neumayeri, in determining growth and tissue metabolic enzyme activities. Individuals were collected from two sites differing dramatically in concentration of dissolved oxygen (DO), Rwembaita Swamp (annual average DO 1.35 mgO₂ L^-1) and Inlet Stream West (annual average DO 5.58 mgO₂ L^-1) in Kibale National Park, Uganda, and reciprocally transplanted using a cage experiment in the field, allowing us to …

Biosynthesis Of Cyanobacterial Phycobiliproteins In Escherichia Coli: Chromophorylation Efficiency And Specificity Of All Bilin Lyases From Synechococcus Sp. Strain Pcc 7002, Wendy M. Schluchter

Biological Sciences Faculty Publications

No abstract provided.

Effects Of Modified Phycobilin Biosynthesis In The Cyanobacterium Synechococcus Sp. Strain Pcc 7002, Wendy M. Schluchter

Biological Sciences Faculty Publications

No abstract provided.

Attachment Of Non-Cognate Chromophores To Cpca Of Synechocystis Sp. Strain Pcc 6803 Expressed In E. Coli, Wendy M. Schluchter

Biological Sciences Faculty Publications

No abstract provided.

Geographic Variation In The Effects Of Heat Exposure On Maximum Sprint Speed And Hsp70 Abundance In Populations Of The Western Fence Lizard, Scelopolus Occidentalis, D M. Mcmillan, D J. Irschick, Bernard B. Rees

Biological Sciences Faculty Publications

We examined whether western fence lizards Sceloporus occidentalis

occurring in thermally divergent environments display differential

responses to high temperature in locomotor performance

and heat-shock protein (Hsp) expression. We measured

maximum sprint speed in S. occidentalis from four populations

at paired latitudes and elevations before and after exposure to

an experimental heat treatment and then quantified hind-limb

muscle Hsp70 expression. Lizards collected from northern or

high-elevation collection sites suffered a greater reduction in

sprint speed after heat exposure than lizards collected from

southern or low-elevation sites. In addition, lizards from northern

collection sites also exhibited an increase in Hsp70 expression

after …

Oxygen Limitation And Tissue Metabolic Potential Of The African Fish Barbus Neumayeri: Roles Of Native Habitat And Acclimatization, Martinez M. L, Raynard E. L, Bernard B. Rees, Chapman L. J

Biological Sciences Faculty Publications

Background: Oxygen availability in aquatic habitats is a major environmental factor influencing the ecology, behaviour, and physiology of fishes. This study evaluates the contribution of source population and hypoxic acclimatization of the African fish, Barbus neumayeri, in determining growth and tissue metabolic enzyme activities. Individuals were collected from two sites differing dramatically in concentration of dissolved oxygen (DO), Rwembaita Swamp (annual average DO 1.35 mgO2 L-1) and Inlet Stream West (annual average DO 5.58 mgO2 L-1) in Kibale National Park, Uganda, and reciprocally transplanted using a cage experiment in the field, allowing us to maintain individuals under natural conditions of …

Exercise-And Hypoxia-Induced Anaerobic Metabolism And Recovery: A Student Laboratory Exercise Using Teleost Fish, Bernard Rees

Biological Sciences Faculty Publications

Anaerobic metabolism is recruitedin vertebrates under conditions of intense exercise or lowered environmental oxygen availability (hypoxia), typically resulting in the accumulation of lactate in blood and tissues. Lactate will be cleared over time after the reoxygenation of tissues, eventually returning to control levels. Here, we present a laboratory exercise developed as part of an upper-level vertebrate physiology class that demonstrates the effects of exercise and hypoxia exposure on blood lactate in fish and the subsequent decrease in lactate during recovery. Typically, the results obtained by students demonstrate that both treatments cause significant increases in blood lactate concentrations (two to three …

Optimality In A Partitioned Task Performed By Social Insects, Jerome J. Howard

Biological Sciences Faculty Publications

Biologists have long been aware that adaptations should not be analysed in isolation from the function of the whole organism. Here, we address the equivalent issue at the scale of a social insect colony: the optimality of component behaviours in a partitioned sequence of tasks. In colonies of Atta colombica, a leaf-cutting ant, harvested leaf tissue is passed from foragers to nest workers that distribute, clean, shred and implant the tissue in fungal gardens. In four laboratory colonies of A. colombica, we found that the highest colony-wide rate of leaf tissue processing in the nest was achieved when leaf fragment …

Vertebrate Osmoregulation: A Student Laboratory Exercise Using Teleost Fish, Bernard Rees

Biological Sciences Faculty Publications

Anaerobic metabolism is recruited in vertebrates under conditions of intense exercise or lowered environmental oxygen availability (hypoxia), typically resulting in the accumulation of lactate in blood and tissues. Lactate will be cleared over time after the reoxygenation of tissues, eventually returning to control levels. Here, we present a laboratory exercise developed as part of an upper-level vertebrate physiology class that demonstrates the effects of exercise and hypoxia exposure on blood lactate in fish and the subsequent decrease in lactate during recovery. Typically, the results obtained by students demonstrate that both treatments cause significant increases in blood lactate concentrations (two to …

Fundulus As The Premier Teleost Model In Environmental Biology: Opportunities For New Insights Using Genomics, Bernard Rees

Biological Sciences Faculty Publications

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these …

Effects Of Long-Term Hypoxia On Enzymes Of Carbohydrate Metabolism In The Gulf Killifish, Fundulus Grandis, Bernard Rees

Biological Sciences Faculty Publications

The goal of the current study was to generate a comprehensive, multi-tissue perspective of the effects of chronic hypoxic exposure on carbohydrate metabolism in the Gulf killifish Fundulus grandis. Fish were held at approximately 1.3 mg l(-1) dissolved oxygen (approximately 3.6 kPa) for 4 weeks, after which maximal activities were measured for all glycolytic enzymes in four tissues (white skeletal muscle, liver, heart and brain), as well as for enzymes of glycogen metabolism (in muscle and liver) and gluconeogenesis (in liver). The specific activities of enzymes of glycolysis and glycogen metabolism were strongly suppressed by hypoxia in white skeletal muscle, …

Effects Of Long-Term Hypoxia On Enzymes Of Carbohydrate Metabolism In The Gulf Killifish, Fundulus Grandis, M L. Manning, C A. Landry, R D. Boehm, S Manning, A O. Cheek, Bernard B. Rees

Biological Sciences Faculty Publications

The goal of the current study was to generate a comprehensive, multi-tissue perspective of the effects of chronic hypoxic exposure on carbohydrate metabolism in the Gulf killifish Fundulus grandis. Fish were held at approximately 1.3·mg·l–1 dissolved oxygen (~3.6·kPa) for 4·weeks, after which maximal activities were measured for all glycolytic enzymes in four tissues (white skeletal muscle, liver, heart and brain), as well as for enzymes of glycogen metabolism (in muscle and liver) and gluconeogenesis (in liver). The specific activities of enzymes of glycolysis and glycogen metabolism were strongly suppressed by hypoxia in white skeletal muscle, which may reflect decreased energy …

Structure And Sequence Conservation Of A Putative Hypoxia Responsive Element In The Lactate Dehydrogenase-B Gene Of Fundulus, Bernard B. Rees, John A.L. Bowman, P M. Schulte

Biological Sciences Faculty Publications

No abstract provided.

Molecular Mechanisms Of Metabolic Arrest: Life In Limbo (Book Review), Bernard Rees

Biological Sciences Faculty Publications

No abstract provided.

Assembly Of Reca-Like Recombinases: Distinct Roles For Mediator Proteins In Mitosis And Meiosis, Stephen L. Gasior

Biological Sciences Faculty Publications

Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. …

Acclimation To Hypoxia Increases Survival Time Of Zebrafish, Danio Rerio, During Lethal Hypoxia, Bernard B. Rees, F Sudradjat, J W. Love

Biological Sciences Faculty Publications

Survivorship of zebrafish, Danio rerio, was measured during lethal hypoxic stress after pretreatment in water at either ambient oxygen or at a lowered, but nonlethal, level of oxygen. Acclimation to nonlethal hypoxia (pO2 @ 15 Torr; ca. 10% air-saturation) for 48 hr significantly extended survival time during more severe hypoxia (pO2 @ 8 Torr; ca. 5% air-saturation) compared to survival of individuals with no prior hypoxic exposure. The magnitude of the acclimation effect depended upon the sex of the fish: hypoxia pretreatment increased the survival times of males by a factor of approximately 9 and that of females by a …

Tid1/Rdh54 Promotes Colocalization Of Rad51 And Dmc1 During Meiotic Recombination, Stephen L. Gasior

Biological Sciences Faculty Publications

Two RecA homologs, Rad51 and Dmc1, assemble as cytologically visible complexes (foci) at the same sites on meiotic chromosomes. Time course analysis confirms that co-foci appear and disappear as the single predominant form. A large fraction of co-foci are eliminated in a red1 mutant, which is expected as a characteristic of the interhomolog-specific recombination pathway. Previous studies suggested that normal Dmc1 loading depends on Rad51. We show here that a mutation in TID1/RDH54, encoding a RAD54 homolog, reduces Rad51-Dmc1 colocalization relative to WT. A rad54 mutation, in contrast, has relatively little effect on RecA homolog foci except when strains also …