164 results on '"J. Thomas Beatty"'
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2. Purification and preparation of Rhodobacter sphaeroides reaction centers for photocurrent measurements and atomic force microscopy characterization
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Daniel Jun, Sylvester Zhang, Adrian Jan Grzędowski, Amita Mahey, J. Thomas Beatty, and Dan Bizzotto
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Biophysics ,Atomic Force Microscopy (AFM) ,Microbiology ,Microscopy ,Protein Biochemistry ,Protein expression and purification ,Science (General) ,Q1-390 - Abstract
Summary: The formation of defined surfaces consisting of photosynthetic reaction centers (RCs) in biohybrid solar cells is challenging. Here, we start with the production of engineered RCs for oriented binding. RCs are deposited onto gold electrodes, and 6-mercapto-1-hexanol (MCH) is used to displace multilayers and non-specifically adsorbed RCs. The resulting electrode surfaces are analyzed for photocurrent generation using an intensity-modulated light and lock-in amplifier. Atomic force microscopy (AFM) is used to characterize the surface and the formation of RC structural assemblies.For complete details on the use and execution of this profile, please refer to Jun et al. (2021).
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- 2022
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3. Correlating structural assemblies of photosynthetic reaction centers on a gold electrode and the photocurrent - potential response
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Daniel Jun, Sylvester Zhang, Adrian Jan Grzędowski, Amita Mahey, J. Thomas Beatty, and Dan Bizzotto
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Chemistry ,Electrochemistry ,Surface chemistry ,Surface science ,Science - Abstract
Summary: The use of biomacromolecules is a nascent development in clean alternative energies. In applications of biosensors and biophotovoltaic devices, the bacterial photosynthetic reaction center (RC) is a protein-pigment complex that has been commonly interfaced with electrodes, in large part to take advantage of the long-lived and high efficiency of charge separation. We investigated assemblies of RCs on an electrode that range from monolayer to multilayers by measuring the photocurrent produced when illuminated by an intensity-modulated excitation light source. In addition, atomic force microscopy and modeling of the photocurrent with the Marcus-Hush-Chidsey theory detailed the reorganization energy for the electron transfer process, which also revealed changes in the RC local environment due to the adsorbed conformations. The local environment in which the RCs are embedded significantly influenced photocurrent generation, which has implications for electron transfer of other biomacromolecules deposited on a surface in sensor and photovoltaic applications employing a redox electrolyte.
- Published
- 2021
- Full Text
- View/download PDF
4. Genomic diversity of bacteriophages infecting Rhodobacter capsulatus and their relatedness to its gene transfer agent RcGTA
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Jackson Rapala, Brenda Miller, Maximiliano Garcia, Megan Dolan, Matthew Bockman, Mats Hansson, Daniel A. Russell, Rebecca A. Garlena, Steven G. Cresawn, Alexander B. Westbye, J. Thomas Beatty, Richard M. Alvey, and David W. Bollivar
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Medicine ,Science - Abstract
The diversity of bacteriophages is likely unparalleled in the biome due to the immense variety of hosts and the multitude of viruses that infect them. Recent efforts have led to description at the genomic level of numerous bacteriophages that infect the Actinobacteria, but relatively little is known about those infecting other prokaryotic phyla, such as the purple non-sulfur photosynthetic α-proteobacterium Rhodobacter capsulatus. This species is a common inhabitant of freshwater ecosystems and has been an important model system for the study of photosynthesis. Additionally, it is notable for its utilization of a unique form of horizontal gene transfer via a bacteriophage-like element known as the gene transfer agent (RcGTA). Only three bacteriophages of R. capsulatus had been sequenced prior to this report. Isolation and characterization at the genomic level of 26 new bacteriophages infecting this host advances the understanding of bacteriophage diversity and the origins of RcGTA. These newly discovered isolates can be grouped along with three that were previously sequenced to form six clusters with four remaining as single representatives. These bacteriophages share genes with RcGTA that seem to be related to host recognition. One isolate was found to cause lysis of a marine bacterium when exposed to high-titer lysate. Although some clusters are more highly represented in the sequenced genomes, it is evident that many more bacteriophage types that infect R. capsulatus are likely to be found in the future.
- Published
- 2021
5. Persulfide-Responsive Transcription Factor SqrR Regulates Gene Transfer and Biofilm Formation via the Metabolic Modulation of Cyclic di-GMP in Rhodobacter capsulatus
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Takayuki Shimizu, Toma Aritoshi, J. Thomas Beatty, and Tatsuru Masuda
- Subjects
transcriptional regulation ,gene transfer ,persulfide ,redox signaling ,cyclic GMP ,Biology (General) ,QH301-705.5 - Abstract
Bacterial phage-like particles (gene transfer agents—GTAs) are widely employed as a crucial genetic vector in horizontal gene transfer. GTA-mediated gene transfer is induced in response to various stresses; however, regulatory mechanisms are poorly understood. We found that the persulfide-responsive transcription factor SqrR may repress the expression of several GTA-related genes in the photosynthetic bacterium Rhodobacter capsulatus. Here, we show that the sqrR deletion mutant (ΔsqrR) produces higher amounts of intra- and extracellular GTA and gene transfer activity than the wild type (WT). The transcript levels of GTA-related genes are also increased in ΔsqrR. In spite of the presumption that GTA-related genes are regulated in response to sulfide by SqrR, treatment with sulfide did not alter the transcript levels of these genes in the WT strain. Surprisingly, hydrogen peroxide increased the transcript levels of GTA-related genes in the WT, and this alteration was abolished in the ΔsqrR strain. Moreover, the absence of SqrR changed the intracellular cyclic dimeric GMP (c-di-GMP) levels, and the amount of c-di-GMP was correlated with GTA activity and biofilm formation. These results suggest that SqrR is related to the repression of GTA production and the activation of biofilm formation via control of the intracellular c-di-GMP levels.
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- 2022
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6. Porphyrin Excretion Resulting From Mutation of a Gene Encoding a Class I Fructose 1,6-Bisphosphate Aldolase in Rhodobacter capsulatus
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Hao Ding, Rafael G. Saer, and J. Thomas Beatty
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porphyrin excretion ,coproporphyrinogen III ,HemN ,fructose 1,6-bisphosphate aldolase ,class I FBA ,moonlight activity ,Microbiology ,QR1-502 - Abstract
This paper describes a mutant (called SB1707) of the Rhodobacter capsulatus wild type strain SB1003 in which a transposon-disrupted rcc01707 gene resulted in a ∼25-fold increase in the accumulation of coproporphyrin III in the medium of phototrophic (anaerobic) cultures grown in a yeast extract/peptone medium. There was little or no stimulation of pigment accumulation in aerobic cultures. Therefore, this effect of rcc01707 mutation appears to be specific for the anaerobic coproporphyrinogen III oxidase HemN as opposed to the aerobic enzyme HemF. The protein encoded by rcc01707 is homologous to Class I fructose 1,6-bisphosphate aldolases, which catalyze a glycolytic reaction that converts fructose 1, 6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, precursors of pyruvate. There were significant differences in coproporphyrin III accumulation using defined media with individual organic acids and sugars as the sole carbon source: pyruvate, succinate and glutamate stimulated accumulation the most, whereas glucose suppressed coproporphyrin III accumulation to 10% of that of succinate. However, although quantitatively lesser, similar effects of carbon source on the amount of accumulated pigment in the culture medium were seen in a wild type control. Therefore, this mutation appears to exaggerate effects also seen in the wild type strain. It is possible that mutation of rcc01707 causes a metabolic bottleneck or imbalance that was not rectified during growth on the several carbon sources tested. However, we speculate that, analogous to other fructose 1,6-bisphosphate aldolases, the rcc01707 gene product has a “moonlighting” activity that in this case is needed for the maximal expression of the hemN gene. Indeed, it was found that the rcc01707 gene is needed for maximal expression of a hemN promoter-lacZ reporter. With the decrease in hemN expression due to the absence of the rcc01707 gene product, coproporphyrinogen III accumulates and is released from the cell, yielding the spontaneous oxidation product coproporphyrin III.
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- 2019
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7. A Photovoltaic Device Using an Electrolyte Containing Photosynthetic Reaction Centers
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Ali Mahmoudzadeh, John D.W. Madden, J. Thomas Beatty, Rafael Saer, and Arash Takshi
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reaction center ,Rhodobacter sphaeroides ,mediators ,photocurrent ,bio-photovoltaic (bio-PV) ,Technology - Abstract
The performance of bio-photovoltaic devices with a monolayer of the immobilized photosynthetic reaction center (RC) is generally low because of weak light absorption and poor charge transfer between the RC and the electrode. In this paper, a new bio-photovoltaic device is described in which the RC is dissolved in the electrolyte of an electrochemical cell. The charges generated by the illuminated RC are transferred to electrodes via mediators. The difference between the reaction rates of two types of mediator at the electrode surfaces determines the direction of the photocurrent in the device. Experimental results show that the magnitude of the photocurrent is proportional to the incident light intensity, and the current increases nonlinearly with an increase in the RC concentration in the electrolyte. With further optimization this approach should lead to devices with improved light absorption.
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- 2010
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8. Extracellular Polysaccharide Receptor and Receptor-Binding Proteins of the Rhodobacter capsulatus Bacteriophage-like Gene Transfer Agent RcGTA
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Nawshin T. B. Alim, Sonja Koppenhöfer, Andrew S. Lang, and J. Thomas Beatty
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Genetics ,Genetics (clinical) ,Rhodobacter ,gene transfer agent ,RcGTA ,horizontal gene transfer ,phage ,virus ,head spike ,tail fiber - Abstract
A variety of prokaryotes produce a bacteriophage-like gene transfer agent (GTA), and the alphaproteobacterial Rhodobacter capsulatus RcGTA is a model GTA. Some environmental isolates of R. capsulatus lack the ability to acquire genes transferred by the RcGTA (recipient capability). In this work, we investigated the reason why R. capsulatus strain 37b4 lacks recipient capability. The RcGTA head spike fiber and tail fiber proteins have been proposed to bind extracellular oligosaccharide receptors, and strain 37b4 lacks a capsular polysaccharide (CPS). The reason why strain 37b4 lacks a CPS was unknown, as was whether the provision of a CPS to 37b4 would result in recipient capability. To address these questions, we sequenced and annotated the strain 37b4 genome and used BLAST interrogations of this genome sequence to search for homologs of genes known to be needed for R. capsulatus recipient capability. We also created a cosmid-borne genome library from a wild-type strain, mobilized the library into 37b4, and used the cosmid-complemented strain 37b4 to identify genes needed for a gain of function, allowing for the acquisition of RcGTA-borne genes. The relative presence of CPS around a wild-type strain, 37b4, and cosmid-complemented 37b4 cells was visualized using light microscopy of stained cells. Fluorescently tagged head spike fiber and tail fiber proteins of the RcGTA particle were created and used to measure the relative binding to wild-type and 37b4 cells. We found that strain 37b4 lacks recipient capability because of an inability to bind RcGTA; the reason it is incapable of binding is that it lacks CPS, and the absence of CPS is due to the absence of genes previously shown to be needed for CPS production in another strain. In addition to the head spike fiber, we found that the tail fiber protein also binds to the CPS.
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- 2023
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9. A physiological perspective on the origin and evolution of photosynthesis
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William F Martin, Donald A Bryant, and J Thomas Beatty
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- 2017
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10. DNA Gyrase Inhibitors Increase the Frequency of Bacteriophage-like RcGTA-Mediated Gene Transfer in
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Rachel, Bernelot-Moens and J Thomas, Beatty
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Bacterial Proteins ,Topoisomerase II Inhibitors ,Bacteriophages ,Gene Expression Regulation, Bacterial ,Rhodobacter capsulatus ,Novobiocin ,Ecosystem ,Anti-Bacterial Agents - Published
- 2022
11. Formal recognition and classification of gene transfer agents as viriforms
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Roman Kogay, Sonja Koppenhöfer, J Thomas Beatty, Jens H Kuhn, Andrew S Lang, and Olga Zhaxybayeva
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Virology ,Microbiology - Abstract
Morphological and genetic features strongly suggest that gene transfer agents (GTAs) are caudoviricete-derived entities that have evolved in concert with cellular genomes to such a degree that they should not be considered viruses. Indeed, GTA particles resemble caudoviricete virions, but, in contrast to caudoviricetes (or any viruses), GTAs can encapsidate at best only part of their own genomes, are induced solely in small subpopulations of prokaryotic host cells, and are transmitted vertically as part of cellular genomes during replication and division. Therefore, the lifecycles of GTAs are analogous to virus-derived entities found in the parasitoid wasps, which have recently been recognized as non-virus entities and therefore reclassified as viriforms. We evaluated three distinct, independently exapted GTA groups, for which the genetic basis for GTA particle production has been established. Based on the evidence, we outline a classification scheme for these viriforms.
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- 2022
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12. Intracytoplasmic-membrane development in alphaproteobacteria involves the homolog of the mitochondrial crista-developing protein Mic60
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Sergio A. Muñoz-Gómez, Lawrence Rudy Cadena, Alastair T. Gardiner, Michelle M. Leger, Shaghayegh Sheikh, Louise B. Connell, Tomáš Bilý, Karel Kopejtka, J. Thomas Beatty, Michal Koblížek, Andrew J. Roger, Claudio H. Slamovits, Julius Lukeš, and Hassan Hashimi
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General Agricultural and Biological Sciences ,General Biochemistry, Genetics and Molecular Biology - Published
- 2023
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13. The development of intracytoplasmic membranes in alphaproteobacteria involves the conserved mitochondrial crista-developing protein Mic60
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Sergio A. Muñoz-Gómez, Lawrence Rudy Cadena, Alastair T. Gardiner, Michelle M. Leger, Shaghayegh Sheikh, Louise Connell, Tomáš Bilý, Karel Kopejtka, J. Thomas Beatty, Michal Koblížek, Andrew J. Roger, Claudio H. Slamovits, Julius Lukeš, and Hassan Hashimi
- Abstract
Mitochondrial cristae expand the surface area of respiratory membranes and ultimately allow for the evolutionary scaling of respiration with cell volume across eukaryotes. The discovery of Mic60 homologs among alphaproteobacteria, the closest extant relatives of mitochondria, suggested that cristae might have evolved from bacterial intracytoplasmic membranes (ICMs). Here, we investigated the predicted structure and function of alphaproteobacterial Mic60, and a protein encoded by an adjacent gene Orf52, in two distantly related purple alphaproteobacteria,Rhodobacter sphaeroidesandRhodopseudomonas palustris. In addition, we assessed the potential physical interactors of Mic60 and Orf52 inR. sphaeroides. We show that the three α-helices of mitochondrial Mic60’s mitofilin domain, as well as its adjacent membrane-binding amphipathic helix, are present in alphaproteobacterial Mic60. The disruption of Mic60 and Orf52 caused photoheterotrophic growth defects, which are most severe under low light conditions, and both their disruption and overexpression led to enlarged ICMs in both studied alphaproteobacteria. We also found that alphaproteobacterial Mic60 physically interacts with BamA, the homolog of Sam50, one of the main physical interactors of eukaryotic Mic60. This interaction, responsible for making contact sites at mitochondrial envelopes, has been conserved in modern alphaproteobacteria despite more than a billion years of evolutionary divergence. Our results suggest a role for Mic60 in photosynthetic ICM development and contact site formation at alphaproteobacterial envelopes. Overall, we provide support for the hypothesis that mitochondrial cristae evolved from alphaproteobacterial ICMs, and therefore have improved our understanding of the nature of the mitochondrial ancestor.
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- 2022
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14. Structure and mechanism of DNA delivery of a gene transfer agent
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J. Thomas Beatty, T. Fuzik, Pavol Bárdy, D. Hrebik, Roman Pantůček, and Pavel Plevka
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0301 basic medicine ,DNA, Bacterial ,Gene Transfer, Horizontal ,Base pair ,viruses ,Science ,030106 microbiology ,General Physics and Astronomy ,Phage biology ,Siphoviridae ,Virus-host interactions ,General Biochemistry, Genetics and Molecular Biology ,Article ,Rhodobacter capsulatus ,03 medical and health sciences ,chemistry.chemical_compound ,Podoviridae ,Cryoelectron microscopy ,Bacteriophages ,lcsh:Science ,Gene ,Multidisciplinary ,Rhodobacter ,biology ,Gene Transfer Techniques ,General Chemistry ,Periplasmic space ,Gene Expression Regulation, Bacterial ,Virus structures ,biology.organism_classification ,Gene transfer agent ,030104 developmental biology ,chemistry ,Biophysics ,lcsh:Q ,DNA - Abstract
Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm., Gene transfer agents (GTAs) are phage-like particles that mediate lateral gene exchange. Here, the authors provide the structure of the GTA of Rhodobacter capsulatus (RcGTA), which resembles a tailed phage, and describe the conformational changes required for DNA ejection.
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- 2020
15. Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins
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Amita Mahey, Tomas Richardson-Sanchez, Rachel C. Fernandez, Daniel Jun, J. Thomas Beatty, and Michael E. P. Murphy
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0106 biological sciences ,Photosynthetic reaction centre ,0303 health sciences ,biology ,Operon ,Chemistry ,Chloroflexus aurantiacus ,Biomedical Engineering ,General Medicine ,biology.organism_classification ,medicine.disease_cause ,Photosynthesis ,7. Clean energy ,01 natural sciences ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Metabolic engineering ,03 medical and health sciences ,Rhodobacter sphaeroides ,Biochemistry ,010608 biotechnology ,medicine ,Escherichia coli ,Integral membrane protein ,030304 developmental biology - Abstract
Quinones are redox-active molecules that transport electrons and protons in organelles and cell membranes during respiration and photosynthesis. In addition to the fundamental importance of these processes in supporting life, there has been considerable interest in exploiting their mechanisms for diverse applications ranging from medical advances to innovative biotechnologies. Such applications include novel treatments to target pathogenic bacterial infections and fabricating biohybrid solar cells as an alternative renewable energy source. Ubiquinone (UQ) is the predominant charge-transfer mediator in both respiration and photosynthesis. Other quinones, such as menaquinone (MK), are additional or alternative redox mediators, for example in bacterial photosynthesis of species such as Thermochromatium tepidum and Chloroflexus aurantiacus. Rhodobacter sphaeroides has been used extensively to study electron transfer processes, and recently as a platform to produce integral membrane proteins from other species. To expand the diversity of redox mediators in R. sphaeroides, nine Escherichia coli genes encoding the synthesis of MK from chorismate and polyprenyl diphosphate were assembled into a synthetic operon in a newly designed expression plasmid. We show that the menFDHBCE, menI, menA, and ubiE genes are sufficient for MK synthesis when expressed in R. sphaeroides cells, on the basis of high performance liquid chromatography and mass spectrometry. The T. tepidum and C. aurantiacus photosynthetic reaction centers produced in R. sphaeroides were found to contain MK. We also measured in vitro charge recombination kinetics of the T. tepidum reaction center to demonstrate that the MK is redox-active and incorporated into the QA pocket of this heterologously expressed reaction center.
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- 2020
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16. Genomic diversity of bacteriophages infecting Rhodobacter capsulatus and their relatedness to its gene transfer agent RcGTA
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Maximiliano Garcia, Alexander B. Westbye, Mats Hansson, J. Thomas Beatty, Megan Dolan, Brenda Miller, Rebecca A. Garlena, Richard M Alvey, Daniel A. Russell, David Bollivar, Steven G. Cresawn, Matthew R. Bockman, and Jackson Rapala
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Science ,Microbial Genomics ,Viral Structure ,Biology ,Microbiology ,Biochemistry ,Genome ,Rhodobacter capsulatus ,Actinobacteria ,Bacteriophage ,Bacterial Proteins ,Gene Types ,Virology ,DNA-binding proteins ,Genetics ,Capsids ,Caudovirales ,Bacteriophages ,Gene Prediction ,Gene ,Viral Genomics ,Multidisciplinary ,Rhodobacter ,Phylum ,Gene Transfer Techniques ,Organisms ,Genetic Variation ,Biology and Life Sciences ,Proteins ,Computational Biology ,Gene Expression Regulation, Bacterial ,Genomics ,Genome Analysis ,biology.organism_classification ,Gene transfer agent ,Structural Genes ,Viruses ,Horizontal gene transfer ,Medicine ,Research Article - Abstract
The diversity of bacteriophages is likely unparalleled in the biome due to the immense variety of hosts and the multitude of viruses that infect them. Recent efforts have led to description at the genomic level of numerous bacteriophages that infect the Actinobacteria, but relatively little is known about those infecting other prokaryotic phyla, such as the purple non-sulfur photosynthetic α-proteobacterium Rhodobacter capsulatus. This species is a common inhabitant of freshwater ecosystems and has been an important model system for the study of photosynthesis. Additionally, it is notable for its utilization of a unique form of horizontal gene transfer via a bacteriophage-like element known as the gene transfer agent (RcGTA). Only three bacteriophages of R. capsulatus had been sequenced prior to this report. Isolation and characterization at the genomic level of 26 new bacteriophages infecting this host advances the understanding of bacteriophage diversity and the origins of RcGTA. These newly discovered isolates can be grouped along with three that were previously sequenced to form six clusters with four remaining as single representatives. These bacteriophages share genes with RcGTA that seem to be related to host recognition. One isolate was found to cause lysis of a marine bacterium when exposed to high-titer lysate. Although some clusters are more highly represented in the sequenced genomes, it is evident that many more bacteriophage types that infect R. capsulatus are likely to be found in the future.
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- 2021
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17. Highly Sensitive Method to Isolate Photocurrent Signals from Large Background Redox Currents on Protein‐Modified Electrodes
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Dan Bizzotto, J. Thomas Beatty, and Daniel Jun
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Photocurrent ,Photosynthetic reaction centre ,Electron transfer ,Chemistry ,Electrode ,Electrochemistry ,Photochemistry ,Redox ,Catalysis ,Highly sensitive - Published
- 2019
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18. Photosynthetic Reaction Centres Assembled on a Gold Electrode and the Photocurrent - Potential Response
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Daniel Jun, Adrian Grzedowski, J. Thomas Beatty, and Dan Bizzotto
- Abstract
The photosynthetic reaction centre (RC) from Rhodobacter sphaeroides has been studied for use in biohybrid solar cells. Much of the previous work has focussed on improving photocurrent generation by loading the electrode surface with many copies of the protein resulting in multilayers. The primary disadvantage with this approach is the random orientation of proteins, with some supposedly oriented properly. We used RCs with Cys for covalent attachment to a gold electrode and for proper orientation. Areas of bare electrode surface and RCs bound non-specifically (i.e. not bound via the Cys) were competitively displaced by an insulating, non-redox layer of mercaptohexanol (MCH). The adsorbed monolayer of RCs was imaged using atomic force microscopy to detail the distribution of RCs on the gold surface for surfaces prepared with different RC deposition concentrations. Photocurrents were measured for all RC modified surfaces using a LED modulation method which enabled measurement of photocurrent in the presence of large faradaic currents from the sacrificial reactant (hydroquinone) at a variety of applied potentials.[1] The photocurrents generated from a monolayer composed of RCs and MCH resulted in consistent photocurrent currents. which enabled modeling of the photocurrent generation using the Marcus-Hush-Chidsey theory to extract a reorganization energy for this process. Multilayers of adsorbed RCs were distinctly different and revealed that the local environment in which the RCs are embedded significantly influenced photocurrent generation. [1] Jun, D.; Beatty, J. T.; Bizzotto, D. Highly Sensitive Method to Isolate Photocurrent Signals From Large Background Redox Currents on Protein‐Modified Electrodes. ChemElectroChem 2019, 6 (11), 2870–2875.
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- 2022
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19. Correlating structural assemblies of photosynthetic reaction centers on a gold electrode and the photocurrent - potential response
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J. Thomas Beatty, Adrian Jan Grzędowski, Dan Bizzotto, Amita Mahey, Daniel Jun, and Sylvester Zhang
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0301 basic medicine ,Photosynthetic reaction centre ,Materials science ,Science ,02 engineering and technology ,Electrolyte ,Electrochemistry ,7. Clean energy ,Article ,03 medical and health sciences ,Electron transfer ,Monolayer ,Photocurrent ,Multidisciplinary ,Biophotovoltaic ,business.industry ,021001 nanoscience & nanotechnology ,Surface chemistry ,3. Good health ,Chemistry ,Surface science ,030104 developmental biology ,Electrode ,Optoelectronics ,0210 nano-technology ,business - Abstract
Summary The use of biomacromolecules is a nascent development in clean alternative energies. In applications of biosensors and biophotovoltaic devices, the bacterial photosynthetic reaction center (RC) is a protein-pigment complex that has been commonly interfaced with electrodes, in large part to take advantage of the long-lived and high efficiency of charge separation. We investigated assemblies of RCs on an electrode that range from monolayer to multilayers by measuring the photocurrent produced when illuminated by an intensity-modulated excitation light source. In addition, atomic force microscopy and modeling of the photocurrent with the Marcus-Hush-Chidsey theory detailed the reorganization energy for the electron transfer process, which also revealed changes in the RC local environment due to the adsorbed conformations. The local environment in which the RCs are embedded significantly influenced photocurrent generation, which has implications for electron transfer of other biomacromolecules deposited on a surface in sensor and photovoltaic applications employing a redox electrolyte., Graphical abstract, Highlights • Controlled the formation of monolayer or multilayers of RCs on a gold electrode • Correlated electrochemical and photocurrent responses to the RC adsorbed conformations measured using AFM • Adsorbed RC conformations affect photocurrent generation • Photocurrent-potential response influenced by local redox mediator concentration and pH environment experienced by the adsorbed RCs, Chemistry; Electrochemistry; Surface chemistry; Surface science
- Published
- 2021
20. The CckA-ChpT-CtrA Phosphorelay Controlling Rhodobacter capsulatus Gene Transfer Agent Production Is Bidirectional and Regulated by Cyclic di-GMP
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J. Thomas Beatty, Purvikalyan Pallegar, Andrew S. Lang, Reynold G. Farrera-Calderon, Christina L. Wiesmann, and Alexander B. Westbye
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Cyclic di-GMP ,Gene Transfer, Horizontal ,Histidine Kinase ,Phosphatase ,Biology ,Microbiology ,Rhodobacter capsulatus ,Phosphotransferase ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Protein phosphorylation ,Phosphorylation ,Cyclic GMP ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Rhodobacter ,030306 microbiology ,Phosphotransferases ,Autophosphorylation ,Histidine kinase ,Gene Transfer Techniques ,biology.organism_classification ,Recombinant Proteins ,Cell biology ,Response regulator ,Amino Acid Substitution ,chemistry ,hormones, hormone substitutes, and hormone antagonists ,Research Article ,Signal Transduction ,Transcription Factors - Abstract
Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus, these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads. IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus. This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphotransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.
- Published
- 2021
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21. Bio-Phototransistors with Immobilized Photosynthetic Proteins
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J. Thomas Beatty, Arash Takshi, Daniel Jun, and Houman Yaghoubi
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Photosynthetic reaction centre ,Absorption spectroscopy ,Computer Networks and Communications ,Photodetector ,lcsh:TK7800-8360 ,Electron donor ,02 engineering and technology ,010402 general chemistry ,Photosynthesis ,Photochemistry ,01 natural sciences ,bio-phototransistor ,Rhodobacter sphaeroides ,chemistry.chemical_compound ,rhodobacter sphaeroides ,Electrical and Electronic Engineering ,biology ,Chemistry ,reaction center ,lcsh:Electronics ,021001 nanoscience & nanotechnology ,biology.organism_classification ,0104 chemical sciences ,Hardware and Architecture ,Control and Systems Engineering ,Signal Processing ,Photosynthetic bacteria ,ISFET ,0210 nano-technology - Abstract
The efficient mechanism of light capture by photosynthetic proteins allows for energy transfer and conversion to electrochemical energy at very low light intensities. In this work, reaction center (RC) proteins, or a core complex consisting of the RC encircled by light harvesting (LH1) proteins (RC-LH1) from photosynthetic bacteria, were immobilized on an insulating layer of an ion-sensitive field-effect transistor (ISFET) to build bio-photodetectors. The orientation of the RC proteins was controlled via application of a hybrid linker made of 10-carboxydecylphosphonic acid and cytochrome c that anchored the RCs to their electron donor side. Bio-phototransistors consisting of either the core RC or the RC-LH1 core complex were tested under white and monochromic light. The difference between the dark and light currents at different wavelengths are well-matched with the absorption spectrum of the photosynthetic proteins. The results show potential for the use of photosynthetic proteins in photodetectors.
- Published
- 2020
22. Introduction of the Menaquinone Biosynthetic Pathway into
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Daniel, Jun, Tomas, Richardson-Sanchez, Amita, Mahey, Michael E P, Murphy, Rachel C, Fernandez, and J Thomas, Beatty
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Electron Transport ,Kinetics ,Spectrometry, Mass, Electrospray Ionization ,Photobleaching ,Bacterial Proteins ,Metabolic Engineering ,Ubiquinone ,Photosynthetic Reaction Center Complex Proteins ,Membrane Proteins ,Vitamin K 2 ,Rhodobacter sphaeroides ,Chromatography, High Pressure Liquid ,Plasmids - Abstract
Quinones are redox-active molecules that transport electrons and protons in organelles and cell membranes during respiration and photosynthesis. In addition to the fundamental importance of these processes in supporting life, there has been considerable interest in exploiting their mechanisms for diverse applications ranging from medical advances to innovative biotechnologies. Such applications include novel treatments to target pathogenic bacterial infections and fabricating biohybrid solar cells as an alternative renewable energy source. Ubiquinone (UQ) is the predominant charge-transfer mediator in both respiration and photosynthesis. Other quinones, such as menaquinone (MK), are additional or alternative redox mediators, for example in bacterial photosynthesis of species such as
- Published
- 2020
23. A physiological perspective on the origin and evolution of photosynthesis
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Donald A. Bryant, William Martin, and J. Thomas Beatty
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0301 basic medicine ,Photosynthetic reaction centre ,Zn-tetrapyrroles ,030106 microbiology ,Review Article ,Biology ,Cyanobacteria ,Photosynthesis ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,hydrothermal light ,Botany ,Autotroph ,Ferredoxin ,photothiotrophy ,Phototroph ,Carbon fixation ,Pigments, Biological ,15. Life on land ,Archaea ,Biological Evolution ,lateral gene transfer ,Anoxygenic photosynthesis ,reaction center evolution ,Infectious Diseases ,chemistry ,Chlorophyll - Abstract
The origin and early evolution of photosynthesis are reviewed from an ecophysiological perspective. Earth's first ecosystems were chemotrophic, fueled by geological H2 at hydrothermal vents and, required flavin-based electron bifurcation to reduce ferredoxin for CO2 fixation. Chlorophyll-based phototrophy (chlorophototrophy) allowed autotrophs to generate reduced ferredoxin without electron bifurcation, providing them access to reductants other than H2. Because high-intensity, short-wavelength electromagnetic radiation at Earth's surface would have been damaging for the first chlorophyll (Chl)-containing cells, photosynthesis probably arose at hydrothermal vents under low-intensity, long-wavelength geothermal light. The first photochemically active pigments were possibly Zn-tetrapyrroles. We suggest that (i) after the evolution of red-absorbing Chl-like pigments, the first light-driven electron transport chains reduced ferredoxin via a type-1 reaction center (RC) progenitor with electrons from H2S; (ii) photothioautotrophy, first with one RC and then with two, was the bridge between H2-dependent chemolithoautotrophy and water-splitting photosynthesis; (iii) photothiotrophy sustained primary production in the photic zone of Archean oceans; (iv) photosynthesis arose in an anoxygenic cyanobacterial progenitor; (v) Chl a is the ancestral Chl; and (vi), anoxygenic chlorophototrophic lineages characterized so far acquired, by horizontal gene transfer, RCs and Chl biosynthesis with or without autotrophy, from the architects of chlorophototrophy—the cyanobacterial lineage., Questions of how and where chlorophyll-based photosynthesis (chlorophototrophy) arose and how the process subsequently spread among bacteria are typically investigated using phylogenetic trees, but in prokaryotes horizontal gene transfer decouples physiology from phylogeny; here we address the evolution of photosynthesis not from the perspective of gene or lineage phylogenies, but from the physiological perspective of chemical processes.
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- 2017
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24. The Rhodobacter capsulatus gene transfer agent is induced by nutrient depletion and the RNAP omega subunit
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Alexander B. Westbye, Zoe R. O’Neill, Tegan Schellenberg-Beaver, and J. Thomas Beatty
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0301 basic medicine ,Gene Transfer, Horizontal ,Protein subunit ,030106 microbiology ,medicine.disease_cause ,Microbiology ,Rhodobacter capsulatus ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,RNA polymerase ,medicine ,Amino Acids ,Promoter Regions, Genetic ,Amino acid synthesis ,chemistry.chemical_classification ,Regulation of gene expression ,Mutation ,Rhodobacter ,biology ,DNA-Directed RNA Polymerases ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,Carbon ,Gene transfer agent ,Biochemistry ,chemistry ,DNA ,Plasmids - Abstract
Small bacteriophage-like particles called gene transfer agents (GTAs) that mediate DNA transfer between cells are produced by a variety of prokaryotes. The model GTA, produced by the alphaproteobacterium Rhodobacter capsulatus (RcGTA), is controlled by several cellular regulators, and production is induced upon entry into the stationary phase. We report that RcGTA production and gene transfer are stimulated by nutrient depletion. Cells depleted of organic carbon or blocked for amino acid biosynthesis increased RcGTA production and release from cells. Furthermore, cells lacking the sole RelA-SpoT homologue produced decreased levels of RcGTA, and the RNA polymerase omega (ω) subunit was required for appreciable production of RcGTA.
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- 2017
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25. Induction of Rhodobacter capsulatus Gene Transfer Agent Gene Expression Is a Bistable Stochastic Process Repressed by an Extracellular Calcium-Binding RTX Protein Homologue
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Andrew S. Lang, Marc P. Grüll, J. Thomas Beatty, Martin E. Mulligan, and Hao Ding
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0303 health sciences ,Rhodobacter ,biology ,Cell division ,030306 microbiology ,Bacterial genome size ,biology.organism_classification ,Microbiology ,Gene transfer agent ,Cell biology ,03 medical and health sciences ,Quorum sensing ,Horizontal gene transfer ,Gene expression ,Molecular Biology ,Gene ,030304 developmental biology - Abstract
Bacteriophage-like gene transfer agents (GTAs) have been discovered in both of the prokaryotic branches of the three-domain phylogenetic tree of life. The production of a GTA (RcGTA) by the phototrophic alphaproteobacterium Rhodobacter capsulatus is regulated by quorum sensing and a phosphorelay homologous to systems in other species that control essential functions such as the initiation of chromosome replication and cell division. In wild-type strains, RcGTA is produced in IMPORTANCE GTAs catalyze horizontal gene transfer (HGT), which is important for genomic evolution because the majority of genes found in bacterial genomes have undergone HGT at some point in their evolution. Therefore, it is important to determine how the production of GTAs is regulated to understand the factors that modulate the frequency of gene transfer and thereby specify the tempo of evolution. This work describes a new type of genetic regulation in which an extracellular calcium-binding protein homologue represses the induction of the Rhodobacter capsulatus GTA, RcGTA.
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- 2019
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26. Ion-sensitive field-effect transistors with Si3N4 and TaO2 gate insulator for studying self-assembly of photosynthetic proteins
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Houman Yaghoubi, Fatemeh Khorramshahi, Daniel Jun, Arash Takshi, and J. Thomas Beatty
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Materials science ,business.industry ,Ion sensitive ,Gate insulator ,Optoelectronics ,Field-effect transistor ,Self-assembly ,Photosynthesis ,business - Published
- 2019
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27. Induction of
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Hao, Ding, Marc P, Grüll, Martin E, Mulligan, Andrew S, Lang, and J Thomas, Beatty
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Stochastic Processes ,Gene Transfer, Horizontal ,Whole Genome Sequencing ,Calcium-Binding Proteins ,Quorum Sensing ,Gene Expression Regulation, Bacterial ,Rhodobacter capsulatus ,Luminescent Proteins ,Bacterial Proteins ,Genes, Reporter ,Mutagenesis ,Mutation ,DNA Transposable Elements ,Escherichia coli ,Calcium ,Cell Division ,Phylogeny ,Plasmids ,Research Article - Abstract
Bacteriophage-like gene transfer agents (GTAs) have been discovered in both of the prokaryotic branches of the three-domain phylogenetic tree of life. The production of a GTA (RcGTA) by the phototrophic alphaproteobacterium Rhodobacter capsulatus is regulated by quorum sensing and a phosphorelay homologous to systems in other species that control essential functions such as the initiation of chromosome replication and cell division. In wild-type strains, RcGTA is produced in
- Published
- 2019
28. Electron Transfer in Bacterial Reaction Centers with the Photoactive Bacteriopheophytin Replaced by a Bacteriochlorophyll through Coordinating Ligand Substitution
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Jie Pan, Rafael G. Saer, Neal W. Woodbury, Su Lin, and J. Thomas Beatty
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0301 basic medicine ,Photosynthetic reaction centre ,Stereochemistry ,Photosynthetic Reaction Center Complex Proteins ,Ligands ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Electron Transport ,03 medical and health sciences ,Rhodobacter sphaeroides ,chemistry.chemical_compound ,Residue (chemistry) ,Electron transfer ,Bacteriochlorophylls ,Histidine ,chemistry.chemical_classification ,biology ,Chemistry ,Pheophytins ,biology.organism_classification ,Electron transport chain ,0104 chemical sciences ,Amino acid ,Kinetics ,030104 developmental biology ,Bacteriochlorophyll - Abstract
The influence of amino acid substitutions at position M214 (M-subunit, residue 214) on the rate and pathway of electron transfer involving the bacteriopheophytin cofactor, HA, in a bacterial photosynthetic reaction center has been explored in a series of Rhodobacter sphaeroides mutants. The M214 leucine (L) residue of the wild type was replaced with histidine (H), glutamine (Q), and asparagine (N), creating the mutants M214LH, M214LQ, and M214LN, respectively. As has been reported previously for M214LH, each of these mutations resulted in a bacteriochlorophyll molecule in place of a bacteriopheophytin in the HA pocket, forming so-called β-type mutants (in which the HA cofactor is called βA). In addition, these mutations changed the properties of the surrounding protein environment in terms of charge distribution and the amino acid side chain volume. Electron transfer reactions from the excited primary donor P to the acceptor QA were characterized using ultrafast transient absorption spectroscopic techniques. Similar to that of the previously characterized M214LH (β mutant), the strong energetic mixing of the P(+)BA(-) and P(+)βA(-) states (the mixed anion is denoted I(-)) increased the rate of charge recombination between P(+) and I(-) in competition with the I(-) → QA forward reaction. This reduced the overall yield of charge separation forming the P(+)QA(-) state. While the kinetics of the primary electron transfer forming P(+)I(-) were essentially identical in all three β mutants, the rates of the βA(-) (I(-)) → QA electron transfer in M214LQ and M214LH were very similar but quite different from that of the M214LN mutant. The observed yield changes and the differences in kinetics are correlated more closely with the volume of the mutated amino acid than with their charge characteristics. These results are consistent with those of previous studies of a series of M214 mutants with different sizes of amino acid side chains that did not alter the HA cofactor composition [Pan, J., et al. (2013) J. Phys. Chem. B 117, 7179-7189]. Both studies indicate that protein relaxation in this region of the reaction center plays a key role in stabilizing charge-separated states involving the HA or βA cofactor. The effect is particularly pronounced for reactions occurring on time scales of tens and hundreds of picoseconds (forward transfer to the QA and charge recombination).
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- 2016
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29. The SOS Response Master Regulator LexA Regulates the Gene Transfer Agent of Rhodobacter capsulatus and Represses Transcription of the Signal Transduction Protein CckA
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Cedric A. Brimacombe, Kevin S. Kuchinski, Hao Ding, J. Thomas Beatty, and Alexander B. Westbye
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0301 basic medicine ,Transcription, Genetic ,Molecular Sequence Data ,030106 microbiology ,Mutant ,Microbiology ,Rhodobacter capsulatus ,03 medical and health sciences ,Transduction (genetics) ,Bacterial Proteins ,Phosphorylation ,SOS response ,SOS Response, Genetics ,Molecular Biology ,Prophage ,Regulation of gene expression ,Genetics ,Rhodobacter ,Base Sequence ,biology ,Serine Endopeptidases ,Gene Expression Regulation, Bacterial ,Articles ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Gene transfer agent ,enzymes and coenzymes (carbohydrates) ,Mutation ,bacteria ,Repressor lexA ,Signal Transduction - Abstract
The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a genetic exchange element that combines central aspects of bacteriophage-mediated transduction and natural transformation. RcGTA particles resemble a small double-stranded DNA bacteriophage, package random ∼4-kb fragments of the producing cell genome, and are released from a subpopulation (R. capsulatus cells, and the DNA is integrated into the recipient genome though a process that requires homologs of natural transformation genes and RecA-mediated homologous recombination. Here, we report the identification of the LexA repressor, the master regulator of the SOS response in many bacteria, as a regulator of RcGTA activity. Deletion of the lexA gene resulted in the abolition of detectable RcGTA production and an ∼10-fold reduction in recipient capability. A search for SOS box sequences in the R. capsulatus genome sequence identified a number of putative binding sites located 5′ of typical SOS response coding sequences and also 5′ of the RcGTA regulatory gene cckA , which encodes a hybrid histidine kinase homolog. Expression of cckA was increased >5-fold in the lexA mutant, and a lexA cckA double mutant was found to have the same phenotype as a Δ cckA single mutant in terms of RcGTA production. The data indicate that LexA is required for RcGTA production and maximal recipient capability and that the RcGTA-deficient phenotype of the lexA mutant is largely due to the overexpression of cckA . IMPORTANCE This work describes an unusual phenotype of a lexA mutant of the alphaproteobacterium Rhodobacter capsulatus in respect to the phage transduction-like genetic exchange carried out by the R. capsulatus gene transfer agent (RcGTA). Instead of the expected SOS response characteristic of prophage induction, this lexA mutation not only abolishes the production of RcGTA particles but also impairs the ability of cells to receive RcGTA-borne genes. The data show that, despite an apparent evolutionary relationship to lambdoid phages, the regulation of RcGTA gene expression differs radically.
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- 2016
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30. Guest editorial: Mobile genetic elements and horizontal gene transfer in prokaryotes
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J. Thomas Beatty, Andrew S. Lang, and Phoebe A. Rice
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0301 basic medicine ,Microbiology (medical) ,Gene Transfer, Horizontal ,Genomic Islands ,Computational biology ,Microbiology ,Genes, Archaeal ,Bacterial genetics ,03 medical and health sciences ,Plasmid ,Gene ,Bacteria ,biology ,Interspersed Repetitive Sequences ,biology.organism_classification ,Archaea ,030104 developmental biology ,Infectious Diseases ,Genes, Bacterial ,Horizontal gene transfer ,DNA Transposable Elements ,Mobile genetic elements ,Plasmids - Published
- 2017
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31. The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution
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John S. Klassen, Franck Duong, Lucien Fabre, Jun Li, Zhiyu Zhao, Irvin Wason, Daniel Jun, Jianing Li, Isabelle Rouiller, John W. Young, Allan Mills, Michael Carlson, Harveer Singh Dhupar, and J. Thomas Beatty
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0301 basic medicine ,Scaffold protein ,QH301-705.5 ,Science ,Structural Biology and Molecular Biophysics ,Detergents ,Porins ,Peptide ,Micelle ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Biochemistry and Chemical Biology ,structural biology ,membrane protein ,Biology (General) ,Nanodisc ,Micelles ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,General Immunology and Microbiology ,General Neuroscience ,Escherichia coli Proteins ,E. coli ,Membrane Proteins ,Water ,General Medicine ,self-assembly ,Lipids ,peptide ,Tools and Resources ,030104 developmental biology ,Structural biology ,Membrane protein ,chemistry ,Solubility ,Biophysics ,Medicine ,nanoparticles ,ATP-Binding Cassette Transporters ,Target protein ,Peptides ,SEC Translocation Channels ,Bacterial Outer Membrane Proteins - Abstract
Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this ‘one size fits all’ method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during ‘on-column’, ‘in-gel’, and ‘on-bead’ reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies., eLife digest Surrounding every living cell is a biological membrane that is largely impermeable to water-soluble molecules. This hydrophobic (or “water-hating”) barrier preserves the contents of the cell and also regulates how the cell interacts with its environment. This latter function is critical and relies on a class of proteins that are embedded within the membrane and are also hydrophobic. The hydrophobic nature of membrane proteins is however inconvenient for biochemical studies which usually take place in water-based solutions. Therefore, membrane proteins are under-represented in biological research compared to the water-soluble ones, even though roughly one quarter of a cell’s proteins are membrane proteins. Researchers have developed a few tricks to keep membrane proteins soluble after they have been extracted from the membrane. An old but popular technique makes use of detergents, which are chemicals with opposing hydrophobic and hydrophilic properties (hydrophilic literally means “water-loving”). However, even mild detergents can damage membrane proteins and will sometimes lead to experimental artifacts. More recent tricks to stabilize membrane proteins without detergents have been described but remain laborious, costly or difficult to perform. To overcome these limitations, Carlson et al. developed a simple method to stabilize membrane proteins without detergent. Called the “peptidisc”, the method uses multiple copies of a unique peptide – a short sequence of the building blocks of protein – that had been redesigned to have optimal hydrophobic and hydrophilic properties. The idea was that the peptides would wrap around the hydrophobic parts of the membrane protein, and shield them from the watery solution. Indeed, when Carlson et al. mixed this peptide with five different membrane proteins from bacteria, all were perfectly soluble and functional without detergent. The ideal ratio of peptide needed to form a peptidisc around each membrane protein was reached automatically, without having to test many different conditions. This indicates that the peptidisc acts like a “one size fits all” scaffold. The peptidisc is a new tool that will allow more researchers, including those who are not expert biochemists, to study membrane proteins. This will yield a better understanding of the structure of a cell’s membrane and how it interacts with the environment. Since the approach is both simple and easy to apply, more membrane proteins can now also be included in high-throughput searches for potential new drugs for various medical conditions.
- Published
- 2018
32. The Protease ClpXP and the PAS Domain Protein DivL Regulate CtrA and Gene Transfer Agent Production in Rhodobacter capsulatus
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Hao Ding, Alexander B. Westbye, J. Thomas Beatty, Lukas Kater, Christina L. Wiesmann, and Calvin K. Yip
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0301 basic medicine ,Gene Transfer, Horizontal ,030106 microbiology ,Genetics and Molecular Biology ,Applied Microbiology and Biotechnology ,Rhodobacter capsulatus ,03 medical and health sciences ,Bacterial Proteins ,Protein Domains ,PAS domain ,Phosphorylation ,Kinase activity ,Rhodobacter ,Ecology ,biology ,Caulobacter crescentus ,Histidine kinase ,Endopeptidase Clp ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,Gene transfer agent ,Cell biology ,Rhodobacterales ,Response regulator ,Food Science ,Biotechnology - Abstract
Several members of the Rhodobacterales ( Alphaproteobacteria ) produce a conserved horizontal gene transfer vector, called the gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is the Rhodobacter capsulatus GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the Alphaproteobacteria and is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production in R. capsulatus . We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in R. capsulatus and identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the Rhodobacterales . IMPORTANCE Members of the Rhodobacterales are abundant in ocean and freshwater environments. The conserved GTA produced by many Rhodobacterales may have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA in R. capsulatus extends the model of CtrA regulation from Caulobacter crescentus to a member of the Rhodobacterales . We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.
- Published
- 2018
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33. Author response: The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution
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Isabelle Rouiller, Michael Carlson, Jun Li, Irvin Wason, Harveer Singh Dhupar, Allan Mills, Franck Duong, Zhiyu Zhao, Lucien Fabre, Jianing Li, John W. Young, John S. Klassen, Daniel Jun, and J. Thomas Beatty
- Subjects
Membrane protein ,Simple (abstract algebra) ,Chemistry ,Biophysics ,Free solution - Published
- 2018
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34. Applications of ZnO Nanowires as Electrode Materials in Photosynthetic Bio-Photoelectrochemical Cells
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J. Thomas Beatty, Khan, Houman Yaghoubi, Anand Kumar Santhanakrishn, and Arash Takshi
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Photocurrent ,Auxiliary electrode ,Materials science ,Working electrode ,Chemical engineering ,law ,Open-circuit voltage ,Electrode ,Solar cell ,Photoelectrochemical cell ,Electrochemistry ,law.invention - Abstract
Harvesting solar energy, is only one of the incentives of incorporating photosynthetic proteins in electrochemical devices. Understanding the interface of photosynthetic protein complexes and organic\inorganic underlying electrodes can give rise to development of new generation of nano-bioelectronics for other applications such as sensing, as well. Previous approaches in fabricating photosynthetic bio-hybrid electrochemical solar cells were mainly based on metallic electrodes with protein complexes attached, either directly or through linker molecules. Due to the energy band structure in semiconductors, they potentially can be useful for selective charge transfer in an electrochemical device. In the current study, a two terminal sealed bio-hybrid solar cell device was fabricated comprising of hydrothermally grown ZnO nanowires on fluorine doped tin oxide (FTO) glass working electrode, a Pt counter electrode, and methyl viologen (MV) as a single diffusible redox mediator. The ZnO working electrode was initially characterized using scanning electron microscopy (XRD) and X-ray diffraction (XRD). A solution of dimeric Rhodobacter sphaeroides – light harvesting 1 (RC-LH1) core complexes and redox electrolyte was injected into the cavity between working and counter electrodes. Such structure resulted in ∼0.64 µA.cm-2 photocurrent density and ∼0.24 V open circuit potential difference in the dark and under illumination. Additionally, the device stability tests demonstrated that the current response of such devices remained unchanged after 33 hours storage in the dark.
- Published
- 2015
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35. Electrochemical Field-Effect Transistor Utilization to Study the Coupling Success Rate of Photosynthetic Protein Complexes to Cytochrome c
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J. Thomas Beatty, Arash Takshi, Houman Yaghoubi, Jing Wang, and Daniel Jun
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Photosynthetic reaction centre ,Cytochrome ,Transistors, Electronic ,Clinical Biochemistry ,Photosynthetic Reaction Center Complex Proteins ,02 engineering and technology ,010402 general chemistry ,Photochemistry ,7. Clean energy ,01 natural sciences ,Article ,law.invention ,protein-protein interaction ,Electron transfer ,law ,rhodobacter sphaeroides ,biology ,bio-photosensor ,business.industry ,Chemistry ,Cytochrome c ,reaction center ,Transistor ,charge density ,Cytochromes c ,General Medicine ,Electrochemical Techniques ,021001 nanoscience & nanotechnology ,Electron transport chain ,0104 chemical sciences ,Threshold voltage ,biology.protein ,Optoelectronics ,Field-effect transistor ,0210 nano-technology ,business ,Protein Binding - Abstract
Due to the high internal quantum efficiency, reaction center (RC) proteins from photosynthetic organisms have been studied in various bio-photoelectrochemical devices for solar energy harvesting. In vivo, RC and cytochrome c (cyt c; a component of the biological electron transport chain) can form a cocomplex via interprotein docking. This mechanism can be used in vitro for efficient electron transfer from an electrode to the RC in a bio-photoelectrochemical device. Hence, the success rate in coupling RCs to cyt c is of great importance for practical applications in the future. In this work, we use an electrochemical transistor to study the binding of the RC to cytochrome. The shift in the transistor threshold voltage was measured in the dark and under illumination to estimate the density of cytochrome and coupled RCs on the gate of the transistor. The results show that ~33% of the cyt cs on the transistor gate were able to effectively couple with RCs. Due to the high sensitivity of the transistor, the approach can be used to make photosensors for detecting low light intensities.
- Published
- 2017
36. Guaranteeing a captive audience: coordinated regulation of gene transfer agent (GTA) production and recipient capability by cellular regulators
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Alexander B. Westbye, Andrew S. Lang, and J. Thomas Beatty
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0301 basic medicine ,Microbiology (medical) ,Gene Transfer, Horizontal ,030106 microbiology ,Bacterial genome size ,Computational biology ,Microbiology ,Rhodobacter capsulatus ,03 medical and health sciences ,Transduction (genetics) ,chemistry.chemical_compound ,Biological Factors ,Transduction, Genetic ,Gene ,Rhodobacter ,biology ,business.industry ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,DNA Transformation Competence ,Gene transfer agent ,Biotechnology ,Quorum sensing ,Response regulator ,Infectious Diseases ,chemistry ,Transformation, Bacterial ,business ,DNA - Abstract
Gene transfer agents (GTAs) are bacteriophage-like particles produced by many prokaryotes. Several members of the Alphaproteobacteria produce a class of genetically-related GTAs that is best studied in Rhodobacter capsulatus. DNA transfer by the R. capsulatus GTA (RcGTA) combines aspects of both transduction and natural transformation, as recipient cells require a natural transformation-like system to incorporate donated DNA. The genes involved in RcGTA production and recipient capability are located at multiple loci in the bacterial genome; however, a conserved phosphorelay containing the response regulator CtrA and a quorum sensing system regulate both RcGTA production and recipient capability. This review highlights recent discoveries in RcGTA biology, and focuses on the co-regulation of genes involved in RcGTA production and recipient capability.
- Published
- 2017
37. A DNA-Directed Light-Harvesting/Reaction Center System
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Su Lin, J. Thomas Beatty, Symon Levenberg, Neal W. Woodbury, Palash K. Dutta, Yan Liu, Andrey Loskutov, Daniel Jun, Rafael G. Saer, and Hao Yan
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Models, Molecular ,Photosynthetic reaction centre ,Nanostructure ,Photosynthetic Reaction Center Complex Proteins ,Rhodobacter sphaeroides ,Conjugated system ,Photochemistry ,Biochemistry ,Catalysis ,Colloid and Surface Chemistry ,Molecule ,A-DNA ,Coloring Agents ,biology ,business.industry ,Chemistry ,DNA ,General Chemistry ,biology.organism_classification ,Nanostructures ,Energy Transfer ,Optoelectronics ,Antenna (radio) ,Photonics ,business - Abstract
A structurally and compositionally well-defined and spectrally tunable artificial light-harvesting system has been constructed in which multiple organic dyes attached to a three-arm-DNA nanostructure serve as an antenna conjugated to a photosynthetic reaction center isolated from Rhodobacter sphaeroides 2.4.1. The light energy absorbed by the dye molecules is transferred to the reaction center, where charge separation takes place. The average number of DNA three-arm junctions per reaction center was tuned from 0.75 to 2.35. This DNA-templated multichromophore system serves as a modular light-harvesting antenna that is capable of being optimized for its spectral properties, energy transfer efficiency, and photostability, allowing one to adjust both the size and spectrum of the resulting structures. This may serve as a useful test bed for developing nanostructured photonic systems.
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- 2014
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38. Illuminating Microbial Dark Matter in Meromictic Sakinaw Lake
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Steven J. Hallam, Kishori M. Konwar, J. Thomas Beatty, and Esther A. Gies
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Molecular Sequence Data ,DNA, Ribosomal ,Applied Microbiology and Biotechnology ,Microbial Ecology ,Phylogenetics ,Cluster Analysis ,Phylogeny ,Bacteria ,British Columbia ,Ecology ,biology ,Phylum ,Lake ecosystem ,Community structure ,Eukaryota ,Sequence Analysis, DNA ,biology.organism_classification ,Archaea ,Biota ,Lakes ,Chloroflexi (class) ,Metagenomics ,Pyrosequencing ,Food Science ,Biotechnology - Abstract
Despite recent advances in metagenomic and single-cell genomic sequencing to investigate uncultivated microbial diversity and metabolic potential, fundamental questions related to population structure, interactions, and biogeochemical roles of candidate divisions remain. Numerous molecular surveys suggest that stratified ecosystems manifesting anoxic, sulfidic, and/or methane-rich conditions are enriched in these enigmatic microbes. Here we describe diversity, abundance, and cooccurrence patterns of uncultivated microbial communities inhabiting the permanently stratified waters of meromictic Sakinaw Lake, British Columbia, Canada, using 454 sequencing of the small-subunit rRNA gene with three-domain resolution. Operational taxonomic units (OTUs) were affiliated with 64 phyla, including more than 25 candidate divisions. Pronounced trends in community structure were observed for all three domains with eukaryotic sequences vanishing almost completely below the mixolimnion, followed by a rapid and sustained increase in methanogen-affiliated (∼10%) and unassigned (∼60%) archaeal sequences as well as bacterial OTUs affiliated with Chloroflexi (∼22%) and candidate divisions (∼28%). Network analysis revealed highly correlated, depth-dependent cooccurrence patterns between Chloroflexi , candidate divisions WWE1, OP9/JS1, OP8, and OD1, methanogens, and unassigned archaeal OTUs indicating niche partitioning and putative syntrophic growth modes. Indeed, pathway reconstruction using recently published Sakinaw Lake single-cell genomes affiliated with OP9/JS1 and OP8 revealed complete coverage of the Wood-Ljungdahl pathway with potential to drive syntrophic acetate oxidation to hydrogen and carbon dioxide under methanogenic conditions. Taken together, these observations point to previously unrecognized syntrophic networks in meromictic lake ecosystems with the potential to inform design and operation of anaerobic methanogenic bioreactors.
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- 2014
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39. Hybrid Wiring of the Rhodobacter sphaeroides Reaction Center for Applications in Bio-photoelectrochemical Solar Cells
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Zhi Li, J. Thomas Beatty, Rudy Schlaf, Arash Takshi, Houman Yaghoubi, Xiaomei Jiang, Evan Lafalce, and Daniel Jun
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Photosynthetic reaction centre ,Working electrode ,biology ,Chemistry ,biology.organism_classification ,Photochemistry ,Electrochemistry ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Electrochemical cell ,Electron transfer ,Rhodobacter sphaeroides ,General Energy ,Electrode ,Monolayer ,Physical and Theoretical Chemistry - Abstract
The growing demand for nonfossil fuel-based energy production has drawn attention to the utilization of natural proteins such as photosynthetic reaction center (RC) protein complexes to harvest solar energy. The current study reports on an immobilization method to bind the wild type Rhodobacter sphaeroides RC from the primary donor side onto a Au electrode using an immobilized cytochrome c (cyt c) protein via a docking mechanism. The new structure has been assembled on a Au electrode by layer-by-layer deposition of a carboxylic acid-terminated alkanethiol (HOOC (CH2)5S) self-assembled monolayer (SAM), and layers of cyt c and RC. The Au|SAM|cyt c|RC working electrode was applied in a three-probe electrochemical cell where a peak cathodic photocurrent density of 0.5 μA cm–2 was achieved. Further electrochemical study of the Au|SAM|cyt c|RC structure demonstrated ∼70% RC surface coverage. To understand the limitations in the electron transfer through the linker structure, a detailed energy study of the SAM a...
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- 2014
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40. Rhodobacter capsulatus DprA is essential for RecA-mediated gene transfer agent (RcGTA) recipient capability regulated by quorum-sensing and the CtrA response regulator
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J. Thomas Beatty, Hao Ding, and Cedric A. Brimacombe
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Genetics ,0303 health sciences ,education.field_of_study ,Rhodobacter ,biology ,030306 microbiology ,Population ,biology.organism_classification ,medicine.disease_cause ,Microbiology ,Gene transfer agent ,Homologous Recombination Pathway ,03 medical and health sciences ,Response regulator ,chemistry.chemical_compound ,chemistry ,medicine ,education ,Molecular Biology ,Gene ,Escherichia coli ,DNA ,030304 developmental biology - Abstract
Gene transfer agents (GTAs) are genetic exchange elements that resemble small DNA bacteriophages that transfer random pieces of the producing cell's genome to recipient cells. The best-studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA-mediated gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA-protecting protein A) homologue is essential for RcGTA-mediated gene acquisition, and that dprA expression is induced by gtaI-dependent quorum-sensing and non-phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C-terminal region that resembles a dsDNA-binding protein domain. Purified His-tagged R. capsulatus DprA protein bound to both single-stranded (ss)DNA and double-stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single-cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation.
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- 2014
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41. Photoactive Electrodes Incorporating Electrosprayed Bacterial Reaction Centers
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Mehr Negar Mirvakili, Daniel Jun, Seyed M. Mirvakili, J. Thomas Beatty, Ashwin R Usgaocar, Joanna E. Slota, John D. W. Madden, and Ali Mahmoudzadeh
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Photocurrent ,Photosynthetic reaction centre ,Materials science ,biology ,Analytical chemistry ,Quantum yield ,Condensed Matter Physics ,Photochemistry ,biology.organism_classification ,Electronic, Optical and Magnetic Materials ,law.invention ,Biomaterials ,Rhodobacter sphaeroides ,law ,Yield (chemistry) ,Solar cell ,Electrochemistry ,Pyrolytic carbon ,Photosystem - Abstract
Highly efficient light absorption and charge separation within the photosystem and reaction center (RC) complexes of photosynthetic plants and bacteria are of great interest for solar cell and photo detector applications, since they offer almost unity quantum yield and expected ultimate power conversion efficiencies of more than 18% and 12%, respectively. In addition, the charge separated states created by these protein complexes are very long lived compared to conventional semiconductor solar cells. In this work, a novel technique is presented for the deposition of photosynthetic protein complexes, by electrospraying RCs of Rhodobacter sphaeroides onto highly ordered pyrolytic graphite (HOPG) electrodes. Remarkably, it is shown that the RCs not only survive exposure to the high electric fields but also yield peak photocurrent densities of up to 7 μA cm−2, which is equal to the highest value reported to date.
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- 2014
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42. Cover Feature: Highly Sensitive Method to Isolate Photocurrent Signals from Large Background Redox Currents on Protein‐Modified Electrodes (ChemElectroChem 11/2019)
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Dan Bizzotto, Daniel Jun, and J. Thomas Beatty
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Photocurrent ,Electron transfer ,Materials science ,business.industry ,Feature (computer vision) ,Electrode ,Electrochemistry ,Optoelectronics ,Cover (algebra) ,business ,Redox ,Catalysis ,Highly sensitive - Published
- 2019
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43. The Protein Environment of the Bacteriopheophytin Anion Modulates Charge Separation and Charge Recombination in Bacterial Reaction Centers
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Jie Pan, Rafael G. Saer, Neal W. Woodbury, Zhi Guo, Su Lin, and J. Thomas Beatty
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Anions ,Models, Molecular ,Alanine ,Photosynthetic reaction centre ,Methionine ,biology ,Stereochemistry ,Kinetics ,Pheophytins ,Primary charge separation ,Rhodobacter sphaeroides ,Photochemistry ,biology.organism_classification ,Surfaces, Coatings and Films ,Electron Transport ,chemistry.chemical_compound ,Electron transfer ,Bacterial Proteins ,chemistry ,Materials Chemistry ,Physical and Theoretical Chemistry ,Cysteine - Abstract
The kinetics and pathway of electron transfer has been explored in a series of reaction center mutants from Rhodobacter sphaeroides, in which the leucine residue at M214 near the bacteriopheophytin cofactor in the A-branch has been replaced with methionine, cysteine, alanine, and glycine. These amino acids have substantially different volumes, both from each other and, except for methionine, from the native leucine. Though the mutation site of M214 is close to the bacteriopheophytin cofactor, which is involved in the electron transfer, none of the mutations alter the cofactor composition of the reaction center and the primary charge separation reaction is essentially undisturbed. However, the kinetics of electron transfer from HA(-) → QA becomes both slower and substantially heterogeneous in three of the four mutants. The decreased HA(-) → QA electron transfer rate allows charge recombination between P(+) and HA(-) to compete with the forward reaction, resulting in a drop in the overall yield of charge separation. Both the yield change and the variation in kinetics correlate well with the volume of the mutant amino acid side chains. Analysis of the kinetics suggests that the introduction of a smaller side chain at M214 results in greater protein structural heterogeneity and dynamics on multiple time scales, resulting in perturbation of the electronic environment and its evolution in the vicinity of the early charge-separated radical pair, P(+)HA(-), and the subsequent acceptor QA, affecting both the extent and time scale of dielectric relaxation. It appears that the reaction center has been optimized not only in terms of its static structure-function relationships, but also finely tuned to favor particular reaction pathways on particular time scales by adjusting protein dynamics.
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- 2013
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44. Quorum-sensing regulation of a capsular polysaccharide receptor for theRhodobacter capsulatusgene transfer agent (RcGTA)
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Aaron Stevens, Cedric A. Brimacombe, Ryan G. Mercer, Andrew S. Lang, J. Thomas Beatty, and Daniel Jun
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Bacterial capsule ,Bacteriophage ,Quorum sensing ,Rhodobacter ,biology ,Biochemistry ,Mutant ,Mutagenesis (molecular biology technique) ,biology.organism_classification ,Molecular Biology ,Microbiology ,Gene ,Gene transfer agent - Abstract
The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) resembles a small tailed bacteriophage that packages almost random genomic DNA segments that may be transferred to other R. capsulatus cells. Gene transfer agents are produced by a number of prokaryotes; however, no receptors have been identified. We investigated the RcGTA recipient capability of wild-type R. capsulatus cells at different culture growth phases, and found that the frequency of RcGTA-dependent acquisition of an allele increases as cultures enter the stationary phase. We also found that RcGTA adsorption to cells follows a similar trend. RcGTA recipient capability and adsorption were found to be dependent on the GtaR/I quorum-sensing (QS) system. Production of an extracellular polysaccharide was found to be regulated by GtaR/I QS, as was production of the cell capsule. A number of QS-regulated putative polysaccharide biosynthesis genes were identified, and mutagenesis of two of these genes, rcc01081 and rcc01932, yielded strains that lack a capsule. Furthermore, these mutants were impaired in RcGTA recipient capability and adsorption, as was a non-encapsulated wild-type isolate of R. capsulatus. Overall, our results indicate that capsular polysaccharide is a receptor for the gene transfer agent of R. capsulatus, RcGTA.
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- 2013
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45. Mutation-Induced Changes in the Protein Environment and Site Energies in the (M)L214G Mutant of the Rhodobacter sphaeroides Bacterial Reaction Center
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Rafael G. Saer, Ryszard Jankowiak, Olga Rancova, Adam Kell, Jinhai Chen, Darius Abramavicius, and J. Thomas Beatty
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Photosynthetic reaction centre ,Models, Molecular ,Mutant ,Photosynthetic Reaction Center Complex Proteins ,Electrons ,Rhodobacter sphaeroides ,010402 general chemistry ,Photosynthesis ,01 natural sciences ,Spectral line ,Cofactor ,0103 physical sciences ,Materials Chemistry ,Computer Simulation ,Physical and Theoretical Chemistry ,Ferricyanides ,010304 chemical physics ,biology ,Chemistry ,Spectrum Analysis ,Pheophytins ,Temperature ,Chromophore ,biology.organism_classification ,0104 chemical sciences ,Surfaces, Coatings and Films ,Crystallography ,Absorption band ,Mutation ,biology.protein ,Mutagenesis, Site-Directed ,Quantum Theory ,Protons ,Oxidation-Reduction - Abstract
This work focuses on the low-temperature (5 K) photochemical (transient) hole-burned (HB) spectra within the P870 absorption band, and their theoretical analysis, for the (M)L214G mutant of the photosynthetic Rhodobacter sphaeroides bacterial reaction center (bRC). To provide insight into system-bath interactions of the bacteriochlorophyll a (BChl a) special pair, i.e., P870, in the mutated bRC, the optical line shape function for the P870 band is calculated numerically. On the basis of the modeling studies, we demonstrate that (M)L214G mutation leads to a heterogeneous population of bRCs with modified (increased) total electron-phonon coupling strength of the special pair BChl a and larger inhomogeneous broadening. Specifically, we show that after mutation in the (M)L214G bRC a large fraction (∼50%) of the bacteriopheophytin (HA) chromophores shifts red and the 800 nm absorption band broadens, while the remaining fraction of HA cofactors retains nearly the same site energy as HA in the wild-type bRC. Modeling using these two subpopulations allowed for fits of the absorption and nonresonant (transient) HB spectra of the mutant bRC in the charge neutral, oxidized, and charge-separated states using the Frenkel exciton Hamiltonian, providing new insight into the mutant's complex electronic structure. Although the average (M)L214G mutant quantum efficiency of P(+)QA(-) state formation seems to be altered in comparison with the wild-type bRC, the average electron transfer time (measured via resonant transient HB spectra within the P870 band) was not affected. Thus, mutation in the vicinity of the electron acceptor (HA) does not tune the charge separation dynamics. Finally, quenching of the (M)L214G mutant excited states by P(+) is addressed by persistent HB spectra burned within the B band in chemically oxidized samples.
- Published
- 2016
46. The Role of Gold-Adsorbed Photosynthetic Reaction Centers and Redox Mediators in the Charge Transfer and Photocurrent Generation in a Bio-Photoelectrochemical Cell
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Joanna E. Slota, Daniel Jun, Rudy Schlaf, Zhi Li, John D. W. Madden, Arash Takshi, Rafael G. Saer, Houman Yaghoubi, M. M. Beerbom, and J. Thomas Beatty
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Photosynthetic reaction centre ,Photocurrent ,Working electrode ,biology ,Chemistry ,macromolecular substances ,Photoelectrochemical cell ,Photochemistry ,biology.organism_classification ,Purple bacteria ,Redox ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,General Energy ,Charge carrier ,Physical and Theoretical Chemistry ,Cyclic voltammetry - Abstract
Bacterial photosynthetic reaction centers (RCs) are promising materials for solar energy harvesting, due to their high quantum efficiency. A simple approach for making a photovoltaic device is to apply solubilized RCs and charge carrier mediators to the electrolyte of an electrochemical cell. However, the adsorption of analytes on the electrodes can affect the charge transfer from RCs to the electrodes. In this work, photovoltaic devices were fabricated incorporating RCs from purple bacteria, ubiquinone-10 (Q2), and cytochrome c (Cyt c) (the latter two species acting as redox mediators). The adsorption of each of these three species on the gold working electrode was investigated, and the roles of adsorbed species in the photocurrent generation and the cycle of charge transfer were studied by a series of photochronoamperometric, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and cyclic voltammetry (CV) tests. It was shown that both redox mediators were required for photocurrent gene...
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- 2012
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47. Regulatory systems controlling motility and gene transfer agent production and release in Rhodobacter capsulatus
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Ryan G. Mercer, Stephan Noll, Andrew S. Lang, Matthew Quinlan, J. Thomas Beatty, and Alexandra R. Rose
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Gene Transfer, Horizontal ,Mutant ,Motility ,Microbiology ,DNA-binding protein ,Rhodobacter capsulatus ,Bacterial Proteins ,Caulobacter crescentus ,Gene expression ,Genetics ,Molecular Biology ,Transcription factor ,Regulation of gene expression ,Rhodobacter ,biology ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,United States ,Cell biology ,DNA-Binding Proteins ,Flagella ,Genes, Bacterial ,bacteria ,Locomotion ,Transcription Factors - Abstract
Production of the gene transfer agent of Rhodobacter capsulatus, RcGTA, is dependent upon several cellular regulatory systems, including a putative phosphorelay involving the CtrA and CckA proteins. These proteins are also involved in flagellar motility in R. capsulatus. The interactions of proteins in this system are best understood in Caulobacter crescentus where CtrA is activated by phosphorylation by the CckA-ChpT phosphorelay. CtrA~P activity is further controlled by SciP, which represses ctrA transcription and CtrA activation of transcription. We show that R. capsulatus chpT and cckA mutants both have greatly reduced motility and RcGTA activity. Unlike the ctrA mutant where RcGTA gene transcription is absent, the decrease in RcGTA activity is because of reduced release of RcGTA from the cells. The sciP mutant is not affected for RcGTA production but our results support the C. crescentus model of SciP repression of flagellar motility genes. We show that both unphosphorylated and phosphorylated CtrA can activate RcGTA gene expression, while CtrA~P seems to be required for release of the particle and expression of motility genes. This has led us to a new model of how this regulatory system controls motility and production of RcGTA in R. capsulatus.
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- 2012
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48. Electron Transfer in Rhodobacter sphaeroides Reaction Centers Containing Zn-Bacteriochlorophylls: A Hole-Burning Study
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Rafael G. Saer, J. Thomas Beatty, Paul R. Jaschke, Ryszard Jankowiak, Mike Reppert, and Bhanu Neupane
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Photosynthetic reaction centre ,Binding Sites ,biology ,Photosynthetic Reaction Center Complex Proteins ,Temperature ,Electron donor ,Rhodobacter sphaeroides ,biology.organism_classification ,Photochemistry ,Recombinant Proteins ,Surfaces, Coatings and Films ,Electron Transport ,Zinc ,chemistry.chemical_compound ,Electron transfer ,chemistry ,Spectrophotometry ,Mutagenesis, Site-Directed ,Materials Chemistry ,Bacteriochlorophyll ,Physical and Theoretical Chemistry ,Bacteriochlorophylls - Abstract
Nonresonant and resonant transient, photochemical hole-burned (HB) spectra are presented for primary electron donor states of a novel bacterial reaction center (Zn-RC) of Rhodobacter sphaeroides, containing six Zn-bacteriochlorophylls (Zn-BChls). A "Zn-β-RC" in which the Zn-BChl in the bacteriopheophytin (BPhe)-binding site on the A side (H(A)) has the Zn penta-coordinated, was also studied. The fifth ligand comes from a histidine introduced by site-directed mutagenesis. Formation of the P(+)Q(A)(-) state was observed in both types of RC, although under identical experimental conditions a significantly deeper P(-) band (corresponding to the lower-energy, special pair, excitonic component) was revealed in the Zn-RC. Assuming a similar lifetime of the P(+)Q(A)(-) state, the quantum yield of P(+)Q(A)(-) formation decreased by ~60% in the Zn-β-RC (compared to the Zn-RC), as was seen in a comparison of analogous (Mg) BChl-containing wild type and β-RCs of Rb. sphaeroides [Kirmaier et al. Science1991, 251, 922]. However, the average (weakly frequency-dependent) low-temperature electron transfer (ET) rates of the Zn-RC and Zn-β-RC (measured from zero phonon holes in resonant transient HB spectra) were both ~1 ps and similar to a rate previously measured in the Rb. sphaeroides native RC [Johnson et al. J. Phys. Chem. 1989, 93, 5953]. Electron transfer rates observed in this work on the Zn-RC yielded a P870* decay rate in good agreement with recent room-temperature, time-domain data [Lin et al. Proc. Natl. Acad. Sci. 2009, 106, 8537]. A lack of correlation observed between the holes near 810 and 883 nm, accounting for electrochromically induced shifts of the Zn-BChl transitions in the B(A,B) and H(A,B) binding sites, produced by formation of the P(+)BHQ(A)(-) state, indicates that the 810 nm bleach does not correspond to the P(+) (upper excitonic component of the dimer) band and is mostly contributed to by a shift of the B(B) absorption band. ZPH-action spectra indicated inhomogeneous broadening (Γ(inh)) of ~110 cm(-1) (Zn-RC) and ~130 cm(-1) (Zn-β-RC). Experimentally determined Γ(inh) decreased the number of variables in theoretical fits of the absorption and frequency-dependent shapes of resonant HB spectra, leading to more reliable Huang-Rhys factors for both low-frequency phonons and a pseudolocalized phonon, ω(SP), often referred to as the special pair marker mode.
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- 2012
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49. The GtaR protein negatively regulates transcription of the gtaRI operon and modulates gene transfer agent (RcGTA) expression in Rhodobacter capsulatus
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J. Thomas Beatty, Molly M. Leung, George B. Spiegelman, and Cedric A. Brimacombe
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Rhodobacter ,biology ,Operon ,Gene expression ,Mutant ,Transcriptional regulation ,Promoter ,biology.organism_classification ,Molecular Biology ,Microbiology ,Molecular biology ,Gene ,Transcription factor - Abstract
The gtaI gene of Rhodobacter capsulatus encodes an N-acyl-homoserine lactone (acyl-HSL) synthase. Immediately 5′ of the gtaI gene is ORF rcc00328 that encodes a potential acyl-HSL receptor protein. A combination of genetic and biochemical approaches showed that rcc00328 (renamed gtaR) modulates the production of a genetic exchange element called the gene transfer agent (RcGTA), and regulates the transcription of gtaI. Although gtaI mutants exhibited decreased levels of RcGTA production, mutagenesis of gtaR did not, whereas a gtaR/gtaI double mutant produced wild-type levels of RcGTA. Because mutagenesis of gtaR suppressed the effect of the gtaI mutation, we suggest that the GtaR protein is a negative transcriptional regulator of RcGTA gene expression. We discovered that the gtaR and gtaI genes are co-transcribed, and also negatively regulated by the GtaR protein in the absence of acyl-HSL. A His-tagged GtaR protein was purified, and DNA-binding experiments revealed a binding site in the promoter region of the gtaRI operon. This GtaR protein did not bind to the RcGTA promoter region, and therefore modulation of RcGTA production appears to require at least one additional factor. We found that RcGTA production was stimulated by spent media from other species, and identified exogenous acyl-HSLs that induce RcGTA.
- Published
- 2012
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50. Characterization of a newly discovered Mu-like bacteriophage, RcapMu, in Rhodobacter capsulatus strain SB1003
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Andrew S. Lang, Alexander P. Hynes, Elizabeth L. Digby, Paul C. M. Fogg, and J. Thomas Beatty
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Transposable element ,DNA, Bacterial ,Replicative transposition ,viruses ,Genome, Viral ,Genome ,Rhodobacter capsulatus ,Bacteriophage ,Siphoviridae ,03 medical and health sciences ,Open Reading Frames ,Viral Proteins ,Virology ,Bacteriophages ,Amino Acid Sequence ,030304 developmental biology ,Genetics ,0303 health sciences ,Rhodobacter ,biology ,Base Sequence ,030306 microbiology ,Sequence Analysis, DNA ,biology.organism_classification ,Gene transfer agent ,DNA, Viral ,DNA Transposable Elements ,bacteria ,Bacteriophage Mu - Abstract
The α-proteobacterium Rhodobacter capsulatus is a model organism for the study of bacterial photosynthesis and the bacteriophage-like gene transfer agent. Characterization of phages that infect Rhodobacter is extremely rare, and scarce for the α-proteobacteria in general. Here, we describe the discovery of the only functional Mu-like transposing phage to have been identified in the α-proteobacteria, RcapMu, resident in the genome-sequenced R. capsulatus SB1003 strain. RcapMu packages ~ 42 kb of total DNA, including
- Published
- 2011
- Full Text
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