Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum

Orf, Gregory S.; Redding, Kevin E.

doi:10.1007/s11120-019-00672-3

Title: Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum

Abstract

The heliobacterial photochemical reaction center (HbRC) from the chlorophototrophic Firmicutes bacterium Heliobacterium modesticaldum is the only homodimeric type I RC whose structure is known. Using genetic techniques recently established in our lab, we have developed a rapid heterologous expression system for the HbRC core polypeptide PshA. Our system relies on rescue of the non-chlorophototrophic ΔpshA::cbp2p-aph3 strain of Hbt. modesticaldum by expression of a heterologous pshA gene from a replicating shuttle vector. Furthermore, we constructed two tagged variants of PshA, one with an N-terminal octahistidine tag and one with an internal hexahistidine tag, which facilitate rapid purification of pure, active HbRC cores in milligram quantities. We constructed a suite of shuttle vectors bearing untagged or tagged versions of pshA driven by various promoters. Surprisingly, we found that the eno and gapDH_2 promoters from Clostridium thermocellum drive better expression of pshA than fragments of DNA derived from the region upstream of the pshA locus on the Hbt. modesticaldum genome. This “pshA rescue” strategy additionally provided a useful window into how Hbt. modesticaldum regulates pigment synthesis and growth rate when chlorophototrophic output decreases.

Authors:

^[1];

^[1]

Arizona State Univ., Tempe, AZ (United States)

Publication Date:: 2019-09-21

Research Org.:: Arizona State Univ., Tempe, AZ (United States)

Sponsoring Org.:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division

OSTI Identifier:: 1631822

Grant/Contract Number:: SC0010575

Resource Type:: Accepted Manuscript

Journal Name:: Photosynthesis Research

Additional Journal Information:: Journal Volume: 142; Journal Issue: 3; Journal ID: ISSN 0166-8595

Publisher:: Springer

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; reaction center; heliobacteria; epitope-tagging; membrane protein, mutagenesis

Citation Formats


                    Orf, Gregory S., and Redding, Kevin E. Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum.  United States: N. p., 2019. 
Web.  doi:10.1007/s11120-019-00672-3.

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                    Orf, Gregory S., & Redding, Kevin E. Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum.  United States.  https://doi.org/10.1007/s11120-019-00672-3

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                    Orf, Gregory S., and Redding, Kevin E. Sat .  
"Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum".  United States.  https://doi.org/10.1007/s11120-019-00672-3.  https://www.osti.gov/servlets/purl/1631822.

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@article{osti_1631822,

  title        = {Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum},

  author       = {Orf, Gregory S. and Redding, Kevin E.},

  abstractNote = {The heliobacterial photochemical reaction center (HbRC) from the chlorophototrophic Firmicutes bacterium Heliobacterium modesticaldum is the only homodimeric type I RC whose structure is known. Using genetic techniques recently established in our lab, we have developed a rapid heterologous expression system for the HbRC core polypeptide PshA. Our system relies on rescue of the non-chlorophototrophic ΔpshA::cbp2p-aph3 strain of Hbt. modesticaldum by expression of a heterologous pshA gene from a replicating shuttle vector. Furthermore, we constructed two tagged variants of PshA, one with an N-terminal octahistidine tag and one with an internal hexahistidine tag, which facilitate rapid purification of pure, active HbRC cores in milligram quantities. We constructed a suite of shuttle vectors bearing untagged or tagged versions of pshA driven by various promoters. Surprisingly, we found that the eno and gapDH_2 promoters from Clostridium thermocellum drive better expression of pshA than fragments of DNA derived from the region upstream of the pshA locus on the Hbt. modesticaldum genome. This “pshA rescue” strategy additionally provided a useful window into how Hbt. modesticaldum regulates pigment synthesis and growth rate when chlorophototrophic output decreases.},

  doi          = {10.1007/s11120-019-00672-3},

  journal      = {Photosynthesis Research},

  number       = 3,

  volume       = 142,

  place        = {United States},

  year         = {2019},

  month        = {9}

}

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Title: Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum

Abstract

Citation Formats

A heterogeneous tag-attachment to the homodimeric type 1 photosynthetic reaction center core protein in the green sulfur bacterium Chlorobaculum tepidum journal, July 2011

A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum journal, August 2019

Prokaryotic photosynthesis and phototrophy illuminated journal, November 2006

Silver staining of proteins in polyacrylamide gels journal, November 2006

Evolution of Oxygenic Photosynthesis journal, June 2016

Complete nucleotide sequence and genetic organization of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens journal, March 1988

Structure of a symmetric photosynthetic reaction center–photosystem journal, July 2017

Rapid isolation of bacterial photosynthetic reaction centers with an engineered poly-histidine tag journal, September 1996

Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii journal, January 2008

A modular system for Clostridium shuttle plasmids journal, July 2009

Heliobacterial photosynthesis journal, April 2007

Resolution and Reconstitution of a Bound Fe−S Protein from the Photosynthetic Reaction Center of Heliobacterium modesticaldum † journal, July 2005

Identification and Characterization of PshB, the Dicluster Ferredoxin that Harbors the Terminal Electron Acceptors F A and F B in Heliobacterium modesticaldum † , ‡ journal, March 2007

Chlorophyll breakdown in higher plants journal, August 2011

Light-driven quinone reduction in heliobacterial membranes journal, March 2018

Heliobacterium modesticaldum, sp. nov., a thermophilic heliobacterium of hot springs and volcanic soils journal, April 1995

Menaquinone as the Secondary Electron Acceptor in the Type I Homodimeric Photosynthetic Reaction Center of Heliobacterium modesticaldum journal, June 2015

FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method journal, January 2011

RNA-stabilization factors in chloroplasts of vascular plants journal, February 2018

ESR Signal of the Iron−Sulfur Center F X and Its Function in the Homodimeric Reaction Center of Heliobacterium modesticaldum † , ‡ journal, May 2006

An Electron Spin-Polarized Signal of the P800 + A 1 (Q) − State in the Homodimeric Reaction Center Core Complex of Heliobacterium modesticaldum † journal, April 2008

Identifying promoters for gene expression in Clostridium thermocellum journal, December 2015

Evolution of photosynthetic reaction centers: insights from the structure of the heliobacterial reaction center journal, March 2018

Engineered biosynthesis of bacteriochlorophyll gF in Rhodobacter sphaeroides journal, July 2018

Modulation of the fluorescence yield in heliobacterial cells by induction of charge recombination in the photosynthetic reaction center journal, December 2013

Purification of the photosynthetic reaction center from Heliobacterium modesticaldum journal, March 2012

The Genome of Heliobacterium modesticaldum, a Phototrophic Representative of the Firmicutes Containing the Simplest Photosynthetic Apparatus journal, April 2008

Tricine–SDS-PAGE journal, May 2006

Genome-wide Transcriptional Response during the Shift to N2-fixing Conditions in Heliobacterium modesticaldum journal, January 2018

In-gel digestion for mass spectrometric characterization of proteins and proteomes journal, December 2006

Arabidopsis STAY-GREEN , Mendel’s Green Cotyledon Gene, Encodes Magnesium-Dechelatase journal, September 2016

High-performance liquid chromatography of chloroplast pigments journal, January 1988

Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid journal, May 1992

Highly Purified Thermo-Stable Oxygen-Evolving Photosystem II Core Complex from the Thermophilic Cyanobacterium Synechococcus elongatus Having His-Tagged CP43 journal, January 1999

Carbon Flow of Heliobacteria Is Related More to Clostridia than to the Green Sulfur Bacteria journal, August 2010

Isolation of a photoactive photosynthetic reaction center-core antenna complex from Heliobacillus mobilis journal, December 1989

Purification and properties of an antenna-reaction center complex from heliobacteria journal, February 1990

Identification of 81-hydroxychlorophyll a as a functional reaction center pigment in heliobacteria journal, July 1991

Transient electron paramagnetic resonance spectroscopy on green-sulfur bacteria and heliobacteria at two microwave frequencies journal, December 1998

Heliobacterium modesticaldum, sp. nov., a thermophilic heliobacterium of hot springs and volcanic soils journal, April 1995

Chlorophyll breakdown in higher plants. text, January 2011

A heterogeneous tag-attachment to the homodimeric type 1 photosynthetic reaction center core protein in the green sulfur bacterium Chlorobaculum tepidum
journal, July 2011

A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum
journal, August 2019

Prokaryotic photosynthesis and phototrophy illuminated
journal, November 2006

Silver staining of proteins in polyacrylamide gels
journal, November 2006

Evolution of Oxygenic Photosynthesis
journal, June 2016

Complete nucleotide sequence and genetic organization of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens
journal, March 1988

Structure of a symmetric photosynthetic reaction center–photosystem
journal, July 2017

Rapid isolation of bacterial photosynthetic reaction centers with an engineered poly-histidine tag
journal, September 1996

Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii
journal, January 2008

A modular system for Clostridium shuttle plasmids
journal, July 2009

Heliobacterial photosynthesis
journal, April 2007

Resolution and Reconstitution of a Bound Fe−S Protein from the Photosynthetic Reaction Center of Heliobacterium modesticaldum ^†
journal, July 2005

Identification and Characterization of PshB, the Dicluster Ferredoxin that Harbors the Terminal Electron Acceptors F _A and F _B in Heliobacterium modesticaldum ^† ^, ^‡
journal, March 2007

Chlorophyll breakdown in higher plants
journal, August 2011

Light-driven quinone reduction in heliobacterial membranes
journal, March 2018

Heliobacterium modesticaldum, sp. nov., a thermophilic heliobacterium of hot springs and volcanic soils
journal, April 1995

Menaquinone as the Secondary Electron Acceptor in the Type I Homodimeric Photosynthetic Reaction Center of Heliobacterium modesticaldum
journal, June 2015

FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method
journal, January 2011

RNA-stabilization factors in chloroplasts of vascular plants
journal, February 2018

ESR Signal of the Iron−Sulfur Center F _X and Its Function in the Homodimeric Reaction Center of Heliobacterium modesticaldum ^† ^, ^‡
journal, May 2006

An Electron Spin-Polarized Signal of the P800 ⁺ A ₁ (Q) ⁻ State in the Homodimeric Reaction Center Core Complex of Heliobacterium modesticaldum ^†
journal, April 2008

Identifying promoters for gene expression in Clostridium thermocellum
journal, December 2015

Evolution of photosynthetic reaction centers: insights from the structure of the heliobacterial reaction center
journal, March 2018

Engineered biosynthesis of bacteriochlorophyll gF in Rhodobacter sphaeroides
journal, July 2018

Modulation of the fluorescence yield in heliobacterial cells by induction of charge recombination in the photosynthetic reaction center
journal, December 2013

Purification of the photosynthetic reaction center from Heliobacterium modesticaldum
journal, March 2012

The Genome of Heliobacterium modesticaldum, a Phototrophic Representative of the Firmicutes Containing the Simplest Photosynthetic Apparatus
journal, April 2008

Tricine–SDS-PAGE
journal, May 2006

Genome-wide Transcriptional Response during the Shift to N2-fixing Conditions in Heliobacterium modesticaldum
journal, January 2018

In-gel digestion for mass spectrometric characterization of proteins and proteomes
journal, December 2006

Arabidopsis STAY-GREEN , Mendel’s Green Cotyledon Gene, Encodes Magnesium-Dechelatase
journal, September 2016

High-performance liquid chromatography of chloroplast pigments
journal, January 1988

Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid
journal, May 1992

Highly Purified Thermo-Stable Oxygen-Evolving Photosystem II Core Complex from the Thermophilic Cyanobacterium Synechococcus elongatus Having His-Tagged CP43
journal, January 1999

Carbon Flow of Heliobacteria Is Related More to Clostridia than to the Green Sulfur Bacteria
journal, August 2010

Isolation of a photoactive photosynthetic reaction center-core antenna complex from Heliobacillus mobilis
journal, December 1989

Purification and properties of an antenna-reaction center complex from heliobacteria
journal, February 1990

Identification of 81-hydroxychlorophyll a as a functional reaction center pigment in heliobacteria
journal, July 1991

Transient electron paramagnetic resonance spectroscopy on green-sulfur bacteria and heliobacteria at two microwave frequencies
journal, December 1998

Heliobacterium modesticaldum, sp. nov., a thermophilic heliobacterium of hot springs and volcanic soils
journal, April 1995

Chlorophyll breakdown in higher plants.
text, January 2011