Zn2+-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor

Pérez, Adam A.; Gajewski, John P.; Ferlez, Bryan H.; Ludwig, Marcus; Baker, Carol S.; Golbeck, John H.; Bryant, Donald A.

doi:10.1128/aem.02491-16

Zn²⁺-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor

Journal Article · Tue Jan 17 00:00:00 EST 2017 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/aem.02491-16· OSTI ID:1536858

Pérez, Adam A. ^[1]; Gajewski, John P. ^[2]; Ferlez, Bryan H. ^[2]; Ludwig, Marcus ^[2]; Baker, Carol S. ^[2]; Golbeck, John H. ^[2]; ^[3]

Pennsylvania State Univ., University Park, PA (United States); DOE/OSTI
Pennsylvania State Univ., University Park, PA (United States)
Pennsylvania State Univ., University Park, PA (United States); Montana State Univ., Bozeman, MT (United States)

Synechococcus sp. strain PCC 7002 has been gaining significance as both a model system for photosynthesis research and for industrial applications. Until recently, the genetic toolbox for this model cyanobacterium was rather limited and relied primarily on tools that only allowed constitutive gene expression. This work describes a two-plasmid, Zn²⁺-inducible expression platform that is coupled with a zurA mutation, providing enhanced Zn²⁺uptake. The control elements are based on the metal homeostasis system of a class II metallothionein gene (smtA₇₉₄₂) and its cognate SmtB₇₉₄₂repressor fromSynechococcus elongatusstrain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA₆₈₀₃) promoter ofSynechocystissp. strain PCC 6803. This metal-inducible expression system inSynechococcussp. strain PCC 7002 allowed the titratable gene expression of YFP that was up to 19-fold greater than the background level. This system was utilized successfully to control the expression of theDrosophila melanogasterβ-carotene 15,15'-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter inSynechococcussp. strain PCC 7002. Together, these properties establish this metal-inducible system as an additional useful tool that is capable of controlling gene expression for applications ranging from basic research to synthetic biology inSynechococcussp. strain PCC 7002.

IMPORTANCEThis is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn²⁺-inducible system, which is based on thesmtA₇₉₄₂operator/promoter and smtB₇₉₄₂repressor, as a versatile gene expression platform that expands the genetic toolbox ofSynechococcussp. strain PCC 7002.

View Accepted Manuscript (DOE)

Research Organization:: Pennsylvania State Univ., University Park, PA (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: FG02-05ER46222

OSTI ID:: 1536858

Journal Information:: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 3 Vol. 83; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

References (42)

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of Proteins Manns, Joanne M. Current Protocols in Microbiology, Vol. 22, Issue 1 https://doi.org/10.1002/9780471729259.mca03ms22	journal	August 2011
Expression of Genes in Cyanobacteria: Adaptation of Endogenous Plasmids as Platforms for High-Level Gene Expression in Synechococcus sp. PCC 7002 Xu, Yu; Alvey, Richard M.; Byrne, Patrick O. Methods in Molecular Biology, p. 273-293 https://doi.org/10.1007/978-1-60761-925-3_21	book	September 2010
Evidence for genetic transformation in blue-green alga Anacystis nidulans Shestakov, S. V.; Khyen, Nguyen Than MGG Molecular & General Genetics, Vol. 107, Issue 4 https://doi.org/10.1007/BF00441199	journal	January 1970
Plastocyanin: Structural and functional analysis Redinbo, Matthew R.; Yeates, Todd O.; Merchant, Sabeeha Journal of Bioenergetics and Biomembranes, Vol. 26, Issue 1 https://doi.org/10.1007/BF00763219	journal	February 1994
Cyanobacteria carrying ansmt-lux transcriptional fusion as biosensors for the detection of heavy metal cations Erbe, J. L.; Adams, A. C.; Taylor, K. B. Journal of Industrial Microbiology, Vol. 17, Issue 2 https://doi.org/10.1007/BF01570047	journal	August 1996
Comparative analysis of idiA and isiA transcription under iron starvation and oxidative stress in Synechococcus elongatus PCC 7942 wild-type and selected mutants Yousef, Naeima; Pistorius, Elfriede K.; Michel, Klaus-Peter Archives of Microbiology, Vol. 180, Issue 6 https://doi.org/10.1007/s00203-003-0618-4	journal	December 2003
Structural Determinants of Metal Selectivity in Prokaryotic Metal-responsive Transcriptional Regulators Pennella, Mario A.; Giedroc, David P. BioMetals, Vol. 18, Issue 4 https://doi.org/10.1007/s10534-005-3716-8	journal	August 2005
Zn, Cu and Co in cyanobacteria: selective control of metal availability Cavet, Jennifer S.; Borrelly, Gilles P. M.; Robinson, Nigel J. FEMS Microbiology Reviews, Vol. 27, Issue 2-3 https://doi.org/10.1016/S0168-6445(03)00050-0	journal	June 2003
The SmtB/ArsR family of metalloregulatory transcriptional repressors: structural insights into prokaryotic metal resistance Busenlehner, Laura S.; Pennella, Mario A.; Giedroc, David P. FEMS Microbiology Reviews, Vol. 27, Issue 2-3 https://doi.org/10.1016/S0168-6445(03)00054-8	journal	June 2003
A High-Throughput Biological Calorimetry Core Yennawar, Neela H.; Fecko, Julia A.; Showalter, Scott A. Methods in Enzymology https://doi.org/10.1016/bs.mie.2015.07.024	book	January 2016
Chemical rescue of a site-modified ligand to a [4Fe–4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I Antonkine, Mikhail L.; Maes, Estelle M.; Czernuszewicz, Roman S. Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1767, Issue 6 https://doi.org/10.1016/j.bbabio.2007.02.003	journal	June 2007
Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803 Chen, Que; van der Steen, Jeroen B.; Dekker, Henk L. Metabolic Engineering, Vol. 35 https://doi.org/10.1016/j.ymben.2016.02.001	journal	May 2016
SmtB−DNA and Protein−Protein Interactions in the Formation of the Cyanobacterial Metallothionein Repression Complex: Zn ²⁺ Does Not Dissociate the Protein−DNA Complex in Vitro ^† Kar, Sambit R.; Lebowitz, Jacob; Blume, Scott Biochemistry, Vol. 40, Issue 44 https://doi.org/10.1021/bi011289f	journal	November 2001
Coordination Chemistry of Bacterial Metal Transport and Sensing Ma, Zhen; Jacobsen, Faith E.; Giedroc, David P. Chemical Reviews, Vol. 109, Issue 10 https://doi.org/10.1021/cr900077w	journal	October 2009
Synthetic Biology Toolbox for Controlling Gene Expression in the Cyanobacterium Synechococcus sp. strain PCC 7002 Markley, Andrew L.; Begemann, Matthew B.; Clarke, Ryan E. ACS Synthetic Biology, Vol. 4, Issue 5 https://doi.org/10.1021/sb500260k	journal	September 2014
Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria Zhou, Jie; Zhang, Haifeng; Meng, Hengkai Scientific Reports, Vol. 4, Issue 1 https://doi.org/10.1038/srep04500	journal	March 2014
Multiple bacteria encode metallothioneins and SmtA-like zinc fingers: Bacterial metallothioneins and zinc fingers Blindauer, Claudia A.; Harrison, Mark D.; Robinson, Andrea K. Molecular Microbiology, Vol. 45, Issue 5 https://doi.org/10.1046/j.1365-2958.2002.03109.x	journal	September 2002
Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes Allaway, David; Schofield, Neil A.; Leonard, Mary E. Environmental Microbiology, Vol. 3, Issue 6 https://doi.org/10.1046/j.1462-2920.2001.00205.x	journal	June 2001
Analysis of the blind Drosophila mutant ninaB identifies the gene encoding the key enzyme for vitamin A formation invivo von Lintig, J. Proceedings of the National Academy of Sciences, Vol. 98, Issue 3 https://doi.org/10.1073/pnas.031576398	journal	January 2001
Transformation in Agmenellum quadruplicatum Stevens, S. E.; Porter, R. D. Proceedings of the National Academy of Sciences, Vol. 77, Issue 10 https://doi.org/10.1073/pnas.77.10.6052	journal	October 1980
An SmtB-like repressor from Synechocystis PCC 6803 regulates a zinc exporter Thelwell, C.; Robinson, N. J.; Turner-Cavet, J. S. Proceedings of the National Academy of Sciences, Vol. 95, Issue 18 https://doi.org/10.1073/pnas.95.18.10728	journal	September 1998
Vipp1 Is Essential for the Biogenesis of Photosystem I but Not Thylakoid Membranes in Synechococcus sp. PCC 7002 Zhang, Shuyi; Shen, Gaozhong; Li, Zhongkui Journal of Biological Chemistry, Vol. 289, Issue 23 https://doi.org/10.1074/jbc.M114.555631	journal	April 2014
Assembly of Photosystem I: I. INACTIVATION OF THE Shen, Gaozhong; Zhao, Jindong; Reimer, Susan K. Journal of Biological Chemistry, Vol. 277, Issue 23 https://doi.org/10.1074/jbc.M201103200	journal	March 2002
SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene smtA : identification of a Zn inhibited DNA-protein complex Morby, Andrew P.; Turner, Jennifer S.; Huckle, James W. Nucleic Acids Research, Vol. 21, Issue 4 https://doi.org/10.1093/nar/21.4.921	journal	January 1993
Network analysis of transcriptomics expands regulatory landscapes in Synechococcus sp. PCC 7002 McClure, Ryan S.; Overall, Christopher C.; McDermott, Jason E. Nucleic Acids Research, Vol. 44, Issue 18 https://doi.org/10.1093/nar/gkw737	journal	August 2016
Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria Stanier, Roger Y.; Deruelles, Josette; Rippka, Rosmarie Microbiology, Vol. 111, Issue 1, p. 1-61 https://doi.org/10.1099/00221287-111-1-1	journal	March 1979
Isolation of a prokaryotic metallothionein locus and analysis of transcriptional control by trace metal ions Huckle, James W.; Morby, Andrew P.; Turner, Jennifer S. Molecular Microbiology, Vol. 7, Issue 2 https://doi.org/10.1111/j.1365-2958.1993.tb01109.x	journal	January 1993
The Production of Hydrogen Peroxide by Blue-Green Algae: a Survey Stevens, S. E.; Patterson, C. O. Pat; Myers, Jack Journal of Phycology, Vol. 9, Issue 4 https://doi.org/10.1111/j.1529-8817.1973.tb04116.x	journal	December 1973
Prokaryotic carbonic anhydrases Smith, Kerry S.; Ferry, James G. FEMS Microbiology Reviews, Vol. 24, Issue 4 https://doi.org/10.1111/j.1574-6976.2000.tb00546.x	journal	October 2000
Characterization of psal and psaL Mutants of Synechococcus sp. Strain PCC 7002: A New Model for State Transitions in Cyanobacteria Schluchter, Wendy M.; Shen, Gaozhong; Zhao, Jindong Photochemistry and Photobiology, Vol. 64, Issue 1 https://doi.org/10.1111/j.1751-1097.1996.tb02421.x	journal	July 1996
A Drosophila Complementary DNA Resource Rubin, G. M. Science, Vol. 287, Issue 5461 https://doi.org/10.1126/science.287.5461.2222	journal	March 2000
Luminescent Whole-Cell Cyanobacterial Bioreporter for Measuring Fe Availability in Diverse Marine Environments Boyanapalli, R.; Bullerjahn, G. S.; Pohl, C. Applied and Environmental Microbiology, Vol. 73, Issue 3 https://doi.org/10.1128/AEM.01670-06	journal	December 2006
Purification and properties of unicellular blue-green algae (order Chroococcales). Stanier, R. Y.; Kunisawa, R.; Mandel, M. Bacteriological Reviews, Vol. 35, Issue 2 https://doi.org/10.1128/MMBR.35.2.171-205.1971	journal	January 1971
Purification and properties of unicellular blue-green algae (order Chroococcales) Stanier, R. Y.; Kunisawa, R.; Mandel, M. Bacteriological Reviews, Vol. 35, Issue 2 https://doi.org/10.1128/br.35.2.171-205.1971	journal	June 1971
Unlocking the Constraints of Cyanobacterial Productivity: Acclimations Enabling Ultrafast Growth Bernstein, Hans C.; McClure, Ryan S.; Hill, Eric A. mBio, Vol. 7, Issue 4 https://doi.org/10.1128/mBio.00949-16	journal	July 2016
Development of a Univariate Membrane-Based Mid-Infrared Method for Protein Quantitation and Total Lipid Content Analysis of Biological Samples Strug, Ivona; Utzat, Christopher; Cappione, Amedeo Journal of Analytical Methods in Chemistry, Vol. 2014 https://doi.org/10.1155/2014/657079	journal	January 2014
Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803 Guerrero, Fernando; Carbonell, Verónica; Cossu, Matteo PLoS ONE, Vol. 7, Issue 11 https://doi.org/10.1371/journal.pone.0050470	journal	November 2012
Group 3 sigma factors in the marine cyanobacterium Synechococcus sp. strain PCC 7002 are required for growth at low temperature Inoue-Sakamoto, Kaori; Gruber, Tanja M.; Christensen, Suzanne K. The Journal of General and Applied Microbiology, Vol. 53, Issue 2 https://doi.org/10.2323/jgam.53.89	journal	January 2007
Transcription Profiling of the Model Cyanobacterium Synechococcus sp. Strain PCC 7002 by Next-Gen (SOLiD™) Sequencing of cDNA Ludwig, Marcus; Bryant, Donald A. Frontiers in Microbiology, Vol. 2 https://doi.org/10.3389/fmicb.2011.00041	journal	January 2011
Bacteriochlorophyll f: properties of chlorosomes containing the “forbidden chlorophyll” Vogl, Kajetan; Tank, Marcus; Orf, Gregory S. Frontiers in Microbiology, Vol. 3 https://doi.org/10.3389/fmicb.2012.00298	journal	August 2012
Synthetic biology of cyanobacteria: unique challenges and opportunities Berla, Bertram M.; Saha, Rajib; Immethun, Cheryl M. Frontiers in Microbiology, Vol. 4 https://doi.org/10.3389/fmicb.2013.00246	journal	January 2013
Fur-type transcriptional repressors and metal homeostasis in the cyanobacterium Synechococcus sp. PCC 7002 Ludwig, Marcus; Chua, Tiing Tiing; Chew, Chyue Yie Frontiers in Microbiology, Vol. 6 https://doi.org/10.3389/fmicb.2015.01217	journal	October 2015

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