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

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

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

Appl Environ Microbiol. 2017 Jan 17;83(3):e02491-16. doi: 10.1128/AEM.02491-16. Print 2017 Feb 1.

Authors

Adam A Pérez¹, John P Gajewski¹, Bryan H Ferlez¹, Marcus Ludwig¹, Carol S Baker¹, John H Golbeck^{1

2}, Donald A Bryant^{3

4}

Affiliations

¹ Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
² Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, USA.
³ Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA dab14@psu.edu.
⁴ Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, USA.

Abstract

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 SmtB₇₉₄₂ repressor from Synechococcus elongatus strain PCC 7942. Under optimal induction conditions, yellow fluorescent protein (YFP) levels were about half of those obtained with the strong, constitutive phycocyanin (cpcBA₆₈₀₃) promoter of Synechocystis sp. strain PCC 6803. This metal-inducible expression system in Synechococcus sp. 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 the Drosophila melanogaster β-carotene 15,15'-dioxygenase, NinaB, which is toxic when constitutively expressed from a strong promoter in Synechococcus sp. 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 in Synechococcus sp. strain PCC 7002.

Importance: This 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 the smtA₇₉₄₂ operator/promoter and smtB₇₉₄₂ repressor, as a versatile gene expression platform that expands the genetic toolbox of Synechococcus sp. strain PCC 7002.

Keywords: Zn2+-inducible promoter; cyanobacteria; gene expression platform; metallothionein; ninaB; photosynthesis.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Animals, Genetically Modified / genetics
Bacterial Proteins / genetics*
Bacterial Proteins / metabolism
Drosophila melanogaster / genetics
Gene Expression Regulation, Bacterial*
Metallothionein / genetics*
Metallothionein / metabolism
Operator Regions, Genetic*
Promoter Regions, Genetic*
Synechococcus / genetics*

Substances

Bacterial Proteins
smtA protein, Synechococcus
Metallothionein

Grants and funding

P40 OD010949/OD/NIH HHS/United States