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Research Article
17 January 2017

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

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.

INTRODUCTION

Synechococcus sp. strain PCC 7002 is a euryhaline cyanobacterial model organism with a very high growth rate, natural transformability, tolerance to high-light irradiance, a fully sequenced genome, and an expanding genetic toolbox. Over the past 30 years, this cyanobacterium has been extensively used in the study of photosynthesis and for synthetic biology applications (14). Despite its importance as an industrially relevant organism, genetic tools in Synechococcus sp. strain PCC 7002 were rather limited until recently, mostly comprising a few constitutive promoters and a single repressible promoter system (57). Markley et al. (2) made considerable additions to the genetic toolbox for Synechococcus sp. strain PCC 7002 by expanding the collection of constitutive promoters, a ribosome binding site library to improve translation initiation, and the generation of a tightly controlled isopropyl-β-d-1-thiogalactopyranoside (IPTG) induction system. Their work provides a platform from which to continue optimizing these tools to improve the versatility of gene expression control, which is a key element to the field of synthetic biology in cyanobacteria (6). The present study provides the use of a robust two-component, metal ion-inducible expression system as a promising addition to the engineering tools in Synechococcus sp. strain PCC 7002.
Metal ion-responsive promoters are among the gene regulation systems that have been used to control gene expression in cyanobacteria; however, their use has been hindered by factors including low sensitivity, limited distribution, and nonorthogonality (2, 6). These endogenous systems are derived from ubiquitous metal homeostasis genes that are regulated by metalloregulatory proteins, such as the ArsR/SmtB family (8). Among the known assortment of ArsR/SmtB-regulated systems in cyanobacteria, the smtA7942 promoter/operator of the small, cysteine-rich metallothionein, SmtA7942, from Synechococcus elongatus strain PCC 7942, has been the most completely characterized (9). SmtA7942 sequesters metal ions, such as cadmium, copper, and zinc (10), and its promoter is regulated by the divergently transcribed SmtB7942 transcriptional repressor, which exhibits preferential affinity for Zn2+ (11). The smtA7942 operator-promoter and SmtB7942 repressor have been tested for metal ion-inducible expression in S. elongatus strain PCC 7942, in which this system was used to control the expression of the luxCDABE operon. The resulting strain was used as a fluorescent biosensor for environmental heavy metal contamination with marginal success (12). The smtA7002 promoter of Synechococcus sp. strain PCC 7002 was tested in Synechocystis sp. strain PCC 6803 for its ability to control ethylene production with similarly limited success (13).
To our knowledge, only one heterologous metal-responsive gene expression system has been implemented in Synechococcus sp. strain PCC 7002 (14). The iron-responsive isiAB operator-promoter was fused to the luxAB genes of Vibrio harveyi, and the resulting strain was used as a biosensor for soluble Fe3+ in marine environments. Challenges surrounding metal-responsive expression systems can be attributed to the potential disruption of metal homeostasis, which is a critical aspect of the physiology of processes related to photosynthesis in cyanobacteria. Disruption of metal homeostasis can impose severe growth constraints on the modified strains (15). For example, copper is essential for electron transfer when bound to plastocyanin (16), iron is an integral component in the iron-sulfur clusters of the electron transfer chain of photosystem I (17), and zinc is an important component for carbon fixation because it is an essential cofactor in the active site of carbonic anhydrases (18). Despite these hurdles, metal ion-responsive systems for gene expression have been sought as inducible expression systems with inexpensive inducers. Furthermore, inducible promoter systems (e.g., Plac) are frequently based on Escherichia coli promoters, which use a transcription machinery that is distinct from that found in cyanobacteria and which often lead to little or no activity when implemented in cyanobacteria (19). It is, therefore, advantageous to focus on the use and optimization of endogenous cyanobacterial metabolite- and metal-responsive systems to minimize the incompatibility between promoters and the transcriptional machinery (6).
In a recent study of metal homeostasis in Synechococcus sp. strain PCC 7002, deletion of the zurA gene resulted in the constitutive expression of an ABC transporter for Zn2+ that is normally induced in response to Zn2+ limitation (15). This Zn2+ transport system presumably has a high affinity for Zn2+, but there was no discernible growth defect associated with the zurA mutation under standard growth conditions for this cyanobacterium (15). We hypothesized that the zurA mutant may be more responsive to the concentration of Zn2+ in the growth medium, and we further hypothesized that this strain may be a suitable background to construct a Zn2+-inducible expression platform. To test these hypotheses, the yfp gene, encoding yellow fluorescent protein (YFP), was transcriptionally fused to the class II metallothionein promoter (PsmtA7942) from S. elongatus strain PCC 7942 in the Synechococcus sp. strain PCC 7002 pAQ1Ex system (5), and the gene encoding the cognate smtB7942 transcriptional repressor was coexpressed under the Synechocystis sp. strain PCC 6803 psbA26803 promoter (the promoter for the D1 protein of photosystem II) in pAQ3Ex (5). The zurA mutant strain was used as the background strain to enable enhanced metal sensing by the recombinant SmtB7942 transcriptional regulator. In combination, these manipulations provided a versatile Zn2+-inducible system with a titratable response that increased linearly in response to the concentration of exogenous Zn2+ and that allowed for high-level expression of the YFP reporter. This expression system also exhibited tight regulation and allowed the successful expression of a gene encoding a protein that can be toxic to Synechococcus sp. strain PCC 7002 when expressed at excessive levels.

RESULTS

The smtA7942 promoter from S. elongatus strain PCC 7942 requires its cognate SmtB7942 repressor.

Synechococcus sp. strain PCC 7002 contains a class II metallothionein for metal homeostasis that is homologous to that found in S. elongatus strain PCC 7942 (20). Although the SmtB transcriptional repressors of these cyanobacteria are homologous and similar in sequence (64% identity and 80% similarity) (Fig. 1), the yfp gene in the recombinant pAQ1Ex-PsmtA7942[yfp] system (Fig. 2A) is not regulated by the native Synechococcus sp. strain PCC 7002 repressor, as evidenced by the similarly high levels of YFP produced with and without Zn2+ induction (Fig. 2B). In contrast, the coexpression of SmtB7942 from S. elongatus strain PCC 7942 (Fig. 3A) led to the repression of YFP fluorescence in the A+(−Zn2+) growth medium (see the Materials and Methods), which contains less than 1 μM Zn2+ (Fig. 3B). The addition of buffered ZnCl2 to final concentrations ranging from 50 to 100 μM caused the induction of yfp expression when PsmtA7942[yfp] was coexpressed with the cognate SmtB7942 from S. elongatus strain PCC 7942 (Fig. 3B).
FIG 1
FIG 1 Amino acid sequence alignment of cyanobacterial SmtB and SmtB-like apoproteins. The SmtB and SmtB-like apoprotein amino acid sequences of Synechocystis sp. strain PCC 6803 (SYNPCC6803_sll0792), Synechococcus elongatus strain PCC 7942 (SYNPCC7942_1291), and Synechococcus sp. strain PCC 7002 (SYNPCC7002_A2564) were aligned for comparison of DNA and metal-binding regions. The putative α3N and α5 metal-binding sites are shaded in teal and yellow, respectively. Letters in red font denote predicted DNA-binding regions. The key to the symbols in the consensus alignment is as follows: an asterisk (*) represents a fully conserved residue, a colon (:) represents a strongly conserved residue within a similar functional group, and a period (.) indicates a residue with weakly similar properties.
FIG 2
FIG 2 Introduction and evaluation of pAQ1Ex-PsmtA7942[yfp] in strain ML001. (A) Scheme showing how the PsmtA7942 promoter of S. elongatus strain PCC 7942 was transcriptionally fused to the yfp fluorescent reporter gene in pAQ1EX. (B) In vivo fluorescence emission spectrum showing YFP production in the AAPZN01 strain. Samples were normalized to an OD730 of 1.0 using growth medium A+. Synthesis of YFP was unchanged in growth conditions, including 50 μM Zn2+, denoted by the dashed line, and with only trace amounts of Zn2+ from modified A+, denoted by the solid line. The dotted line shows the fluorescence emission from strain ML001 that served as a negative control.
FIG 3
FIG 3 Introduction and evaluation of pAQ3Ex-PpsbA26803[smtB7942] in strain AAPZN01. (A) Scheme showing how the psbA2 promoter (PpsbA26803) of Synechocystis sp. strain PCC 6803 was transcriptionally fused to the smtB7942 gene, which encodes the SmtB7942 repressor from S. elongatus strain PCC 7942 in pAQ3Ex. (B) In vivo fluorescence emission showing YFP synthesis in strain AAPZN02. Samples were normalized to an OD730 of 1.0 using growth medium A+. Synthesis of YFP was repressed under growth conditions with only trace amounts of Zn2+ (<1 μM) from modified A+ when SmtB7942 was coexpressed (solid line). The dashed line shows derepression of the YFP fluorescent reporter when 50 μM Zn2+ was added to the growth medium. The dotted line shows the fluorescence emission spectrum of strain ML001 that served as a negative control.

PsmtA 7942 induction is optimal at a final concentration of 40 to 60 μM Zn2+.

Strain AAPZN02 (pAQ1Ex-PsmtA7942[yfp] pAQ3Ex-PpsbA26803[smtB7942] ΔzurA::ermC; Table 1) did not produce YFP when Zn2+ was present at concentrations of ≤30 μM (results not shown). Based on the YFP fluorescence emission, an induction response was only observed when the final concentrations of Zn2+ were in the range of 40 to 60 μM (Fig. 4C and 5). Induction at higher concentrations of ZnCl2 not only showed lowered fluorescence of YFP but also caused impaired growth of the cells (Fig. 4D, E, and F). At a final Zn2+ concentration of 150 μM (Fig. 4F), no growth or expression of YFP occurred. Based on these results, it was determined that the optimal balance of expression and cell viability occurred when cells were induced during the late exponential phase (optical density at 730 nm [OD730] = 0.7 to 0.8) at a final Zn2+ concentration of 60 μM. The maximum YFP production under these conditions was recorded 24 h after induction when cells were grown under standard conditions (Fig. 5).
TABLE 1
TABLE 1 Bacterial strains and plasmids used in this study
Plasmid/strainRelevant characteristic(s)Reference or source
Plasmids
    pAQ1ExpGEM-7zf pMB1 vector backbone with Synechococcus sp. strain PCC 7002 pAQ1 flanking sites, Spr5
    pAQ1Ex-PcpcBA6803[yfp]Constitutive expression of YFP, Spr5
    pAQ1Ex-PsmtA7942[yfp]Zn2+-regulated expression of YFP, SprThis study
    pAQ1Ex-PsmtA7942[ninaB]Zn2+-regulated expression of NinaB, SprThis study
    pAQ1Ex-PcpcBA6803[ninaB]Constitutive expression of NinaB, SprThis study
    pAQ1Ex-PcpcBA9.4%[ninaB]Weak constitutive expression of NinaB (9.4% of PcpcBA6803), SprThis study
    pAQ3ExpGEM-7zf pMB1 vector backbone with Synechococcus sp. strain PCC 7002 pAQ3 flanking sites, Kmr5
    pAQ3Ex-PpsbA26803[smtB7942]Expression of SmtB, KmrThis study
    pOT2Promoter probe vector with promoterless gfpUV, Cmr41
Strains
    Synechococcus elongatus strain PCC 794230
    Synechocystis sp. strain PCC 680328
    Synechococcus sp. strain PCC 700228
    Escherichia coli TOP10F'F'[lacIq, Tn10 (Tetr)] mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara leu)7697 galU galK rpsL (Strr) endA1 nupGInvitrogen
Constructed strains
    ML001ΔzurA::ermC15
    AAPZN01pAQ1Ex-PsmtA7942[yfp] ΔzurA::ermCThis study
    AAPZN02pAQ1Ex-PsmtA7942[yfp] pAQ3Ex-PpsbA26803[smtB7942] ΔzurA::ermCThis study
    AAPZN03pAQ1Ex-PcpcBA6803[yfp] ΔzurA::ermCThis study
    AAPZN04pAQ3Ex-PpsbA26803[smtB7942] ΔzurA::ermCThis study
    AAPZN05pAQ1Ex-PcpcBA6803 [ninaB] pAQ3Ex-PpsbA26803[smtB7942] ΔzurA::ermCThis study
    AAPZN06pAQ1Ex-PcpcBA9.4%[ninaB] pAQ3Ex-PpsbA26803[smtB7942] ΔzurA::ermCThis study
    AAPZN07pAQ1Ex-PsmtA7942[ninaB] pAQ3Ex-PpsbA26803[smtB7942] ΔzurA::ermCThis study
FIG 4
FIG 4 Zn2+ titration analysis for the determination of optimal conditions for induction of YFP synthesis in strain AAPZN02. Cultures were grown photoautotrophically under standard growth conditions and were assayed up to 29 h after induction at time 0 h. Cultures were induced with various ZnCl2 concentrations at the late exponential phase (OD730, ∼0.8); the data presented are the averages of the three replicates, and the standard deviation is shown by the error bars. Samples were assayed for growth by measuring OD730 (solid black line), and the fluorescence amplitude for YFP (fluorescence emission) was measured at 4-h intervals after induction (0 h). Fluorescence emission was recorded at the 527-nm emission maximum of YFP and plotted as bar graphs (gray bars); the data are the average values for three replicate cultures, and the standard deviations for the measurements are shown by the error bars. Samples were induced as follows: no ZnCl2 added (A), 25 μM ZnCl2 (B), 50 μM ZnCl2 (C), 75 μM ZnCl2 (D), 100 μM ZnCl2 (E), and 150 μM ZnCl2 (F).
FIG 5
FIG 5 Fluorescence emission spectra for whole cells of strain AAPZN02 showing YFP fluorescence emission when the yfp gene was under the control of PsmtA7942. Samples were normalized to an OD730 of 1.0 using the growth medium A+(–Zn2+). Samples were induced with ZnCl2 at the specified final concentrations when cells reached late exponential phase (OD730, ∼0.8). The dotted line denotes fluorescence emission in the absence of added ZnCl2. The dashed line denotes induction with 40 μM ZnCl2. The dot and dash line denotes induction with 50 μM ZnCl2, and the solid line denotes induction with 60 μM ZnCl2.

The PsmtA7942 inducible promoter exhibits titratable expression of YFP.

Purification of YFP by metal chelation affinity chromatography (Fig. 6A) from uninduced (<1 μM Zn2+) samples yielded an estimated 0.14 mg of YFP from 1 g (wet weight) of cells. Induction with a 40 μM final Zn2+ concentration yielded 0.64 mg of YFP per 1.0 g of cells, a 4.5-fold increase compared to an uninduced sample. When the PsmtA7942[yfp] system was induced with 50 μM and 60 μM final Zn2+ concentrations, the yield of YFP increased 11-fold and 19-fold per 1.0 g of cells, respectively. At optimal induction conditions (i.e., a 60 μM Zn2+ final concentration), the total yield of YFP from the PsmtA7942[yfp] construct was ∼48% of the YFP level produced by the strong constitutive PcpcBA6803 promoter of Synechocystis sp. strain PCC 6803 (Table 2) (21) in strain AAPZN03. Interestingly, as determined by the fluorescence emission in whole cells, the YFP level produced by the PsmtA7942[yfp] construct was lower, only 36%, than the YFP level produced by PcpcBA6803[yfp] (data not shown). These data indicate that fluorescence emission is not a reliable quantitative determinant of yfp expression, possibly because some of the YFP produced may not carry a chromophore. Qualitative evidence of titratable expression was evident when the purified YFP fractions were exposed to UV excitation (Fig. 6B) and SDS-PAGE analysis (Fig. 6C). The increased fluorescence emission of the solutions, and a more prominent YFP band around 30 kDa upon SDS-PAGE analysis, correlated positively with the increasing final Zn2+ concentration within the range that did not inhibit growth (0 to 60 μM exogenous Zn2+).
FIG 6
FIG 6 Purification and evaluation of YFP expressed by the smtA7942/SmtB7942 system. (A) SDS-PAGE analysis of fractions derived from the purification of N-terminal (His)10-tagged YFP from strain AAPZN02: uninduced cells, lane 1; 60 μM Zn2+-induced cells, lane 2; lysate, lane 3; clarified lysate after ultracentrifugation, lane 4; insoluble fraction after ultracentrifugation, lane 5; flowthrough fraction from chelation chromatography, lane 6; wash fraction with Tergitol NP-40 detergent, lane 7; wash fraction without detergent, lane 8; purified YFP after elution, lane 9. For additional details, see the text. (B) Purified YFP fractions from strain AAPZN02 compared using a long-wavelength UV transilluminator to visualize the YFP fluorescence, (C) SDS-PAGE analysis of cells: no added Zn2+, lane 1; 40 μM Zn2+, lane 2; 50 μM Zn2+, lane 3; 60 μM Zn2+, lane 4; YFP purified from strain AAPZN03 as a positive control, lane 5. Molecular mass markers are shown at the left, and sizes are indicated in kilodaltons.
TABLE 2
TABLE 2 Total YFP purified from strains AAPZN02 and AAPZN02 under the specified conditions
Condition (μM Zn2+PsmtA7942)YFP (mg/1 g cells)a
PcpcBA 68035.74
00.14
400.64
501.53
602.67
a
Values were obtained from a single purification trial.

Expression and characterization of the ninaB gene in Synechococcus sp. strain PCC 7002.

When attempts were made to express the ninaB gene of Drosophila melanogaster under the control of the strong PcpcBA6803 promoter, no transformants were ever obtained (hypothetical strain AAPZN05). A significantly weaker constitutive promoter with ∼9.4% of the activity of the wild-type (PcpcBA9.4%) or the tightly regulated PsmtA7942/SmtB7942 system was required to obtain transformants on selective plates (results not shown).
Induction of NinaB production using the PsmtA7942/SmtB7942 system in strain AAPZN07 resulted in chlorosis of cells when the final Zn2+ concentration was ≥50 μM. In contrast, a control strain lacking the ninaB gene but including the plasmid for SmtB7942 production (strain AAPZN04) showed a higher tolerance to Zn2+ (Fig. 7) and only exhibited chlorosis at Zn2+ concentrations of ≥75 μM during induction. These results and the failure to obtain transformants when ninaB was transcriptionally fused to the strong constitutive PcpcBA6803 promoter from Synechocystis sp. strain PCC 6803 strongly imply that NinaB is toxic to Synechococcus sp. strain PCC 7002 when expressed at high levels.
FIG 7
FIG 7 Comparison of cell culture appearance for strains AAPZN04 and AAPZN07 after induction with the Zn2+ concentrations indicated. Aliquots of a 50 mM ZnCl2 stock buffered in 50 mM MES (pH 6.5) were used to induce samples in 25 μM increments (0, 25, 50, 75, and 100 μM final Zn2+ concentrations). The cultures at times 0 h, 20 h, and 40 h of incubation under standard photoautotrophic growth conditions are shown. The induced expression of the ninaB gene at Zn2+ concentrations of ≥50 μM led to chlorosis and/or cell death. Zn2+ concentrations of ≥75 μM led to chlorosis and/or cell death for strain AAPZN04.
NinaB expression was not detected by immunoblotting in extracts of uninduced PsmtA7942/SmtB7942 cells or in a control strain with the same genetic background but excluding the plasmid for the expression of ninaB. However, NinaB production was readily detected upon induction with Zn2+ (Fig. 8). NinaB production was also detected when the much weaker, constitutive promoter PcpcBA9.4% was used (Fig. 8).
FIG 8
FIG 8 Immunoblot analysis of NinaB expression using an anti-NinaB primary antibody. A band of ∼71 kDa, the theoretical mass of NinaB, is expected for positive expression. Proteins are derived from strain AAPZN07 cells, to which no exogenous Zn2+ was added (lane 1) or 50 μM Zn2+ was added for induction (lane 2). Controls included proteins from strain AAPZN04 as a negative control (lane 3) and from strain AAPZN06 as a positive control (lane 4). The left lane shows the positions of marker proteins whose masses are indicated in kilodaltons.

DISCUSSION

An alignment of the sequence of the SmtB7942 transcriptional regulator of S. elongatus strain PCC 7942 with that of its homolog in Synechococcus sp. strain PCC 7002 shows that these two transcriptional repressors share 64% amino acid sequence and 80% similarity (20) (Fig. 1). An alignment of these Zn2+ responsive transcriptional regulators to their ziaR (RefSeq accession number sll0792 ) counterpart in Synechocystis sp. strain PCC 6803 (22) (Fig. 1) shows conservation of the putative α3N and α5 metal-binding amino acids and their DNA-binding regions (23). Interestingly, however, the native SmtB7002 repressor from Synechococcus sp. strain PCC 7002 was unable to control the PsmtA7942 promoter from S. elongatus strain PCC 7942 (Fig. 2B). These results are similar to those of Guerrero et al. (13), who showed that the PsmtA7002 promoter from Synechococcus sp. strain PCC 7002 was not controlled by the endogenous ziaR repressor of Synechocystis sp. strain PCC 6803. The divergence in promoter binding between the SmtB homologs in cyanobacteria can probably be attributed to the amino acid differences of the α3N motifs in their N-terminal regions (Fig. 1). Differences in the smtA operator and promoter regions among these cyanobacterial species are presumably responsible for their observed incompatibility with other members of the SmtB family of transcriptional regulators (20). Alternatively, the expression levels of the native SmtB7002 repressor in Synechococcus sp. strain PCC 7002 may have been inadequate to regulate the high-copy-number plasmid pAQ1Ex-PsmtA7942[yfp].
Because Synechococcus sp. strain PCC 7002 has an endogenous Zn2+ homeostasis mechanism, which is based on a class II metallothionein (SmtA7002) (15), high concentrations of exogenous Zn2+ would be required to elicit an induction response, and this can cause toxicity effects that would interfere with the desired induction response (results not shown). To circumvent this problem, a ΔzurA (Cyanobase accession number SYNPCC7002_A2498) strain was used as the genetic background for the heterologous PsmtA7942 expression system. Previous studies by Ludwig et al. (15) demonstrated that the zurA gene product is not essential under standard growth conditions and that a null mutation in this gene increases the relative transcript abundance for a putative ABC transporter for Zn2+. Additionally, in this prior study, no discernible responses to reactive oxygen species under high light conditions or stress under a broad range of exogenous Zn2+ concentrations were observed for the ΔzurA mutant or the wild type. Our study confirmed these results, but we found that the use of ZnCl2 dissolved in Tris-HCl, pH 8.0, led to precipitate formation in the medium, which may affect the actual final concentration of Zn2+. Based on the studies presented here, we suggest the use of 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 6.5, which allowed for the preparation of fresh solutions of ZnCl2 up to 50 mM without precipitation.
To achieve SmtB-mediated repression of the PsmtA7942 promoter from S. elongatus strain PCC 7942, coexpression of the cognate SmtB7942 repressor from the same strain was required (Fig. 3). Interestingly, introduction of the overlapping, divergent operator/promoter of the smt locus (24), from which smtB7942 is controlled by its native promoter in the minus DNA strand when PsmtA7942 was genetically fused to YFP in the plus DNA strand, did not allow for SmtB7942-regulated repression of YFP synthesis (results not shown). It was necessary to provide SmtB7942 constitutively to produce a system in which exogenous Zn2+ relieves repression of the PsmtA7942[yfp] construct. The use of the intermediate-strength, light-induced PpsbA26803 promoter (5) within the moderately high-copy-number plasmid pAQ3Ex (Fig. 3A) was necessary to increase the concentration of SmtB7942 to achieve the desired controlled response with the Zn2+ inducer (Fig. 4). The low sensitivity of previously reported metal-responsive expression systems (6) is one of the major hurdles of their use. Future optimization may involve the introduction of similar-strength promoters, which function independently of light or other factors, to control SmtB7942 in tandem with a gene of interest under the control of PsmtA7942.
The cpcBA promoters of Synechocystis sp. strain PCC 6803 and Synechococcus sp. strain PCC 7002 are among the strongest promoters known in cyanobacteria (1, 5, 21). Interestingly, compared to the PcpcBA6803 promoter from Synechocystis sp. strain PCC 6803, the PsmtA7942 promoter system described here produced ∼48% as much YFP per cell (Table 2) and did so in a titratable fashion. This makes this repressible and titratable promoter a promising system for controlled gene expression in Synechococcus sp. strain PCC 7002. The data also suggest that this promoter is tightly regulated, yielding little or no expression in the absence of an added inducer (i.e., exogenous Zn2+) (Fig. 6), unlike systems that rely on more traditional IPTG induction with the lacY repressor (2).
A frequently encountered problem with unregulated gene expression systems occurs when the gene product is toxic to cells. For example, attempts to express the Drosophila melanogasterninaB gene (25) in Synechococcus sp. strain PCC 7002, which encodes 15,15′ β-carotene dioxygenase, by using the pAQ1Ex::PcpcBA6803 system were unsuccessful, possibly because of toxicity effects associated with overproduction of retinal or depletion of β-carotene in such strains. Whatever the actual mechanism, stable transformants were never obtained using this expression platform. Thus, we decided to use the PsmtA7942/SmtB7942 system described here to express the ninaB gene. Stable transformants were obtained by selecting for transformants on a medium lacking exogenous Zn2+. After induction by adding Zn2+, the production of poly-(His)10-tagged NinaB was detected by immunoblotting (Fig. 8). This example shows the potential utility of this system for the inducible expression of potentially toxic products of genes that can cause severe growth impairment of Synechococcus sp. strain PCC 7002. Furthermore, this strategy may be beneficial for some biotechnological applications. The cleavage of β-carotene by NinaB to produce retinal can improve the activation of heterologously expressed opsins, which use retinal as chromophore, to introduce an additional light-energy conversion module that can improve the growth of cyanobacteria (26).
Although this system offers many advantages over previous attempts to use metal ions as regulatory elements to control gene expression, further studies will be required to establish how this Zn2+-inducible system affects the global transcriptional network of Synechococcus sp. strain PCC 7002 and whether there are unanticipated changes that may interfere with synthetic biology applications. Because Zn2+ is hazardous to the environment, its suitability for large-scale applications requires further evaluation, and postinduction strategies to eliminate this heavy metal from the growth medium may be necessary. In spite of these potential limitations, this expression system, based on the pAQ1Ex and pAQ3Ex systems (5), combines most of the beneficial properties sought for an inducible promoter, including little or no activation in the presence of low concentrations of soluble Zn2+ and good specificity with no discernible induction by Co2+ or Cu2+ at the concentrations found in the growth medium (27). The PsmtA7942/SmtB7942 system produces a graded expression response to increasing concentrations of Zn2+ that are below the level that is toxic for Synechococcus sp. strain PCC 7002. Lastly, the cost, stability, and availability of Zn2+ as an inducer make it a cost-effective alternative, in large-scale applications, to traditional inducible systems that employ more expensive inducers, such as IPTG.

MATERIALS AND METHODS

Strains, culture conditions, and transformation procedure.

All of the cyanobacterial strains utilized in this study were obtained from the Pasteur Culture Collection, Institut Pasteur, Paris, France (28). A zurA (zinc uptake regulator) deletion mutant strain of Synechococcus sp. strain PCC 7002 was previously constructed (15) and is designated strain ML001. All Synechococcus sp. strain PCC 7002 strains in this study are derived from strain ML001. Table 1 summarizes the description of these strains and the plasmids constructed in this study. Constructed strains of Synechococcus sp. strain PCC 7002 were grown in medium A supplemented with 1 mg NaNO3 ml−1 (designated medium A+), which contains 4 μg cyanocobalamin liter−1 (27, 29). To decrease the amount of Zn2+ in medium A+ to trace levels (<1 μM based on the amount of contaminating Zn in compounds used in the growth medium), ZnCl2 was omitted from the trace metal mixture. This modified medium is denoted as A+(–Zn2+) and was used as the basal medium for the growth of all Synechococcus sp. PCC 7002 strains in this study. Wild-type S. elongatus strain PCC 7942 (30) was grown in medium BG-11 (31, 32). S. elongatus strain PCC 7942 and Synechococcus sp. strain PCC 7002 isolates (described below) were grown photoautotrophically in 20-mm culture tubes containing medium BG-11 or A+(–Zn2+), respectively, at 38°C with continuous, cool white fluorescent illumination at 250 μmol photons m–2 s–1 and slow sparging with 1% CO2 (vol/vol) in air, otherwise referred to as “standard conditions” (1). For constructed strains, the following antibiotic concentrations were supplied as appropriate: 50 μg spectinomycin ml–1, 100 μg kanamycin ml–1, and 20 μg erythromycin ml–1. Transformation of Synechococcus sp. strain PCC 7002 was performed as described previously (5).

PCR amplification, restriction digestions, and ligations.

The PCR primers utilized in this study are listed in Table 3. All PCR amplifications in this work were performed using Phusion high-fidelity DNA polymerase (catalog number M0530S; New England BioLabs Inc., Ipswich, MA). Purification of the PCR products prior to digestion was performed as described previously (5). Genomic DNA was isolated as described previously (33). Digested DNA fragments were purified after electrophoresis on 0.8% (wt/vol) agarose gels using the EZ-10 spin column DNA extraction kit (catalog number BS353; Bio Basic Inc., Amherst, NY). Ligations with equimolar concentrations of DNA fragments (volume, 20 μl) were performed with T4 DNA ligase for 8 h at 16°C. Routine recombinant DNA procedures were performed using chemically competent Escherichia coli strain TOP10F' cells (34).
TABLE 3
TABLE 3 Oligonucleotide primers utilized in this study
NameSize (bases)Sequencea
PsmtA_F325′-AAA AAA GAA TTC GCA GCA CTG GTT TTG TCA TGA-3′ (EcoRI)
PsmtA_R305′-AAA AAA CCA TGG CAG CAA CTC CTT TGA ATA-3′ (NcoI)
SmtB_F345′-AAA AAA AAA ACA TAT GAC AAA ACC AGT GCT GCA G-3′ (NdeI)
SmtB_R315′-AAA AAA GGA TCC AGC CGA TTT CTG CCT AAG G-3′ (BamHI)
PpsbA2_F375′- GTT TAC CCG CGG AAA AAA CGA CAA TTA CAA GAA AGT A-3′ (SacII)
PpsbA2_R325′-TCG TGG CCA TGG GGT TAT AAT TCC TTA TGT AT-3′ (NcoI)
NinaB_F245′-GGG CAT ATG GCA GCC GGT GTC TTC-3′ (NdeI)
NinaB_R215′-CCC TGC AGC TAA ATG GCA TTG-3′ (PstI)
a
Underlining indicates introduced restriction site (noted at the end of the oligonucleotide sequence).

Expression of yfp under the control of the metal-regulated smtA promoter.

Genomic DNA from S. elongatus strain PCC 7942 was used as a template to amplify a 141-bp amplicon containing the smtA7942 class II metallothionein promoter region (PsmtA7942) (35) using primers PsmtA_F and PsmtA_R, which introduce 5′ EcoRI and 3′ NcoI sites, respectively (Table 3). The PsmtA7942 amplicon was ligated to a DNA fragment encoding the N-terminal decahistidine-tagged yellow fluorescent protein reporter gene (yfp) in the endogenous plasmid-based pAQ1Ex system, which contains an aadA spectinomycin/streptomycin resistance gene, using the unique EcoRI and NcoI restriction sites (5). The pAQ1Ex-PsmtA7942[yfp] construct was transformed into strain ML001 as previously described (5) to generate strain AAPZN01. Strain AAPZN01 was grown in medium A+(–Zn2+) with the appropriate antibiotics for several transfers to dilute any residual Zn2+ in the cells and/or the medium. Expression from the PsmtA7942 promoter in this strain was induced using aliquots of a ZnCl2 stock solution (50 mM ZnCl2 buffered with 50 mM MES at pH 6.5) to produce final Zn2+ concentrations of 25 μM, 50 μM, 75 μM, 100 μM, and 150 μM during the late exponential phase of growth (OD730 = 0.7 to 0.8). The whole-cell fluorescence emission was measured 12 to 16 h after induction for cells incubated under standard growth conditions. Fluorescence emission amplitudes for uninduced control cells and induced cells were normalized on the basis of equal cell numbers using an OD730 of 1.0 (see reference 36). The fluorescence emission amplitude of YFP when excited at 488 nm was determined using an SLM-Aminco 8100C fluorometer that has been modernized for computerized data acquisition by On-Line Instrument Systems (Bogart, GA).

Coexpression of cognate PsmtA7942 and the SmtB7942 repressor.

Genomic DNA isolated from S. elongatus strain PCC 7942 was used as a template to amplify a 420-bp amplicon containing the smtB7942 gene, encoding the metalloregulatory transcriptional repressor of PsmtA7942 (37), by using primers SmtB_F and SmtB_R to introduce the 5′ NdeI and 3′ BamHI sites (Table 3). Genomic DNA from Synechocystis sp. strain PCC 6803 was used as a template to amplify a 183-bp amplicon containing the promoter for the D1 protein of photosystem II (PpsbA26803) using primers PpsbA2_F and PpsbA2_R, which introduce the 5′ SacII and 3′ NcoI sites. The PpsbA26803 promoter of Synechocystis sp. strain PCC 6803 was ligated into the pAQ3Ex expression platform with the aphAII kanamycin resistance cassette (5) using the unique SacII and NcoI restriction sites. The smtB7942gene was introduced into the resulting pAQ3Ex construct as a transcriptional fusion to the PpsbA26803 promoter using the unique NdeI and BamHI restriction sites. The resulting plasmid, pAQ3Ex-PpsbA26803[smtB7942], was transformed into strain AAPZN01 to generate strain AAPZN02.

Zn2+ induction of the PsmtA7942 system.

Strain AAPZN02 was grown to an OD730 of 0.7 to 0.8 in medium A+(–Zn2+) supplemented with the antibiotics spectinomycin and kanamycin to maintain plasmids pAQ1Ex-PsmtA7942[yfp] and pAQ3Ex-PpsbA26803[smtB7942], respectively (Table 1). The expression of yfp was induced by the addition of buffered ZnCl2 to the desired final Zn2+ concentrations. Each induction experiment was conducted as three biological replicates, which were incubated and screened for a period of 29 h under standard growth conditions from the point of induction (t = 0 h). The OD730 and fluorescence emission amplitude of YFP at 527 nm when excited at 488 nm were recorded for each biological sample at 4- to 5-h intervals.

Purification of YFP.

Strain AAPZN02 was grown in medium A+(–Zn2+) (100 ml) with appropriate antibiotics under standard conditions for each induction trial. Cultures were induced by adding Zn2+ when the OD730 reached 0.7 to 0.8, and cultures were then incubated for an additional 24-h period under standard growth conditions prior to harvest. Strain ML001 was transformed with pAQ1Ex-PcpcBA6803[yfp] (5) to generate strain AAPZN03, which served as a positive control. In this strain, the yfp gene was expressed from pAQ1Ex under the control of the strong, constitutive cpcBA6803 promoter from Synechocystis sp. strain PCC 6803 (5). Strain AAPZN03 was grown to an OD730 of 1.0 prior to cell harvest, the condition under which the maximal YFP production has been reported previously (5).
Harvested cells were washed twice using lysis/binding buffer comprising 100 mM potassium phosphate buffer, pH 7.4, 500 mM NaCl, 10 mM 2-mercaptoethanol, and 5 mM imidazole. Each induced sample was normalized on the basis of the wet weight (g) of cells per milliliter. Cells were resuspended in lysis/binding buffer at a ratio of 1.0 g of wet weight cells per 5.0 ml of buffer. Purification of YFP was performed using the same volume for each sample after normalization. Samples were supplemented with 1 mM (each) protease inhibitors, pepstatin A and leupeptin (Sigma-Aldrich, St. Louis, MO), prior to disruption by three passages through a chilled French pressure cell operated at 128 MPa. The lysate was treated with 1 mM phenylmethylsulfonyl fluoride (PMSF) protease inhibitor, and the detergent Tergitol NP-40 (nonyl phenoxypolyethoxylethanol; Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 0.1% (vol/vol). The lysate was clarified by ultracentrifugation at 90,000 × g in a Beckman 70 Ti rotor for 1 h at 4°C. To purify the N-terminal His10-tagged YFP from the clarified lysate, HisPur cobalt spin columns (1.0 ml) (product number 89968; Thermo Scientific, Rockford, IL) were equilibrated at room temperature with a lysis/binding buffer with 0.1% (vol/vol) Tergitol NP-40 detergent. The clarified lysate of each sample was added to the HisPur cobalt-immobilized metal affinity chromatography resin and allowed to mix for 1 h at room temperature before proceeding with the washing and elution steps. Elution was performed with a lysis/binding buffer containing 150 mM imidazole. Wash and elution fractions were examined for YFP content by visual observation of fluorescence using long-wavelength UV excitation. Samples from each purification step and eluted fractions were also analyzed using a 12% SDS-PAGE gel (38). The fluorescence amplitude of purified YFP was measured at its emission maximum at 527 nm after excitation at 488 nm. The YFP concentration was determined using a Direct Detect infrared spectrometer (EMD Millipore, Merck, Darmstadt, Germany). YFP protein concentrations were determined using the amide 1 region of the infrared (IR) spectrum (39, 40).

Expression of Drosophila melanogaster NinaB.

Plasmid vector pOT2 (41) containing Drosophila melanogaster ninaB cDNA (25, 42) was obtained from the Drosophila Genomics Resource Center (Flybase identification number FBcl0129473). Primers NinaB_F and NinaB_R (Table 3) were used to produce an 1,877-bp ninaB amplicon with 5′ NdeI and 3′ PstI sites, respectively. The unique NdeI and PstI sites of pAQ1Ex-PcpcBA6803[yfp] (5) were used to fuse ninaB to the strong constitutive cpcBA6803 promoter of Synechocystis sp. strain PCC 6803 to produce the control plasmid pAQ1Ex-PcpcBA6803[ninaB]. Similarly, these unique sites were used to generate two additional ninaB expression plasmids: pAQ1Ex-PcpcBA9.4%[ninaB], containing a reduced activity PcpcBA6803 promoter variant (∼9.4% activity of the wild type) developed by Markley et al. (2), and pAQ1Ex-PsmtA7942[ninaB], which contains the ninaB gene under the control of the Zn2+-inducible smtA7942 promoter (Table 1). The three plasmids were individually transformed into strain AAPZN04, a strain resulting from the introduction of pAQ3Ex-PpsbA26803[smtB7942] into strain ML001. Introduction of pAQ1Ex-PcpcBA6803[ninaB] into strain ML001 generated strain AAPZN05, introduction of pAQ1Ex-PcpcBA9.4%[ninaB] into strain ML001generated strain AAPZN06, and introduction of pAQ1Ex-PsmtA7942[ninaB] into strain ML001generated strain AAPZN07 (Table 1).

Zn2+ induction, purification, and detection of NinaB.

Strain AAPZN07 was grown in medium A+(–Zn2+) with appropriate antibiotics. Replicates (25-ml cultures) of this mutant were grown in parallel for individual treatment with ZnCl2 to final concentrations of 0, 25, 50, 75, and 100 μM when the culture reached an OD730 of 0.7 to 0.8 (t = 0 h for induction). Samples were screened for phenotypic changes at 0, 20, and 40 h. Strain AAPZN04 was included in this induction experiment as the control.
Strain AAPZN06 was grown in 4 liters of medium A+(–Zn2+) with appropriate antibiotics to an OD730 of 1.0 prior to harvesting cells by centrifugation. Strain AAPZN04, as a negative control, was also grown as described above. Strain AAPZN07 was grown in medium A+(–Zn2+) (4 liters) with appropriate antibiotics under standard conditions. Cultures were induced with a final concentration of 50 μM ZnCl2 at an OD730 of 0.7 to 0.8 and then incubated for 24 h under standard growth conditions prior to harvesting the cells by centrifugation.
Harvested cells were washed twice using lysis/binding buffer, comprising 100 mM potassium phosphate buffer (pH 8.0), 500 mM NaCl, 25% (vol/vol) glycerol, 1 mM dithiothreitol (DTT), and 5 mM imidazole. Each sample was normalized on the basis of grams of cells per milliliter as described above. Purification of NinaB was performed using the same volume for each sample after normalization to equal cell density. Samples were supplemented with 1 mM of the protease inhibitors pepstatin A and leupeptin (Sigma-Aldrich, St. Louis, MO) prior to disruption by three passages through a chilled French pressure cell operated at 128 MPa. The lysate was treated with PMSF protease inhibitor (1 mM), and the detergent Tergitol NP-40 (nonyl phenoxypolyethoxylethanol) was added to a final concentration of 0.1% vol/vol (Sigma-Aldrich, St. Louis, MO). The lysate was clarified by ultracentrifugation at 90,000 × g in a Beckman 70 Ti rotor for 1 h at 4°C. All subsequent steps were performed at 4°C. To purify the N-terminal His-tagged NinaB, the clarified lysate was batch-loaded onto 1.8-mm Protino Ni-NTA agarose resin (Macherey-Nagel GmbH & Co., Düren, Germany) during 3 h of incubation at 4°C. The Ni-NTA resin was gently pelleted by centrifugation at 700 × g for 8 min, the unbound lysate was decanted, and the resin was poured into a small chromatography column. The settled resin was washed with 10 column volumes of lysis/binding buffer supplemented with PMSF, DTT, and Tergitol NP-40 detergent. A second washing step with 10 column volumes of lysis/binding buffer without detergent was then performed. A third wash with 10 column volumes of lysis buffer supplemented with imidazole to 40 mM, PMSF, and DTT was then performed. Elution occurred with a new buffer: 50 mM Tris-HCl (pH 8.0) at 4°C, 100 mM NaCl, 500 mM imidazole, glycerol at 25% (vol/vol), 5 mM DTT, and 1 mM PMSF. Fractions equivalent to one-half of a column volume were collected and analyzed by Bradford assay (Bio-Rad Laboratories, Hercules, CA). The fractions enriched for protein were concentrated 20-fold by using a 30-kDa MWCO Nanosep microcentrifuge concentrator (Pall Corporation, Port Washington, NY) at 10,000 × g and 4°C in steps of 10 min. NinaB was detected by immunoblotting as described previously (43) using a rabbit and polyclonal antiserum raised against recombinant NinaB from the moth Galleria mellonella as the primary antibody (kindly provided by J. von Lintig, Case Western Reserve University, Cleveland, OH). A horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (Rockland Antibodies & Assays, Gilbertsville, PA) was utilized for detection, together with an Amersham ECL Western blotting (GE Healthcare, Little Chalfont, United Kingdom) detection system; the resulting blot was exposed to HyBlot CL autoradiography film (Denville Scientific, Metuchen, NJ).

ACKNOWLEDGMENTS

We are grateful to Johannes von Lintig for helpful discussions as well as providing the anti-NinaB primary antibody and to David Gilmore for providing the ninaB gene (obtained from the Drosophila Genome Resource Project, which is supported by NIH grant 2P40OD010949-10A1). We acknowledge Neela Yennawar at The Pennsylvania State University Huck Institutes of the Life Sciences Automated Biological Calorimetry Facility for use of the Direct Detect infrared spectrometer for protein concentration measurements.
This work was supported by DOE contract DE-FG-02-05-ER46222 and NSF grants MCB-1021725 and MCB-1613022 to D.A.B. and by NSF grant MCB-1359634 to J.H.G.

REFERENCES

1.
Ludwig M and Bryant DA. 2011. Transcription profiling of the model cyanobacterium Synechococcus sp. strain PCC 7002 by Next-Gen (SOLiD™) sequencing of cDNA. Front Microbiol2:41.
2.
Markley AL, Begemann MB, Clarke RE, Gordon GC, and Pfleger BF. 2015. Synthetic biology toolbox for controlling gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002. ACS Synth Biol4:595–603.
3.
Bernstein HC, McClure RS, Hill EA, Markillie LM, Chrisler WB, Romine MF, McDermott JE, Posewitz MC, Bryant DA, Konopka AE, Fredrickson JK, and Beliaev AS. 2016. Unlocking the constraints of cyanobacterial productivity: acclimations enabling ultrafast growth. mBio7:e00949-16.
4.
McClure RS, Overall CC, McDermott JE, Hill E, Markillie LM, McCue LA, Taylor RC, Ludwig M, Bryant DA, and Beliaev AS. 2016. Network analysis of transcriptomics expands regulatory landscapes in Synechococcus sp. PCC 7002. Nucleic Acids Res44:8810–8825.
5.
Xu Y, Alvey RM, Byrne PO, Graham JE, Shen G, and Bryant DA. 2011. Expression of genes in cyanobacteria: adaptation of endogenous plasmids as platforms for high-level gene expression in Synechococcus sp. PCC 7002. Methods Mol Biol684:273–293.
6.
Berla BM, Saha R, Immethun CM, Maranas CD, Moon TS, and Pakrasi HB. 2013. Synthetic biology of cyanobacteria: unique challenges and opportunities. Front Microbiol4:246.
7.
Zhang S, Shen G, Li Z, Golbeck JH, and Bryant DA. 2014. Vipp1 is essential for the biogenesis of photosystem I but not thylakoid membranes in Synechococcus sp. PCC 7002. J Biol Chem289:15904–15914.
8.
Ma Z, Jacobsen FE, and Giedroc DP. 2009. Coordination chemistry of bacterial metal transport and sensing. Chem Rev109:4644–4681.
9.
Blindauer CA, Harrison MD, Robinson AK, Parkinson JA, Bowness PW, Sadler PJ, and Robinson NJ. 2002. Multiple bacteria encode metallothioneins and SmtA-like zinc fingers. Mol Microbiol45:1421–1432.
10.
Cavet JS, Borrelly GP, and Robinson NJ. 2003. Zn, Cu and Co in cyanobacteria: selective control of metal availability. FEMS Microbiol Rev27:165–181.
11.
Huckle JW, Morby AP, Turner JS, and Robinson NJ. 1993. Isolation of a prokaryotic metallothionein locus and analysis of transcriptional control by trace metal ions. Mol Microbiol7:177–187.
12.
Erbe JL, Adams AC, Taylor KB, and Hall LM. 1996. Cyanobacteria carrying an smt-lux transcriptional fusion as biosensors for the detection of heavy metal cations. J Ind Microbiol17:80–83.
13.
Guerrero F, Carbonell V, Cossu M, Correddu D, and Jones PR. 2012. Ethylene synthesis and regulated expression of recombinant protein in Synechocystis sp. PCC 6803. PLoS One7:e50470.
14.
Boyanapalli R, Bullerjahn GS, Pohl C, Croot PL, Boyd PW, and McKay RM. 2007. Luminescent whole-cell cyanobacterial bioreporter for measuring Fe availability in diverse marine environments. Appl Environ Microbiol73:1019–1024.
15.
Ludwig M, Chua TT, Chew CY, and Bryant DA. 2015. Fur-type transcriptional repressors and metal homeostasis in the cyanobacterium Synechococcus sp. PCC 7002. Front Microbiol6:1217.
16.
Redinbo MR, Yeates TO, and Merchant S. 1994. Plastocyanin: structural and functional analysis. J Bioenerg Biomembr26:49–66.
17.
Antonkine ML, Maes EM, Czernuszewicz RS, Breitenstein C, Bill E, Falzone CJ, Balasubramanian R, Lubner C, Bryant DA, and Golbeck JH. 2007. Chemical rescue of a site-modified ligand to a [4Fe–4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to photosystem I. Biochim Biophys Acta1767:712–724.
18.
Smith KS and Ferry JG. 2000. Prokaryotic carbonic anhydrases. FEMS Microbiol Rev24:335–366.
19.
Heidorn T, Camsund D, Huang HH, Lindberg P, Oliveira P, Stensjo K, and Lindblad P. 2011. Synthetic biology in cyanobacteria engineering and analyzing novel functions. Methods Enzymol497:539–579.
20.
Shelake RM, Aibara K, Hayashi H, Abe S, and Morita EH. 2013. Comparative analysis of heavy-metal ion sensing mechanisms with transcription factors, SmtBs, from freshwater Synechococcus sp. PCC 7942, and marine Synechococcus sp. PCC 7002: evolutionary and structural aspects. Int J Sci Technol Res2:274–284.
21.
Zhou J, Zhang H, Meng H, Zhu Y, Bao G, Zhang Y, Li Y, and Ma Y. 2014. Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria. Sci Rep4:4500.
22.
Thelwell C, Robinson NJ, and Turner-Cavet JS. 1998. An SmtB-like repressor from Synechocystis PCC 6803 regulates a zinc exporter. Proc Natl Acad Sci U S A95:10728–10733.
23.
Pennella MA and Giedroc DP. 2005. Structural determinants of metal selectivity in prokaryotic metal-responsive transcriptional regulators. Biometals18:413–428.
24.
Kar SR, Lebowitz J, Blume S, Taylor KB, and Hall LM. 2001. SmtB-DNA and protein-protein interactions in the formation of the cyanobacterial metallothionein repression complex: Zn2+ does not dissociate the protein-DNA complex in vitro. Biochemistry40:13378–13389.
25.
von Lintig J, Dreher A, Kiefer C, Wernet MF, and Vogt K. 2001. Analysis of the blind Drosophila mutant ninaB identifies the gene encoding the key enzyme for vitamin A formation in vivo. Proc Natl Acad Sci U S A98:1130–1135.
26.
Chen Q, van der Steen JB, Dekker HL, Ganapathy S, de Grip WJ, and Hellingwerf KJ. 2016. Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803. Metab Eng35:83–94.
27.
Stevens SE and Porter RD. 1980. Transformation in Agmenellum quadruplicatum. Proc Natl Acad Sci U S A77:6052–6056.
28.
Rippka R, Deruelles J, Waterbury JB, Herdman M, and Stanier RY. 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol111:1–61.
29.
Stevens SE, Patterson CO, and Myers J. 1973. The production of hydrogen peroxide by blue-green algae: a survey. J Phycol9:427–430.
30.
Shestakov SV and Khyen NT. 1970. Evidence for genetic transformation in blue-green alga Anacystis nidulans. Mol Gen Genet107:372–375.
31.
Yousef N, Pistorius EK, and Michel KP. 2003. Comparative analysis of idiA and isiA transcription under iron starvation and oxidative stress in Synechococcus elongatus PCC 7942 wild-type and selected mutants. Arch Microbiol180:471–483.
32.
Stanier RY, Kunisawa R, Mandel M, and Cohen-Bazire G. 1971. Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev35:171–205.
33.
Schluchter WM, Shen G, Zhao J, and Bryant DA. 1996. Characterization of psal and psaL mutants of Synechococcus sp. strain PCC 7002: a new model for state transitions in cyanobacteria. Photochem Photobiol64:53–66.
34.
Vogl K, Tank M, Orf GS, Blankenship RE, and Bryant DA. 2012. Bacteriochlorophyll f: properties of chlorosomes containing the “forbidden chlorophyll”. Front Microbiol3:298.
35.
Morby AP, Turner JS, Huckle JW, and Robinson NJ. 1993. SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene smtA: identification of a Zn inhibited DNA-protein complex. Nucleic Acids Res21:921–925.
36.
Inoue-Sakamoto K, Gruber TM, Christensen S, Arima H, Sakamoto T, and Bryant DA. 2007. Group 3 sigma factors in the marine cyanobacterium Synechococcus sp. strain PCC 7002 are required for growth at low temperature. J Gen Appl Microbiol53:89–104.
37.
Busenlehner LS, Pennella MA, and Giedroc DP. 2003. The SmtB/ArsR family of metalloregulatory transcriptional repressors: structural insights into prokaryotic metal resistance. FEMS Microbiol Rev27:131–143.
38.
Manns J. 2011. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins. Curr Protoc Microbiol22:A.3M.1–A.3M.13.
39.
Strug I, Utzat C, Cappione A III, Gutierrez S, Amara R, Lento J, Capito F, Skudas R, Chernokalskaya E, and Nadler T. 2014. Development of a univariate membrane-based mid-infrared method for protein quantitation and total lipid content analysis of biological samples. J Anal Methods Chem2014:657079.
40.
Yennawar NH, Fecko JA, Showalter SA, and Bevilacqua PC. 2016. A high-throughput biological calorimetry core: steps to startup, run, and maintain a multiuser facility. Methods Enzymol567:435–460.
41.
Allaway D, Schofield NA, Leonard ME, Gilardoni L, Finan TM, and Poole PS. 2001. Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes. Environ Microbiol3:397–406.
42.
Rubin GM, Hong L, Brokstein P, Evans-Holm M, Frise E, Stapleton M, and Harvey DA. 2000. A Drosophila complementary DNA resource. Science287:2222–2224.
43.
Shen G, Zhao J, Reimer SK, Antonkine ML, Cai Q, Weiland SM, Golbeck JH, and Bryant DA. 2002. Assembly of photosystem I. I. Inactivation of the rubA gene encoding a membrane-associated rubredoxin in the cyanobacterium Synechococcus sp. PCC 7002 causes a loss of photosystem I activity. J Biol Chem277:20343–20354.

Information & Contributors

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Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 83Number 31 February 2017
eLocator: e02491-16
Editor: Frank E. Löffler, University of Tennessee and Oak Ridge National Laboratory
PubMed: 27836841

History

Received: 28 August 2016
Accepted: 7 November 2016
Published online: 17 January 2017

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Keywords

  1. Zn2+-inducible promoter
  2. cyanobacteria
  3. gene expression platform
  4. metallothionein
  5. ninaB
  6. photosynthesis

Contributors

Authors

Adam A. Pérez
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA
John P. Gajewski
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA
Bryan H. Ferlez
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA
Marcus Ludwig
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA
Present address: Marcus Ludwig, Jennewein Biotechnologie GmbH, Rheinbreitbach, Germany.
Carol S. Baker
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA
John H. Golbeck
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, USA
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA
Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana, USA

Editor

Frank E. Löffler
Editor
University of Tennessee and Oak Ridge National Laboratory

Notes

Address correspondence to Donald A. Bryant, [email protected].

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