a novel expression vector for the cyanobacterium, synechococcus

DNA RESEARCH 1, 181-189 (1994)

A Novel Expression Vector for the Cyanobacterium,Synechococcus PCC 6301

Yasunobu TAKESHIMA,1* Masahiro SUGIURA,2 and Hideaki HAGIWARA1

Hagiwara Institute of Health, Asazuma-cho, Kasai Hyogo 679-01, Japan1 and Center for Gene Research,Nagoya University, Chitose-ku, Nagoya 464-01, Japan2

(Received 5 August 1994)

Abstract

A cyanobacterial expression vector was constructed using ribulose-l,5-bisphosphate carboxy-lase/oxygenase (RuBisCO) promoter and terminator sequences derived from Synechococcus PCC 6301. Therecombinant plasmid, designated pARUB19, has an ampicillin-resistant (ApR) gene as a selectable markerand four unique restriction sites to allow the insertion of foreign genes. Using this vector, the luciferasegene from the firefly, Photinus pyralis, was introduced into Synechococcus PCC 6301 cells. The luciferaseexpression vector could be maintained stably in the host cells. Light production of luciferin/luciferase wasdetected in the transformants. Luciferase amounted to 1.2% of the total soluble protein. This plasmid mayfacilitate higher levels of foreign gene expression in Synechococcus PCC 6301.

Key words: cyanobacterium; expression vector; luciferase gene; plasmid replication

1. Introduction

Cyanobacteria are autotrophic prokaryotes which per-form oxygenic photosynthesis similar to that of higherplants. Since the organisms have a relatively simple ge-nomic organization, they are suitable for the study ofplant-type photosynthesis at a molecular level.

Several cyanobacterial species, such as SynechococcusPCC 7942, Synechococcus PCC 7002 and SynechocystisPCC 6803 have the ability to take up exogenous DNAinto the cells, allowing them to be transformed easilywith exogenous DNA.1'2'3 Taking advantage of this abil-ity, a number of different shuttle cloning vectors capableof replicating in both Escherichia coli and cyanobacteriahave been constructed.4 Concerning the development ofthe expression vectors for cyanobacteria, however, onlya limited number of reports have appeared.5'6'7 The lackof a system for expression of foreign genes in cyanobac-terial cells has restricted the genetic manipulation ofcyanobacteria. We have constructed a small shuttle vec-tor which contains cyanobacterial promoter and termina-tor sequences of the rbcL-S operon, and expressed fireflyluciferase gene in Synechococcus PCC 6301 cells using theplasmid.

Communicated by Mituru Takanami* To whom correspondence should be addressed. Tel. +81-790-

47-1581, Fax. +81-790-47-1585

2. Materials and Methods

2.1. Bacterial strain and transformationE. coli (strains HB101 and JM109) was grown in L-

broth or 2YT at 37°C. Ampicillin (Ap) was added at50 /xg/ml when required. Transformation of E. coli cellswith plasmid DNA was performed as described by Mani-atis et al.8

Synechococcus PCC 7942 (Anacystis nidulans R2) andSynechococcus PCC 6301 (A. nidulans 6301) were cul-tured in 100 ml of BG-11 liquid medium at 28°C undercool white fluorescent light and subcultured at the mid-exponential phase of growth. To 1.0 ml of cell suspensioncontaining 2xlO8 cells (for strain PCC 7942 A730=0.5)of 109 cells (for strain PCC 6301, A730=2.5), which werecultured at the mid-exponential phase of growth, 0.5 or1.0 fig of donor DNA (in 10 mM Tris/1 mM EDTA, pH8.0) was added, and the mixture was incubated in thedark at 26°C overnight. After incubation for a further 6h in the light, the transformants were directly selected onBG-11 agar plates containing 1.5% agar, 1 mM sodiumthiosulfate and 0.5 /ig/mL Ap. The transformation fre-quency was calculated by counting the number of trans-formants after 15 days.

2.2. Construction of shuttle cloning vectorsThe multiple cloning site (MCS, EcoRl-Hindlll frag-

ment) derived from pUCl8 was introduced between theEcoRI and Hindlll sites of pBR322. This construct wasdesignated as pBR322M (Fig. lb). To the 2.5-kb de-phosphorylated Pvull-EcoA7Ill (PEC) fragment of plas-

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182 Expression Vector for Synechococcus PCC 6301 [Vol. 1,

a. pBA1(7.8 kb)

b. pBR322M(4.3 kb)

Hindlll Bglll Hpal Pvull Xhol BglllI I I LJ I

BamHI OriS Pvull H P a l

Pvull MCS

P BamHIECO47III

BamHI

BamHI Tc QriE AP

c. pBASiO(10.1 kb)

d. pBASiOR(10.1 kb)

BamHI

Bglll Xhol Pvull Hpal BamHI MCS

Hpal Pvull OriS OriE Ap

Hpal

1

OriS

Pvull

Pvull

Xhol

•

Bglll

I1

Hpal ONE

MCS

Ap

e. PBAS18(12 kb)

f. pBAXIO(8.8 kb)

Bglll Xhol Pvull Hpal Hindlll Pvull

-IBamHI

TBamHI Hpal Pvull OriS BamHI OriE Ap

Pvull Pvull Hpal Hindlll Pvull EcoRI

EcoRI

OriS OriE Ap B a m H I

g. pBAXiOR(8.8 kb)

h. pBAX18(6.9 kb)

Hindlll Hpal Pvull Pvull Pvull EcoRI

OriS

Pvull Pvull Hpal Hindlll. 1 1 | '

OriE Ap B a m H I

OriS OriE Ap

i. PBAX18R(6.9 kb)

Hindlll Hpal Pvull PvullI I I I

MCS

OriS OriE Ap

Figure 1. Schematic presentation of the new shuttle vectors. The plasmid construction is described in the text. \SiXmm<^\, SynechococcuspBAl sequences; K$$$$$$$$i, pBR322 sequences; B B B H > pBR322M sequences; OriS, putative Synechococcus replication origin; OriE,E. coli replication origin; MCS, multicloning sites of pUC18.


No. 4] Y. Takeshima, M. Sugiura, and H. Hagiwara 183

mid pBR322M, the BamHl fragment of pBAl9 (Fig. la)which originated from Synechococcus PCC 6301 was lig-ated after filling in the ends with T4 DNA polymerase.The hybrid plasmids were designated as pBASlO andpBASlOR (Figs, lc and d, respectively). pBASlO andpBASlOR differ in the orientation of the BamHl frag-ment of pBAl.

After filling in the ends, the 4.4-kb BamHl-Xhol(BAX) fragment of pBAl was introduced in the BamHlsite of pBR322, the ends of which were filled withT4 DNA polymerase. The constructs were named aspBAXlO and pBAXlOR (Figs. If and g, respectively).pBAXlO and pBAXlOR differ in the orientation of theBAX fragment of pBAl. To the 2.5-kb dephosphorylatedPEC fragment, the BAX fragment was ligated after fillingin the ends. The plasmid was designated pBAX18 andpBAX18R (Figs, lh and i, respectively). pBAX18 andpBAX18R differ in the orientation of the BAX fragmentof pBAl.

2.3. Construction of an expression vector, pARUB19The construction procedures are summarized in Fig.

2. The promoter region of rbcL was prepared frompANE1810 by digestion with Sad and Pstl. The Sad-Pstl fragment was introduced between the Sad and Pstlsites of pUC18. Recutting with EcoRl and Pstl, the 353-bp EcoRl-Pstl fragment was obtained as a promoter se-quence. The 353-bp EcoRl-Pstl fragment was ligated toa synthesized 74-bp Pstl-Hindlll linker fragment to con-struct the 423-bp EcoRl-Hindlll fragment. The termi-nator region of rbc was prepared from pANP115510 bydigestion with Eco52l and Pstl. After filling in the endswith T4 DNA polymerase, the 294-bp Eco52l-Pstl frag-ment was inserted into the Smal site of pUC13. Fromthe construct, the 318-bp rbc terminator region, fromthe filled in BamHl site to the EcoRl site, was obtained.To this 318-bp rbc terminator region, MCS of whichthe .SeoRI site was filled in was fused, and the 373-bpHindlll-EcoRl fragment was obtained. The 423-bp rbcpromoter region and the 373-bp terminator region wereligated at the Hindlll site and after filling in both theEcoRl ends with T4 DNA polymerase, the 786-bp expres-sion cassette was constructed. The expression cassettewas introduced between the EcoRl and Nofl sites of theshuttle cloning vector pBAX18R by blunt-end ligation.

2.4- Analysis of stability of the chimeric plasmids in thehost cells

Each single colony of Ap-resistant (ApR) Synechococ-cus PCC 6301 cells transformed with respective plas-mids was cultured in BG-11 liquid medium contain-ing 10 ^g/ml Ap and plasmid DNA was isolated fromthe culture by the alkaline-SDS method. The plasmidDNAs were digested with EcoRl, electrophoresed in 1.0%

agarose gel and transferred to a nitrocellulose membrane(Hybond-C, Amersham, UK) as described.8 Blot hy-bridization was carried out according to the instructionmanual of Labezyme-POD (Wako Pure Chemical Indus-tries Japan) with peroxidase-labeled pBR322. The blot-ted membrane was incubated at 37° C for 1 h in a blockingsolution containing 50% formamide, lOxDenhardt's so-lution, 0.1% SDS and 4xSET [SET: 0.15 M NaCl, 1 mMEDTA and 30 mM Tris-HCl (pH 8.0)]. Prehybridiza-tion was performed at 37° C for 1 h in 50% formamide,2xDenhardt's solution, 4xSET, 6% polyethylene gly-col 6000, 0.1% SDS and 100 ^g/ml denatured, frag-mented salmon sperm DNA. Peroxidase-labeled pBR322was added to the prehybridization solution, and hy-bridization was carried out at 37°C for 10 h. The mem-brane was washed 3 times at 37° C for 20 min in 50%formamide, 0.4% SDS and 0.5xSSC (SSC: 0.15 M NaCland 15 mM sodium citrate, pH 7.0) and 3 times at roomtemperature for 20 min in 2xSSC. The chimeric plasmidDNAs which hybridized with peroxidase-labeled pBR322were visualized by enzymatic color development throughincubation with 4-chloro-l-naphthol.

2.5. Expression of firefly lucif eraseThe firefly, Photinus pyralis, luciferase gene was pre-

pared from the cassette vector (Toyo Inki-Seizo Co,Japan) by digestion with BamHl and Xhol. The lu-ciferase expression vector, pARUB-/wc, was constructedby insertion of the 1.7-kb luciferase gene, the ends ofwhich were filled with T4 DNA polymerase, into theblunted Sphl site of pARUB19.

A single colony of ApR Synechococcus PCC 6301 cellstransformed with pARUB-Zttc was cultured in BG-11medium containing 50 /xg/ml Ap under cool white flu-orescent light (4000 lux) for 10 days. The cells werewashed and resuspended in 0.5 M Tris-succinate (pH 7.7)containing 3 mM dithiothreitol, and then broken by son-ication. .Total soluble proteins were analyzed by elec-trophoresis in a 2-15% gradient SDS-polyacrylamide gelfollowed by staining with Coomassie blue.

The luciferase activity was detected in transformedcells using Camlight™ 500 (Analytical LuminescenceLaboratory, USA). Portions (100 fA) of the cells weredistributed into 18 Well Tilt Tray (Analytical Lumines-cence Laboratory). The tray was exposed by contactwith Polaroid film Type 612 for 5 min.

3. Results and Discussion

3.1. Modification of hybrid plasmidsA shuttle cloning vector pBAS18, which includes

pBR322 and an endogenous plasmid pBAl from Syne-chococcus PCC6301, has previously been constructed.9

However, the transformation of Synechococcus PCC 7942



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792 bpEcoRI

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pBAX18R(6.9kb)

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Figure 2. Schematic representation of the construction of a new expression vector with four unique restriction sites, Sphl, Xbal, BamHIand Smal. BAP, bacterial alkaline phosphatase.



CO3UOooo•sQ)

00 0 0 O O O O O O 0 0 0 0 0 0

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kb

23.13

9 . 4 2 -

6 .56 -

4.36 .— 4.26

Figure 3. Southern blot analysis of plasmid DNAs isolated from Synechococcus PCC 6301 and transformants; 0.2 jig each of plasmidDNAs were digested with EcoRl, electrophoresed in 1.0% agarose gel and transferred to a nitrocellulose membrane. Southernhybridization was performed using peroxidase-labeled pBR322 as a probe. Synechococcus, (S) and (E) indicate endogenous plasmidsfrom Synechococcus PCC 6301, and plasmid DNAs recovered from Synechococcus PCC 6301 and E. coli, respectively. Size markersused are A DNA ffindlll digests and A DNA Styl digests.

with pBAS18 occurred at a relatively low frequency, andpBAS18 could not be maintained stably in both the Syne-chococcus PCC 7942 and PCC 6301. To improve thisdisadvantage of pBAS18, we have constructed various

chimeric plasmids and compared their properties withregard to the stability in transformants of SynechococcusPCC 6301.

The endogenous plasmid pBAl9 (7.8 kb, Fig. la)



SaclSmalBamHIXbalSailHindlll

UC

Sail

Promoter

SaclBglll

Hpal

Hindlll

Figure 4. The physical map of the expression vector of the fireflyluciferase gene, pARUB-/«c.

from Synechococcus PCC 6301 has a restriction mapclosely related to that of plasmid pUH24 (or pANS)of Synechococcus PCC 7942 and can be cleaved intotwo pieces by digestion with BamHI and Xhol.11 Thelarger 4.65 kb BarriHI-Xhol fragment of pUH24 has beenreported to support replication in Synechococcus PCC7942.12 This fragment, when present in recombinantplasmids, is able to transform Synechococcus PCC 7942and maintain plasmid replication in the absence of theother pUH24 segment.13 Based on these findings, thesmaller 3.3-kb Xhol-BamEl segment of pBAl was deletedfrom the chimeric plasmid pBAS18 (pBAXlO, pBAXlOR,pBAX18 and pBAX18R) (Fig. 1).

The entire sequence of pBR322 is contained inpBAS18, and the pBR322 moiety contains two remov-able genes: rop, which is involved in copy number regu-lation of pBR322,14 and the tetracycline-resistant (TcR)gene. Between the two selectable markers, ApR andTcR, in pBR322, TcR is unsuitable for the selectionof the transformants of cyanobacterial cells because ofthe instability of Tc under light. Thus, the 1.8-kbEco471II-PvuII segment, which contains the TcR geneand rop of pBR322, was deleted from the chimeric plas-mid (pBASlO, pBASlOR, pBAX18 and pBAX18R) (Fig.

3.2. Stabilization of chimeric plasmids in Synechococ-cus cells

To examine the stability of the constructed chimericplasmids within Synechococcus PCC 6301, plasmid DNAswere recovered from the transformed cells, digested withEcoRI and analyzed by Southern hybridization usingperoxidase-labeled pBR322 as a probe. Figure 3 showsthat the patterns of the plasmid DNAs recovered fromthe cells transformed with pBASlO, pBASlOR, pBAX18and pBAX18R were identical to those of each plasmidDNA used for transformation [Fig. 3, lanes pBAS10(S),pBASlOR(S), pBAX18(S) and pBAX18R(S)]. The re-sult suggests that pBASlO, pBASlOR, pBAX18 andpBAX18R exist in the host cells without alteration dur-ing replication. In contrast, the plasmid DNA fractionswhich were recovered from the cells transformed withpBAS18, pBAXlO and pBAXlOR under the same con-ditions yielded some extra bands unrelated to the origi-nal plasmids [Fig. 2, lanes pBAS18(S), pBAX10(S) andpBAXlOR(S)]. This result suggests that the recombi-nants which contained the entire sequence of pBR322 areunstable in the host cells, and that the deletion of the1.8-kb Eco47III-PvulI segment of pBR322 results in sta-bilization of replication in the Synechococcus PCC 6301cells.

3.3. A novel expression vector, pARUB19The minimal region of pUH24 fully capable of support-

ing autonomous replication has been narrowed down toa 3.6-kb DNA region.15 Besed on this report, the 3.7-kb Xhol-Notl fragment of pBAl, which corresponds tothe 3.6-kb minimal region of pUH24 was used as thecyanobacterial origin of replication.

To express foreign genes efficiently in cyanobacterialcells, a promoter that strongly functions in cyanobacte-rial cells is required. Because RuBisCO is one of themajor soluble proteins in Synechococcus PCC 6301 cells,we used the promoter of the rbcL-S operon,16 and therbc terminator sequence was placed downstream for sta-ble translation of the foreign genes and stable replicationof the shuttle vector. To facilitate the insertion of foreigngenes, MCS derived from pUC18 was introduced betweenthe RuBisCO promoter and terminator sequences.

Many cyanobacterial genes have Shine-Dalgarno (SD)-like sequences, which are putative ribosomal bindingsites, upstream from the ATG start codon of thegenes.17'18 We identified an SD-like sequence, GGAG,upstream from the ffindlll site of MCS. In addition, tofacilitate the ligation of foreign genes and further mod-ification of around the SD-like sequence, the Taql sitelocated 24 bp upstream from the SD-like sequence10 wasmodified to an Sail site. Consequently, the expressionvector became 6.9 kb in size and carried four unique re-striction sites (BamHI, Smal, Sphl and Xbal) that allow


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188

the insertion of foreign genes.

Expression Vector for Synechococcus PCC 6301 [Vol. 1,

3.4- Stability of expression vector in host cellsTo examine the ability of pARUB19 as an expression

vector, the firefly luciferase gene was inserted in the SphIsite of the MCS (Fig. 4). The constructed luciferaseexpression vector was designated pARUB-Juc. Syne-chococcus PCC 6301 was transformed with pARUB19and pARUB-Zwc, respectively. The transformation fre-quencies were 10 to 102 cells//ig plasmid DNA. From eachof the transformed cells, irnact hybrid plasmids capableof transforming Synechococcus PCC 6301 and E. coli wereisolated. The plasmid DNAs recovered from the transfor-mants were digested with Sail (Fig. 5A). SynechococcusPCC 6301 contains cryptic plasmids, 7.8-kb (pBAl) and48.8-kb.4 The restriction patterns show that the plas-mid DNAs recovered from pARUB19 and pARUB-/«ctransformants were identical to those of each plasmidDNA used for transformation (Fig. 5A). Transformantshave been stably maintained for over 6 months by re-peated subculturing in the presence of Ap at 50 ^jg/ml.These results suggest that pARUB19 and pARUB-fec arenot altered during DNA uptake and replication in Syne-chococcus PCC 6301 cells. The mechanism is not knownby which the recombinants are more stably maintainedin the cells, although the same repL^tion origin of theendogenous plasmid pBAl has been used in both trans-formants.

In cyanobacterial cells, it is well known that chimericDNA containing the cyanobacterial chromosomal DNAintegrate into the recipient DNA by homologous recom-bination at high frequencies.19 The expression vector,pARUB19 carries the cyanobacterial RuBisCO promoterand terminator sequences. Nevertheless, pARUB19 wasstably maintained in the host cells without homologousrecombination.

3.5. Expression of firefly luciferase in host cellsThe expression of luciferase in cells transformed with

the expression vector, pARUB-Zuc, was demonstrated bySDS-PAGE (Fig. 5B, lane pARUB-fec). By densitomet-ric analysis of SDS-polyacrylamide gel, the expressionlevels of the protein reached about 1.2% of the total sol-uble protein. In the control, the luciferase protein wasabsent from the cells transformed with pARUB19 (lanePARUB19).

The firefly luciferase gene is useful as a transcriptionalreporter in cyanobacterial cells because SynechococcusPCC 6301 has the ability to take up luciferin into thecells. Therefore, we could directly determine the activityof luciferase expressed in the host cells without destruc-tion of the cells. The cells transformed with pARUB- luc,and with pARUB19 were incubated with several con-centrations of luciferin. Light production was detected

in the transformants carrying pAKUB-luc, but no lu-ciferase activity was detected in the cells transformedwith pARUB19 (Fig. 5C).

The cyanobacterial RuBisCO promoter functionsstrongly in the host cells. Therefore, the novel expres-sion vector, pARUB19, would facilitate higher levels offoreign gene expression in Synechococcus PCC 6301 andstrain PCC 7942. This vector is expected to be useful foranalyzing the function of foreign genes in SynechococcusPCC 6301 and strain PCC 7942 cells.

Acknowledgments: We are grateful to Drs. Henry K.W. Fong and Y. Aotsuka for their helpful advice in thepreparation of this manuscript, and N. Takatsugu and T.Kinshi for their technical assistance. We appreciate Dr.Y. Hagiwara for providing the research funds (YH fund)and for his encouragement.

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a novel expression vector for the cyanobacterium, synechococcus

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