# A single vector-based strategy for marker-less gene replacement in Synechocystis sp. PCC 6803

Stefania Viola, Thilo Rühle, Dario Leister

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Stefania Viola, Thilo Rühle, Dario Leister. A single vector-based strategy for marker-less gene replacement in Synechocystis sp. PCC 6803. Microbial Cell Factories, 2014, 13 (1), pp.4. 10.1186/1475-2859-13-4. hal-04004153

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# A single vector-based strategy for marker-less gene replacement in *Synechocystis* sp. PCC 6803

Stefania Viola, Thilo Rühle and Dario Leister*

**Abstract**

**Background:** The cyanobacterium *Synechocystis* sp. PCC 6803 is widely used for research on photosynthesis and circadian rhythms, and also finds application in sustainable biotechnologies. *Synechocystis* is naturally transformable and undergoes homologous recombination, which enables the development of a variety of tools for genetic and genomic manipulations. To generate multiple gene deletions and/or replacements, marker-less manipulation methods based on counter-selection are generally employed. Currently available methods require two transformation steps with different DNA plasmids.

**Results:** In this study, we present a marker-less gene deletion and replacement strategy in *Synechocystis* sp. PCC 6803 which needs only a single transformation step. The method utilizes an *nptl-sacB* double selection cassette and exploits the ability of the cyanobacterium to undergo two successive genomic recombination events via double and single crossing-over upon application of appropriate selective procedures.

**Conclusions:** By reducing the number of cloning steps, this strategy will facilitate gene manipulation, gain-of-function studies, and automated screening of mutants.

**Keywords:** Genetic engineering, Gene replacement, Homologous recombination, *Synechocystis*

**Background**

Cyanobacteria, also known as blue-green algae, are Gram-negative, photosynthetic prokaryotes. Moreover, they are the evolutionary ancestors of plastids [1] and the only prokaryotes that exhibit a classical circadian clock mechanism [2]. Thanks to their ability to adapt to extreme environmental conditions, cyanobacteria are widely distributed. These features, and their recent exploitation for biotechnological purposes [3,4], explain why cyanobacteria have been extensively studied. Over the past few decades, numerous cyanobacterial genomes have been sequenced (http://genome.microbedb.jp/cyanobase) and many molecular tools for their genetic manipulation have been developed. *Synechocystis* sp. PCC 6803 (hereafter designated as *Synechocystis*) is an excellent model organism, because it has a small (3.6 Mb), sequenced genome [5], is capable of spontaneous uptake of exogenous DNA and can integrate it via homologous recombination into its genome. In addition, a spontaneous glucose-tolerant mutant is available [6] that can

grow heterotrophically in the presence of glucose and is thus useful for the study of oxygenic photosynthesis. Photosynthetic *Synechocystis* mutants have been extensively used to study photosystems I (PSI) [7,8] and II (PSII) [9,10] and to investigate the functionality of photosynthesis-related proteins from higher plants in cyanobacteria [11,12]. All these studies have employed gene deletions or replacements.

The classical means of deleting a target gene or inserting exogenous genetic material into the *Synechocystis* genome via homologous recombination involves the use of a resistance marker [9,13]. In this approach, the insertion cassette (marker plus exogenous DNA) is flanked by sequences homologous to the targeted site in the genome, and the whole segment is cloned into a suicide vector, which is unable to replicate in the host cell. After integration of the insertion cassette into the host genome, the resistance-mediating marker allows for positive selection of the mutant cells in which integration has occurred. However, to perform additional genomic modifications, one must use other resistance markers. Consequently, the number of available markers restricts the number of genetic manipulations. To overcome this

<footnote>* Correspondence: leister@lmu.de
Department Biology I, Ludwig-Maximilians-Universität München,
Großhaderner Str. 2, Planegg, Martinsried D-82152, Germany</footnote>
limitation, so called marker-less strategies have been de-
veloped, which allow for the removal of the integrated
marker. The first marker-less method was established in
Gram-negative bacteria using the *nptI-sacB* double se-
lection cassette [14]. Here, the *nptI* gene confers resist-
ance to the antibiotic kanamycin, while expression of the
*sacB* gene from *Bacillus subtilis* [15,16] is toxic to
Gram-negative bacteria grown on sucrose-containing
media. SacB is a levansucrase that hydrolyzes sucrose,
and utilizes the fructose unit for the production of
levans [17]. The polymers are toxic to Gram-negative
bacteria, and cells harbouring the *sacB* gene die when
grown in presence of 5% sucrose, although the under-
lying mechanism is not fully understood. In cyanobac-
teria, the *nptI-sacB* cartridge was first used to establish
marker-less gene replacement in *Anabaena* sp. PCC
7120 [18]. Gene replacement requires two homologous
recombination events, each of which requires a
suicide vector and a bacterial transformation step (see
Additional file 1). 1) The target genomic sequence is re-
placed by the double-selection cassette, and the mutants
in which the homologous recombination has taken place
are positively selected by their ability to grow in the
presence of the antibiotic. 2) A second transformation
and homologous recombination event leads to the exci-
sion of the *nptI-sacB* selection cartridge and its con-
comitant replacement by the exogenous sequence. The
desired mutants can be selected on the basis of their
resistance to sucrose and sensitivity to kanamycin. An
alternative version of double selection was developed for
gene replacement in *Synechococcus elongatus* PCC 7942
[19]. In this approach, the background strain carries a
mutated form of the *rps12* gene (which codes for subunit
S12 of the 30S ribosome) which confers resistance to spec-
tinomycin [20,21]. Since the mutation is recessive, the
double selection cassette carries a kanamycin resistance
gene and - as an alternative negative selection marker - a
wild-type copy of *rps12*, which results in a dominant
spectinomycin-sensitive phenotype. A drawback of the
*rps12* marker system is the need to perform all multiple
replacements in the *rps12* mutant background, which in-
troduces the need of an additional manipulation step. A
general weakness of the marker-less gene replacement
methods described above is that two time-consuming
transformation events and cloning of two different suicide
vectors are required. Alternative double recombination
strategies have been developed that rely on initial integra-
tion of the whole vector into the target genomic sequence
via single crossing-over, as in the case of allelic exchange
[22,23]. However, in this case, the second recombination,
leading to the excision of the integrated vector, gives rise
to both (reverted) wild-type cells and bacteria that retain
the desired mutation, so that this population of recombi-
nants must be screened by PCR.

The aim of the present study was to develop a time-
saving strategy for marker-less gene replacement in
*Synechocystis* which requires only a single vector and a
single bacterial transformation event. The strategy finally
chosen leads to a population which consists exclusively
of mutants. Therefore, the method offers a number of
technical advantages over the classical approaches and is
applicable to other bacterial species capable of undergo-
ing gene transfer, irrespective of the selective markers
employed.

Results

Design of the single-step double-recombination vectors

A vector designed for a single-step double-recombination
approach (see Figure 1) must include several essential
elements: 1. an origin of replication for *E. coli* that is non-
functional in *Synechocystis*; 2. flanking sequences homolo-
gous to *Synechocystis* chromosomal sites to allow stable
integration of the constructs into the cyanobacterial gen-
ome (designated as HR1 and HR2); 3. the *nptI-sacB*
double selection cassette that allows both positive and
negative selection of the *Synechocystis* recombinants; 4.
the exogenous gene of interest (GOI), split into two, par-
tially overlapping (shaded box) segments (5' GOI and 3'
GOI) that are separated by the *nptI-sacB* cassette; 5. a
*Synechocystis* promoter for expression of the introduced
recombinant gene(s).

When *Synechocystis* cells are transformed with such
a vector, the initial recombination event takes place
(Figure 1, upper panel) via a double crossover event be-
tween the two flanks of the construct, HR1 and HR2,
and the corresponding homologous sequences present in
the cyanobacterial genome. Mutants that have integrated
the construct in this way contain the *nptI-sacB* cassette
in the target locus and can be positively selected on
kanamycin. These recombinants are resistant to the anti-
biotic but sensitive to sucrose. In these mutants the GOI
is not functional, because its sequence is disrupted by
the *nptI-sacB* cassette. Moreover, complete segregation
of the first-round ["prim(ary)"] recombinants leads to
loss of the endogenous target gene. The loss of the target
gene also proves that integration of the construct hap-
pened via a double crossover event on HR1 and HR2
and not via a single crossover event involving only one
flanking sequence. After complete segregation, the posi-
tive selective pressure is released and the mutants are
grown in the absence of selection in order to allow the
second recombination event to occur (Figure 1, lower
panel), without the need for a further transformation
step. The second crossover event occurs between the in-
ternal overlapping sequences of the foreign DNA and
leads to the excision of the double selection cassette, re-
storing the integrity of the introduced gene(s). Mutants
in which the second recombination is incomplete can be
<figure><img src="image_2.png" /></figure>

**Figure 1 Schematic depiction of the single-step double recombination strategy.** The first recombination step (upper panel), involving a double crossover between the homologous regions HR1 and HR2 of the vector and the genomic target sequence, leads to genomic integration of the construct. Note that the integrated gene of interest (GOI) is split into two parts, 5' and 3', the sequences of which partially overlap (shaded box). The 5' and 3' GOI segments are separated from each other by the *nptl-sacB* selection cassette, which renders the first recombinant mutants resistant to kanamycin and sensitive to sucrose. After complete segregation of the replacement under positive selection in the presence of kanamycin, which ensures the total elimination of the endogenous target gene function, release of the selective pressure allows the second recombination to take place (lower panel). In this step, a crossover involving the overlapping regions of the split GOI leads to the excision of the *nptl-sacB* cassette. Negative selection on sucrose yields colonies that have lost the entire *sacB* marker and carry the intact, functional GOI in place of the endogenous target gene.

efficiently counter-selected on sucrose, because only
those recombinants that have lost the whole *nptl-sacB*
cassette will survive. In consequence, in the population
generated by the second round of recombination ["sec
(ondary) recombinants"], the target gene is replaced by
the transgenic DNA of choice and no selection marker
remains in the genome.

**Confirmation of the strategy: 1. Introduction of the
luciferase reporter system**

WT *Synechocystis* cells were transformed with the vector pDSlux (Figure 2A). The mutants obtained after the first round of recombination, *lux*<sup>prim</sup>, harboured an inter-rupted *luxAB* operon inserted in the *Synechocystis slr0168* ORF. After complete segregation, all the analyzed strains were knockouts for the *slr0168* target locus, thus confirming the correct double crossover event in the first recombination step. In the mutants obtained after the second recombination round, named *lux*<sup>sec</sup>, excision of the double selection cassette led to the reconstitution of the intact *luxAB* operon under the control of P<sub>psbA2</sub>.

The frequencies of the first and second recombination
events were calculated for *lux* mutants. Ten independent
*lux*<sup>prim</sup> strains obtained after the first recombination
were selected and allowed to undergo a second recom-
bination. The transformation frequency was 4 × 10<sup>-5</sup>,
which is comparable to the values reported in the litera-
ture [24]. The frequency of the second recombination
varied between 2 × 10<sup>-5</sup> and 1 × 10<sup>-7</sup> (Table 1), with an
average value of 9 × 10<sup>-6</sup>, revealing that it occurs with a
frequency about ten times lower than the first. All the
analyzed second-round recombinants were not able to
grow in the presence of kanamycin, confirming that the
sucrose resistance was due to loss of the double selec-
tion cassette and not to inactivation of the *sacB* gene by
spontaneous mutations. Five of the original ten inde-
pendent *lux*<sup>prim</sup> first recombinants and two *lux*<sup>sec</sup> second
recombinants for each of the five *lux*<sup>prim</sup> clones were se-
lected for further analyses.

To confirm the correct segregation of the expected
genetic variants in these mutants, genomic PCR was
performed using three primer pairs. In all five *lux*prim
mutants, but in none of the *lux*sec strains, the primer
pairs P1 + P2 and P3 + P4 generated amplicons (2.7 and
<figure><img src="image_3.png" /></figure>

**Figure 2** Analysis of the *lux* strains. **A.** Schematic depiction of the mutant strains after the first and second rounds of recombination. In *luxprim*, the *nptl-sacB* cassette interrupts the *luxB* gene. In *luxsec*, loss of the cassette leads to the reconstitution of the entire *luxAB* operon under the regulation of the *psbA2* promoter. Annealing sites of the primers used for genotyping (P1-6) and of the *DS* probe used for Southern hybridization as well as the positions of the *Xmal* restriction sites are indicated. **B.** Complete segregation of the *Synechocystis lux* strains generated by the genetic manipulations represented in A. Genotyping PCR was performed on five independent *luxprim* first-round recombinants and, for each of them, two second-round recombinants. Note that *luxprim#2-5* behaved like *luxprim#1*. Expected sizes of the amplicons generated by the used primer pairs were: P1/P2, 2.7 kbps; P3/P4, 2.3 kbps; P1/P4, 2.3 kbps; P5/P6, 1 kbps. Negative control (n.c.) was included. **C.** Southern analysis of genomic DNA from *Synechocystis* WT and *lux* mutants. The *Xmal* restriction map of the *slr0168* genomic region in the analyzed strains and the probe used for hybridization are shown in A. Five µg of DNA were loaded per lane. Genomic fragments F1 (7.5 kbps) and F2 (8.3 kbps) were detected in *luxprim*, the fragment F3 (11 kbps) in the *luxsec* strains. Note that *luxprim#2-5* behaved like *luxprim#1*. The lane corresponding to WT is also shown. **D.** Drop test of *lux* mutants on selective media. Liquid cultures (OD~730~: 0.4) were washed with BG11 without glucose and spotted (15 µl each) onto BG11 medium containing either 100 µg/ml kanamycin or 5% sucrose or no supplement. Colonies from the non-selective plate were incubated with 1 mM decanal and luminescence was determined to quantify luciferase activity.
<table><caption>Table 1 Frequency of the second recombination event in independent *lux*<sup>prim</sup> strains</caption><thead><tr><th>lux<sup>sec</sup></th><th>#1</th><th>#2</th><th>#3</th><th>#4</th><th>#5</th><th>#6</th><th>#7</th><th>#8</th><th>#9</th><th>#10</th></tr></thead><tbody><tr><td></td><td>7×10<sup>-6</sup></td><td>5×10<sup>-7</sup></td><td>7×10<sup>-6</sup></td><td>4×10<sup>-6</sup></td><td>7×10<sup>-6</sup></td><td>1×10<sup>-7</sup></td><td>2×10<sup>-6</sup></td><td>2×10<sup>-5</sup></td><td>2×10<sup>-5</sup></td><td>2×10<sup>-5</sup></td></tr></tbody></table>

2.3 kbps, respectively) spanning the 5' or 3' regions respectively of the *nptI-sacB* cassette and the flanking segment of the interrupted *luxAB* operon. When combined, primers P1 and P3 amplified the reconstituted *luxAB* operon, generating a PCR product of 2.3 kbps that was detectable only in the *lux*<sup>sec</sup> second recombinants. The *slr0168* downstream region (HR2, ~1 kbps), used as a positive control, could be amplified in all the samples using the primer pair P5 + P6 (Figure 2B).

Gene replacement via homologous recombination is sometimes prone to error, and may result in non-homologous DNA integration into the genome, thus generating mutants with multiple or non-targeted genomic insertions. However, when Southern blots bearing genomic XmaI digests from WT and the selected *lux* mutants were probed with a fragment of the *luxAB* recombinant operon (Figure 2C), two labelled bands (F1 and F2, with 7.5 and 8.3 kbps respectively) corresponding to the split *luxAB* operon were found in the *lux*<sup>prim</sup> mutants, while the detection of a single band of 11 kbps (F3) in the *lux*<sup>sec</sup> recombinants confirmed the presence of the intact operon. The absence of any signal of unexpected size indicated that homologous recombination had occurred at the correct target locus in all cases.

In order to phenotypically characterize the *lux* mutants, they were grown on various BG11-based media (Figure 2D). When spotted on BG11 plates with 5 mM glucose and cultured without selection pressure, all *lux*<sup>prim</sup> and *lux*<sup>sec</sup> mutant strains grew normally. The *lux*<sup>prim</sup> cells resulting from the first round of recombinants grew in the presence of kanamycin. Furthermore, the presence of 5% sucrose in the medium was lethal for the *lux*<sup>prim</sup> strains, confirming the functionality of the negative selection marker. Conversely, all the *lux*<sup>sec</sup> strains were sensitive to kanamycin, as they had lost the resistance marker, and grew on BG11 supplemented with 5% sucrose because they no longer harboured the *sacB* gene. None of the strains could grow on BG11 supplemented with both kanamycin and sucrose, as expected. This confirms that no integration of the whole vector, also bearing a kanamycin resistance gene on the backbone, via a single crossover event, took place during the first recombination step.

The *luxAB* operon codes for two proteins that are both required for the expression of luminescence, a phenotype not naturally present in *Synechocystis*. In *lux*<sup>prim</sup> mutants the full-length *luxB* gene is not fused to a functional promoter; therefore, the *LuxB* subunit of the luciferase cannot be synthesized. The second

recombination (in *lux*<sup>sec</sup> mutants) is expected to reconstitute the entire *luxAB* operon under the regulation of *P<sub>psbA2</sub>*), thus allowing the expression and assembly of the functional enzyme. To confirm this, all the spotted strains were tested for luminescence (Figure 2B, lower panel). Strong luciferase activity was detectable only in the *lux*<sup>sec</sup> strains carrying the reconstituted *luxAB* operon, and almost absent in all the *lux*<sup>prim</sup> mutants. A faintly luminescent background was always detectable in the first-round recombinants, possibly due to read-through transcription of *luxB* from the *nptI* promoter. To better quantify the luciferase activity, light emission was measured for the ten *lux*<sup>prim</sup> strains used for second recombination and ten *lux*<sup>sec</sup> independent recombinants for each of them (Table 2). The luminescence values relative to the untransformed WT *Synechocystis* strain clearly indicated a gain of function in all the *lux*<sup>sec</sup> strains, albeit with a certain inter-strain variability. The level of luciferase activity in the *lux*<sup>prim</sup> transformants ranged between 1.5- and 2.5-fold higher than in the WT, while in the second-round recombinants it was at least ten times higher than in WT.

## Confirmation of the strategy: 2. The *psaA* gene

As photosynthesis is one of the core research areas in which cyanobacteria are extensively used, the same strategy was used to replace the *Synechocystis psaA* (*Syn psaA*) gene with the corresponding homolog from the green plant *Arabidopsis thaliana* (*At psaA*). The *psaA* gene codes for PsaA, a core subunit of PSI [25], and in both cyanobacteria and plants it is part of the *psaA/psaB* operon. The two genes are regulated by the *P<sub>psaA</sub>* promoter located upstream of *psaA*, and are separated by an intergenic region of 246 base pairs. By transforming the glucose-tolerant WT *Synechocystis* strain with the vector pDSpsaA, analogously to the Lux experiment described above, knockout (*psaA*<sup>prim</sup>) and replacement (*psaA*<sup>sec</sup>) mutants were subsequently obtained (Figure 3A). The first recombination event yielded colonies in which the integrated construct replaced the endogenous *psaA* gene, generating the knockout line *psaA*<sup>prim</sup>. The replacement in *psaA*<sup>sec</sup> affected only the *psaA* coding sequence and left the surrounding genomic regions unchanged, thus retaining all the endogenous regulatory elements intact. In the *psaA*<sup>prim</sup> mutant, the integrated *At psaA* gene was split into two parts and therefore non-functional. Moreover, also expression of PsaB can be expected to be impaired, because (i) the construct integrated between the common promoter
<table><caption>Table 2. Luciferase activity relative to OD$_{730}$ in *Synechocystis lux$^{prim}$* and *lux$^{sec}$* mutants</caption><thead><tr><th></th><th>#1</th><th>#2</th><th>#3</th><th>#4</th><th>#5</th><th>#6</th><th>#7</th><th>#8</th><th>#9</th><th>#10</th></tr></thead><tbody><tr><td><i>lux</i><sup>prim</sup></td><td>2.6 ± 1.4</td><td>2.3 ± 0.4</td><td>1.8 ± 0.4</td><td>2 ± 1.2</td><td>2.1 ± 0.7</td><td>1.8 ± 0.5</td><td>2.2 ± 1.1</td><td>1.7 ± 0.6</td><td>1.5 ± 0.8</td><td>1.5 ± 1</td></tr><tr><td><i>lux</i><sup>sec</sup> (1–10)</td><td>17.3 ± 3.9</td><td>61.8 ± 11.3</td><td>16.6 ± 3.6</td><td>24.1 ± 6.5</td><td>15.3 ± 5.1</td><td>23.2 ± 9.6</td><td>22.7 ± 9.6</td><td>10.4 ± 3.2</td><td>10.6 ± 2.9</td><td>13.1 ± 3.8</td></tr></tbody></table>

For each strain, the first recombinant and ten independent second recombinants were analyzed. Each suspension was measured in duplicate and the assay was repeated twice with independently grown cultures. Average luminescence values of the mutants are relative to background signal in the untransformed WT *Synechocystis* strain, which value was set to 1.

and the *psaB* gene, and (ii) disruption of *psaA* leads any-
how to the complete lack of any functional PSI, as both
PsaA and PsaB cannot accumulate [26]. In consequence,
the *psaA*<sup>prim</sup> strain showed a severe phenotype, since it
lacks the PsaA and PsaB proteins, the loss of which pre-
vents the accumulation of any PSI complex [26] and ac-
counts for the turquoise-blue colour of the cells, their
high light sensitivity and inability to perform photosyn-
thesis. The absorption spectrum of the mutants showed
a complete depletion of chlorophyll *a* (Chl) with the
only presence of the phycobilisome chromophore
phycocyanin (PC), responsible for the “blue” phenotype
(Figure 3B). After complete segregation of the first re-
placement, removal of the selective pressure led to the
second recombination event and subsequent negative se-
lection on sucrose-containing medium yielded colonies
that had lost the *nptI-sacB* cassette and carried the
reconstituted *At psaA* gene in place of the endogenous
one. These *psaA*<sup>sec</sup> mutants were also bluish, having a
drastically reduced Chl/PC ratio with respect to the
wild-type, but they were able to accumulate more
chlorophyll than the *psaA*<sup>prim</sup> strain (Figure 3B). The
second-round recombinants were not as light sensitive
as the first-round recombinants, and they could grow
under normal light conditions.

To confirm correct segregation of the introduced se-
quences in these mutants, genomic PCR was performed
using three primer pairs (Figure 3C). The region (2 kbps)
spanning the upstream homologous region used for inte-
gration of the construct (HR1) and the endogenous *psaA*
gene could be amplified with the primer pair P7 + P8
only in WT, confirming that in both mutant strains the
knockout was complete. In *psaA*<sup>prim</sup>, but not in WT and
in the *psaA*<sup>sec</sup> mutant, the primer pair P7 + P2 generated
an amplicon (3 kbps) spanning HR1, the flanking 5' *At
psaA* segments and the 5' region of the *nptI-sacB* cas-
sette. Primers P9 and P10 amplified the fully reconsti-
tuted *psaA* gene from *A. thaliana*, generating a PCR
product of 2.2 kbps that was detectable only in the
*psaA*<sup>sec</sup> recombinants.

Phenotypic analysis was performed by spotting strains
onto different BG11-based media (Figure 3D). The
*psaA*<sup>prim</sup> mutant was able to grow on BG11 supple-
mented with kanamycin, as it harboured the *nptI* resist-
ance gene but, being PSI-deficient, it only grew
heterotrophically in presence of 5 mM glucose and

under Light Activated Heterotrophic Growth (LAHG)
conditions. In LAHG, bacteria are grown heterotrophi-
cally in the dark except for a single short exposure to
dim light every 24 h, which was suggested to promote
the cell division or the progression through the cell cycle
[27]. In contrast, under continuous bright light and in
the absence of glucose growth of *psaA*<sup>prim</sup> was com-
pletely inhibited. The *psaA*<sup>sec</sup> recombinant was unable to
grow on kanamycin but could grow on BG11 containing
sucrose, having lost the *nptI-sacB* cassette, whereas the
presence of 5% sucrose was lethal to the knockout strain.
None of the mutants, as expected, was viable in presence
of both kanamycin and 5% sucrose. Interestingly, *psaA*<sup>sec</sup>
was not light sensitive and was able to grow photoauto-
trophically on BG11 agar plates containing no glucose.
From these results it can be concluded that the At PsaA
protein can partially functionally replace the *Synechocys-
tis* protein, at least to the extent of restoring light toler-
ance and the ability to grow photoautotrophically.

Discussion

Targeted gene and genome manipulation via homolo-
gous recombination in bacteria relies on the use of
marker genes. In marker-less gene replacement, a nega-
tive and a positive selection marker are employed, in a
process that normally involves two transformation steps
with two DNA suicide vectors. In this work, we devel-
oped an alternative strategy for marker-less gene re-
placement in *Synechocystis*, based on the use of a single
plasmid and a single transformation step. The technique
was tested by inserting the *luxAB* operon from *Vibrio
fischeri* into the neutral receptor site *slr0168*. To this
end the wild-type strain was transformed with the vector
pDSlux and the transformation efficiency with this inte-
gration vector was in the range of 10<sup>-5</sup>, while the average
frequency of the second recombination event was 10<sup>-6</sup>
(see Table 1). This ten-fold difference could be due to
the fact that the first recombination event involves a
double crossover between the genomic DNA and the
plasmid vector, whereas the second recombination pre-
sumably involves a single intra-genomic crossover [23].
Thus, a difference in efficiency between these two re-
combination mechanisms could explain the results ob-
tained. Secondary recombinants were easily obtained
with 509 bps (*psaA*) and 1055 bps (*luxAB*) of homolo-
gous regions; however, the exact impact of the length of
<figure><img src="image_4.png" /></figure>

**Figure 3** Analysis of the *psaA* strains. **A.** Schematic depiction of mutant strains after the first and second rounds of recombination following transformation with the *psaA* construct. The *psaA* construct is integrated in the *Synechocystis psaA* gene. In *psaA*prim the nptI-sacB* cassette interrupts the *At psaA* gene. In *psaA*sec, loss of the cassette leads to the reconstitution of the entire *At psaA* under the control of the native cyanobacterial promoter. Positions of the primers used for genotyping (P2, P7-10) are indicated. B. In vivo absorption spectra of WT, *psaA*prim and *psaA*sec *Synechocystis* strains. The peaks at 438 and 681 nm correspond to the maxima of Chl *a* absorption, the peak at 628 nm corresponds to the absorption maximum of PC. The spectra were normalized to the absorbance at 730 nm. **C.** Complete segregation of the generated *Synechocystis psaA* strains, as demonstrated by PCR analysis. Primer positions are given in A. Expected sizes of the amplicons generated by the used primer pairs were: P7/P8, 2 kbps; P7/P2, 3 kbps; P9/P10, 2.3 kbps. Negative control (n.c.) was included. **D.** Drop test of WT, *psaA*prim and *psaA*sec *Synechocystis* strains on selective media and under different light conditions. Liquid cultures at OD730 of 0.4 were washed with BG11 without glucose and spotted (15 µl each) on non-selective BG11 medium or on BG11 containing 100 µg/ml kanamycin or 5% sucrose. When tested for autotrophic growth, cells were grown in continuous light at 30 µmol photons m-2 s-1 on BG11 without glucose. When grown in Light Activated Heterotrophic Growth (LAHG) conditions, the cells were incubated in the dark on BG11 supplemented with 5 mM glucose, and exposed to light for 5 min every 24 hours.
the homologous region on recombination frequency remains to be determined. In addition, other factors like position and relative concentration of the homology regions might contribute to the difference in efficiency [28].

In comparison with standard procedures, the single-step marker-less gene replacement strategy described here has several advantages. The requirement of only one vector for each gene replacement makes the cloning procedure faster and more facile, especially when one needs to sequentially replace many genes. Our single-step approach merely requires alternative selective growth conditions in addition to the transformation step, although this flexibility requires that the constructs must be assembled from numerous DNA fragments - six (plus the vector backbone) in the case of pDSlux, for instance. However, the increased complexity of the plasmid vectors required can easily be accommodated by using large-scale modular cloning technologies like the Golden Gate [29], the Gibson [30] or the BioBrick [31] assembly systems. Another issue that can arise during gene manipulation concerns the cloning of genes that are toxic to *E. coli*. Also in this regard, our strategy is advantageous, because the interruption of the GOI sequence by the intervening *nptI-sacB* cassette inactivates it, thus avoiding the problem of toxicity. Only the second recombination in the *Synechocystis* host cell reconstructs both the gene sequence and function. Also for studies of biological functions for which two genes are necessary, the single-step gene replacement strategy can constitute a suitable experimental tool, as shown for the *luxAB* operon. In the *lux*<sup>prim</sup> mutants, interruption of this operon by the double selection cassette leads to impairment of the luciferase activity, as *luxB* cannot be transcribed and therefore the heterodimeric enzyme cannot be assembled. Only after removal of the cassette by the second recombination event is the integrity of the dicistronic *luxAB*, and with it the luciferase activity restored, as shown in Figure 2A and Table 2. Some background luminescence was observed in the *lux*<sup>prim</sup> recombinants, which could be due to read-through transcription of *luxB* from the *nptI* promoter. Thus, by using only one vector and performing a single transformation step it was possible to show that both *luxA* and *luxB* are necessary for luciferase function, as previously described [32,33]. These results confirm that, when our strategy is used to analyse a gain of function, the new function can be expected to arise only in the second recombinants and not in the first mutant strains, which can therefore serve as ideal controls.

The single-step strategy was also employed to replace the *psaA* gene from *Synechocystis* with the homolog from *Arabidopsis thaliana*, which resides in the plastid genome of the plant. The PsaA subunit, together with PsaB, constitutes the dimeric core of PSI, and is highly

conserved among photosynthetic organisms from cyanobacteria to flowering plants [34]. PsaA is an essential component of PSI and is necessary for its assembly, accumulation and function, and therefore essential for photoautotrophic growth of both plants and cyanobacteria. Knockout of the *psaA* gene is lethal to higher plants, while genetic inactivation of the PSI reaction centre in *Synechocystis* generates mutants that are able to survive heterotrophically under LAHG conditions, although they have a severe phenotype and are unable to survive in the light. In the *psaA*<sup>sec</sup> mutants, replacement of the endogenous *psaA* gene with the *A. thaliana* homolog led to a partial complementation of the *psaA*<sup>prim</sup> knockout phenotype (Figure 3B,D). Because this is most likely due to a partial functional substitution of the endogenous PsaA protein by its *Arabidopsis* counterpart, the degree of PSI assembly and function the *psaA*<sup>sec</sup> mutants needs to be analysed in future experiments. Also in this case, using the single-step gene replacement strategy, both the knockout and replacement lines were generated using a single vector and a single transformation. In the case of essential genes like *psaA* the one-vector strategy confers an additional advantage because mutants with severe phenotypes can be difficult to grow in liquid cultures and therefore their transformation can be challenging. This alternative gene replacement strategy makes it possible to obtain a second recombination event simply by releasing the positive selective pressure on the segregated transformants and applying negative selection in an alternating manner.

## Conclusions

Taken together, the implementation of a double recombination event by a simple stepwise change of growth conditions can be exploited to address various biological issues and could also be employed for large-scale gene replacement approaches. Thanks to the tight counter-selection against incomplete second-round recombinants and transformants bearing multiple genomic integrations, the method could indeed be applied in partially automated systems.

## Methods

### Bacterial strains and growth conditions

The bacterial strains used are described in Table 3. *E. coli* DH5α cultures were grown in lysogeny broth (LB) medium at 37°C and shaken at 225 rpm. Unless otherwise indicated, wild-type (glucose-tolerant) and mutant *Synechocystis* sp. PCC 6803 strains were grown at 25°C and under continuous illumination at 30 µmol m⁻² s⁻¹ in BG11 medium containing 5 mM glucose [35]. Liquid cultures were shaken at 120 rpm. For growth on plates, 1.5% (w/v) agar and 0.3% (w/v) sodium thiosulfate were added to the BG11 medium. The *psaA*<sup>prim</sup> mutant strain
<table><caption>Table 3 Bacterial strains and plasmids used in this study</caption><thead><tr><th>Strain/plasmid</th><th>Characteristics</th><th>Selection markers</th><th>Source</th></tr></thead><tbody><tr><td colspan="4"><strong>E. coli</strong></td></tr><tr><td>DH5α</td><td>Competent cells</td><td></td><td></td></tr><tr><td colspan="4"><strong>Synechocystis</strong></td></tr><tr><td>WT</td><td>WT <em>Synechocystis</em> sp PCC 6803, glucose tolerant</td><td>kan<sup>S</sup>, suc<sup>R</sup></td><td>H. Pakrasi (Washington University, St. Louis)</td></tr><tr><td>lux<sup>prim</sup></td><td><em>luxAB</em> operon, interrupted by <em>nptl-sacB</em> cassette, replacing <em>slr0168</em> ORF</td><td>kan<sup>R</sup>, suc<sup>S</sup></td><td>This study</td></tr><tr><td>lux<sup>sec</sup></td><td>Intact <em>luxAB</em> operon replacing <em>slr0168</em> ORF</td><td>kan<sup>S</sup>, suc<sup>R</sup></td><td>This study</td></tr><tr><td>psaA<sup>prim</sup></td><td>At <em>psaA</em> gene, interrupted by <em>nptl-sacB</em> cassette, replacing endogenous <em>psaA</em></td><td>kan<sup>R</sup>, suc<sup>S</sup>, heterotroph</td><td>This study</td></tr><tr><td>psaA<sup>sec</sup></td><td>Intact At <em>psaA</em> gene replacing endogenous <em>psaA</em></td><td>kan<sup>S</sup>, suc<sup>R</sup>, photoautotroph</td><td>This study</td></tr><tr><td colspan="4"><strong>Plasmids</strong></td></tr><tr><td>pGEM-T Easy</td><td>Backbone for pDSpsaA</td><td>amp<sup>R</sup></td><td>Promega, Madison, WI</td></tr><tr><td>pRL250</td><td><em>nptl-sacB</em> double selection cassette, <em>sacB</em> gene from <em>Bacillus subtilis</em></td><td>kan<sup>R</sup>, suc<sup>S</sup></td><td>P. Wolk (Michigan State University)</td></tr><tr><td>pICH69822</td><td>Destination vector for Golden Gate cloning</td><td>kan<sup>R</sup></td><td>E. Weber (Icon Genetics GmbH, Halle)</td></tr><tr><td>pRL1063a</td><td><em>luxAB</em> operon from <em>Vibrio fischeri</em></td><td>Sm<sup>R</sup></td><td>P. Wolk (Michigan State University)</td></tr><tr><td>pDSlux</td><td>pICH69822 with <em>nptl-sacB</em> cassette from pRL250, <em>luxAB</em> operon from pRL1063a, <em>Syn psbA2</em> promoter and <em>slr0168</em> flanking regions</td><td>kan<sup>R</sup>, suc<sup>S</sup></td><td>This study</td></tr><tr><td>pDSpsaA</td><td>pICH69822 with <em>nptl-sacB</em> cassette from pRL250, At <em>psaA</em> gene and <em>Syn psbA2</em> promoter and <em>slr0168</em> flanking regions</td><td>kan<sup>R</sup>, suc<sup>S</sup></td><td>This study</td></tr></tbody></table>

Abbreviations: Syn Synechocystis, At Arabidopsis, R resistant, S sensitive.

was grown under Light-Activated Heterotrophic Growth (LAHG) conditions (i.e. in the presence of glucose and in darkness, interrupted only by a 5-min exposure light each day) as described [27].

For positive selection of mutants, increasing concentrations of kanamycin (10 to 100 µg/ml) were added to the medium. For negative selection, BG11 containing 5% (w/v) sucrose was used.

## Generation of plasmids

All DNA techniques such as plasmid isolation, restriction and ligation were performed according to standard protocols [36]. *Synechocystis* sequences were obtained from Cyanobase (http://genome.kazusa.or.jp/cyanobase/Synechocystis). All the plasmids used are listed in Table 3. The fragments used in the construction of the plasmids pDSlux and pDSpsaA were amplified by PCR and purified from 1% agarose gel. Primers are listed in Additional file 2.

In case of pDSlux, the amplified segments were assembled into the final construct using the one-step Golden Gate Shuffling cloning strategy [29] and the plasmid pICH69822 as destination vector. The *Synechocystis slr0168* ORF [37] [GenBankID: 954899] was used as a neutral receptor site for stable integration of the pDSlux vector into the genome. To generate the plasmid, the genomic regions upstream and downstream of *slr0168* (~1 kbps each, HR1 and HR2 respectively) were

amplified by PCR and used as flanking regions for homologous recombination. The dicistronic *luxAB* operon from *Vibrio fischeri*, derived from the pRL1063a vector [38], is composed of the *luxA* and *luxB* genes, and encodes the heterodimeric luciferase enzyme which, in presence of its substrate analogue decanal, produces a luminescent product. The operon was placed under the control of the strong *Synechocystis psbA2* promoter (*P<sub>psbA2</sub>* [39] by inserting it upstream of *luxA*. Two *luxAB* amplicons were generated, the first starting at position +1 of *luxA* and ending at position +460 of *luxB* and the second starting at position +856 of *luxA* and ending at the 3' end of *luxB*. Thus, the sequence overlap between the two fragments is 1055 base pairs long. In the final construct, the two amplicons are separated by the *nptl-sacB* double selection cassette, amplified from plasmid pRL250.

The pDSpsaA plasmid was constructed, using overlapping PCR and subsequent standard cloning steps, to replace the *Synechocystis psaA* gene with the chloroplast-encoded homolog from *Arabidopsis thaliana* (*At psaA*). To generate the vector, the *nptl-sacB* cassette was excised from the pRL250 plasmid with BamHI and cloned into the pGEM-T Easy vector (Promega, Madison, Wisconsin) according to the manufacturer's instructions. The regions upstream and downstream (~500 bps each, HR1 and HR2 respectively) of the *Synechocystis psaA* gene (*slr1834*) [GenBank ID: 954060]
To assay luciferase activity on solid medium, the de-
canal solution was added to cells grown on agar plates,
and luminescence was detected with the FUSION FXT
imaging system (Peqlab, http://www.peqlab.com/).

Bacterial whole-cell absorbance spectra

Absorbance spectra of whole *Synechocystis* cells were re-
corded using a spectrophotometer. Cells were harvested,
washed and resuspended in BG11 liquid medium to a
final OD<sub>730</sub> of 0.5. Their absorbance spectra were re-
corded between 350 and 750 nm and corrected for the
light scattering at 730 nm.

**Additional files**

Additional file 1: Schematic depiction of the classical double recombination strategy.

**Additional file 2: List of primers used in this study.** Restriction sites are indicated by bold characters. The *Bsa*I-generated sticky ends used for Golden Gate Shuffling assembly of the pDSlux constructs are underlined. In the case of the pDSPsaA plasmid, regions used for assembly with overlapping PCR are indicated in italics. Abbreviations: *Syn*, *Synechocystis*; *At*, *Arabidopsis*.

Competing interests

The authors declare that they have no competing financial interests.

**Authors' contributions**

SV carried out the experimental work and drafted together with TR and DL the manuscript. All authors participated in the design of the study. DL conceived the study. All authors read and approved the final manuscript.

Acknowledgements

We thank Prof. Dr. Peter Wolk (MSU-DOE Plant Research Laboratory Michigan State University, U.S.A.) for providing the vector pRL250, Dr. Sylvestre Marillonet (Icon Genetics GmbH, Halle, Germany) for providing the Golden Gate vector pLCH69822 and Prof. Dr. Annegret Wilde (Molecular Genetics, Albert-Ludwigs-Universität Freiburg, Germany) for providing the *luxAB* operon from the vector pRL1063a. We thank Paul Hardy for critical reading of the manuscript.

Received: 4 November 2013 Accepted: 3 January 2014

Published: 8 January 2014

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doi:10.1186/1475-2859-13-4

Cite this article as: Viola et al.: A single vector-based strategy for marker-less gene replacement in *Synechocystis* sp. PCC 6803. *Microbial Cell Factories* 2014 **13**:4.

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