DesC1 and DesC2, Δ9 Fatty Acid Desaturases of Filamentous Cyanobacteria: Essentiality and Complementarity

Abstract

DesC1 and DesC2, which are fatty acid desaturases found in cyanobacteria, are responsible for introducing a double bond at the Δ9 position of fatty-acyl chains, which are subsequently esterified to the sn-1 and sn-2 positions of the glycerol moiety, respectively. However, since the discovery of these two desaturases in the Antarctic cyanobacterium Nostoc sp. SO-36, no further research has been reported. This study presents a comprehensive characterization of DesC1 and DesC2 through targeted mutagenesis and transformation using two cyanobacteria strains: Anabaena sp. PCC 7120, comprising both desaturases, and Synechocystis sp. PCC 6803, containing a single Δ9 desaturase (hereafter referred to as DesCs) sharing similarity with DesC1 in amino acid sequence. The results suggested that both DesC1 and DesC2 were essential in Anabaena sp. PCC 7120 and that DesC1, but not DesC2, complemented DesCs in Synechocystis sp. PCC 6803. In addition, DesC2 from Anabaena sp. PCC 7120 desaturated fatty acids esterified to the sn-2 position of the glycerol moiety in Synechocystis sp. PCC 6803.

Introduction

The cyanobacteria belonging to the Gram-negative bacteria possess thylakoid membranes within their cells and can be morphologically categorized as unicellular and filamentous. Cyanobacterial cells resemble chloroplasts of higher plants and algae in both morphology and photosynthetic functions. Lipids and proteins present in thylakoid membranes influence their function in facilitating photosynthetic reactions in cyanobacteria (Awai 2016). Cyanobacterial thylakoid and cytoplasmic membranes comprise four major glycerolipids: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) (Wada and Murata 2009). While MGDG constitutes 50% of thylakoid membrane lipids as the major lipid, DGDG, SQDG and PG constitute just 5–25%. The cyanobacteria also contain another glycerolipid, GlcDG, at a low level, as it serves as an intermediate in MGDG synthesis (Sato and Murata 1982, Awai et al. 2006, 2014).

Most cyanobacteria synthesize fatty acids with carbon chain lengths of 16–18 and contain saturated, monounsaturated and polyunsaturated fatty acids. Saturated fatty acids include palmitic (C16:0), stearic (C18:0) and myristic acids (C14:0) in some cyanobacteria, while the unsaturated fatty acids include palmitoleic (C16:1 Δ9), oleic (C18:1 Δ9), vaccenic (C18:1 Δ11), linoleic (C18:2 Δ9,12), α-linolenic (C18:3 Δ91215), γ-linolenic (C18:3 Δ6,9,12) and stearidonic acids (C18:4 Δ6,9,1215) (Murata et al. 1992).

Cyanobacteria have been classified into four groups in terms of the presence and absence of monounsaturated and polyunsaturated fatty acids (Kenyon 1972, Kenyon et al. 1972, Los and Mironov 2015). Group 1 cyanobacteria contain monounsaturated fatty acids (e.g. palmitoleic and oleic acids) and saturated fatty acids. Strains in this group include Thermosynechococcus vulcanus, Thermosynechococcus elongatus, Synechococcus sp. PCC 6301, Synechococcus elongatus PCC 7942 and Prochlorothrix hollandica. Group 2 cyanobacteria feature the presence of polyunsaturated fatty acids, such as α-linolenic acid, at the sn-1 position and palmitoleic acid at the sn-2 position of the glycerol moiety. Strains in this group include Anabaena sp. PCC 7120 (hereafter Anabaena 7120), Anabaena variabilis, Nostoc sp. SO-36, Gloeobacter violaceus, Nostoc punctiforme, Synechococcus sp. PCC 7002 and Trichodesmium erythraeum. Group 3 cyanobacteria feature the presence of γ-linolenic acid at the sn-1 position and the absence of unsaturated fatty acid at the sn-2 position. Strains in this group include Arthrospira (Spirulina) platensis and Prochlorococcus marinus. Group 4 cyanobacteria share a similar fatty acid composition with Group 3, in addition to including α-linolenic and stearidonic acids at the sn-1 position. The strain in this group is Synechocystis sp. PCC 6803 (hereafter, Synechocystis 6803).

Desaturation is a process that introduces a double bond between two carbons of the fatty acid chain—this process is catalyzed by a group of enzymes called fatty acid desaturases (hereafter, desaturases). Cyanobacterial desaturases are termed ‘acyl-lipid desaturases’ because they desaturate fatty acids esterified to acyl glycerolipids (Wada and Murata 2007). Four desaturase types are classified based on their specificity toward the position of the fatty carbon chain and the sn-position of glycerol moiety. The first type (DesC) targeting saturated fatty acid introduces double bonds in these acids at the Δ9 position, converting palmitic to palmitoleic acid and stearic to oleic acid. DesA, the second type, introduces a double bond at the Δ12 position after DesC initiates the first unsaturation at the Δ9 position, resulting in the synthesis of linoleic acid. The third type (DesD) introduces double bonds at the Δ6 position and synthesizes γ-linolenic acid. The fourth type (DesB) introduces a double bond at the ω3 position and forms α-linolenic acid (Los and Murata 1999). Among these desaturases, DesC-inducing desaturation at the Δ9 position is present in all strains in the four cyanobacteria types (Sakamoto et al. 1994, Murata and Wada 1995).

To comprehend the precise function of these cyanobacterial desaturases, researchers have employed insertional mutagenesis to deactivate them, succeeding in mutating the desA and desB genes in Synechocystis 6803 (Sakamoto et al. 1994). However, the mutation of the desC gene remains unachieved (Tasaka et al. 1996, Mendez-Perez et al. 2014).

Chintalapati et al. (2006) first identified two desC-like genes that were homologous to each other in Nostoc sp. SO-36. One was very similar to the already characterized desC gene of cyanobacteria in Groups 1, 3 and 4, with an amino acid sequence identity above 60%: the other was less similar to the desC gene with an amino acid sequence identity approximately 50%. Chintalapati et al. (2006) termed them desC1 and desC2 genes, respectively, and characterized the desC2 gene and its product DesC2 by transforming Synechocystis 6803. Their results demonstrated that DesC2 was active in desaturating palmitic to palmitoleic acid at the sn-2 of the glycerol moiety. Previously, Sakamoto et al. (1994) and Kiseleva et al. (2000) experimentally confirmed that the desC gene homologous to desC1 was present in Anabaena variabilis (Group 2), Arthrospira (Spirulina) platensis (Group 3), Synechocystis 6803 (Group 4) and Thermosynechococcus vulcanus (Group 1). However, the desC2 gene was found only in Group 2 cyanobacteria. Uncertainty remains regarding whether DesC2 acts only on fatty acids esterified at the sn-2 position or on both the sn-1 and sn-2 positions. Uncertainty also remains regarding whether DesC2 and its product, unsaturated fatty acids, at the sn-2 position are essential for Group 2 cyanobacteria.

This study aimed to clarify the essential roles of DesC1 and DesC2 in Anabaena 7120 and how these desaturases from Anabaena 7120 could complement DesC in Synechocystis 6803. Insertional mutagenesis and antibiotic-assisted transformation were applied to these desaturase genes to achieve this goal.

Results and Discussion

Attempt to mutate the desC1 and desC2 genes in Anabaena 7120

To understand the specific function of DesC1 and DesC2, we attempted to mutate their genes using the tri-parental mating method (Elhai and Wolk 1988) in a filamentous cyanobacterium Anabaena 7120. Fig. 1A shows the design for mutating the desC1 and desC2 genes by inserting a spectinomycin-resistance gene cassette. The degree of mutation was examined by PCR analysis (Fig. 1B). A pair of primers #22 and #2 produced a fragment of 1766 bp for desC1 or another pair of primers #22 and #4 produced a fragment of that of 1746 bp for desC2 in desC1- or desC2-mutated cells, respectively. However, such a fragment was undetected in the wild-type cells. Since these fragments corresponded to the size of DNA fragments, which included a part of the spectinomycin-resistant gene cassette and a part of the desC1 or desC2 gene, these results indicated that genes for these desaturases were mutated. The resultant mutants were further analyzed by PCR with a pair of primers #1 and #2 for desC1 or another pair of primers #3 and #4 for desC2. The results revealed that the mutant cells, as well as the wild-type cells, produced a fragment at 819 bp or 859 bp, respectively. These results indicated that the native desC1 or desC2 gene was present in the mutated cells of Anabaena 7120. Since cyanobacteria contain several copies of genome within a cell (Wang et al. 2018), these results suggested that the mutated and unmutated copies of the genome co-existed in the respective mutant cells. Despite conducting several culturing on plates with sufficient spectinomycin concentration, the native desC1- or desC2-containing copies were not segregated. Overall, both DesC1 and DesC2 are essential in Anabaena 7120 but lack complementarity.

Transformation of Synechocystis 6803 with desC1 and desC2 genes from Anabaena 7120

The specific functions of DesC1 and DesC2 were examined by overexpressing the desC1 and desC2 genes from Anabaena 7120 in Synechocystis 6803 under the promoter of the psbA2 gene (Matsumoto and Awai 2020). Fig. 2A shows the design for inserting the desC1 and desC2 genes into the neutral site of the Synechocystis 6803 genome, and Fig. 2B shows the results of the PCR analysis of the introduced desC1 and desC2 genes. The PCR with a pair of primers #1 and #2, internal to the desC1 gene, produced a fragment of 819 bp, which corresponded to the open-reading frame of the desC1 gene, in the transformed cell genome but not in the wild-type cell genome. Another pair of primers, #7 and #8, internal to the neutral site, produced a 1426-bp fragment in the wild-type cell genome and a 3386-bp fragment in the desC1-transformed cell genome. This increase in the fragment size from 1426 to 3386 bp corresponded to the insertion of the desC1 gene from Anabaena 7120 and the erythromycin-resistant gene cassette into the neutral site. Another PCR analysis with a pair of primers that had been annealed to the middle region of the neutral site of the wild-type cell genome, namely, #9 and #8, produced an amplicon of 724 bp only from this genome. These results confirmed the successful introduction of the desC1 gene into the neutral site of all genome copies of the transformed cells. A similar analysis by PCR was performed on the transformed cells, in which the desC2 gene had been inserted to the neutral site of the Synechocystis 6803 genome, essentially yielding the same result as in the desC1-transormed cells. This result demonstrated the successful introduction of the desC2 gene in the Synechocystis 6803 genome as designed.

Changes in fatty acid composition due to transformation of Synechocystis 6803 with the external desC1 and desC2 genes from Anabaena 7120

Gas chromatography-flame ionization detector (GC-FID) analysis of the fatty acid composition of membrane lipids in the wild-type and desC1- and desC2-transformed cells of Synechocystis 6803 (Tables 1–4) showed that the transformation with the desC1 gene did not change the fatty acid composition in any of the membrane lipid classes. In contrast, the transformation with the desC2 gene decreased the level of C16:0 and increased the level of C16:1 Δ9 in the major membrane lipid, MGDG. Such a change in fatty acid composition was observed in one of the minor lipids, DGDG, but to a lesser extent, but was absent in the other minor lipids, SQDG and PG. These results suggested that the introduced desC2 gene from Anabaena 7120 was activated in Synechocystis 6803, resulting in DesC2 desaturating 16:0 to 16:1 in MGDG. The introduced desC1 gene was also activated, leading to DesC1 synthesis, but it did not affect fatty acid composition. In addition, DesC, native in Synechocystis 6803 (hereafter, DesCs), was active enough in desaturating 18:0 to 18:1, and the addition of activity due to the introduced DesC1 did not affect fatty acid composition under the experimental conditions.

To investigate the specific distribution of fatty acids within the membrane lipids, the Rhizopus lipase method was used to determine the sn-positional distribution of fatty acids in the four membrane lipid classes from transformed cells of Synechocystis 6803 (Tables 5–12). The wild-type cells of Synechocystis 6803 and its negative-control cells contained a very low level of 16:1 fatty acid at the sn-2 position of MGDG (Tables 5–6). In cells transformed with the desC2 gene, but not the desC1, the level of 16:0 decreased considerably and that of 16:1Δ9 increased to a high level (Tables 5–6). Such a significant change in fatty acid unsaturation occurred only at the sn-2 position of MGDG. In DGDG, a similar change in unsaturation was observed but to a much lesser extent. Insignificant changes were detected in the SQDG and PG. These results demonstrated that DesC2 was synthesized in the transformed cells and desaturated 16:0 that was esterified to the sn-2 position of MGDG. Overall, this finding shows that DesC2, but not DesC1, acts on 16:0 at the sn-2 position of MGDG.

Chintalapati et al. (2006) performed a similar experiment using another strain of cyanobacteria in Group 2, Nostoc sp. SO-36. They overexpressed the desC2 gene from this cyanobacterium in Synechocystis 6803 and observed that C16:0 was desaturated to 16:1Δ9 at the sn-2 position of MGDG in this transformant. The results in the present study corresponded to their research.

Mutation of the desCs gene in Synechocystis 6803 that had been transformed with the desC1 or desC2 gene from Anabaena 7120

To further characterize the specific function of DesC1 and DesC2 of Anabaena 7120, the complementarity of these desaturases toward the DesCs of Synechocystis 6803 was examined. For this purpose, the internal desCs gene in Synechocystis 6803 was mutated in desC1-transformed cells. Fig. 3A shows the design used for mutating the desCs gene in Synechocystis 6803 by inserting the spectinomycin-resistant gene cassette (Spe^r). After numerous rounds of segregation on plates that contained spectinomycin, a colony was isolated as the mutant of the desCs gene for further experiments. The mutation was analyzed by PCR with a pair of primers #5 and #6 that was internal to the desCs gene, which produced a fragment of 974 bp, corresponding to the open-reading frame of the desCs gene, in the wild-type cells, but not in the desC1-transformed cells. This result suggested that the desCs gene was disrupted in all copies of the genome in the desC1-transformed cells. The PCR analysis with another pair of primers #10 and #13 that were external to the desCs gene produced a fragment of 1841 bp in the wild-type cell genome and a fragment of 2981 bp in the desC1-trasformed cell genome. This increase in the fragment size corresponded to the insertion of the spectinomycin-resistant gene cassette in the desCs gene. These results indicated that the desCs gene was mutated in all copies of the genome in the desC1-transformed cells.

A different result was obtained for the mutation of the desCs gene in the desC2-transformed cells. The PCR analysis with the pair of primers #5 and #6 produced a 974-bp fragment in both wild-type and desC2-transformed cell genomes, indicating that the desCs gene was not completely disrupted in all genome copies of the desC2-transformed cells. The PCR analysis with the pair of primers #10 and #13 produced an 1841-bp fragment, but not the fragment of 2981 bp, in both wild-type and desC2-transformed cell genomes. These results suggested that the desCs gene was not mutated at all genome copies of the desC2-transformed cells; namely, the desC2 gene of Anabaena 7120 could not complement the desCs gene of Synechocystis 6803. Overall, the desC1 gene, but not the desC2 gene, from Anabaena 7120 complemented the desCs gene of Synechocystis 6803. This may be related to the finding that the desCs gene is homologous to the desC1 gene from Anabaena 7120 with a similarity of 60% in the amino acid sequence, whereas it is less homologous to the desC2 gene from Anabaena 7120 with a similarity of 40%. DesC2 may have failed to desaturate 18:0 to 18:1 at the sn-1 position.

The fatty acid composition was examined in Synechocystis cells that had been transformed with the desC1 gene from Anabaena 7120 and mutated at the internal desCs gene. The results in Tables 1–12 revealed that the mutation of the internal desCs gene had no significant effect on the unsaturation of fatty acids in any of the four lipid classes. These results suggested that DesC1 of Anabaena 7120 complemented the internal DesCs of Synechocystis 6803.

Physiological and biochemical changes due to transformation with external desC1 and desC2 genes and mutation of the internal desCs gene in Synechocystis 6803

As mentioned above, the fatty acid composition was different between the transformant with the desC2 gene and all the other strains examined, such as wild-type, desC1-transformed, and desCs-mutated strains: namely, the C16:0 saturated fatty acid at the sn-2 position of MGDG was replaced by the C16:1Δ9 unsaturated fatty acid in the desC2-transformed cells. We studied whether such changes in fatty acid unsaturation would affect the physiological activities and biochemical composition in Synechocystis 6803. The growth and oxygen-evolution rates were practically unaffected by the transformation and mutation (Fig. 4). The contents of chlorophyll a and carotenoids were essentially the same among the wild-type, transformant and mutant cells (Fig. 5). No remarkable difference was observed among the strains in the maximum quantum yield of PSII (Fv/Fm) and the other parameters of the quantum yield of PSII (Fv’/Fm’) under different light conditions (Fig. 6). These results suggested that the change in fatty acid unsaturation and, in particular, the C16 fatty acid at the sn-2 position in Synechocystis 6803 cells by transformation with the external desC2 gene did not affect the physiological activity and biochemical composition under the conditions employed in the present study. So far, the essentiality of desC2 remains unclear and will be addressed in future investigations.

Distribution and possible role of DesC1 and DesC2

We revisited the phylogenetic analysis of desC1 and desC2 genes by Chintalapati et al. (2006) and found that orthologs of the desC2 gene exist in Nostocales but not in the other cyanobacterial order except for the filamentous genus Plectonema of Oscillatoriales. The desC2 gene is absent in all filamentous and in all diazotrophic cyanobacteria, indicating that the desaturation of fatty acids at the sn-2 position is not important in these morphological and physiological functions. We also examined whether Group 2 cyanobacteria have both desC1 and desC2 genes. In genomes of Synechococcus sp. PCC 7002 and Trichodesmium erythraeum, we only found an ortholog of the desC1 but not desC2 gene, indicating that these cyanobacteria might adopt a system different from that of Nostocales for desaturation of fatty acids at the sn-2 position. Another Group 2 cyanobacterium Gloeobacter violaceus seems to have six homologs of the desC gene whose amino acid sequences cannot be classified into neither desC1 nor desC2 (Chi et al. 2008). Thus, it is also likely that G. violaceus uses a mechanism different from that of Nostocales for desaturation of fatty acids at the sn-2 position.

In our previous study (Effendi et al. 2022), we compared the cold sensitivity of the Antarctic strain (Nostoc sp. SO-36) and Anabaena sp. PCC 7120 and found that Nostoc sp. SO-36 is a psychrotolerant cyanobacterium, whereas Anabaena sp. PCC 7120 is sensitive to low temperature, and both have the desC2 gene. Therefore, we still wonder whether desaturation of fatty acids at the sn-2 position is related to the cold tolerance in Nostocales. It is of interest to elucidate physiological function of unsaturated fatty acids at the sn-2 position in Nostocales.

Materials and Methods

Growth conditions of cyanobacteria strains

Cyanobacteria Anabaena sp. PCC 7120 (Anabaena 7120) and Synechocystis sp. PCC 6803 (Synechocystis 6803) were grown at 30^oC in BG-11 medium (Stanier et al. 1971) supplemented with 0.1 m TES buffer (pH 7.5). These cells were incubated under continuous light from a fluorescent lamp at 50 μmol photon m⁻² s⁻¹ on a rotary shaker at 120 rpm, as described previously (Awai et al. 2007).

Mutational inactivation of the desC1 and desC2 genes in Anabaena sp. PCC 7120

We attempted to mutate the desC1 (all1599) and desC2 (all4991) genes in Anabaena 7120, as they exhibited similarities in the deduced amino acid sequence to the desC1 and desC2 genes of Nostoc sp. SO-36. The vector for the insertional mutation of both genes was constructed as follows: DNA fragments upstream of desC1 and desC2 were amplified by PCR with KOD plus neo DNA polymerase (Toyobo Co. Ltd., Osaka Japan) using the wild-type genome of Anabaena 7120 as a template with a pair of primers, #14 and #15, for desC1 and another pair of primers, #18 and #19, for desC2. Resultant amplicons were subcloned into the SmaI site of pMobΩ1, a vector that contained spectinomycin- and streptomycin-resistance gene cassette (Supplementary Table 1) (Saito and Awai 2020), to make pMobΩ1/desC1 5′and pMobΩ1/desC2 5′ using a hot fusion cloning system (Fu et al. 2014). DNA fragments downstream of desC1 and desC2 were amplified with a pair of primers, #16 and #17, for desC1 and another pair of primers, #20 and #21, for desC2. Resultant amplicons were subcloned into the ApaI site of pMobΩ1/desC1 5′and pMobΩ1/desC2 5′ using the hot fusion cloning system. Resultant plasmids that contained the flanking region of desC1 or desC2 genes were designated as pMobΩ1/desC1 and pMobΩ1/desC2. The constructed plasmid vectors were introduced consecutively into wild-type cells of Anabaena 7120 by the tri-parental mating method (Elhai and Wolk 1988): cells of the Escherichia coli J53 (RP4) and E. coli strain HB101 (pRL623) that contained pMobΩ1/desC1 or pMobΩ1/desC2 were mixed with wild-type cells of Anabaena 7120. After the mating process of E. coli and Anabaena 7120, the mixture was plated on a nitrocellulose membrane (HATF 08520, Merck Millipore Ltd., Tokyo, Japan) on solid agar medium of BG-11 and incubated at 30°C under continuous light of 50 μmol photon m⁻² s⁻¹ for 2 days. After incubation, the membrane was transferred to a solid BG-11 medium supplemented with 20 μg mL⁻¹ spectinomycin and 5% sucrose for the isolation of mutant cells. Spectinomycin-resistant colonies were segregated on BG-11 agar plates (Xu et al. 2015). After five times of the segregation procedure, the genomic DNA was extracted from the mutant cells by glass bead method (Kindle et al. 1991) and genotyped by PCR using the primers described in Supplementary Table 2. The mutant of the desC1 or desC2 gene was genotyped using a pair of primers #2 and #22 for amplifying the spectinomycin-resistant gene for candidates of the mutant of desC1 and a pair of primers #4 and #22 for candidates of the mutant of desC2. The candidate of the mutant of the internal desC1 in Anabaena 7120 was genotyped using the primer pair #1 and #2 for amplifying full length of desC1 and a pair of primers #3 and #4 for desC2; those primer pairs were used for detecting the deletion of the central part of desC1 and desC2 genes, respectively.

Transformation of Synechocystis sp. PCC 6803 with desC1 and desC2 genes from Anabaena sp. PCC 7120

DNA sequences of the desC1 (all1599) and desC2 (all4991) genes of Anabaena 7120 and of the desC gene (sll0541) of Synechocystis 6803 (hereafter termed desCs) were obtained from Cyanobase (genome.microbedb.jp/cyanobase/). The vector for the overexpression of the desC1 and desC2 genes of Anabaena 7120 in Synechocystis 6803 was constructed as follows: both DNA fragments covering the desC1 and desC2 genes were amplified with the genomic DNA from wild-type cells of Anabaena 7120 as a template using a pair of primers #1 and #2 for desC1 and another pair of primers #3 and #4 for desC2. Amplified fragments were subcloned separately into the vector pSEM3 (Matsumoto and Awai 2020), a vector that had been designed to introduce the respective gene into the neutral site 2 of the genome of Synechocystis 6803 (slr2031) under the strong promoter of the psbA2 gene (slr1371) from Synechocystis 6803. DNA fragments were introduced into the SmaI site of the vector pSEM3 by the hot fusion cloning system (Fu et al. 2014), and the sequence of the resultant vector was confirmed. These vectors were used to introduce desC1 and desC2 genes from Anabaena 7120 in Synechocystis 6803 by double homologous recombination (Kufryk et al. 2002). For that purpose, wild-type cells of Synechocystis 6803 and the constructed vectors were mixed and incubated for 6 h at 30°C under illumination from fluorescence lamps at 50 μmol photon m⁻² s⁻¹. For isolation of transformants, the mixture was plated on the nitrocellulose membrane on a solid BG-11 agar medium and incubated at 30°C under illumination from fluorescence lamps at 50 μmol photon m⁻² s⁻¹ for 2 days. After incubation, the membrane was transferred to a solid BG-11 agar medium containing erythromycin at 20 μg mL⁻¹ to isolate the transformed cells. Erythromycin-resistant colonies were segregated on a solid BG-11 medium (Xu et al. 2015). After five times of the segregation procedure, the genome DNA was extracted from the transformed cells by glass bead methods (Kindle et al. 1991) and genotyped by PCR using the primers in Supplementary Table 2 using Hybripol DNA polymerase (Bioline, London, UK). Genotyping of transformant with the desC1 or desC2 gene was performed with a pair of primers #7 and #8 for amplifying the neutral site and another pair of primers #8 and #9 for the middle region of the neutral site, and a pair of primers #1 and # 2 for amplifying the desC1 gene and another pair of primers #3 and #4 for amplifying the desC2 gene.

Mutational inactivation of the desCs gene in Synechocystis sp. PCC 6803 that had been transformed with desC1 and desC2 genes from Anabaena sp. PCC 7120

The DNA sequence of the desCs gene of Synechocystis 6803 (sll0541) was obtained from Cyanobase (genome.microbedb.jp/cyanobase/). The vector for mutating the desCs gene in Synechocystis 6803 was constructed as follows: a DNA fragment upstream of the desCs gene was amplified by PCR with KOD plus neo DNA polymerase using the genome of Synechocystis 6803 as a template and with a pair of primers #10 and #11. The resultant amplicon was subcloned into the SmaI site of pMobΩ1 to make pMobΩ1/desCs 5′ using the hot fusion cloning system. DNA fragments downstream of the desCs gene were amplified by PCR as described above with another pair of primers #12 and #13. The amplified fragments were subcloned into the ApaI site of pMobΩ1/desCs 5′ using a hot fusion cloning system. The resultant plasmid that contained the flanking regions of the desCs gene was designated as pMobΩ1/desCs. Then, this vector was introduced into the transformed Synechocystis 6803, in which the desC1 or desC2 gene had been introduced, as mentioned earlier. For the isolation of mutants, the mixture was plated on the nitrocellulose membrane (HATF 08520, Merck Millipore Ltd., Tokyo, Japan) on a solid BG-11 agar medium and incubated at 30°C under continuous light 50 μmol photon m⁻² s⁻¹ for 2 days. After incubation, the membrane was transferred to a solid BG-11 agar medium containing spectinomycin at 20 μg mL⁻¹ to isolate the candidates of the mutants. Spectinomycin-resistant colonies were segregated on a solid BG-11 medium (Xu et al. 2015). After five times of the segregation procedure, genome DNA was extracted from the mutant cells by glass bead method (Kindle et al. 1991) and genotyped by PCR using the primers in Supplementary Table 2, using Hybripol DNA polymerase (Bioline, London, UK). Genotyping of the desCs mutant of Synechocystis 6803 was performed using a pair of primers #5 and #6 for amplifying full length of desCs to confirm the deletion of the central part of the desCs gene.

Analysis of fatty acids in transformed and mutated cells of Synechocystis sp. PCC 6803

Total lipids were extracted from cyanobacterial cells with a chloroform:methanol (2:1) solution, as described previously (Bligh and Dyer 1959). Lipid classes were separated by thin-layer chromatography (TLC) with a solvent system chloroform/methanol/25%ammonium hydroxide (65:35:5), as described previously (Sato and Murata 1988). Separated lipids were stained with a primuline solution (0.01 g primuline diluted in 80% acetone) and observed under UV light. For positional analysis of fatty acids, each lipid was treated with lipase from Rhizopus oryzae (Merck Sigma Aldrich Ltd., Tokyo, Japan), and the lyso-lipids were separated by TLC with a solvent system chloroform/methanol/dH₂O (170:30:2) as described previously (Sato and Murata 1988). Fatty acids of separated lipids and lyso-lipids were analyzed with a GC-FID on a capillary column (BPX90, 60 metre × 0.25 mm, SGE Analytical Science) after methylation treatment, as described previously (Awai et al. 2014).

Measurement of cell growth, chlorophyll content, oxygen-evolving activity and chlorophyll fluorescence of transformed and mutated cells of Synechocystis sp. PCC 6803

A spectrophotometer (UV-2600, Shimadzu) was used to measure cell growth and chlorophyll content. For growth-curve analysis, both wild-type and transformant cells were inoculated into fresh BG-11 medium at an initial optical density of 0.1 at a wavelength of 730 nm. The chlorophyll and carotenoid contents were measured by extracting these pigments using 100% methanol, and their levels were calculated following previously established methods (Arnon et al. 1974).

The photosynthetic activity of transformed and mutated cells of Synechocystis 6803 was analyzed via oxygen evolution using a 2 mL cell suspension at an initial optical density of 0.1 with the Clark-type electrode (Hansatech Instruments, Norfolk, UK). Synechocystis cells were illuminated using a LED lamp with infrared cutoff filters. The chlorophyll fluorescence was measured using a Dual-Pam system (Heinz Walz GmbH, Germany) in cell suspensions at OD₇₃₀ of 1.0. All the samples were acclimated to a dark environment for 30 min before the measurement. The Fv/Fm and Fv’/Fm’ parameters were measured after 2 min illumination of either 34 µmol m⁻² s⁻¹ (OL) or 212 µmol m⁻² s⁻¹ (HL) (Kobayashi et al. 2020).

Supplementary Data

Supplementary Data are available at PCP online.

Data Availability

The data underlying this article are available in the article and in its online supplementary material.

Funding

MEXT/JSPS KAKENHI Grant Number 18H03941 and 20K06683 to K.A.

Disclosures

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References