Hajime Wada

Cloning of omega 3 desaturase from cyanobacteria and its use in altering the degree of membrane-lipid unsaturation.

Cyanobacteria respond to a decrease in temperature by desaturating fatty acids of membrane lipids to compensate for the decrease in membrane fluidity. Among various desaturation reactions in cyanobacteria, the desaturation of the ω3 position of fatty acids is the most sensitive to the change in temperature. In the present study, we isolated a gene, designated desB, for the ω3 desaturase from the cyanobacterium, Synechocystis sp. PCC 6803. The desB gene encodes a protein a 359 amino-acid residues with molecular mass of 41.9 kDa. The desB gene is transcribed as a monocistronic operon that produced a single transcript of 1.4 kb. The level of the desB transcript in cells grown at 22°C was 10 times higher than that in cells grown at 34°C. In order to manipulate the fatty-acid unsaturation of membrane lipids, the desB gene in Synechocystis sp. PCC 6803 was mutated by insertion of a kanamycin-resistance gene cartridge. The resultant mutant was unable to desaturate fatty acids at the ω3 position. The desA gene, which encodes the Δ12 desaturase of Synechocystis sp. PCC 6803, and the desB gene were introduced into Synechococcus sp. PCC 7942. Whilst the parent cyanobacterium can only desaturate membrane lipids at the Δ9 position of fatty acids, the resultant transformant was able to desaturate fatty acids of membrane lipids at the Δ9, Δ12 and ω3 positions. These results confirm the function of the desB gene and demonstrate that it is possible to genetically manipulate the fatty-acid unsaturation of membrane lipids in cyanobacteria.

Site-directed mutagenesis of histidine residues in the Δ12 acyl-lipid desaturase of Synechocystis

In the cyanobacterium Synechocystis sp. PCC 6803, there are four acyl‐lipid desaturases that are, respectively, specific to the Δ6, Δ9, Δ12 and ω3 positions of fatty acids. The desA gene for the Δ12 acyl‐lipid desaturase was modified by site‐directed mutagenesis, such that four of the histidine residues that are conserved in the four desaturases and one histidine residue that is not conserved were replaced by arginine, and the mutated desA genes were overexpressed in Escherichia coli. All of these mutations eliminated the Δ12 desaturase activity. These results demonstrate that the five histidine residues are essential for the activity of the Δ12 desaturase, perhaps by providing the ligands for the catalytic Fe center.

In vitro ferredoxin-dependent desaturation of fatty acids in cyanobacterial thylakoid membranes.

Thylakoid membranes isolated from the cyanobacterium Synechocystis sp. strain PCC6803 were capable of desaturating the acyl groups in monogalactosyl diacylglycerol. This desaturation reaction required the reduced form of ferredoxin.

Glycinebetaine enhances and stabilizes the evolution of oxygen and the synthesis of ATP by cyanobacterial thylakoid membranes

Glycinebetaine (betaine), an osmoregulant in halophilic plants, stabilized the evolution of oxygen and the synthesis of ATP by thylakoid membranes from the cyanobacterium Synechocystis PCC6803 when it was present during the preparation and incubation of the thylakoid membranes. Moreover, betaine enhanced the evolution of oxygen and the synthesis of ATP when present during assays. When betaine at 1.0 M was present during the preparation of thylakoid membranes and during the measurement of activity, the rate of evolution of oxygen was equivalent to that of intact cells.

Identification of conserved domains in the Δ12 desaturases of cyanobacteria

Cyanobacterial genes for enzymes that desaturate fatty acids at the Δ12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by crosshybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the Δ12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of ω3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.

Heat shock protein synthesis of the cyanobacterium Synechocystis PCC 6803: purification of the GroEL-related chaperonin

Synechocystis PCC 6803 cells could be induced to synthesize four major HSPs with apparent molecular sizes of 70, 64, 15 and 14 kDa. Heat stress at 42.5 °C appeared to be the optimum temperature for HSP formation in cells grown at 30 °C.The relative rate of synthesis of HSP70 and HSP15 reached a maximum at 30 min after the temperature shift-up whereas the capability of cells to accumulate HSP64 and HSP14 continued through 2 h.The two most abundant HSPs, HSP70 and HSP64, were recognized on western blots by antibodies raised against authentic DnaK and GroEL from Escherichia coli. To furnish sufficient evidence for the assumption that HSP64 is a GroEL-related chaperonin, this protein was purified to homogeneity. There was a 76% sequence identity between the amino acid sequence of HSP64 and the corresponding protein in Synechococcus PCC 7942. Moreover, the purified HSP64 cross-reacted to anti-E. coli GroEL antibody. To our knowledge, this is the first report about the purification and partial protein sequencing of a cyanobacterial chaperonin.

A New Type of Cytochrome c from Synechocystis PCC6803

Summary A gene encoding a c-type cytochrome, designated cytM , was discovered 3.4 kbp downstream from the desA gene in the genome of Synechocystis PCC6803. The amino acid sequence derived from the nucleotide sequence exhibits about 35 % similarity to the amino acid sequences of cytochromes c -553 from other cyanobacteria and eukaryotic algae, but it also includes regions typical of soluble c -type cytochromes from eukaryotic mitochondria. The molecular mass of the putative mature protein was estimated to be 8.3 kDa, and the isoelectric point was calculated to be 7.3. The amino-terminal region of the putative product of the cytM gene is hydrophobic, suggesting that this domain may be a transit peptide or a membrane anchor. Both photosynthesis and respiration in a mutant with a disrupted cytM gene were as efficient as they were in the wild-type strain.

Structure of a cyanobacterial gene encoding the 50S ribosomal protein L9

The rplI gene encoding the ribosomal protein L9 was found 4 kbp downstream from the desA gene, but on the opposite strand, in the genome of the cyanobacterium Synechocystis PCC6803. The deduced amino acid sequence is homologous to the sequences of the L9 proteins from Escherichia coli and chloroplasts of Arabidopsis and pea. The gene is present as a single copy in the chromosome and is transcribed as a mRNA of 0.64 kb. An open reading frame of unknown function (ORF291) was found in the upstream region of the rplI gene.

The Cyanobacterial Desaturases: Aspects of Their Structure and Regulation

Desaturases introduce double bonds into fatty acids. They are important in the regulation of the degree of unsaturation of membrane glycerolipids and, thus, in the ability of certain organisms to tolerate low temperatures [1,2,3,4,5]. There are three types of desaturase, as follows. (1) Acyl-CoA desaturases introduce double bonds into fatty acids bound to coenzyme A, and these enzymes are bound to the endoplasmic reticulum in animal, yeast and fungal cells. (2) Acyl-ACP desaturases introduce double bonds into fatty acids that are bound to acyl-carrier protein and they are present in the stroma in plant chloroplasts. (3) Acyl-lipid desaturases introduce double bonds into fatty acids that have been esterified to glycerolipids, and they are bound to the membranes of plant and cyanobacterial cells. This last type of desaturase is the most efficient regulator of the extent of the unsaturation of membrane lipids in response to changes in temperature.

The Molecular Mechanism of the Low- Temperature Tolerance of Plants Studied by Gene Technology of Membrane Lipids

It has been stated that the fatty-acid unsaturation of membrane lipids is related to the tolerance (or sensitivity) of plants to high- and low-temperature stresses. This claim has not been supported directly in previous studies because the level of unsaturation has always been altered indirectly by changing the growth temperature. The change in growth temperature may be expected to alter not only the lipids, but also a number of other cellular components and enzymes. Therefore, to study the relationship between temperature tolerance and the lipid, it is necessary to establish an experimental system in which only the fatty-acid unsaturation of membrane lipids is altered, while the growth temperature remains constant. Such a system is now available through gene technology that modifies membrane lipids using genes for the desaturation of fatty acids. Recently, we have isolated genes termed desA and desB) for fatty-acid desaturation at the Δ12 and Δ15 positions from the cyanobacterium Synechocystis PCC6803. By mutagenesis and transformation with desaturase genes, we obtained strains of this cyanobacterium that had discretely different levels of fatty-acid unsaturation of their membrane lipids. The characteristic features of temperature tolerance of these strains suggest that the fatty-acid unsaturation of membrane lipids is related to the tolerance against low- temperature stress, but not to that against high-temperature stress. A second line of research has been the gene-technological modification of fatty-acid unsaturation of phosphatidylglycerol, one of the membrane lipids of higher- plant chloroplasts. In these studies, we cloned genes for acyl-ACP:glycerol- 3-phosphate acyltransferase from Arabidopsis thaliana and Cucurbita moschata (squash) and introduced them into Nicotiana tabacum (tobacco). Leaves of the resultant tobacco revealed distinctly different levels of unsaturation of the phosphatidylglycerol. By comparing the tolerance of the transformants against low-temperature stress, we have obtained evidence that low-temperature tolerance is enhanced by unsaturation of the membrane lipid.