Yoshikazu Kawata

Effective transformation of the cyanobacterium Spirulina platensis using electroporation

Although Spirulina (Arthrospira) is expected to be a suitableorganism for producing recombinant proteins, a gene transfer system hasnot yet been established, due to a lack of suitable vectors and because Spirulina appears refractory to common genetic manipulations. As theinitial stages of the development of recombinant DNA methodology, weexamined the effects on transformation efficiency of electroporationconditions such as electric-field strength (2, 4, 6, 8, 10, 12kV cm-1) and time constant (2.5, 5 ms). At a time constant of2.5 ms, few transformants were observed regardless of the field strength.The longer time constant of 5.0 ms reproducibly yielded transformants atthe middle field strength of 4 - 8 kV cm-1, but gave high killingand no transformation at the higher field strength of 10 - 12kV cm-1. Chloramphenicol acetyltransferase (CAT) activities wereincreased only in the transformants from 2–6 kV cm-1 and 5.0 ms.The density of the transformants was significantly correlated with therelative value of CAT activity (r = 0.89, n = 11, p < 0.01),suggesting that the chloramphenicol resistance was due to CAT activity. Weconcluded that transformation of S. platensis was most effective at apulse duration 5.0 ms with an electric field of 4 kV cm-1, and thatforeign genes can be expressed in this organism.

Expression of salmon growth hormone in the cyanobacteriumAgmenellum quadruplicatum

SummaryThe salmon growth hormone gene was introduced into the cyanobactriumAgmenellum quadruplicatum PR-6 by plasmid transformation. The gene expressed the hormone under thetrp promoter ofEscherichia. coli. The amount was estimated to be approximately 0.1% of the total cell protein.

News & notes. Efficient library construction with a TA vector and its application to cloning of the phytoene synthase gene from the cyanobacterium Spirulina platensis.

Abstract. An efficient and simple method for constructing a genomic DNA library is presented by use of a TA cloning vector. It is based on sonicative cleavage of genomic DNA and modification of the fragment ends with Taq DNA polymerase, followed by ligation with a TA vector. This method was successfully applied to cloning of the phytoene synthase gene crtB from Spirulina platensis. The method is useful when the genomic DNA is not well digested with restriction enzymes owing to methylation or other reasons.

Cloning and sequencing of the allophycocyanin genes fromSpirulina maxima (Cyanophyta)

The genes coding for the α-and β-subunit of allophycocyanin (apcA andapcB) from the cyanophyteSpirulina maxima were cloned and sequenced. The results revealed 44.4% of nucleotide sequence similarity and 30.4% of similarity of deduced amino acid sequence between them. The amino acid sequence identities betweenS. maxima andS. platensis are 99.4% for α subunit and 100% for β subunit.

Transposable genetic elements inSpirulina and potential applications for genetic engineering

Transposable elements in cyanobacteria are briefly reviewed. Evidence is presented to show that transposable elements inSpirulina platensis is actually reflected on the phenotype change, i e., helical to straight filaments. Transposition intermediates of DNA were isolated from the extrachromosome and the transposition was related to helical variations inSpirulina. Uses of transposable elements for microalgal recombination are discussed based on the transposition mechanism.

Salinity-dependent copy number change of endogenous plasmids in Synechococcus sp. strain PCC 7002

Marine cyanobacterium Synechococcus sp. strain. PCC 7002 has at least six endogenous plasmids. When it was cultured in 1 m NaCl medium, the copy numbers of the smallest (4.5 kb) and the second smallest (9.7 kb) plasmids decreased to one-third and one-tenth of those in control culture (0.3 m NaCl) respectively. In medium without NaCl, the copy numbers of those plasmids also decreased but the changes were much smaller. On the other hand, copy numbers of 15.4-kb, 30.0-kb, and 36.9-kb plasmids did not change among the three different NaCl concentrations (0 m, 0.3 m, 1 m). The copy number changes of the two plasmids were reversible. A similar copy number change was also observed in medium with 0.3 m NaCl+0.7 m KCl. These observations suggest that copy number controls are different among endogenous plasmids, and some of them are affected by salinity in the medium.

Construction of a genomic DNA library with a TA vector and its application in cloning of the phytoene synthase gene from the cyanobacteriumSpirulina platensis M-135

An efficient and simple method for constructing a genomic DNA library using a TA cloning vector is presented. It is based on the sonicative cleavage of genomic DNA and modification of fragment ends withTaq DNA polymerase, followed by ligation using a TA vector. This method was applied for cloning of the phytoene synthase genecrt B fromSpirulina platensis. This method is useful when genomic DNA cannot be efficiently digested with restriction enzymes, a problem often encountered during the construction of a genomic DNA library of cyanobacteria.

Extracellular release of β-lactamase by osmotic shock inSynechococcus transformant

A unicellular cyanobacteriumSynechococcus sp. strain PCC 7002 was transformed with plasmid pQL1, on which β-lactamase gene (bla) and β-galactosidase gene (lacZ) were encoded. The transformant cells released β-lactamase into medium by an abrupt drop of osmotic pressure. This result indicates that this cyanobacterium recognizes and processes the signal sequence of β-lactamase, and accumulates the enzyme in periplasm. Repeated release of β-lactamase was possible by repeated osmotic shocks wihout, impairing cell viability. On the other hand, most of the β-galactosidase remained in cytoplasm under the osmotic shock.

EXTRACELLULAR RELEASE OF A RECOMBINANT GENE PRODUCT BY OSMOTIC SHOCK FROM IMMOBILIZED MICROALGA IN ELECTROCONDUCTIVE MEMBRANE

Abstract The unicellular blue green alga Synechococcus was immobilized in an electroconductive membrane of polypyrrole-agarose. The Synechococcus was recombinant with a plasmid vector encoding the ampicillin resistance gene. The gene product β-lactamase, a periplasm enzyme of Escherichia coli , was released to medium repeatedly by osmotic shocks. The immobilized cells were stable for over a month and kept their viability under electrical potentials from −0.5 V to 0.6 V vs. Ag/AgCl.