phrA, the major photoreactivating factor in the cyanobacterium Synechocystis sp. strain PCC 6803 codes for a cyclobutane-pyrimidine-dimer-specific DNA photolyase

Abstract

A new broad-host-range plasmid, pSL1211, was constructed for the over-expression of genes in Synechocystis sp. strain PCC 6803. The plasmid was derived from RSF1010 and an Escherichia coli over-expression plasmid, pTrcHisC. Over-expressed protein is made with a removable N-terminal histidine tag. The plasmid was used to over-express the phrA gene and purify the gene product from Synechocystis sp. strain PCC 6803. PhrA is the major ultraviolet-light-resistant factor in the cyanobacterium. The purified PhrA protein exhibited an optical absorption spectrum similar to that of the cyclobutane pyrimidine dimer (CPD) DNA photolyase from Synechocuccus sp. strain PCC 6301 (Anacystis nidulans). Mass spectrometry analysis of PhrA indicated that the protein contains 8-hydroxy-5-deazariboflavin and flavin adenine dinucleotide (FADH2) as cofactors. PhrA repairs only cyclobutane pyrimidine dimer but not pyrimidine (6-4) pyrimidinone photoproducts. On the basis of these results, the PhrA protein is classified as a class I, HDF-type, CPD DNA photolyase.

Genetically engineering Synechocystis sp. Pasteur Culture Collection 6803 for the sustainable production of the plant secondary metabolite p -coumaric acid

“…There were about 29 copies of cargo plasmid per transformant cell, which is equivalent to about 3.3 copies per chromosome (22). Therefore, this transformation approach produces a higher copy number of transgene than does the genome-integration approach.…”

“…Triparental Mating for sam8 Overexpression in Synechocystis 6803. sam8 was inserted into pSL1211, a high-level expression vector (22), between XhoI and HindIII to generate the cargo plasmid pSL1211-sam8. For the triparental mating, three strains were mixed: (i) Synechocystis 6803 wild-type strain; (ii) E. coli HB101 strain containing pRL443 plasmid (21); and (iii) E. coli HB101 strain transformed with pSL1211-sam8.…”

Purification and Characterization of Three Members of the Photolyase/Cryptochrome Family Blue-light Photoreceptors from Vibrio cholerae

“…PCC6803 (22,37,43). Synechocystis contains two members of the photolyase/cryptochrome family, which were named PhrA and PhrB, respectively (44). When purified from its native source PhrA contains deazaflavin as a second chromophore and it is a cyclobutane photolyase (44).…”

“…Synechocystis contains two members of the photolyase/cryptochrome family, which were named PhrA and PhrB, respectively (44). When purified from its native source PhrA contains deazaflavin as a second chromophore and it is a cyclobutane photolyase (44). PhrB was purified as a recombinant protein expressed in E. coli, and it was reported that this protein, which has 38% sequence identity with VcCry1, may represent a new branch of the photolyase/cryptochrome family that do not have a second chromophore, based on the following arguments (43).…”

Phytoplankton as Key Mediators of the Biological Carbon Pump: Their Responses to a Changing Climate

“…Some of these include: active vertical migration using flagella or changes in buoyancy so as to move out of zones of excessive irradiance [80,81]; production of photolyase enzymes to repair UV-damaged DNA [82] and superoxide dismutase enzymes to prevent oxidative stress [83]; synthesis of UV absorbing pigments such as scytonemin (produced by cyanobacteria to reduce the impact of UV); and production of UV screening compounds such as mycosporine-like amino acids (MAAs) [46]. Different species of phytoplankton have varying capacities to produce UV screening compounds and thus, enhanced levels of UV-B radiation can potentially result in changes in species dominance within phytoplankton communities.…”