Effects of modified Phycobilin biosynthesis in the Cyanobacterium Synechococcus sp. Strain PCC 7002

Abstract

The pathway for phycocyanobilin biosynthesis in Synechococcus sp. strain PCC 7002 comprises two enzymes: heme oxygenase and phycocyanobilin synthase (PcyA). The phycobilin content of cells can be modified by overexpressing genes encoding alternative enzymes for biliverdin reduction. Overexpression of the pebAB and HY2 genes, encoding alternative ferredoxin-dependent biliverdin reductases, caused unique effects due to the overproduction of phycoerythrobilin and phytochromobilin, respectively. Colonies overexpressing pebAB became reddish brown and visually resembled strains that naturally produce phycoerythrin. This was almost exclusively due to the replacement of phycocyanobilin by phycoerythrobilin on the phycocyanin α-subunit. This phenotype was unstable, and such strains rapidly reverted to the wild-type appearance, presumably due to strong selective pressure to inactivate pebAB expression. Overproduction of phytochromobilin, synthesized by the Arabidopsis thaliana HY2 product, was tolerated much better. Cells overexpressing HY2 were only slightly less pigmented and blue-green than the wild type. Although the pcyA gene could not be inactivated in the wild type, pcyA was easily inactivated when cells expressed HY2. These results indicate that phytochromobilin can functionally substitute for phycocyanobilin in Synechococcus sp. strain PCC 7002. Although functional phycobilisomes were assembled in this strain, the overall phycobiliprotein content of cells was lower, the efficiency of energy transfer by these phycobilisomes was lower than for wild-type phycobilisomes, and the absorption cross-section of the cells was reduced relative to that of the wild type because of an increased spectral overlap of the modified phycobiliproteins with chlorophyll a. As a result, the strain producing phycobiliproteins carrying phytochromobilin grew much more slowly at low light intensity.

FIG. 1.

Scheme showing the synthesis of linear tetrapyrrole chromophores in cyanobacteria, algae, and plants.

FIG. 2.

The pcyA gene is apparently essential in Synechococcus sp. strain PCC 7002. (A) Colonies of wild-type Synechococcus sp. strain PCC 7002. (B) Colonies of Synechococcus sp. strain PCC 7002 transformed with pcyA::aadA on medium containing spectinomycin. Sectors and colonies with wild-type coloration are obvious. (C) PCR amplification of the pcyA locus from the wild type (lane 1) and the pcyA::aadA transformed strain (lane 2) of Synechococcus sp. strain PCC 7002. The two amplicons visible in lane 2 indicate that both the pcyA and pcyA::aadA alleles are present in the genomic DNA of the transformed cells.

FIG. 3.

Overexpression of pebA and pebB in Synechococcus sp. strain PCC 7002. (A) Colonies of Synechococcus sp. strain PCC 7002 transformed with the pebAB overexpression cassette mostly exhibit a reddish-brown phenotype resembling strains that naturally synthesize PE. A sector that has reverted to the wild-type color phenotype can be seen (arrow). (B) Whole-cell absorption spectra of wild-type Synechococcus sp. strain PCC 7002 (solid line) and the strain overexpressing pebA and pebB (dashed line).

FIG. 4.

Overexpression of pebA and pebB causes PEB to be ligated preferentially to CpcA. (A) Coomassie brilliant blue and zinc-enhanced fluorescence images of a single SDS-PAGE gel. Lane 1, recombinant CpcA carrying PCB; lane 2, recombinant CpcA carrying PEB; lane 3, PBS isolated from wild-type cells of Synechococcus sp. strain PCC 7002; lane 4, PBS isolated from cells of Synechococcus sp. strain PCC 7002 overexpressing pebA and pebB. (B) Absorption spectra of major PBP subunits separated by HPLC from PBS isolated from wild-type Synechococcus sp. strain PCC 7002. (C) Absorption spectra of major PBP subunits separated by HPLC from PBS isolated from cells of Synechococcus sp. strain PCC 7002 overexpressing pebA and pebB. Note that the CpcA subunit mostly carries a PEB chromophore with absorption at ∼560 nm, although some absorption from PCB is also evident.

FIG. 5.

Absorption spectra of Synechococcus sp. strain PCC 7002 expressing HY2. Absorption spectra of Synechococcus sp. strain PCC 7002 wild-type cells (solid line) and of a strain in which HY2 expression is being driven by the cpcBA promoter of Synechocystis sp. strain PCC 6803 (dashed line) are shown.

FIG. 6.

HY2 functionally substitutes for pcyA in Synechococcus sp. strain PCC 7002. (A) Whole-cell absorption spectra of wild-type Synechococcus sp. strain PCC 7002 (solid line) and a strain overexpressing HY2 in which pcyA has been inactivated (dashed line). (B) PCR analysis of the pcyA locus in wild-type Synechococcus sp. strain PCC 7002 (lane 1), a strain overexpressing HY2 in which pcyA has been deleted (lane 2), and the pcyA partial deletion strain (lane 3).

FIG. 7.

The 77 K fluorescence emission spectra of Synechococcus sp. strain PCC 7002 wild-type cells (solid line) and cells of the pcyA deletion strain overexpressing HY2 (ΔpcyA::aadA + HY2 strain) (dashed line). The excitation wavelength was 590 nm.

FIG. 8.

Analysis of isolated PBS from the wild type and a ΔpcyA::aadA strain overproducing HY2. (A and B) SDS-PAGE results with isolated PBS from the wild-type and the ΔpcyA::aadA + HY2 strain (lanes labeled as ΔpcyA::HY2), visualized by Coomassie staining (A) or by zinc-enhanced fluorescence (B). Selected proteins are identified by the arrows on the right. (C to E) Absorption spectra (C), 77 K fluorescence emission (D), and absorption spectra after denaturation in acidic urea (E) of PBS isolated from the wild type (solid lines) and a ΔpcyA::aadA + HY2 strain (dashed lines). The absorption spectra in panel C were normalized at the maximal values to facilitate the comparison. The fluorescence emission spectra were not normalized, and samples of equal absorption at 630 nm or 640 nm were used for the measurement. The excitation wavelength was 590 nm.

FIG. 9.

Growth rate analysis of wild-type Synechococcus sp. strain PCC 7002 and the ΔpcyA::aadA + HY2 strain (labeled as pcyA::HY2). Cells were grown under otherwise standard conditions at high (suprasaturating; 500 μmol photons m⁻² s⁻¹), medium (nearly saturating; 200 μmol photons m⁻² s⁻¹), and low (limiting; 50 μmol photons m⁻² s⁻¹) light intensities. The plotted data are the averages of results for at least three replicate cultures, and the calculated doubling times are indicated.