The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria

Abstract

The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO(2)-requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO(2). Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO(2). These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO(2), glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.

Figure 1.

Pathways of 2-PG metabolism by the plant-like photorespiratory C2 cycle (outer circle, glycolate cycle) or the bacterial-like glycerate pathway (inner branch). The metabolites involved, enzymatic steps, and putative genes encoding the relevant enzymes in Synechocystis (http://www.kazusa.or.jp/cyanobase/Synechocystis/index.html) are shown. Ru-1,5-bisP, Ribulose-1,5-bisphosphate.

Figure 2.

Overexpression of glcD and shm from Synechocystis in E. coli to verify their biochemical activities. A, GlcD from Synechocystis with a molecular mass of 57 kD. B, SHMT from Synechocystis with a molecular mass of 50 kD. The relevant genes were cloned in IBA6, overexpressed, purified by the fused Strep-tag, and the activity tested (see “Materials and Methods”). M, Molecular mass standard (broad range; Bio-Rad); H, n.i., homogenate not induced; H, i., homogenate induced; Control, raw extract from not induced E. coli culture; Syn-GlcD, purified Sll0404; Syn-SHMT, purified Sll1931.

Figure 3.

Segregation of the Synechocystis mutants ΔglcD and ΔgcvT (A) and Δsll1559, Δshm, and Δslr2088 (B) was checked by PCR using total DNA of the mentioned strains and the gene-specific primers given in Table I. Abbreviations in A: M, λ-DNA EcoRI/HindIII; WT, wild-type 1.5 kb or 2.0 kb for glcD and gcvT, respectively. The modified (due to the insertions) glcD is 2.7 kb and gcvT is 3.0 kb, as expected. Abbreviations in B: M, λ-DNA EcoRI/HindIII; WT, wild-type 1.2 kb, 1.3 kb, or 2.0 kb; Δsll1559, 3.2 kb; Δshm, 2.5 kb; Δslr2088, 3.0 kb.

Figure 4.

Influence of carbon limitation on the expression of certain genes in the wild type (WT) and a ΔgcvT mutant of Synechocystis. Total RNA was obtained from cells grown at 5% CO₂ (HC) or air level of CO₂ (LC). The transcript abundance was assessed by RT-PCR using the gene-specific primers given in the “Materials and Methods” section.

Figure 5.

Influence of carbon limitation on growth of wild type (WT) and cells bearing mutations in the indicated genes of Synechocystis. Growth was measured as increase in OD₇₅₀ over time. Means and sds from three independent experiments are shown. Growth rates of the wild-type cells grown under 5% CO₂ or air were set to 100% (0.072 ± 0.007 h⁻¹ and 0.021 ± 0.005 h⁻¹, respectively; *, statistically significant growth difference between wild-type and mutant cells, P ≤ 0.05).

Figure 6.

Glycolate content in the cells of mutant ΔglcD of Synechocystis. Samples were taken 3 or 24 h after transfer of high-CO₂-grown cells into air or after 24 h growth at 5% CO₂, respectively. Low-molecular-mass compounds were isolated from cell pellets, and glycolate was detected and quantified by HPLC.

Figure 7.

The intracellular content of Gly (A), Ser (B), and Lys (C) in cells of the wild type and mutants of Synechocystis grown under high (5%, gray columns) or ambient CO₂ (white columns) concentration. Low-molecular-mass compounds were isolated from cell pellets, and amino acids were detected and quantified by HPLC.