Article Figures & Data
Figures
- 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.
Tables
- Table I.
Strategies applied to inactivate selected ORFs in Synechocystis
The oligonucleotides were used to amplify the sequences encoding the candidate genes together with flanking regions. The insertion sites for the kanamycin (KmR)-, chloramphenicol (CmR)-, or spectinomycin-resistance (SpR) cartridges are given. ORFs sll1349 and slr0458 were obtained as two PCR fragments. The resistance genes were inserted in between added restriction sites leading to deletions (D) of 267 bp in sll1349 and 59 bp in slr0458, respectively. ORF sll0171 was cut with MscI, leading to deletion of 1.0 kb. In the case of ORF slr2088, restriction with SnaBI led to a deletion of 0.24 kb. All other insertion sites were unique. Additionally introduced restriction sites are underlined in the oligonucleotide sequences.
ORF Oligonucleotides Insertion Site Inserted Cartridge sll1349 Fw1 5′-TTG GGC GGA ACG GGC CG-3′ KmR Rev1 5′-GGG GGA TCC AGA ATG CGG TAA TCC CG-3′ BamHI Fw2 5′-TAA CTC TGC TCA ACC AA-3′ D Rev2 5′-GGG CTG CAG ACT AGT AAC CAG GGC ATG-3′ PstI slr0458 Fw1 5′-TGA CCT CGA TGG AGT AT-3′ CmR Rev1 5′-GGG GGA TCC AGA ATG CGG TAA TCC CG-3′ BamHI Fw2 5′-GAG TGG GTT TAT GGT CG-3′ D Rev2 5′-GGG GCA TGC CTC CTC ACT AAA GCT CC-3′ SphI sll0404 Fw 5′-CTC GAG ATG GCC ATT TTC TCC-3′ EcoRV KmR Rev 5′-GAA TTC TCA ATA AAT TTC CTC-3′ sll1559 Fw 5′-CTC GAG ATG GAT AAT AAG CAA-3′ StuI SpR Rev 5′-GAA TTC TTA ACC TTT AGC CAA-3′ sll0171 Fw 5′-AGA CCT GAA GGA AGC TGT AG-3′ MscI, D SpR Rev 5′-GAG GAA GTG GTG CAC AGG TT-3′ sll1931 Fw 5′-GCT ATT ACG GCG GCT GTG AA-3′ SmaI KmR Rev 5′-CCA TGA CGG CCA CAA CTG AA-3′ sll1981 Fw 5′-CCG GAT TCG TTA GGC TAG-3′ AfeI KmR Rev 5′-AGT TAG CGT CGA TTT GGT-3′ slr2088 Fw 5′-CGA TTG AGT TAA AAT TAG-3′ SnaBI, D KmR Rev 5′-GGT CAA GAA AAA CCG AGG-3′ slr0229 Fw 5′-ATA AGT CAG AGA AGT GAA-3′ HpaI KmR Rev 5′-CCA TGT TTA CTC CAG TAA-3′ - Table II.
Sequence comparison of proteins participating in phosphoglycolate turnover with candidate proteins from Synechocystis using PSI- and PHI-BLAST (Altschul et al., 1997)
The similar proteins from bacteria or Arabidopsis are in most cases biochemical characterized.
Protein (Abbreviation) Organism Accession No. Literature Similarity e Value ORF in Synechocystis Phosphoglycolate phosphatase (PGP) A. eutrophus P40852 Schäferjohann et al. (1993) 3e−08 slr0458 9e−06 sll1349 Glycolate dehydrogenase subunit D (GlcD) E. coli AAC76015 Pellicer et al. (1996) 3e−126 sll0404 Ala:glyoxylate aminotransferase (AGT) Arabidopsis At2g13360 Liepman and Olsen (2001) 3e−59 sll1559 GDC, P protein (GcvP) Arabidopsis At2g26080 H. Bauwe (unpublished data) 0 slr0293 GDC, T protein (GcvT) Arabidopsis At1g11860 H. Bauwe (unpublished data) 7e−52 sll0171 GDC, H protein (GcvH) Arabidopsis At2g35120 H. Bauwe (unpublished data) 2e−28 slr0879 GDC, L protein (GcvL) Arabidopsis At3g16950 H. Bauwe (unpublished data) 1e−164 slr1096 Ser hydroxymethyltransferase (SHMT) Arabidopsis At4g37930 Voll et al. (2006) 6e−94 sll1931 Hydroxypyruvate reductase (HPR) Arabidopsis At1g68010 H. Bauwe (unpublished data) 2e−28 sll1908 2e−26 slr1556 Glycerate kinase (GLYK) E. coli AAB93855 Cusa et al. (1999) 3e−64 slr1840 Glyoxylate carboligase (GCL) E. coli AAA23864 Chang et al. (1993) 1e−94 slr2088 3e−58 sll1981 Tartronic semialdehyde reductase (TSR) E. coli P77161 Cusa et al. (1999) 3e−35 slr0229
