Article Figures & Data
Figures
- Fig. 1.
Segregation analysis of the deletion mutants. Genomic DNA from the wild-type, Δsll1625, Δsll0823, and Δsll1625 Δsll0823 strains was used as a template for amplification of the sll1625 andsll0823 genes to verify segregation in the mutants (a). The segregated Δsll1625 strain was used as the background strain for creating the Δsll1625 Δsll0823 strain; therefore, only the sll0823 segregation analysis is shown (a, far right lane). Schematic maps of the deletion constructs used to create Δsll1625 (b) and Δsll0823 (c) are shown with the PCR primers used for segregation analysis represented by bars (Table 1). KmR, kanamycin resistance; CmR, chloramphenicol resistance. In panel b, primer a is 5′sll1625b, and primer b is 3′ sll1625b. In panel c, primer a is 5′ sll0823, and primer b is 3′sll0823.
- Fig. 2.
Transcript analysis. Ethidium bromide-stained gels of RT-PCR products with transcripts isolated from the wild type grown under photoautotrophic conditions when bubbled with air or with a 1% CO2–99% N2 mix (left two lanes) and from the three deletion mutant strains grown photoautotrophically in ambient air (right three lanes). The 0.37-kb band represents amplification of thesll1625 transcript, and the 0.32-kb band represents amplification of the sll0823 transcript.
- Fig. 3.
GC/MS analysis of organic acid derivatives prepared from cell extracts. (Top to bottom) Chromatograms generated from the total impact spectrum (TIC; 60 to 500 m/z), the TBDMS-specific 73-m/z fragment, the bis-TBDMS succinate-specific 289-m/z fragment, the bis-TBDMS fumarate 287-m/zfragment, and the bis-TBDMS adipate 317-m/z fragment. Comparison of the top two traces shows the improvement in the signal/noise ratio by using single-ion-fragment (73m/z)-generated chromatograms.
- Fig. 4.
MS fractionation identification of derivatized compounds. Mass fragment spectra from the peaks at 11.407, 11.743, and 14.258 min (A, B, and C, respectively [upper traces]) were identified as the derivatives of succinate, fumarate, and adipate, respectively, based on similarity of fractionation to standards found within the National Institute of Standards and Technology mass fractionation spectral library (A, B, and C, respectively [lower traces]).
- Fig. 5.
Relative levels of succinate and fumarate in extracts from the wild type and deletion mutants. Single-ion-fragment GC/MS-generated chromatograms for bis-TBDMS succinate (289m/z), bis-TBDMS fumarate (287 m/z), and the bis-TBDMS adipate (317 m/z) internal standard are shown. Organic acids were extracted from strains as indicated. Relative levels of succinate and fumarate in the cells can be determined by comparison of the peak areas of the derivatives of these organic acids with the area of the internal standard peak (adipate). Succinate (s), fumarate (f), and adipate (a) peak designations are indicated at the bottom with arrows.
- Fig. 6.
Changes in chlorophyll fluorescence yield in darkness after addition of KCN. Variable fluorescence (arbitrary units [A.U.]) of the wild type (●) and the Δsll1625 Δsll0823 strain (○) was measured by very weak illumination that did not have any actinic effect. Otherwise, cells were kept in darkness. KCN (1 mM final concentration) was added at t = 0 s. Curves were normalized so that a value of 1 on the y axis corresponds to maximal variable fluorescence yield. Constant fluorescence (F0) has been subtracted.
Tables
- Table 1.
Sequence of primers used in this study and their relative positions within the genome of Synechocystis sp. strain PCC 6803
Primer Sequencea Position 5′sll0823 5′ AAC GCC TTA ATG cAT GCC CAA GGC 3′ 2862235–2862212 3′sll0823 5′ TGG CCA AGG gAA TTC CCT GTG ACC 3′ 2860717–2860740 5′sll1625 5′ GGA AGC GGT GcA TgC GGC CCA GGC 3′ 1320979–1320956 3′sll1625 5′ TTT GGT GGA GAA TTC TCA GGG TGC 3′ 1319117–1319140 5′sll1625b 5′ GTG GAG CCC GGT GCC ACC 3′ 1320469–1320452 3′sll1625b 5′ GGC TCC ATC CCG GGA GTC GG 3′ 1319963–1319982 5′ RT-PCRsll0823 5′ CAC CAT CAG TCC CCT GGG GAA 3′ 2861557–2861537 3′ RT-PCRsll0823 5′ GGG CTT CGA GGC GGG CCT TAG TGG 3′ 2861238–2861261 5′ RT-PCRsll1625 5′ CCG ACT CCC GGG ATG GAG CCA CAG 3′ 1319982–1319959 3′ RT-PCRsll1625 5′ CCG GCG GAA GGT TCA AAG CCC AGG 3′ 1319610–1319633 3′ RTsll0823 5′ GGG CTT CGA GGC GGG CCT 3′ 2861238–2861255 3′ RTsll1625 5′ CCG GCG GAA GGT TCA AAG 3′ 1319610–1319627 ↵a Lowercase letters denote base changes used for initial cloning.
- Table 2.
Succinate and fumarate levels after treatment with organic acidsa
Strain Treatment Peak areab Succinate Fumarate Wild type None 1.61 ± 0.09 1.54 ± 0.10 + Fumarate 6.20 ± 0.12 6.41 ± 0.15 + 2-Oxoglutarate 4.80 ± 0.10 5.0 ± 0.12 Δsll1625 Δsll0823 None 0.28 ± 0.04 0.01 ± 0.04 + Fumarate 0.35 ± 0.05 0.48 ± 0.09 + 2-Oxoglutarate 0.76 ± 0.12 0.02 ± 0.05
