Succinate:quinol oxidoreductases in the cyanobacterium synechocystis sp. strain PCC 6803: presence and function in metabolism and electron transport

Abstract

The open reading frames sll1625 and sll0823, which have significant sequence similarity to genes coding for the FeS subunits of succinate dehydrogenase and fumarate reductase, were deleted singly and in combination in the cyanobacterium Synechocystis sp. strain PCC 6803. When the organic acid content in the Deltasll1625 and Deltasll0823 strains was analyzed, a 100-fold decrease in succinate and fumarate concentrations was observed relative to the wild type. A similar analysis for the Deltasll1625 Deltasll0823 strain revealed that 17% of the wild-type succinate levels remained, while only 1 to 2% of the wild-type fumarate levels were present. Addition of 2-oxoglutarate to the growth media of the double mutant strain prior to analysis of organic acids in cells caused succinate to accumulate. This indicates that succinate dehydrogenase activity had been blocked by the deletions and that 2-oxoglutarate can be converted to succinate in vivo in this organism, even though a traditional 2-oxoglutarate dehydrogenase is lacking. In addition, reduction of the thylakoid plastoquinone pool in darkness in the presence of KCN was up to fivefold slower in the mutants than in the wild type. Moreover, in vitro succinate dehydrogenase activity observed in wild-type membranes is absent from those isolated from the double mutant and reduced in those from the single mutants, further indicating that the sll1625 and sll0823 open reading frames encode subunits of succinate dehydrogenase complexes that are active in the thylakoid membrane of the cyanobacterium.

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 and sll0823 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). Km^R, kanamycin resistance; Cm^R, 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 the sll1625 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/z fragment, 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 (73 m/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 (289 m/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.