Essential roles of iron superoxide dismutase in photoautotrophic growth of Synechocystis sp. PCC 6803 and heterogeneous expression of marine Synechococcus sp. CC9311 copper/zinc superoxide dismutase within its sodB knockdown mutant

Abstract

Synechocystis sp. PCC 6803 possesses only one sod gene, sodB, encoding iron superoxide dismutase (FeSOD). It could not be knocked out completely by direct insertion of the kanamycin resistance cassette. When the promoter of sodB in WT Synechocystis was replaced with the copper-regulated promoter PpetE, a completely segregated PpetE–sodB strain could be obtained. When this strain was cultured in copper-starved BG11 medium, the chlorophyll a content was greatly reduced, growth was seriously inhibited and the strain was nearly dead during the 8 days of growth, whilst the WT strain grew well under the same growth conditions. These results indicated that sodB was essential for photoautotrophic growth of Synechocystis. The reduction of sodB gene copies in the Synechocystis genome rendered the cells more sensitive to oxidative stress produced by methyl viologen and norflurazon. sodB still could not be knocked out completely after active expression of sodC (encoding Cu/ZnSOD) from Synechococcus sp. CC9311 in the neutral site slr0168 under the control of the psbAII promoter, which means the function of FeSOD could not be complemented completely by Cu/ZnSOD. Heterogeneously expressed sodC increased the oxidation and photoinhibition tolerance of the Synechocystis sodB knockdown mutant. Membrane fractionation followed by immunoblotting revealed that FeSOD was localized in the cytoplasm, and Cu/ZnSOD was localized in the soluble and thylakoid membrane fractions of the transformed Synechocystis. Cu/ZnSOD has a predicted N-terminal signal peptide, so it is probably a lumen protein. The different subcellular localization of these two SODs may have resulted in the failure of substitution of sodC for sodB.

Roles of CpcF and CpcG1 in Peroxiredoxin-Mediated Oxidative Stress Responses and Cellular Fitness in the Cyanobacterium Synechocystis sp. PCC 6803

“…In these analyses, we included the ocp deletion mutant, i.e., Δ ocp , which lacks OCP that is responsible for photoprotection through NPQ. MV treatment resulted in a reduction of chl levels (Figure 4A) and slowed growth (Figures 4B–D) in WT, as anticipated (Kobayashi et al, 2004; Ke et al, 2014). The Δ ocp mutant grew similar to WT independent of MV treatment (Figure 4B).…”

A unique porin meditates iron‐selective transport through cyanobacterial outer membranes

“…S4B). It should be noted that omission of copper from the growth medium does not have significant effects on the growth rate and Chl a content of Synechocystis (Ke et al ., 2014). Therefore, decreased growth rate and Chl a content in KD strains can only be ascribed to the knockdown of these two genes.…”

“…Before transfer into Fe‐depleted medium, exponential cells were harvested by centrifugation at 6000 g for 5 min at 30°C and washed twice with Fe‐deplete medium. For copper‐regulated growth experiments, copper sulphate was omitted from the BG11 medium and cells were harvested and washed twice with Cu‐depleted medium (Ke et al ., 2014).…”

“…The knockdown strains were constructed by introducing a copper‐regulated promoter as described previously (Ke et al ., 2014). For the slr1908 knockdown strain, a 540 bp DNA fragment containing the upstream sequence of slr1908 (the putative promoter region) was amplified and cloned into the pMD18‐T vector and then confirmed by sequencing, resulting in the plasmid pHS2601.…”

Metalloproteins in the Biology of Heterocysts

“…Fe–SOD is also present in both cell types in diazotrophic filaments but, in contrast to Mn–SOD, is only located in the cytosol [354,365,370]. While all SODs are soluble enzymes, the cyanobacterial Mn–SOD is the only member to be membrane-anchored by a transmembrane helix [350,354,365,372,375,376]. On the other hand, although the periplasmic and luminal Mn–SODs form homodimers, the soluble cytosolic Mn– and Fe–SOD enzymes form active homodimers and heterodimers [354,365,370,374].…”