The molecular mechanism of menadione resistance in the <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 cyanobacterium

The effect of mutations in the genes encoding dehydrogenases and oxidases on the resistance of the Synechocystis sp. PCC 6803 cyanobacterium to menadione, an oxidative stress inducer, was studied. An enhanced sensitivity to menadione was observed in the mutants carrying inserts in the drgA gene encoding the NAD(P)H:quinone oxidoreductase (NQR) and in the ndhB gene encoding the subunit of NDH-1 complex. The menadione resistance in the mutants lacking oxidases (Ox), succinate dehydrogenase (SDH), and NDH-2 dehydrogenase do not differ from those in wild-type cells. An additional mutation in the drgA gene increased the sensitivity to menadione in the NDH-2 and Ox mutants. The double mutant that lacks both SDH and NQR was not viable. The expression of the drgA gene decreased during cell incubation in the dark but increased in the presence of glucose both in the dark and in light. Under photoautotrophic growth conditions, the dehydrogenase activity of the cells mainly depends on the NQR and NDH-1 functions. The re-reduction rate of the photosystem I reaction center (P700⁺) increased in wild-type and NDH-1 mutants after its oxidation with white light in the presence of DCMU after addition of menadione, and it decreased in the NQR mutant. The reduction of P700⁺ was accelerated in the presence of menadiol in all the strains studied. These results suggest that NQR provides defense of cyanobacterium cells from the toxic effect of menadione via its two-electron reduction to menadiol. An increased sensitivity of the NDH-1 mutant to menadione may result from the inhibition of respiration and the cyclic electron transport in photosystem I.     

Reduction of photosystem I reaction center by recombinant DrgA protein in isolated thylakoid membranes of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

To study the function of soluble NAD(P)H:quinone oxidoreductase of the cyanobacterium Synechocystis sp. PCC 6803 encoded by drgA gene, recombinant DrgA protein carrying 12 histidine residues on the C-terminal end was expressed in Escherichia coli and purified. Recombinant DrgA is a flavoprotein that exhibits quinone reductase and nitroreductase activities with NAD(P)H as the electron donor. Using EPR spectroscopy, it was demonstrated that addition of recombinant DrgA protein and NADPH to DCMU-treated isolated thylakoid membranes of the cyanobacterium increased the dark rereduction rate of the photosystem I reaction center (P700⁺). Thus, DrgA can participate in electron transfer from NADPH to the electron transport chain of the Synechocystis sp. PCC 6803 thylakoid membrane.     

Alpha-tocopherol is essential for acquired chill-light tolerance in the cyanobacterium Synechocystis sp. strain PCC 6803

Unlike Escherichia coli, the cyanobacterium Synechocystis sp. strain PCC 6803 is insensitive to chill (5°C) in the dark but rapidly losses viability when exposed to chill in the light (100 μmol photons m⁻² s⁻¹). Preconditioning at a low temperature (15°C) greatly enhances the chill-light tolerance of Synechocystis sp. strain PCC 6803. This phenomenon is called acquired chill-light tolerance (ACLT). Preconditioned wild-type cells maintained a substantially higher level of α-tocopherol after exposure to chill-light stress. Mutants unable to synthesize α-tocopherol, such as slr1736, slr1737, slr0089, and slr0090 mutants, almost completely lost ACLT. When exposed to chill without light, these mutants showed no or a slight difference from the wild type. When complemented, the slr0089 mutant regained its ACLT. Copper-regulated expression of slr0090 from P_petE controlled the level of α-tocopherol and ACLT. We conclude that α-tocopherol is essential for ACLT of Synechocystis sp. strain PCC 6803. The role of α-tocopherol in ACLT may be based largely on a nonantioxidant activity that is not possessed by other tocopherols or pathway intermediates.     

Mutational analysis of genes involved in pilus structure, motility and transformation competency in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803

The relevance of pilus-related genes to motility, pilus structure on the cell surface and competency of natural transformation was studied by gene disruption analysis in the unicellular motile cyanobacterium SYNECHOCYSTIS: sp. PCC 6803. The genes disrupted in this study were chosen as related to the pil genes for biogenesis of the type IV pili in a Gram-negative bacterium PSEUDOMONAS: aeruginosa. It was found that motility of SYNECHOCYSTIS: cells was lost in the mutants of slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 together with a simultaneous loss of the thick pili on the cell surface. Competency of the natural transformation was lost in the mutants listed above and slr0197-disruptant. The gene slr0197 was previously predicted as a competence gene by a search with sequence-independent DNA-binding structure [Yura et al. (1999) DNA Res. 6: 75]. It was suggested that both DNA uptake for natural transformation and motility are mediated by a specific type IV-like pilus structure, while a putative DNA-binding protein encoded by slr0197 is additionally required for the DNA uptake. Based on the homology with the pil genes in P: aeruginosa, slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 were designated pilB1, pilM, pilN, pilO, pilQ and pilA1, respectively. The gene slr0197 was designated comA.     

Multidisciplinary Evidences that Synechocystis PCC6803 Exopolysaccharides Operate in Cell Sedimentation and Protection against Salt and Metal Stresses

Little is known about the production of exopolysaccharides (EPS) in cyanobacteria, and there are no genetic and physiological evidences that EPS are involved in cell protection against the frequently encountered environmental stresses caused by salt and metals. We studied four presumptive EPS production genes, sll0923, sll1581, slr1875 and sll5052, in the model cyanobacterium Synechocystis PCC6803, which produces copious amounts of EPS attached to cells (CPS) and released in the culture medium (RPS) as shown here. We show that sll0923, sll1581, slr1875 and sll5052 are all dispensable to the growth of all corresponding single and double deletion mutants in absence of stress. Furthermore, we report that sll0923, sll1581 and slr1875 unambiguously operate in the production of both CPS and RPS. Both sll1581 and slr1875 are more important than sll0923 for CPS production, whereas the contrary is true for RPS production. We show that the most EPS-depleted mutant, doubly deleted for sll1581 and slr1875, lacks the EPS mantle that surrounds WT cells and sorbs iron in their vicinity. Using this mutant, we demonstrate for the first time that cyanobacterial EPS directly operate in cell protection against NaCl, CoCl₂, CdSO₄ and Fe-starvation. We believe that our EPS-depleted mutants will be useful tools to investigate the role of EPS in cell-to-cell aggregation, biofilm formation, biomineralization and tolerance to environmental stresses. We also suggest using the fast sedimenting mutants as biotechnological cell factories to facilitate the otherwise expensive harvest of the producer cell biomass and/or its separation from products excreted in the growth media.     

Functional Characterization of the slr1944 Gene of Cyanobacterium Synechocystis sp. PCC 6803

The properties of Slr1944 protein encoded by the slr1944 gene and participating in the metabolism of lipophilic compounds in a cyanobacterium Synechocystis were under study. Located in the periplasm, this protein comprises a conserved pentapeptide G-X-S-X-G characteristic of lipases, acetylcholinesterases, and thioesterases. An attempt to delete the gene from the cyanobacterial genome failed; this fact presumes an essential function of Slr1944 protein under the optimum growth conditions. Expression of the slr1944 gene in Escherichia coli cells demonstrated a high affinity of the product for lipophilic compounds. An enhanced slr1944 expression deprived Synechocystis cells of the ability to restore the activity of the photosynthetic electron-transport chain following photoinactivation. The authors believe that Slr1944 participates in the biogenesis of the lipophilic components of photosynthetic complexes.     

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a ΔFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in ΔDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in ΔFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the ΔDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.     

Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002

Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C₁₈ fatty acids increased in cells grown at lower temperature for all lipid classes, and ω3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the ω3 position of C₁₈ fatty acids increased gradually during a 12-h period after a temperature shift-down to 22°C. However, the mRNA levels of the desA (Δ12 desaturase), desB (ω3 desaturase) and desC (Δ9 desaturase) genes increased within 15 min after a temperature shift-down to 22°C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38°C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22°C and 38°C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium.     

20-Hydroxyecdysone induces the expression of one β-tubulin gene in Drosophila Kc cells

The expression of 56D and 60C β-tubulin genes has been examined in Drosophila melanogaster Kc cells in response to the insect moulting hormone, 20-hydroxyecdysone (20-OH-E). Northern blots probed with β-tubulin subclones show that the 56D β-tubulin gene encodes a 1.8 kb mRNA whose abundance is not affected by 20-OH-E. The 60C gene probe detects two mRNAs: one of 1.8 kb present in untreated and 20-OH-E-treated cells, and one of 2.6 kb present only in 20-OH-E-treated cells; using a 60C 3′-specific probe, only the 2.6 kb is revealed. Hybrid selection translation experiment demonstrates that a 20-OH-E-inducible mRNA homologous to the 60C gene encodes a β-tubulin subunit (P4); this subunit is the so-called β3-tubulin. Translation of size-fractionated mRNA shows that the 20-OH-E-induced β3-tubulin subunit is encoded, in treated cells, by the 2.6 kb mRNA.     

Expression of FTL and FTH genes encoding ferritin subunits in lung and renal carcinomas

Numerous human cancers are accompanied with the increase of ferritin content in blood serum. Ferritin is composed of light and heavy chains, encoded by FTL and FTH genes, respectively. The analysis of EST database showed that the expression of FTL and FTH genes in lung tumors is decreased compared to normal tissues, and is not altered in renal cancer cells. The alteration of mRNA corresponding to FTL and FTH genes was estimated by real-time PCR in primary lung and kidney tumors. A significant and frequent inhibition of FTL and FTH gene expression (on average by 11 and 9 times, in 83% (33/40) and 73% (11/15) of cases, respectively) was detected in primary squamous lung carcinoma. The expression of these genes was not altered so significantly (by 6 and 3 times, in 58% and 27% of samples) in clear cell renal cell carcinoma. Our work reports for the first time the down-regulation of FTL gene expression at the first stage of lung cancer (10/10), and proposes this gene as a potential oncomarker for early diagnosis. The FTL mRNA content may be quantified by non-competitive hybridization on expression DNA microarrays. The possible causes of a serum ferritin increase in lung squamous cell carcinoma and clear cell renal cell carcinoma are discussed.     

Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37° C

We have analyzed the structure of genes encoding the glyoxylate cycle enzyme isocitrate lyase from Brassica napus L. and their expression during embryogeny and postgermination. Restriction mapping, nucleotide sequence, and DNA gel blot hybridization analyses of cDNA and genomic clones indicated that there are approximately six isocitrate lyase genes in the B. napus genome that can be divided into at least two subfamilies based upon their divergence in 5′ and 3′ untranslated regions. We showed previously that isocitrate lyase mRNA accumulates during late embryogeny and postgermination. Here, we present results which indicate that several isocitrate lyase genes are expressed at both stages of development. First, gene-specific probes were used to show that mRNAs encoded by representatives of both gene subfamilies accumulated in both late maturation stage embryos and in seedlings of B. napus. Second, a single B. napus isocitrate lyase gene, together with 3.5 kb and 1.4 kb of 5′ and 3′ flanking regions, respectively, was expressed in both embryos and seedlings of transgenic tobacco plants. The results indicated that accumulation of isocitrate lyase in late embryogeny and postgermination does not result from the alternate expression of distinct members of the gene family.     

Nitrogen fixation genes (nif K,D,H) in the filamentous nonheterocystous cyanobacteriumPlectonema boryanum do not rearrange

The organisation of the structural genes for nitrogen fixation (nif K,D and H) in a nonheterocystous, filamentous cyanobacteriumPlectonema boryanum has been examined in comparison with a heterocystous cyanobacterium,Anabaena torulosa. DNA from repressed (fix-) cultures ofA. torulosa showed a discontinuousnif region spread over approximately 18 kb, an arrangement typical of the vegetative cells of heterocystous cyanobacteria. The region contained a contiguousnif DH separated fromnif K. by nearly 11 kb DNA. The intervening 11 kb DNA harboured the genexis A involved in the rearrangement ofnif K,D,H to form a cluster during differentiation of heterocysts. DNA fromPlectonema boryanum had a small, contiguousnif KDH cluster spanning a region of approximately 4 kb. DNA homologous to the 11 kb excison with its residentxis A was not present.Nif hybridisation patterns of restriction digests of the DNA isolated from repressed (fix^-) or induced (fix^--) cultures ofP. boryanum were completely identical. These results unequivocally demonstrate that in the nonheterocystous cyanobacterium, unlike in the heterocystous strains, no gene rearrangement, either within thenif KDII cluster or in its vicinity, accompanies the expression of nitrogenase activity.