Search

Bacteria exposed to environmental stresses often exhibit superior acclimation abilities to environmental change. Acid treatment causes an increase in the cell length of the cyanobacterium Synechocystis sp. PCC6803 under light conditions. We aimed to elucidate the relationship between acidic stress and cell enlargement. After being synchronized under dark conditions, the cells were cultivated at different pH (pH 8.0 or pH 6.0) levels under light conditions. Synechocystis 6803 cells exhibited only cell growth occurred (cell volume expansion) and slow proliferation under the acidic condition. In the recovery experiment of the enlarged cells, they proliferated normally at pH 8.0, and the cell lengths decreased to the normal cell size under light conditions. Inhibition of cell division might be caused by acidic stress. To understand the effect of acidic stress on cell division, we evaluated the expression of FtsZ via Western blotting. The FtsZ concentration in cells was lower at pH 6.0 than at pH 8.0 and was not sufficient for cell division in the photoautotrophic conditions. ClpXP is well known as a regulator of the Z-ring dynamics in E. coli. The transcriptional level of four clpXP genes was upregulated approximately threefold at pH 6.0 after 24 h compared with that in cells grown at pH 8.0. The lack of FtsZ may be caused by the upregulation of clpXP expression under acidic condition. Therefore, ClpXP may participate in the degradation of FtsZ and be involved in the regulation of cell division via FtsZ under acidic stress in Synechocystis 6803., (© 2020. Springer Nature B.V.)

So far, the molecular mechanisms underlying the acidic-stress responses of plants are complicated and only fragmentally understood. Here, we investigated the mechanisms responsible for acidic-stress acclimation. Previously, DNA microarray analysis identified the sll1558 gene in Synechocystis sp. PCC 6803 (hereafter called Synechocystis 6803) to be upregulated following short-term acid treatment (1 h at pH 3.0). The sll1558 gene encodes uridine diphosphate-glucose pyrophosphorylase (UDP-glucose pyrophosphorylase), which catalyzes the conversion of glucose-1-phosphate into UDP-glucose. We constructed mutant cells for this gene and analyzed their phenotype. The sll1558 gene did not completely segregate in sll1558 mutant cells; thus, Sll1558 is essential for the survival of Synechocystis 6803. Besides, the partially disrupted sll1558 mutant cells were highly sensitive to acidic stress (pH 6.0) as well as other stress conditions (high salt, high osmolality, high/low temperature, and ultraviolet-B stress); the number of sll1558 transcripts increased under these conditions. UDP-glucose is used for the synthesis of various materials, such as glycolipids. From the membrane lipid composition analysis, digalactosyldiacylglycerol decreased and phosphatidylglycerol increased in the partially disrupted sll1558 mutant cells under acidic stress. These results suggest that sll1558 is important not only for the survival of Synechocystis 6803, but also for tolerance under various stress conditions.

Over 50 ATP-binding cassette (ABC) transporter-related genes are detected in the Synechocystis sp. PCC 6803 genome by genome sequence analysis. Deletion mutants of other substrate-unknown ABC transporter genes were screened for their acid stress sensitivities in a low-pH medium to identify ABC transporters involved in acid resistance. We found that a mutant of sll1180 encoding proteins with homology to HlyB in Escherichia coli (E.coli) is more sensitive to acid stress than wild-type (WT) cells and analyzed the abundance of expression of the genes in WT cells under acid stress condition by quantitative real-time reverse transcriptase-polymerase chain reaction. sll1180 expression increased in the WT cells after acid stress treatment. Immunofluorescence revealed that Sll1180 localized in the plasma membrane. These results suggest that Sll1180 has an important role in the growth of Synechocystis sp. PCC 6803 under acid stress conditions. HlyB, HlyD, and TolC complex transport HlyA in E.coli; therefore, we searched for genes corresponding to these in Synechocystis sp. PCC 6803. A BlastP search suggested that HlyA, HlyD, and TolC proteins had homology to Sll1951, Sll1181, and Slr1270. Therefore, we constructed deletion mutant of these genes. sll1181 and slr1270 mutant cells revealed acid stress sensitivity. The bacterial two-hybrid analysis showed that Sll1180 interacted with Sll1181 and Sll1951. Dot blot analysis of Sll1951-His revealed that the sll1180 and sll1181 mutant cells did not transport Sll1951-His from the cytoplasm to the extracellular matrix. These results suggest that Sll1180 and Sll1181 transport Sll1951 and that Sll1951-outside of the cells-might be a key factor in acid stress tolerance.

The ATP-binding cassette (ABC) transporter is a multi-subunit membrane protein complex involved in lipid transport and acid stress tolerance in the cyanobacterium Synechocystis sp. PCC 6803. This organism has two sets of three ABC transporter subunits: Slr1045 and Slr1344, Sll0751 and Sll1002, and Sll1001 and Sll1041. We previously found that Slr1045 is essential for survival under acid stress condition (Tahara et al. 2012). In the present study, we examined the participation of other ABC transporter subunits in acid stress tolerance using a deletion mutant series of Synechocystis sp. PCC 6803. Although Slr1344 is highly homologous to Slr1045, Δslr1344 cells were not susceptible to acid stress. Δsll0751 and Δsll1041 cells displayed acid stress sensitivity, whereas Δsll1001/sll1002 double mutant cells grew normally. Under high- and low-temperature stress conditions, the growth rate of Δslr1344 and Δsll1001/sll1002 cells did not differ from WT cells, whereas Δsll0751 and Δsll1041 cells showed significant growth retardation, as previously observed in Δslr1045 cells. Moreover, nile red staining showed more lipid accumulation in Δslr1045, Δsll0751, and Δsll1041 cells than in WT cells. These results suggest that Slr1045, Sll0751, and Sll1041 function together as a lipid transport complex in Synechocystis sp. PCC 6803 and are essential for growth under various stresses.

Living organisms must defend themselves against various environmental stresses. Extracellular polysaccharide-producing cells exhibit enhanced tolerance toward adverse environmental stress. In Synechocystis sp. PCC6803 (Synechocystis), lipopolysaccharide (LPS) may play a role in this protection. To examine the relationship between stress tolerance of Synechocystis and LPS, we focused on Slr2019 because Slr2019 is homologous to MsbA in Escherichia coli, which is related to LPS synthesis. First, to obtain a defective mutant of LPS, we constructed the slr2019 insertion mutant (slr2019) strain. Sodium deoxycholate-polyacrylamide gel electrophoresis indicated that slr2019 strain did not synthesize normal LPS. Second, to clarify the participation of LPS in acid tolerance, wild type (WT) and slr2019 strain were grown under acid stress; slr2019 strain growth was significantly weaker than WT growth. Third, to examine influences on stress tolerance, slr2019 strain was grown under various stresses. Under salinity and temperature stress, slr2019 strain grew significantly slower than WT. To confirm cell morphology, cell shape and envelope of slr2019 strain were observed by transmission electron microscopy; slr2019 cells contained more electron-transparent bodies than WT cells. Finally, to confirm whether electron-transparent bodies are poly-3-hydroxybutyrate (PHB), slr2019 strain was stained with Nile Blue A, a PHB detector, and observed by fluorescence microscopy. The PHB granule content ratio of WT and slr2019 strain grown at BG-11 pH 8.0 was each 7.18 and 8.41 %. At pH 6.0, the PHB granule content ratio of WT and slr2019 strain was 2.99 and 2.60 %. However, the PHB granule content ratio of WT and slr2019 strain grown at BG-11N-reduced was 10.82 and 0.56 %. Because slr2019 strain significantly decreased PHB under BG-11N-reduced compared with WT, LPS synthesis may be related to PHB under particular conditions. These results indicated that Slr2019 is necessary for Synechocystis survival in various stresses.

Experimental evolution is a powerful tool for clarifying phenotypic and genotypic changes responsible for adaptive evolution. In this study, we isolated acid-adapted Synechocystis sp. PCC 6803 (Synechocystis 6803) strains to identify genes involved in acid tolerance. Synechocystis 6803 is rarely found in habitants with pH < 5.75. The parent (P) strain was cultured in BG-11 at pH 6.0. We gradually lowered the pH of the medium from pH 6.0 to pH 5.5 over 3 months. Our adapted cells could grow in acid stress conditions at pH 5.5, whereas the parent cells could not. We performed whole-genome sequencing and compared the acid-adapted and P strains, thereby identifying 11 SNPs in the acid-adapted strains, including in Fo F1-ATPase. To determine whether the SNP genes responded to acid stress, we examined gene expression in the adapted strains using quantitative reverse-transcription polymerase chain reaction. sll0914, sll1496, sll0528, and sll1144 expressions increased under acid stress in the P strain, whereas sll0162, sll0163, slr0623, and slr0529 expressions decreased. There were no differences in the SNP genes expression levels between the P strain and two adapted strains, except for sll0528. These results suggest that SNPs in certain genes are involved in acid stress tolerance in Synechocystis 6803.