Role of Spermidine in Overwintering of Cyanobacteria

Abstract

Polyamines are found in all groups of cyanobacteria, but their role in environmental adaptation has been barely investigated. In Synechocystis sp. strain PCC 6803, inactivation of spermidine synthesis genes significantly reduced the survivability under chill (5°C)-light stress, and the survivability could be restored by addition of spermidine. To analyze the effects of spermidine on gene expression at 5°C, lacZ was expressed from the promoter of carboxy(nor)spermidine decarboxylase gene (CASDC) in Synechocystis. Synechocystis 6803::PCASDC-lacZ pretreated at 15°C showed a high level of LacZ activity for a long period of time at 5°C; without the pretreatment or with protein synthesis inhibited at 5°C, the enzyme activity gradually decreased. In a spermidine-minus mutant harboring PCASDC-lacZ, lacZ showed an expression pattern as if protein synthesis were inhibited at 5°C, even though the stability of its mRNA increased. Four other genes, including rpoA that encodes the α subunit of RNA polymerase, showed similar expression patterns. The chill-light stress led to a rapid increase of protein carbonylation in Synechocystis. The protein carbonylation then quickly returned to the background level in the wild type but continued to slowly increase in the spermidine-minus mutant. Our results indicate that spermidine promotes gene expression and replacement of damaged proteins in cyanobacteria under the chill-light stress in winter.

Importance: Outbreak of cyanobacterial blooms in freshwater lakes is a worldwide environmental problem. In the annual cycle of bloom-forming cyanobacteria, overwintering is the least understood stage. Survival of Synechocystis sp. strain PCC 6803 under long-term chill (5°C)-light stress has been established as a model for molecular studies on overwintering of cyanobacteria. Here, we show that spermidine, the most common polyamine in cyanobacteria, promotes the survivability of Synechocystis under long-term chill-light stress and that the physiological function is based on its effects on gene expression and recovery from protein damage. This is the first report on the role of polyamines in survival of overwintering cyanobacteria. We also analyzed spermidine synthesis pathways in cyanobacteria on the basis of bioinformatic and experimental data.

FIG 1

Pathways for synthesis of spermidine in prokaryotes. Abbreviations: ADC, arginine decarboxylase; AIH, agmatine iminohydrolase; AUH, agmatine ureohydrolase; CASDC, carboxyspermidine decarboxylase; CASDH, carboxyspermidine dehydrogenase; NCPAH, N-carbamoylputrescine amidohydrolase; SAMDC, S-adenosylmethionine decarboxylase; SPDS, spermidine synthase.

FIG 2

mRNA levels of CASDC in Synechocystis 6803 at different stages of acquisition of chill-light tolerance. (A) Northern blot analysis of expression of CASDC in cells exposed to chill-light stress. (B) qRT-PCR measurement of CASDC mRNA levels in cells grown at 30°C, treated at 15°C, and exposed to chill (5°C)-light stress. (C) qRT-PCR analysis of CASDC mRNA decay at 15°C (■) or under chill-light stress (□).

FIG 3

Content of spermidine in Synechocystis 6803 at different temperatures. (A) Cells grown at 30°C were treated at 15°C for 2 days before exposure to chill-light stress. (B) Cells grown at 30°C were directly exposed to chill-light stress. d, day(s).

FIG 4

Effect of spermidine on the chill-light survivability of Synechocystis 6803. RACLT, relative acquired chill-light tolerance. (A) Requirement of CASDC for the chill-light survivability. (B) Requirement of CASDH for the chill-light survivability.

FIG 5

Effects of spermine, cadaverine, putrescine, and homospermidine on the RACLT of the CASDC mutant of Synechocystis 6803.

FIG 6

Expression of lacZ in Synechocystis 6803::P_CASDC-lacZ under the chill-light stress with (B and D) or without (A and C) preconditioning. The expression of lacZ was determined as shown by the β-galactosidase activity (A and B) or qRT-PCR (C, D). Bars in panels A and B: dark gray, Synechocystis 6803::P_CASDC-lacZ; light gray, Synechocystis 6803::P_CASDC-lacZ supplemented with chloramphenicol upon transfer to 5°C; open, Synechocystis 6803 treated in parallel (background activity).

FIG 7

Assays of β-galactosidase activity and LacZ abundance showed the effect of spermidine on expression of P_CASDC-lacZ in Synechocystis strains. (A) β-Galactosidase activity in Synechocystis 6803::P_CASDC-lacZ (dark gray), Synechocystis 6803 DRHB2193::P_CASDC-lacZ without (light gray), or with (open) exogenous spermidine. (B) Western blot detection of LacZ in Synechocystis 6803::P_CASDC-lacZ (row I) and Synechocystis 6803 DRHB2193::P_CASDC-lacZ (row II). DRHB2193, the CASDC mutant.

FIG 8

Measurements of lacZ mRNA level showing the effect of spermidine on expression of P_CASDC-lacZ in Synechocystis strains. (A) qRT-PCR analysis of lacZ mRNA levels in Synechocystis 6803::P_CASDC-lacZ (dark gray) and Synechocystis 6803 DRHB2193::P_CASDC-lacZ (light gray). (B) qRT-PCR analysis of lacZ mRNA decay in Synechocystis 6803::P_CASDC-lacZ (■) and Synechocystis 6803 DRHB2193::P_CASDC-lacZ (□) under chill-light stress. DRHB2193, the CASDC mutant.

FIG 9

qRT-PCR measurement of mRNA levels showing the role of spermidine synthesis in gene expression under chill-light stress. Bars: dark gray, Synechocystis 6803; light gray, the CASDC mutant. rbp1, RNA-binding protein 1; tsf, EF-Ts; rpoA, α subunit of RNA polymerase; slr0338, probable oxidoreductase.

FIG 10

Comparison of protein carbonylation in the wild type and the CASDC mutant of Synechocystis 6803. Bars: open, wild type; gray, CASDC mutant.

FIG 11

Effect of spermidine on chill-light survivability of Synechococcus 7942.