|Other Abstract||Cyanobacteria often form waterblooms, even producing toxins, in inland eutrophic waters. It has been a worldwide environmental problem that causes public attention. How cyanobacteria overwinter is very important to understanding of next year’s outbreaks of waterblooms in temperate and subtropical regions. However, little is known about the adaptation of cyanobacteria to low temperatures in the range from 0 to 15℃. In regard to the evolutionary history, a cyanobacterium was proposed to be the ancestor of chloroplasts, and about 18% genes of Arabidopsis nuclear genome were found to be derived from the cyanobacterium. Consequently, there are many similarities between cyanobacteria and higher plants in physiological and genetic characters. Cold tolerance genes of cyanobacteria could be a resource of genes for crop genetic engineering.
Synechocystis sp. PCC6803 is a unicelluar mesophillic cyanobacterium, with its optimal growth temperature at 30℃ and a range of favorable temperature from 25 to 40℃. In this research, the author studied the physiological characters at low temperatures from 5~15℃ and found the phenomenon of low temperature enhancement, obtained chill and light-sensitive mutants by random insertion of the genome and identified some genes required to low temperature adaptation. Employing DNA microarray techniques, the author analyzed the changes in transcriptome in response to lowered temperature and the role of genes upregulated at 15℃ in the low temperature enhancement, in the hope to elucidate the mechanism of autumnal acclimation before overwintering of cyanobacteria in temperate and subtropical regions.
The experimentation and results are described in 4 parts:
1. The physiological characters of Synechocystis PCC 6803 at low temperatures. There is no growth at 5℃ and cells grown at 30℃ could survive for up to over 2 months in the dark but within 10 days under illumination of 100μE.m-2.s-1. Transfer of cells grown at 15℃ to 5℃, however, would result in a remarkable increase in cold tolerance no matter with or without light, to which I propose to call ‘low temperature enhancement’.
2. Identification of genes required to cold tolerance in Synechocystis sp. PCC6803. After random insertion of the genome or targeted gene disruption of certain genes with a kanamycin-resistance cassette and targeted genes disruption, mutant clones grown at
30℃ were transferred to 5℃ with light of 100μE.m-2.s-1, 19 cold-sensitive mutants were obtained and the inserted genes were determined by inversed PCR and sequencing of the PCR products. Among them, 4 genes sll0268, sll1913, sll1242 and slr0688 encode unknown or hypothetical proteins, 8 genes sll0158(1,4-alpha-glucan branching enzyme, glgB), sll0726(phosphoglucomutase, pgm), slr0415(Na+/H+ antiportor, nhaS5), slr1908(probable porin), slr0089(gamma-tocopherol methyltransferase), sll0262(6 acyl-lipid desaturase, desD) and sll0273(Na+/H+ antiporter, nhaS2) are similar to known genes. Two genes sll0158 and sll0726 are involved in glycogen synthesis, 3 genes desD, sll0268 and sll0273 are located in the same region of the genome. Physiological studies of the mutants indicated that the sensitivity of sll0158- and sll0726- were mainly caused by light and that the sensitivity of slr0415-, slr1908- and slr0089- to cold requires the presence of glucose in the medium, while Δsll0268, sll1913-, sll1242-, slr0688-, desD- and sll0273- were specifically sensitive to cold. Targeted insertion of these genes and their downstream genes showed that the cold-sensitive phenotype were not due to a polar effect, except for sll0268, whose downstream gene sll0269 was also required for cold tolerance. Integration of the wild type genes into a platform in the genome of sll0269-, sll1913-, sll1242-, desD- and sll0273- respectively restored their cold tolerance. In the dark, these mutants were insensitive to cold. When light intensity decreased from 100μE.m-2.s-1 to 15μE.m-2.s-1, mutants sll1913- and sll0273- partially restored their viability. These mutants grow as the wild type at 30℃ and 20℃, but unable to grow or grow poorly at 15℃. Growth of sll1242- and sll0273- at 15℃ or Δsll0268, sll1913-, sll1242- at 20℃ before transfer to 5℃ with light of 100μE.m-2.s-1 could partially or completely restore their survivability, which implies that low temperature enhancement could alleviate the detrimental effects of defects in certain genes. Growth of desD- and sll1242- in a low phosphate medium could partially alleviate their sensitivity to cold, but the effect of such low phosphate growth on the wild type was hardly visible. Suboptimal growth of a cyanobacterium may enhance cold tolerance to some extent, but the mechanism of low temperature enhancement should be much more than the effect of suboptimal growth.
3. Genome-wide transcriptional analyses of Synechocystis sp. PCC6803. Using DNA microarrays, differences of genome-wide transcriptional patterns were analyzed in cells grown at 30℃ and 15℃, or grown at 15℃ but transferred to 4℃ with light of 100μE.m-2.s-1 for 2h. Among genes upregulated in cells grown at 15℃ (compared with 30℃) are: genes involved in transport of carbonate and CO2; sll1566 encoding glucosylglycerolphosphate synthase, slr1171 encoding NADPH-dependent fatty acid peroxidase, sll0629 encoding an analog of PsaK, sll0856 encoding sigma factor H and some genes of unknown functions such as sll1242 and sll0330. RT-PCR with serially diluted templates showed similar results as DNA microarray analyses. Upon transfer of cells grown at 15℃ to 4℃, nblAl encoding a phycobilisome-degrading protease, crtB and crtD involved in carotenoid synthesis were upregulated, some genes required for transport or metabolism of nitrate were downregulated, some regulatory genes such as sigD and hik34 were upregulated but others such as sll1291 and sll1292 were repressed.
4. The mechanism of low temperature enhancement. Through targeted disruption of genes found to be upregulated with 15℃/30℃ microarrays, their roles in low temperature enhancement were analyzed. sll1566, the glucosylglycerolphosphate synthase gene showed no effect on it, while sll0629 and sll0330 showed remarkable effects on it. The role of slr1171 and desB in low temperature enhancement appeared to be wobbling.
The main conclusions of this study are as follows:
1. Exposure of cells grown at 30℃ to 5℃ with light of 100μE.m-2.s-1 caused decrease in viability of Synechocystis sp. PCC6803, growth at 15℃ before transfer to 5℃ could basically eliminate the effect of chilling under light. To this phenomenon, the author proposes the name ‘low temperature enhancement’.
2. By insertion mutagenesis of Synechocystis sp. PCC6803, chill and light-sensitive mutants were obtained; genes involved in cold adaptation were identified, including sll1242 and desD. Low temperature enhancement could more or less restore the tolerance of these mutants to chill and light conditions.
3. This study proposed a method to evaluate the role of single gene in the low temperature enhancement and directly showed the effects of sll0330, sll0629 and other genes on it.|