Yu Kanesaki

The pathway for perception and transduction of low-temperature signals in Synechocystis

Low temperature is an important environmental factor that has effects on all living organisms. Various low‐temperature‐inducible genes encode products that are essential for acclimation to low temperature, but low‐temperature sensors and signal transducers have not been identified. However, systematic disruption of putative genes for histidine kinases and random mutagenesis of almost all the genes in the genome of the cyanobacterium Synechocystis sp. PCC 6803 have allowed us to identify two histidine kinases and a response regulator as components of the pathway for perception and transduction of low‐temperature signals. Inactivation, by targeted mutagenesis, of the gene for each of the two histidine kinases and inactivation of the gene for the response regulator depressed the transcription of several lowtemperature‐inducible genes.

Cold‐regulated genes under control of the cold sensor Hik33 in Synechocystis

A histidine kinase, Hik33, appears to sense decreases in temperature and to regulate the expression of certain cold‐inducible genes in the cyanobacterium Synechocystis sp. PCC6803. To examine the role of Hik33 in the regulation of gene expression, we analysed a ΔHik33 mutant using the DNA microarray technique. In wild‐type cells, genes that were strongly induced at low temperature encoded proteins that were predominantly subunits of the transcriptional and translational machinery. Most cold‐repressible genes encoded components of the photosynthetic machinery. Mutation of the hik33 gene suppressed the expression of some of these cold‐regulated genes, which could be divided into three groups according to the effect of the mutation of hik33. In the first group, regulation of gene expression by low temperature was totally abolished; in the second group, the extent of such regulation was reduced by half; and, in the third group, such regulation was totally unaffected. These results suggest that expression of the genes in the first group is regulated solely by Hik33, expression of genes in the third group is regulated by an as yet unidentified cold sensor, and expression of genes in the second group is regulated by both these cold sensors.

Salt Stress Inhibits the Repair of Photodamaged Photosystem II by Suppressing the Transcription and Translation of psbA Genes in Synechocystis

Light stress and salt stress are major environmental factors that limit the efficiency of photosynthesis. However, we have found that the effects of light and salt stress on photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 are completely different. Strong light induced photodamage to PSII, whereas salt stress inhibited the repair of the photodamaged PSII and did not accelerate damage to PSII directly. The combination of light and salt stress appeared to inactivate PSII very rapidly as a consequence of their synergistic effects. Radioactive labeling of cells revealed that salt stress inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern- and western-blotting analyses demonstrated that salt stress inhibited the transcription and the translation of psbA genes, which encode D1 protein. DNA microarray analysis indicated that the light-induced expression of various genes was suppressed by salt stress. Thus, our results suggest that salt stress inhibits the repair of PSII via suppression of the activities of the transcriptional and translational machinery.

The histidine kinase Hik33 perceives osmotic stress and cold stress in Synechocystis sp. PCC 6803

The stress imposed on living organisms by hyperosmotic conditions and low temperature appears to be perceived via changes in the physical state of membrane lipids. We compared genome‐wide patterns of transcription between wild‐type Synechocystis sp. PCC 6803 and cells with a mutation in the histidine kinase Hik33 using a DNA microarray. Our results indicated that Hik33 regulated the expression of both osmostress‐inducible and cold‐inducible genes. The respective genes that were regulated by Hik33 under hyperosmotic and low‐temperature conditions were, for the most part, different from one another. However, Hik33 also regulated the expression of a set of genes whose expression was induced both by osmotic stress and by cold stress. These results indicate that Hik33 is involved in responses to osmotic stress and low‐temperature stress but that the mechanisms of the responses differ.

Gene-engineered Rigidification of Membrane Lipids Enhances the Cold Inducibility of Gene Expression in Synechocystis

A sudden decrease in ambient temperature induces the expression of a number of genes in poikilothermic organisms. We report here that the cold inducibility of gene expression inSynechocystis sp. PCC 6803 was enhanced by the rigidification of membrane lipids that was engineered by disruption of genes for fatty acid desaturases. DNA microarray analysis revealed that cold-inducible genes could be divided into three groups according to the effects of the rigidification of membrane lipids. The first group included genes whose expression was not induced by cold in wild-type cells but became strongly cold-inducible upon rigidification of membrane lipids. This group included certain heat-shock genes, genes for subunits of the sulfate transport system, and the hik34gene for a histidine kinase. The second group consisted of genes whose cold inducibility was moderately enhanced by the rigidification of membrane lipids. Most genes in this group encoded proteins of as yet unknown function. The third group consisted of genes whose cold inducibility was unaffected by the rigidification of membrane lipids. This group included genes for an RNA helicase and an RNA-binding protein. DNA microarray analysis also indicated that the rigidification of membrane lipids had no effect on the heat inducibility of gene expression. Hik33, a cold-sensing histidine kinase, regulated the expression of most genes in the second and third groups but of only a small number of genes in the first group, an observation that suggests that the cold-inducible expression of genes in the first group might be regulated by a cold sensor that remains to be identified.

An RNA helicase, CrhR, regulates the low-temperature-inducible expression of heat-shock genes groES, groEL1 and groEL2 in Synechocystis sp. PCC 6803

The crhR gene for RNA helicase, CrhR, was one of the most highly induced genes when the cyanobacterium Synechocystis sp. PCC 6803 was exposed to a downward shift in ambient temperature. Although CrhR may be involved in the acclimatization of cyanobacterial cells to low-temperature environments, its functional role during the acclimatization is not known. In the present study, we mutated the crhR gene by replacement with a spectinomycin-resistance gene cassette. The resultant DeltacrhR mutant exhibited a phenotype of slow growth at low temperatures. DNA microarray analysis of the genome-wide expression of genes, and Northern and Western blotting analyses indicated that mutation of the crhR gene repressed the low-temperature-inducible expression of heat-shock genes groEL1 and groEL2, at the transcript and protein levels. The kinetics of the groESL co-transcript and the groEL2 transcript after addition of rifampicin suggested that CrhR stabilized these transcripts at an early phase, namely 5-60 min, during acclimatization to low temperatures, and enhanced the transcription of these genes at a later time, namely 3-5 h. Our results suggest that CrhR regulates the low-temperature-inducible expression of these heat-shock proteins, which, in turn, may be essential for acclimatization of Synechocystis cells to low temperatures.