SpkH (Sll0005) from Synechocystis sp. PCC 6803 is an active Mn2+-dependent Ser kinase.

Twelve genes for the potential serine-threonine protein kinases (STPKs) have been annotated in the genome of Synechocystis sp. PCC 6803. Based on similarities and distinctive domain organization, they were divided into two clusters: serine/threonine-protein N2-like kinases (PKN2-type) and "activity of bc1 complex" kinases (ABC1-type). While the activity of the PKN2-type kinases have been demonstrated, no ABC1-type kinases activity have hitherto been reported. In this study, a recombinant protein previously annotated as a potential STPK of ABC1-type (SpkH, Sll0005) was expressed and purified to homogeneity. We demonstrated SpkH phosphorylating activity and substrate preference for casein in in vitro assays using [γ-32P]ATP. Detailed analyses of activity showed that Mn2+ had the strongest activation effect. The activity of SpkH was significantly inhibited by heparin and spermine, but not by staurosporine. By means of semi-quantitative mass-spectrometric detection of phosphopeptides, we identified a consensus motif recognized by this kinase - X1X2pSX3E. Thus, we first report here that SpkH of Synechocystis represents a true active serine protein kinase, which shares the properties of casein kinases according to its substrate specificity and sensitivity to some activity effectors.

Systemic analysis of stress transcriptomics of Synechocystis reveals common stress genes and their universal triggers.

Systemic analysis of stress transcriptomes of the cyanobacterium Synechocystis revealed that all stress-induced genes can be separated into two groups: one is clustered around heat-shock- and another - around cold-shock inducible genes. Genes for so-called heat shock proteins (HSPs) are induced by various stressors, e.g. heat, salt, hyperosmotic environment, reactive oxygen species (ROS), changes in light intensity and quality, or in the redox potential of the photosynthetic electron transport chain components. The number of specifically heat-induced genes is rather limited and their functions are mostly unknown. Genes induced by cold overlap with other set of genes induced by all above mentioned stressors with the exception of heat shock. The analysis shows that ROS and redox changes may function as universal triggers for stress responses in cyanobacteria.

[Lessons from cyanobacterial transcriptomics: Universal genes and triggers of stress responses].

A systemic transcriptome analysis of the cyanobacterium Synechocystis sp. PCC 6803 revealed a number of genes whose transcription is induced in response to almost all abiotic stresses (heat shock, salt stress, osmotic stress, oxidative stress, changes in light intensity or spectral composition, and changes in the redox potential of electron transport chain (ETC) components). Heat shock protein (HSP) genes were induced by all types of stress, forming a group of genes that universally react to various changes in the environment. Reactive oxygen species (ROS), including H2O2 in particular, and changes in the redox potential of components of the photosynthetic ETC were assumed to function as universal triggers of stress responses in cyanobacteria.

[Involvement of serine/threonine protein kinases in cold stress response in the cyanobacterium Synechocystis sp. PCC 6803: functional characterization of a protein kinase Spke].

Stress responses of the unicellular cyanobacterium Synechocystis are fulfilled via a number of regulatory systems, namely, two-component systems as well as through negative supercoiling of the genome DNA. We have studied an involvement of serine/threonine protein kinases (STPK) in the cyanobacterium Synechocystis cold stress response. A search for the STPK mutants allowed us to determine four protein kinases, SpkB, SpkD, SpkE and SpkG, which could regulate transcription under the low temperature. According to a proteome analysis, SpkE significantly affects the protein pattern in Synechocystis. Functional activity of the recombinant SpkE was confirmed in in vitro phosphorylation assay with a use of a set of potential protein kinase substrates. It have been demonstrated that the basic proteins are preferable substrates for the recombinant protein kinase SpkE.

[A feedback between membrane fluidity and transcription of the desB gene for the omega3 fatty acid desaturase in the cyanobacterium Synechocystis].

Prokaryotic cells, including cyanobacteria, respond to a decrease in ambient temperature by activation of numerous cold shock genes. Low temperatures cause a decrease in membrane fluidity, which is maintained at some optimal level mainly by fatty acid (FA) desaturases. Here, temperature-dependent expression of the desB gene for the omega3-desaturase in Synechocystis, which synthesized polyunsaturated FAs, and in its mutant, desA-/desD-, which is defective in genes for delta12- and delta6-desaturases and is capable of synthesizing only monounsaturated FAs was studied. Low temperatures caused the increase in the amount of the desB mRNA in the wild-type cells with the maximum observed at 24 degrees C. In the double mutant desA-/desD- cells, the maximum amount of this mRNA was accumulated at 28-30 degrees C. Thus, our studies of the desB gene for the omega3-desaturase demonstrated that temperature-dependent expression of genes, which are responsible for the maintenance of the optimal membrane fluidity, depends on physical state of these membranes and is regulated by a feedback mode.

[Protein sensors and transducers of cold, hyperosmotic and salt stresses in cyanobacteria and plants].

The genome-wide investigation of gene expression at the transcript level with use of DNA microarray has recently allowed to list almost all the genes that are induced by a distinct environmental stress in cyanobacterial and plant cells. Acclimation of living organisms to stress conditions begins with the perception and transduction of the stress signal. The combination of systematic mutagenesis of potential sensors and transducers with DNA microarray analysis in an attempt to identify these components led to significant progress in understanding the mechanisms for perception of environmental stresses in photosynthesizing cells. This review is focused on signaling systems that perceive and transduce the signals of cold, hyperosmotic, and salt stresses in cyanobacteria and plants.

[Comparative expression in Escherichia coli of the native and hybrid genes for acyl-lipid delta(9) desaturase].

Expression of the desC gene coding for acyl-lipid delta(9) desaturase of thermophilic cyanobacterium Synechocystis sp. PCC6803 was studied in Escherichia coli cells. In a hybrid gene constructed (desC-licBM3), a sequence of the native acyl-lipid delta(9) desaturase was fused in frame with the reporter gene coding for thermostable lichenase. Lichenase contained in the hybrid protein simplified selection and analysis of the expression of membrane desaturase in the heterologous host. Comparisons of the expression for the native and hybrid genes in bacterial cells showed that lichenase remained active and thermostable in the hybrid protein, while desaturase retains the capability of introducing a double bound in the corresponding position of fatty acids.

The effect of low-temperature-induced DNA supercoiling on the expression of the desaturase genes in synechocystis.

Temperature-dependent changes in genomic DNA supercoiling might play an important role in temperature perception and responsive gene regulation. In the cyanobacterium Synechocystis, low temperatures induce the expression of the genes for fatty acid desaturases that introduce double bonds into acyl chains of lipid-bound fatty acids, thus regulating the membrane fluidity. I studied the effects of low temperature on supercoiling of the genomic DNA region that contains the regulatory elements of the desB gene for the omega3 desaturase, which is strongly induced by cold. Upon decrease in temperature, the degree of DNA supercoiling increased in this region. Novobiocin, an inhibitor of the DNA gyrase, prevented low-temperature-induced changes in DNA supercoiling and affected the expression of several desaturase genes with the most effect on desB. Decreasing in temperature induces three genes of FA desaturases encoding delta12, delta6 and omega3 desaturases in Synechocystis cells. Novobiocin inhibited completely low-temperature-induced transcription of desB, accumulation of the corresponding protein, and the formation of the omega3 unsaturated fatty acids. In the presence of novobiocin, the novobiocin-resistant mutant cells of Synechocystis responded to the low-temperature treatment in the same way as the wild-type cells in the absence of the antibiotics. Thus, temperature-induced changes in DNA supercoiling might form an essential part of a global regulation pathway leading to low-temperature acclimation in this mesophilic cyanobacterium.

Synechocystis HSP17 is an amphitropic protein that stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-mediated refolding.

The small heat shock proteins (sHSPs) are ubiquitous stress proteins proposed to act as molecular chaperones to prevent irreversible protein denaturation. We characterized the chaperone activity of Synechocystis HSP17 and found that it has not only protein-protective activity, but also a previously unrecognized ability to stabilize lipid membranes. Like other sHSPs, recombinant Synechocystis HSP17 formed stable complexes with denatured malate dehydrogenase and served as a reservoir for the unfolded substrate, transferring it to the DnaK/DnaJ/GrpE and GroEL/ES chaperone network for subsequent refolding. Large unilamellar vesicles made of synthetic and cyanobacterial lipids were found to modulate this refolding process. Investigation of HSP17-lipid interactions revealed a preference for the liquid crystalline phase and resulted in an elevated physical order in model lipid membranes. Direct evidence for the participation of HSP17 in the control of thylakoid membrane physical state in vivo was gained by examining an hsp17(-) deletion mutant compared with the isogenic wild-type hsp17(+) revertant Synechocystis cells. We suggest that, together with GroEL, HSP17 behaves as an amphitropic protein and plays a dual role. Depending on its membrane or cytosolic location, it may function as a "membrane stabilizing factor" as well as a member of a multichaperone protein-folding network. Membrane association of sHSPs could antagonize the heat-induced hyperfluidization of specific membrane domains and thereby serve to preserve structural and functional integrity of biomembranes.

Identification of secreted proteins of the cyanobacterium Synechocystis sp. strain PCC 6803.

We investigated the spectrum of secreted proteins in the cyanobacterium Synechocystis, and identified these proteins by amino-terminal sequencing. In total, seven sequences have been determined that corresponded to the proteins Sll0044, Sll1694, Sll1891, Slr0924, Slr0841, Slr0168, and Slr1855. The protein Sll1694 of 18 kDa that formed one of two major bands on SDS-PAGE was identified as cyanobacterial pilin, PilA. The amino-terminal sequence of another protein that formed a second major band was blocked. The analysis of the data revealed that five of seven proteins had distinct putative leader sequences for secretion.

Expression of the gene for the delta9 acyl-lipid desaturase in the thermophilic cyanobacterium.

A single-copy gene resembling the gene for the delta9 acyl-lipid desaturase (desC) was cloned from the thermophilic cyanobacterium Synechococcus vulcanus. Expression of desC in Escherichia coli confirmed that it encodes the delta9 desaturase. The nucleotide sequence of the desC was characterized by high G+C content that is typical of the sequences of thermophilic bacteria. The deduced amino acid sequence exhibited low Cys content and high Arg/Lys ratio that are the attributes of thermostable enzymes. A low level of the desC mRNA was detected in the cells grown at 55 degrees C, the optimum growth temperature for S. vulcanus. About a 10-fold increase was observed in the levels of the transcript and the protein during the shift in temperature from 55 to 45 degrees C. At 35 degrees C the amount of the desC mRNA and of the enzyme accumulated in the cells, was 3 to 4 times smaller than at 45 degrees C. At both temperatures, however, lipids were desaturated at similar rates. These results suggest that in S. vulcanus the conversion of stearic acid into oleic acid may be controlled not only by the de novo synthesis of the delta9 desaturase but, possibly, by the activation of the pre-existing enzyme.

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.

Perception and transduction of low-temperature signals to induce desaturation of fatty acids.

When cells of the cyanobacterium Synechocystis sp. PCC 6803 are exposed to a low temperature, genes for fatty acid desaturases are expressed with resultant increases in the degree of unsaturation of fatty acids in membrane lipids. However, the sensor and transducers of low-temperature signals had not yet been identified. In order to identify these components we applied to the cyanobacterium Synechocystis sp. PCC 6803 the systematic disruption of all 43 putative genes for histidine kinases and random mutagenesis of the whole genome in conjunction with screening by the transcriptional activity of the promoter of the desB gene for the omega3 desaturase. This allowed us to identify two histidine kinases and a response regulator as components of the perception and transduction of low-temperature signals for the expression of genes for fatty acid desaturases.

Regulation of enzymatic activity and gene expression by membrane fluidity.

Changes in the cellular environment can lead to alterations in the fluidity of the membranes of prokaryotes and eukaryotes. Changes in temperature and osmotic conditions are two of the best-studied stresses that can affect membrane fluidity. Los and Murata discuss the types of sensors that detect these changes in membrane fluidity and the types of signals that are generated.

Responses to cold shock in cyanobacteria.

Acclimation of cyanobacteria to low temperatures involves induction of the expression of several families of genes. Fatty acid desaturases are responsible for maintaining the appropriate fluidity of membranes under stress conditions. RNA-binding proteins, which presumably act analogously to members of the bacterial Csp family of RNA chaperones, are involved in the maintenance of the translation under cold stress. The RNA helicase, whose expression is induced specifically by cold, might be responsible for modifying inappropriate secondary structures of RNAs induced by cold. The cold-inducible family of CIp proteins appears to be involved in the proper folding and processing of proteins. Although genes for cold-inducible proteins in cyanobacteria are heterogeneous, some common features of their untranslated regulatory regions suggest the existence of a common factor(s) that might participate in regulation of the expression of these genes under cold-stress conditions. Studies of the patterns of expression of cold-inducible genes in cyanobacteria have revealed the presence of a cold-sensing mechanism that is associated with their membrane lipids. Available information about cold-shock responses in cyanobacteria and molecular mechanisms of cold acclimation are reviewed in this article.

Structure and expression of fatty acid desaturases.

Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains. They are present in all groups of organisms, i.e., bacteria, fungi, plants and animals, and play a key role in the maintenance of the proper structure and functioning of biological membranes. The desaturases are characterized by the presence of three conserved histidine tracks which are presumed to compose the Fe-binding active centers of the enzymes. Recent findings on the structure and expression of different types of fatty acid desaturase in cyanobacteria, plants and animals are reviewed in this article. Roles of individual desaturases in temperature acclimation and principles of regulation of the desaturase genes are discussed.

Biochemical characterization of a delta12 acyl-lipid desaturase after overexpression of the enzyme in Escherichia coli.

The Delta12 acyl-lipid desaturase of Synechocystis sp. PCC 6803 was overexpressed in Escherichia coli as an active enzyme. The overexpressed protein was associated with cell membranes; it represented about 10% of the total cellular protein and 25% of the total membrane protein. The enzyme in the membrane fraction exhibited strong fatty-acid desaturase activity. The desaturase in salt-washed membranes was stabilized by the presence of sorbitol. Storage of salt-washed membranes in 2 M sorbitol at 4 degrees C and at pH 7-8 for six days resulted in the loss of less than 10% of the desaturase activity. The desaturase activity had a positive temperature coefficient, a result that suggests that the increase in the desaturation of fatty acids at low temperature might not be caused by the activation of desaturases at low temperature but, rather, by the increased synthesis of desaturases de novo.

Differences in the control of the temperature-dependent expression of four genes for desaturases in Synechocystis sp. PCC 6803.

Cyanobacteria are capable of desaturating the fatty acids in their membrane lipids in response to decreases in temperature. The cyanobacterium, Synechocystis sp. PCC 6803, contains four desaturases, which specifically catalyse desaturation at the delta6, delta9, delta12 and omega3 positions of fatty acids. The levels of the mRNAs transcribed from the genes that encode the delta6, delta12 and omega3 desaturases increased about 10-fold, but at different rates, upon a decrease in temperature from 34 degrees C to 22 degrees C, whereas the level of the mRNA for the delta9 desaturase remained constant. The increases in the levels of mRNAs were caused both by the enhanced transcription and by the increased stability of the mRNAs at the low temperature. Western blotting analysis demonstrated that levels of the delta6, delta12 and omega3 desaturases increased at different rates at the low temperature, while that of the delta9 desaturase remained constant. These observations indicate that the expression of the genes for the four desaturases is regulated by temperature in different ways.