Hik33 蓝藻 信号调节

REVIEWHistidine kinase Hik33is an important participant in cold-signal transduction in cyanobacteriaNorio Murata a,*and Dmitry A.Los ba National Institute for Basic Biology,Myodaiji,Okazaki,444-8585,Japanb Institute of Plant Physiology,Russian Academy of Sciences,Botanicheskaya Street35,127276Moscow,RussiaCorrespondence*Corresponding author,e-mail:murata@nibb.ac.jpReceived5September2005;revised17 September2005doi:10.1111/j.1399-3054.2005.00608.x Acclimation of living organisms to cold stress begins with the perception and transduction of the cold signal.However,traditional methods failed to identify the sensors and transducers of cold stress.Therefore,we combined systematic mutagenesis of potential sensors and transducers with DNA microarray analysis in an attempt to identify these components in the cya-nobacterium Synechocystis sp.PCC6803.We identified histidine kinase Hik33as a potential cold sensor and found that Hik33participates in the regulation of the expression of more than60%of the cold-inducible genes. Further study revealed that Hik33is also involved in the perception of hyperosmotic stress and salt stress and transduction of the signals. Complexity of responses to cold and other environmental stresses is discussed.IntroductionDecreases in ambient temperature reduce enzymatic activities and ultimately depress various physiological activities.When the temperature changes suddenly and significantly,organisms often fail to survive.When such change is gradual,organisms can acclimate to their environment by sensing the shift in temperature and expressing large numbers of previously unexpressed genes,with resultant synthesis of specific proteins and metabolites that participate in protection against low temperature(Fig.1).Acclimation begins with the per-ception of the shift in temperature and transduction of the resultant anisms and/or individual cells appear to be equipped with sensors and signal transdu-cers that perceive and transduce cold signals.This review focuses on the initial events in cold-inducible gene expression,describing our analysis of potential sensors and transducers of cold stress in cyanobacteria (also reviews by Los and Murata2002,2004,and by Mikami and Murata2003).Unicellular cyanobacteria are particularly suitable for studies of stress responses at the molecular level.The general features of their plasma and thylakoid mem-branes resemble those of the chloroplasts of higher plants in terms of both lipid composition and assembly of membranes.Thus,cyanobacteria appear to provide powerful model systems for studies of the molecular mechanisms of acclimation to low temperature (Murata and Wada1995).Some strains of cyanobacteria,such as Synecho-cystis sp.PCC6803(hereafter,Synechocystis), Synechococcus sp.PCC7942and Synechococcus sp. PCC7002,are naturally competent,incorporating for-eign DNA that is efficiently integrated into theirAbbreviations–Hik,histidine kinase;Rre,response regulator.This paper is dedicated to Dr Marilyn Griffith,who passed away on February19th,2005,to recognize her outstanding contribution to research on cold stress in plants.Physiologia Plantarum126:17–27.2006CopyrightßPhysiologia Plantarum2006,ISSN0031-9317genomes by homologous recombination (Haselkorn 1991,Williams 1988).Therefore,many researchers have used cyanobacteria for the production of mutants with disrupted genes of interest (for review,see Vermaas 1998).The Synechocystis genome was sequenced in 1996(Kaneko et al.1996)with subsequent publication of the genome sequences of other cyanobacteria (see Murata and Suzuki 2005for a complete list with references).Genome sequences provide basic information that can be exploited for the genome-wide study of gene expres-sion.A commercially available genome-wide DNA microarray for analysis of gene expression in Synechocystis (Takara Bio Co.,Ohtu,Japan)covers 3079of the 3165(97%)genes on the chromosome of Synechocystis (99genes for transposases are excluded from this calculation).There are also 397genes on plasmids harboured by Synechocystis (Kaneko et al.2003).These genes are not included in the above-mentioned DNA microarray.Genome-wide analysis of cold-stress-inducible genes in SynechocystisWe used the DNA microarray from Takara to character-ize the genome-wide expression of genes in responseto environmental stresses,starting with cold stress (Suzuki et al.2001).The expression of a large number of genes was enhanced in response to cold stress,while that of another large number of genes was repressed.Some researchers have inferred that cold stress induces cellular dehydration that is essentially identical to that induced by hyperosmotic stress.To examine whether Synechocystis recognizes these kinds of stress similarly,we compared the effects of cold stress and hyperosmotic stress by DNA microarray analysis (Fig.2;Mikami et al.2002).These two kinds of stress enhanced,in common,the expression of genes for high light-inducible proteins (hliA,hliB,and hliC ),for rare lipoprotein A (rlpA ),for DNA mismatch repair protein (mutS ),for a sigma factor (sigD ),and for proteins with other functions.However,only cold stress enhanced the expression of the rbpA gene for an RNA-binding protein,the ndhD2gene for NADH dehydrogenase subunit 4,the crhR gene for an ATP-dependent RNA helicase,the fus gene for translation elongation factor EF-G,the feoB gene for ferrous iron transport protein,the infB gene for translation initiation factor IF-2,and various genes for proteins of known and unknown function.By contrast,only hyperosmotic stress enhanced the expression of genes for heat-shock proteins (hspA,dnaK2,groEL2,and clpB1),for the synthesis of glucosylglycerol (ggpS and glpD ),for the synthesis of lipids and lipoproteins (fabG and repA ),and for various other proteins (htrA,Cold stress SensormRNAs Proteins - enzymesMetabolitesgenes Signal transducerAcclimation to cold conditionsFig.1.A general scheme for the responses of a cyanobacterial cell to cold stress.rbpA, ndhD2, ndhF,crhR, rpl3, rpl4,fus, feoB, infB,ycf39, desB, nusG secE, folK, slr0082,slr0616, slr0236,slr1927, sll1611,sll0086hspA, clpB1, fabG htrA, spkHsigB, sodB, hik34htpG, dnaK2, dnaJ groES, groEL, groEL2ggpS, glpD gloA, repAsll0528, sll0846, slr1963slr0959, sll1884slr1603, slr1915slr0967, sll0939……4020513111hliA, hliB hliC, rlpA,mutS, sigD,slr1544, sll1541sll1862, sll1863sll1483HyperosmoticColdFig.2.Some cold-stress-inducible genes and hyperosmotic stress-inducible genes are the same,and some are different.The diagram includes genes that are induced during incubation of Synechocystis for 20min after a shift in growth temperature from 34 C to 22 C (cold stress)and after addition of sorbitol at 0.5M (hyperosmotic stress).Adapted,with permission,from Mikami et al.(2002)with inclusion of recent results from authors’laboratory.spkH,sodB,htpG,and gloA).Thus,cold and hypero-smotic stress each induced expression of a number of specific genes,while both stresses induced expression of a relatively small number of common genes(Fig.2), suggesting that Synechocystis recognizes cold stress and hyperosmotic stress as different signals via specific signal-transduction pathways.Two-component systems:positive regulation and negative regulationThe existence of two-component systems has been well-established in Escherichia coli and Bacillus subtilis (Aguilar et al.2001,Stock et al.2000).Each two-component system consists of a histidine kinase(Hik) and a cognate response regulator(Rre).The Hik per-ceives a change in the environment via its sensor domain,and then a conserved histidine residue within the Hik domain is autophosphorylated,with ATP pro-viding phosphate group(Stock et al.2000)that is trans-ferred from the Hik to a conserved aspartate residue in the receiver domain of the cognate Rre.Upon phos-phorylation,the conformation of the Rre changes, allowing the binding of the Rre to promoter regions of genes that are located downstream in the acclimation pathway(Koretke et al.2000).In E.coli and B.subtilis,the genes for each Hik and its cognate Rre are generally located close to one another on the chromosome.Moreover,these genes are often located near functionally related genes.Two-component systems are found also in cyanobac-teria(Mizuno et al.1996).However,genes for Hiks and Rres in Synechocystis are,in most cases,distributed somewhat randomly on the chromosome.Among the 44genes for Hiks,14are located near genes for poten-tially cognate Rres,whereas genes for the other30Hiks are located far from any rre genes.Thus,it is difficult to predict the pairs of Hiks and Rres that might function as individual two-component systems.Therefore,we sys-tematically mutated the genes for all the potential sen-sors and transducers of environmental signals and examined gene expression under various conditions using DNA microarrays.Synechocystis has3661putative genes,of which47 encode Hiks and45encode Rres(http://www.kazusa. or.jp/cyanobase/Synechocystis/index.html).There are 44genes for Hiks on the chromosome,which we named hik1through hik44.We deduced that three putative Hiks,namely,Hik11,Hik17,and Hik37, might be inactive as Hiks,because they lack the con-served histidine residue in the Hik domain.Hik32might also be inactive as a Hik,because the hik32(sll1473) gene is part of a larger gene,namely,sll1473-sll1475,which is interrupted by a transposon(sll1474)in the strain that was used for genome sequencing and sys-tematic mutagenesis(Okamoto et al.1999).The gene for the Hik encoded by pSYSM,a plasmid harboured by Synechocystis,was designated hik45,and the genes for Hiks encoded by pSYSX,another plasmid,were desig-nated hik46and hik47.There are42chromosomal genes for Rres and two and one on plasmids pSYSX and pSYSM,respectively.We designated the42chro-mosomal genes for rre1through rre42.The rre genes on pSYSM and pSYSX were designated rre43and rre44and 45,respectively.We inactivated each putative hik gene in Synechocystis by inserting a spectinomycin-resistance gene cassette to create a gene-knockout library(Suzuki et al.2000;CyanoMutants,http://www.kazusa.or.jp/ cyano/Synechocystis/mutants/).This library has proved to be a powerful tool for the identification of sensors of various stimuli and the corresponding signal transducers in Synechocystis(Marin et al.2003,Paithoonrangsarid et al.2004,Shoumskaya et al.2005,Suzuki et al.2000, Suzuki et al.2004,Yamaguchi et al.2002).We exam-ined the genome-wide expression of genes in wild-type cells and in each line of hik mutant cells with DNA microarrays in an attempt to identify the Hiks involved in the regulation of expression of stress-inducible genes. Regulation of gene expression in response to stress can be positive or negative(for details,see Murata and Suzuki2005).In positive regulation,a two-component system is inactive under non-stress conditions.Genes controlled by such systems are silent or expressed. When cells are exposed to the appropriate environmen-tal stress,the two-component system is activated(by phosphorylation in response to the stress),and then the activated system enhances the expression of genes that are silent under non-stress conditions or represses the expression of genes that are expressed under non-stress conditions(Murata and Suzuki2005).Most stress-inducible gene expression in Synechocystis is positively regulated.In negative regulation,the two-component system is active under non-stress conditions,and the expression of the genes controlled by such a system is either enhanced or repressed.Under appropriate environmen-tal stress,the two-component system becomes inactive. Genes that are expressed or repressed under non-stress conditions become silent or are released from repres-sion,respectively.Levels of expression of genes fall in the former case and rise in the latter.Changes in phenotype due to mutations in Hiks and Rres,which reflect the effects of such two-component systems on gene expression,differ between positive regulation and negative regulation.A knockoutmutation in either the Hik or the Rre in a two-compo-nent system for negative regulation has marked effects on gene expression.The expression of genes controlled by a negatively regulating two-component system is either enhanced or repressed under non-stress condi-tions.Therefore,a specific signal-transduction pathway with a specific Hik and a specific Rre can be identified with relative ease in cases of negative regulation.By contrast,a knockout mutation in a Hik or an Rre in a two-component system that operates via positive reg-ulation does not have a significant effect on gene expression under non-stress conditions.In such a sys-tem,the identification of the Hik and the Rre in a specific signal-transduction pathway requires the screening of knockout libraries of hik and rre genes under individual types of stress.Negative regulation of gene expression Using DNA microarray,we analyzed the effects of muta-tion of each hik gene on gene expression in mutant cells that had been grown under normal conditions.No sig-nificant alterations in gene expression were evident in most of the mutants.However,three mutants,D hik27, D hik34,and D hik20,each with a mutation in the indi-cated Hik,were unique,because,in these mutants,the expression of some genes was enhanced and that of some others was repressed,indicating that Hik27,Hik34,and Hik20might be involved in signal transduction via the negative regulation of gene expression.We compared gene expression in D hik27cells with that in wild-type cells under normal conditions (Yamaguchi et al.2002).Marked changes,with induc-tion factors higher than10,due to mutation of the hik27 gene(slr0640)were recognized only in the expression of three genes,namely,mntC,mntA,and mntB,which constitute the mntCAB operon that encodes subunits of the ABC-type Mn2þtransporter(Bartsevich and Pakrasi 1995,1996).Under normal growth conditions,Hik27 might transduce a signal that represses the expression of the mntCAB operon.Moreover,disappearance of this signal,due to inactivation of Hik27,might allow the expression of the mntCAB operon.This scenario corre-sponds to the scheme outlined for negative regulation. We examined the effects of mutation of various rre genes on gene expression in mutant cells.The mutation in D rre16cells enhanced the expression of only the mntC,mntA,and mntB genes.This phenomenon was very similar to the change in gene expression detected in the D hik27mutant.In its active form,Rre16might have repressed expression of the mntCAB operon.Then, inactivation of Rre16in D rre16-mutant cells eliminated the repressive effect of Rre16,allowing the expression of the mntCAB operon.These findings suggested that Hik27and Rre16might constitute a two-component system,acting as the sensor and signal transducer of Mn2þdeficiency.Ogawa et al.(2002)identified this two-component system independently by‘traditional’methods.Mutation of Hik34altered the genome-wide expres-sion of genes under normal conditions,namely,at a growth temperature of34 C(Suzuki et al.2005).In D Hik34cells,levels of transcripts of heat-shock genes, such as htpG,groES,and groEL1,were elevated,sug-gesting that Hik34might act as a negative regulator of the expression of these genes during normal growth. Because Hik34appeared to be a negative regulator of heat-shock-responsive genes,we postulated that its overexpression should result in repressed expression of these genes.We observed that the effect of overexpres-sion of hik34was the opposite of that of inactivation of hik34on the expression of heat-shock genes at the normal growth temperature(Suzuki et al.2005).This observation is consistent with the hypothesis that Hik34 is important in the regulation of expression of heat-shock genes.Positive regulation by Hik33in response to low temperatureMutation of the42other hik genes did not induces significant changes in gene expression.Therefore,it is likely that the Hiks encoded by these42genes regulate gene expression in a positive manner.To confirm this hypothesis,we screened our library of hik mutants under specific kinds of stress.To monitor the inducibility of the cold-inducible desB gene for the o3desaturase,we generated the pdesB::lux strain of Synechocystis,in which the promoter region of the desB gene was ligated to the coding region of the luxAB gene for a bacterial luciferase(Suzuki et al. 2000).Thus,we could use luciferase activity as an indicator of cold-inducible changes in the activity of the desB promoter.Then we inactivated separately the gene for each Hik in pdesB::lux cells by inserting a spectinomycin-resistance cartridge(Sp r),creating a gene-knockout library(Suzuki et al.2000).We screened the members of this library for loss of cold inducibility of gene expression by monitoring luciferase activity at a low temperature.Only pdesB::lux/D Hik33 and pdesB::lux/D Hik19,with disruption of the genes for Hik33and Hik19,respectively,exhibited reduced ability to activate luciferase at low temperature, suggesting that Hik33and Hik19might be involved in the perception and tranduction of cold signals.DNA microarray analysis of D hik33cells indicated that Hik33regulates the expression of 21of 36cold-inducible genes,with induction factors higher than 3.0(Fig.3;Suzuki et al.2001).These 21genes include ndhD2,hliA,hliB,hliC ,feoB ,crp ,and genes for pro-teins of unknown function.By contrast,15of the 36cold-inducible genes were not regulated by Hik33.Therefore,we can deduce that Synechocystis might have at least one other pathway for cold-signal trans-duction.DNA microarray analysis of the expression of genes in D hik19cells indicated that Hik19is unlikely to be a cold sensor.To identify the Rre that is located downstream of Hik33,we screened an Rre-knockout library by RNA slot-blot hybridization using some of the cold-inducible genes controlled by Hik33as probes.We identified Rre26as a candidate for the Rre that,with Hik33,con-stitutes a two-component system for cold-signal trans-duction (our unpublished results;Fig.3A).Tu et al.(2004)and Hsiao et al.(2004)postulated that Hik33might negatively regulate the expression of a set of photosynthetic and high light-responsive genes.Their hypothesis was based on changes in the global expres-sion of genes under normal conditions and the comple-mentation of the D hik33mutation in Synechocystis by the homologous nblS gene from Synechococcus with respect to the light-induced expression of hli genes,as monitored by Northern blotting.However,because the complementation test did not examine the genome-wide expression of genes,it is possible that their D Hik33mutant cells had,in addition to the mutation in hik33,a further mutation that might have produced changes in gene expression under normal conditions.To confirm our conclusion that Hik33is a positive regulator of signal transduction,we replaced the entire open-reading frame of the hik33gene with a spectino-mycin-resistance gene cassette that contained the O sequence,which is a strong terminator of transcription and inhibits the read-through of inserted genes on both sides of the cassette (our unpublished work).The growth rate of cells with deletion of hik33was similar to that of the wild-type and the insertion mutant of Hik33,contra-dicting the inferences made by Hsiao et al.(2004).DNA microarray analysis of the deletion mutant,comparing the pattern of gene expression with that of the insertion mutant under normal conditions,confirmed the absence of the major changes in gene expression reported by Hsiao et al.(2004).Our results suggest that the Hik33-dependent-signalling pathway involves the positive reg-ulation of gene expression.Responses to salt stress and hyperosmotic stressWe demonstrated previously that the perception of hyperosmotic stress involves Hik33and other unknown components (Mikami et al.2002).Later when we used the DNA microarray to investigate the impact of muta-tions in each Hik on changes in gene expression under salt stress (Marin et al.2003),we found that theCytoplasmic membrane?slr1927sll1611slr0955slr0236sll0494slr1436slr1974Hik33Rre26ndhD2hliA hliB hliC fus ycf39sigDslr1747ssr2016slr0400sll1911slr0401sll0815sll1770crhR rlpA rbpA cbiO cbiQ mutS desB slr0082feoB crtP slr1544sll1483sll1541sll0086slr0616Cold stressTotal: 21Total: 15AhtrAHik10??Hyperosmotic stressHik34Rre1sll0939slr0967Hik41Hik33fabG hliA hliB hliC gloA sigD sll1483slr1544ssr2016ssl3446sll1541rlpA repA glpD sll1863sll1862sll1772slr0581hspA clpB1sodB htpG dnak2spkH groES groEL1groEL2dnaJRre3Rre1Rre17Rre31sigB sll0528slr1119slr0852ssr3188sll0846slr1963slr0959sll1884slr1603slr1915ssl2971slr1413ssr1853slr0112sll0294slr0895slr1501sll0293sll0470Hik2Total: 11Total: 19Total: 5Total: 2Total: 1Total: 14Cytoplasmic membraneBHik16Fig.3.Hypothetical schemes showing the two-component systems that are involved in the transduction of cold stress and hyperosmotic stress,as well as the genes that are under the control of the individual two-component systems.Primary signals are represented by open arrows.Hiks are indicated as ellipses,Rres are indicated as hexagons,and selectively regulated genes are shown in boxes.Uncharacterized mechanisms are represented by question marks.Genes with induction factors higher than 3.0are included in these schemes.Minor discrepan-cies with respect to cold-inducible genes between this figure and Fig.2are due to the use of different versions of the DNA microarray in the respective experiments.(A)Cold stress;adapted originally from Suzuki et al.(2001)and Mikami et al.(2002),with inclusion of more recent results from authors’laboratory.(B)Hyperosmotic stress;adapted from Paithoonrangsarid et al.(2004)and Shoumskaya et al.(2005).inducibility of gene expression by elevated levels of NaCl was significantly affected in D Hik33-,D Hik34-, D Hik16-,and D Hik41-mutant cells.In each of these mutants,several genes were no longer induced by salt, or their inducibility by salt was markedly reduced. Because hyperosmotic stress and salt stress might affect some aspects of the physiology of cyanobacterial cells similarly,we examined whether these Hiks might also regulate gene expression under hyperosmotic stress.DNA microarray analysis demonstrated that they are indeed involved in hyperosmotic-signal trans-duction.Fig.3B shows that they regulate the expression of38of52hyperosmotic stress-inducible genes (Paithoonrangsarid et al.2004).Screening our Rre-knockout library by RNA slot-blot hybridization and with DNA microarray,we identified four Rres,namely,Rre31,Rre1,Rre3,and Rre17,that are involved in hyperosmotic signal transduction. Further analysis with microarray showed that four Hik-Rre systems,namely,Hik33-Rre31,Hik34-Rre1,Hik10-Rre3,and Hik16-Hik41-Rre17,as well as another sys-tem that included Rre1and possibly Hik2,appeared to be involved in the perception of hyperosmotic stress and transduction of the signal(Paithoonrangsarid et al. 2004).Fig.3B shows a hypothetical model of the hyper-osmotic signal-transducing systems that involve these Hiks and Rres,including the hyperosmotic stress-inducible genes that are controlled by the individual Hik-Rre systems.The Hik33-Rre31two-component system regulates the expression of11hyperosmotic stress-inducible genes.Inactivation of either Hik33or Rre31resulted in the elimination of or a marked reduction in the hyper-osmotic stress-inducible expression of these genes,indi-cating that Hik33and Rre31are tightly coupled in the signal-transduction pathway.The Hik10-Rre3two-component system regulates the hyperosmotic stress-inducible expression of only htrA,which encodes a serine protease.The Hik16-Hik41-Rre17system regulates the hyper-osmotic stress-inducible expression of sll0939and slr0967.Inactivation of Hik16,of Hik41,or of Rre17 eliminated the expression of these genes,suggesting that Hik16,Hik41,and Rre17are all essential for the per-ception of hyperosmotic stress and for transduction of the corresponding signal.Hik41probably acts down-stream of Hik16,because Hik41is a hybrid-type Hik that contains both a signal-receiver domain and a Hik domain,whereas Hik16is a typical Hik with a Hik domain and potential sensory domain that,hypotheti-cally,spans the membrane seven times.It is also possi-ble that Hik16and Hik41might perceive hyperosmotic stress as a complex.The Hik34-Rre1system regulates the expression of19 hyperosmotic stress-inducible genes(for heat-shock pro-teins and for proteins of unknown function).The Hik2-Rre1system regulates the expression of five genes that include the sigB gene for a sigma factor.Rre1might perceive hyperosmotic signals from both Hik34and Hik2.However,the regulated genes are specific to either His34or Hik2(Fig.3B;Paithoonrangsarid et al. 2004).DNA microarray analysis revealed that expression of 14of the52hyperosmotic stress-inducible genes was not controlled by any of the five Hiks and four Rres discussed above(Fig.3B).The signals,due to hyperos-motic stress,that induce the expression of these genes are probably perceived by unknown mechanisms that differ from typical Hik-Rre two-component systems. Such signals might act directly to regulate either tran-scription or the stability of the transcripts of these indu-cible genes.Using similar methods,we identified that five two-component systems are involved in the salt-stress signal-transduction pathway.To our surprise,they were iden-tical to those involved in response to hyperosmotic stress.However,the genes controlled by the individual pathways are different(Shoumskaya et al.2005),as discussed below.His33is a multifunctional regulatorAs described above,Hik33is involved in the perception and transduction of the cold signal and the hyperosmotic-stress signal(Fig.3).However,the cold-responsive genes controlled by Hik33are not identical to the hyperosmotic stress-responsive genes controlled by the same system.Fig.4shows that eight genes,including hliA,hliB,hliC,and sigD,are induced by both kinds of stress under the control of Hik33.However,Hik33reg-ulates the expression of eight other genes,including ndhD2,fus,crtP,and feoB,in response to cold stress specifically,and not to hyperosmotic stress,whereas it regulates the expression of three other genes,including fabG and gloA,in response to hyperosmotic stress spe-cifically,and not to cold stress.Thus,cold stress,hyper-osmotic stress,and salt stress are perceived as distinct signals by a sensory system that includes Hik33.In addition,recent studies(Hsiao et al.2004,Tu et al. 2004)suggest that Hik33might be involved in response to light stress.Moreover,a homolog of Hik33in Synechococcus is involved in sensing nutritional deficits (van Waasbergen et al.2002).Another level of compexity reflects the differential involvement of Rres in signal transduction and gene expression.Microarray analysis indicated that Rre26isinvolved in cold-signal tranduction (Fig.3A),whereas Rre31is involved in hyperosmotic-and salt-signal trans-duction (Fig.3B;Paithoonrangsarid et al.2004,Shoumskaya et al.2005).Moreover,eight genes respond,in common,to cold stress and hyperosmotic stress via Rre26and Rre31,respectively.Our observa-tions suggest that the mechanisms for perception of cold stress and hyperosmotic stress by Hik33are complex and that as-yet-unidentified components exist that pro-vide the sensory systems with their respective activities for induction of responses specific to each individual type of stress.Hik33perceives the cold signal via rigidification of membrane lipidsThe Hik33sensory kinase includes a type-P linker,a leucine zipper,and a PAS domain (Fig.5A;Los and Murata 1999,2002,2004,Mikami and Murata 2003).The type-P linker contains two helical regions in tandem that are assumed to transduce stress signals via intramo-lecular structural changes that result from interactions between the two helical regions and lead to intermole-cular dimerization of membrane proteins (Aravind et al.2003,Williams and Stewart 1999).In Hik33,cold stress might promote a conformational change in the type-P linker,with subsequent activation of Hik33via dimeriza-tion of the protein (Fig.5B;Los and Murata 2000,2004).There are two transmembrane domains in the amino-terminal region of Hik33(Los and Murata 2004,Mikami and Murata 2003).Because it has been suggested thatchanges in membrane fluidity might be involved in the sensing of temperature (Los and Murata 2000,Murata and Los 1997),it is likely that the transmembrane domains of Hik33can recognize changes in membrane fluidity at low temperatures (Los and Murata 1999,2000,2004).In 1996,we produced a series of mutants in which the extent of unsaturation of fatty acids is modified in a step-wise manner (Tasaka et al.1996).In one such mutant,the desA and desD genes for the D 12and D 6fatty acid desaturases,respectively,are inactive as a result of targeted mutagenesis.The desA –/desD –-double mutant synthesize only a saturated C16fatty acid and a mono-unsaturated C18fatty acid,regardless of growth temperature,whereas wild-type cells synthesize di-unsaturated and tri-unsaturated C18fatty acids in addi-tion to the mono-unsaturated C18fatty acid (Tasaka et al.1996).FTIR spectrometry revealed that the double mutation of the desA and desD genes rigidified the plasma membrane of Synechocystis at physiological temperatures (Szalontai et al.2000).ndhD2, fus, crtP, ycf39, feoB, slr1747, sll0086, slr0616fabG, gloA,ssl30448381121Cold; Hik33-Rre26Hyperosmotic;Hik33-Rre31hliA, hliB hliC, sigD slr1544ssr2016, sll1483sll1541Fig.4.A schematic representation of genes that are induced by cold stress and hyperosmotic stress under control of Hik33.The diagram includes genes that are induced during incubation for 20min after a shift in growth temperature from 34 C to 22 C (cold stress)and after addition of sorbitol at 0.5M (hyperosmotic stress).TM1TM2Type-PPAS His kinase 13248201217220270310350400600663ABType-P PASHis kinaseH HHLZ LZ Fig.5.A hypothetical scheme for the structure of Hik33.(A)Domain structure of Hik33.(B)A putative dimeric form of Hik33and its relation-ship to the cell membrane.A decrease in temperature rigidifies the membrane,leading to compression of the lipid bilayer,which forces the membrane-spanning domains closer changes the linker conforma-tion and finally causes dimerization and autophosphorylation of histi-dine kinase domains.TM1and TM2,Transmembrane domains;LZ,leucine zipper;Type-P,a type-P linker domain;and PAS,PAS domain that contains amino acid motifs P er,Arnt,S im,and phytochrome (Taylor and Zhulin,1999);H in circles,histidine residues that can be phosphorylated in response to cold stress.。