Histidine kinase Hik33 is an important participant in cold-signal transduction in cyanobacteria

REVIEW

Histidine kinase Hik33 is an important participant in cold-signal transduction in cyanobacteriaNorio Murataa,* and Dmitry A. Losb

aNational Institute for Basic Biology, Myodaiji, Okazaki, 444-8585, JapanbInstitute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, 127276 Moscow, Russia

Correspondence

*Corresponding author,

e-mail: [email protected]

Received 5 September 2005; revised 17

September 2005

doi: 10.1111/j.1399-3054.2005.00608.x

Acclimation of living organisms to cold stress begins with the perception andtransduction of the cold signal. However, traditional methods failed toidentify the sensors and transducers of cold stress. Therefore, we combinedsystematic mutagenesis of potential sensors and transducers with DNAmicroarray analysis in an attempt to identify these components in the cya-nobacterium Synechocystis sp. PCC 6803. We identified histidine kinaseHik33 as a potential cold sensor and found that Hik33 participates in theregulation of the expression of more than 60% of the cold-inducible genes.Further study revealed that Hik33 is also involved in the perception ofhyperosmotic stress and salt stress and transduction of the signals.Complexity of responses to cold and other environmental stresses isdiscussed.

Introduction

Decreases in ambient temperature reduce enzymaticactivities and ultimately depress various physiologicalactivities. When the temperature changes suddenly andsignificantly, organisms often fail to survive. When suchchange is gradual, organisms can acclimate to theirenvironment by sensing the shift in temperature andexpressing large numbers of previously unexpressedgenes, with resultant synthesis of specific proteins andmetabolites that participate in protection against lowtemperature (Fig. 1). Acclimation begins with the per-ception of the shift in temperature and transduction ofthe resultant signal. Organisms and/or individual cellsappear to be equipped with sensors and signal transdu-cers that perceive and transduce cold signals. Thisreview focuses on the initial events in cold-induciblegene expression, describing our analysis of potential

sensors and transducers of cold stress in cyanobacteria(also reviews by Los and Murata 2002, 2004, and byMikami and Murata 2003).

Unicellular cyanobacteria are particularly suitable forstudies of stress responses at the molecular level. Thegeneral features of their plasma and thylakoid mem-branes resemble those of the chloroplasts of higherplants in terms of both lipid composition and assemblyof membranes. Thus, cyanobacteria appear to providepowerful model systems for studies of the molecularmechanisms of acclimation to low temperature(Murata and Wada 1995).

Some strains of cyanobacteria, such as Synecho-cystis sp. PCC 6803 (hereafter, Synechocystis),Synechococcus sp. PCC 7942 and Synechococcus sp.PCC 7002, are naturally competent, incorporating for-eign DNA that is efficiently integrated into their

Abbreviations – Hik, histidine kinase; Rre, response regulator.

This paper is dedicated to Dr Marilyn Griffith, who passed away on February 19th, 2005, to recognize her outstanding

contribution to research on cold stress in plants.

Physiol. Plant. 126, 2006 17

Physiologia Plantarum 126: 17–27. 2006 Copyright � Physiologia Plantarum 2006, ISSN 0031-9317

genomes by homologous recombination (Haselkorn1991, Williams 1988). Therefore, many researchershave used cyanobacteria for the production of mutantswith disrupted genes of interest (for review, see Vermaas1998).

The Synechocystis genome was sequenced in 1996(Kaneko et al. 1996) with subsequent publication of thegenome sequences of other cyanobacteria (see Murataand Suzuki 2005 for a complete list with references).Genome sequences provide basic information that canbe exploited for the genome-wide study of gene expres-sion. A commercially available genome-wide DNAmicroarray for analysis of gene expression inSynechocystis (Takara Bio Co., Ohtu, Japan) covers3079 of the 3165 (97%) genes on the chromosome ofSynechocystis (99 genes for transposases are excludedfrom this calculation). There are also 397 genes onplasmids 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 Synechocystis

We used the DNA microarray from Takara to character-ize the genome-wide expression of genes in response

to environmental stresses, starting with cold stress(Suzuki et al. 2001). The expression of a largenumber of genes was enhanced in response to coldstress, while that of another large number of genes wasrepressed.

Some researchers have inferred that cold stressinduces cellular dehydration that is essentially identicalto that induced by hyperosmotic stress. To examinewhether Synechocystis recognizes these kinds of stresssimilarly, we compared the effects of cold stress andhyperosmotic stress by DNA microarray analysis(Fig. 2; Mikami et al. 2002). These two kinds of stressenhanced, in common, the expression of genes for highlight-inducible proteins (hliA, hliB, and hliC), for rarelipoprotein A (rlpA), for DNA mismatch repair protein(mutS), for a sigma factor (sigD), and for proteins withother functions. However, only cold stress enhanced theexpression of the rbpA gene for an RNA-binding protein,the ndhD2 gene for NADH dehydrogenase subunit 4,the crhR gene for an ATP-dependent RNA helicase, thefus gene for translation elongation factor EF-G, the feoBgene for ferrous iron transport protein, the infB gene fortranslation initiation factor IF-2, and various genes forproteins of known and unknown function. By contrast,only hyperosmotic stress enhanced the expression ofgenes for heat-shock proteins (hspA, dnaK2, groEL2,and clpB1), for the synthesis of glucosylglycerol (ggpSand glpD), for the synthesis of lipids and lipoproteins(fabG and repA), and for various other proteins (htrA,

Cold stress

Sensor

mRNAs

Proteins - enzymes

Metabolites

genes

Signaltransducer

Acclimation to cold conditions

Fig. 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, nusGsecE, folK, slr0082,slr0616, slr0236,slr1927, sll1611,sll0086

hspA, clpB1, fabGhtrA, spkHsigB, sodB, hik34htpG, dnaK2, dnaJgroES, groEL, groEL2ggpS, glpDgloA, repAsll0528, sll0846, slr1963slr0959, sll1884slr1603, slr1915slr0967, sll0939……

4020

5131

11

hliA, hliBhliC, rlpA,mutS, sigD,slr1544, sll1541sll1862, sll1863sll1483

HyperosmoticCold

Fig. 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

20 min after a shift in growth temperature from 34� C to 22� C (cold

stress) and after addition of sorbitol at 0.5 M (hyperosmotic stress).

Adapted, with permission, from Mikami et al. (2002) with inclusion of

recent results from authors’ laboratory.

18 Physiol. Plant. 126, 2006

spkH, sodB, htpG, and gloA). Thus, cold and hypero-smotic stress each induced expression of a number ofspecific genes, while both stresses induced expressionof a relatively small number of common genes (Fig. 2),suggesting that Synechocystis recognizes cold stressand hyperosmotic stress as different signals via specificsignal-transduction pathways.

Two-component systems: positive regulationand negative regulation

The 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 sensordomain, and then a conserved histidine residue withinthe 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 inthe receiver domain of the cognate Rre. Upon phos-phorylation, the conformation of the Rre changes,allowing the binding of the Rre to promoter regions ofgenes that are located downstream in the acclimationpathway (Koretke et al. 2000).

In E. coli and B. subtilis, the genes for each Hik and itscognate Rre are generally located close to one anotheron the chromosome. Moreover, these genes are oftenlocated near functionally related genes.

Two-component systems are found also in cyanobac-teria (Mizuno et al. 1996). However, genes for Hiks andRres in Synechocystis are, in most cases, distributedsomewhat randomly on the chromosome. Among the44 genes for Hiks, 14 are located near genes for poten-tially cognate Rres, whereas genes for the other 30 Hiksare located far from any rre genes. Thus, it is difficult topredict the pairs of Hiks and Rres that might function asindividual two-component systems. Therefore, we sys-tematically mutated the genes for all the potential sen-sors and transducers of environmental signals andexamined gene expression under various conditionsusing DNA microarrays.

Synechocystis has 3661 putative genes, of which 47encode Hiks and 45 encode Rres (http://www.kazusa.or.jp/cyanobase/Synechocystis/index. html). There are44 genes for Hiks on the chromosome, which wenamed hik1 through hik44. We deduced that threeputative Hiks, namely, Hik11, Hik17, and Hik37,might be inactive as Hiks, because they lack the con-served histidine residue in the Hik domain. Hik32 mightalso 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 thestrain that was used for genome sequencing and sys-tematic mutagenesis (Okamoto et al. 1999). The genefor the Hik encoded by pSYSM, a plasmid harboured bySynechocystis, was designated hik45, and the genes forHiks encoded by pSYSX, another plasmid, were desig-nated hik46 and hik47. There are 42 chromosomalgenes for Rres and two and one on plasmids pSYSXand pSYSM, respectively. We designated the 42 chro-mosomal genes for rre1 through rre42. The rre genes onpSYSM and pSYSX were designated rre43 and rre44 and45, respectively.

We inactivated each putative hik gene inSynechocystis by inserting a spectinomycin-resistancegene cassette to create a gene-knockout library (Suzukiet al. 2000; CyanoMutants, http://www.kazusa.or.jp/cyano/Synechocystis/mutants/). This library has provedto be a powerful tool for the identification of sensors ofvarious stimuli and the corresponding signal transducersin Synechocystis (Marin et al. 2003, Paithoonrangsaridet 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-typecells and in each line of hik mutant cells with DNAmicroarrays in an attempt to identify the Hiks involvedin the regulation of expression of stress-inducible genes.

Regulation of gene expression in response to stresscan be positive or negative (for details, see Murata andSuzuki 2005). In positive regulation, a two-componentsystem is inactive under non-stress conditions. Genescontrolled by such systems are silent or expressed.When cells are exposed to the appropriate environmen-tal stress, the two-component system is activated (byphosphorylation in response to the stress), and then theactivated system enhances the expression of genes thatare silent under non-stress conditions or represses theexpression of genes that are expressed under non-stressconditions (Murata and Suzuki 2005). Most stress-inducible gene expression in Synechocystis is positivelyregulated.

In negative regulation, the two-component system isactive under non-stress conditions, and the expressionof the genes controlled by such a system is eitherenhanced or repressed. Under appropriate environmen-tal stress, the two-component system becomes inactive.Genes that are expressed or repressed under non-stressconditions become silent or are released from repres-sion, respectively. Levels of expression of genes fall inthe former case and rise in the latter.

Changes in phenotype due to mutations in Hiks andRres, which reflect the effects of such two-componentsystems on gene expression, differ between positiveregulation and negative regulation. A knockout


mutation in either the Hik or the Rre in a two-compo-nent system for negative regulation has marked effectson gene expression. The expression of genes controlledby a negatively regulating two-component system iseither enhanced or repressed under non-stress condi-tions. Therefore, a specific signal-transduction pathwaywith a specific Hik and a specific Rre can be identifiedwith relative ease in cases of negative regulation.

By contrast, a knockout mutation in a Hik or an Rre ina two-component system that operates via positive reg-ulation does not have a significant effect on geneexpression under non-stress conditions. In such a sys-tem, the identification of the Hik and the Rre in aspecific signal-transduction pathway requires thescreening of knockout libraries of hik and rre genesunder 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 cellsthat had been grown under normal conditions. No sig-nificant alterations in gene expression were evident inmost of the mutants. However, three mutants, Dhik27,Dhik34, and Dhik20, each with a mutation in the indi-cated Hik, were unique, because, in these mutants, theexpression of some genes was enhanced and that of someothers was repressed, indicating that Hik27, Hik34, andHik20 might be involved in signal transduction via thenegative regulation of gene expression.

We compared gene expression in Dhik27 cells withthat in wild-type cells under normal conditions(Yamaguchi et al. 2002). Marked changes, with induc-tion factors higher than 10, due to mutation of the hik27gene (slr0640) were recognized only in the expressionof three genes, namely, mntC, mntA, and mntB, whichconstitute the mntCAB operon that encodes subunits ofthe ABC-type Mn2þ transporter (Bartsevich and Pakrasi1995, 1996). Under normal growth conditions, Hik27might transduce a signal that represses the expression ofthe mntCAB operon. Moreover, disappearance of thissignal, due to inactivation of Hik27, might allow theexpression of the mntCAB operon. This scenario corre-sponds to the scheme outlined for negative regulation.We examined the effects of mutation of various rregenes on gene expression in mutant cells. The mutationin Drre16 cells enhanced the expression of only themntC, mntA, and mntB genes. This phenomenon wasvery similar to the change in gene expression detectedin the Dhik27 mutant. In its active form, Rre16 mighthave repressed expression of the mntCAB operon. Then,inactivation of Rre16 in Drre16-mutant cells eliminatedthe repressive effect of Rre16, allowing the expression of

the mntCAB operon. These findings suggested thatHik27 and Rre16 might constitute a two-componentsystem, acting as the sensor and signal transducer ofMn2þ deficiency. Ogawa et al. (2002) identified thistwo-component system independently by ‘traditional’methods.

Mutation of Hik34 altered the genome-wide expres-sion of genes under normal conditions, namely, at agrowth temperature of 34� C (Suzuki et al. 2005). InDHik34 cells, levels of transcripts of heat-shock genes,such as htpG, groES, and groEL1, were elevated, sug-gesting that Hik34 might act as a negative regulator ofthe expression of these genes during normal growth.Because Hik34 appeared to be a negative regulator ofheat-shock-responsive genes, we postulated that itsoverexpression should result in repressed expression ofthese genes. We observed that the effect of overexpres-sion of hik34 was the opposite of that of inactivation ofhik34 on the expression of heat-shock genes at thenormal growth temperature (Suzuki et al. 2005). Thisobservation is consistent with the hypothesis that Hik34is important in the regulation of expression of heat-shock genes.

Positive regulation by Hik33 in response tolow temperature

Mutation of the 42 other hik genes did not inducessignificant changes in gene expression. Therefore, it islikely that the Hiks encoded by these 42 genes regulategene expression in a positive manner. To confirm thishypothesis, we screened our library of hik mutantsunder specific kinds of stress.

To monitor the inducibility of the cold-inducible desBgene for the o3 desaturase, we generated the pdesB::luxstrain of Synechocystis, in which the promoter region ofthe desB gene was ligated to the coding region ofthe luxAB gene for a bacterial luciferase (Suzuki et al.2000). Thus, we could use luciferase activity as anindicator of cold-inducible changes in the activity ofthe desB promoter. Then we inactivated separatelythe gene for each Hik in pdesB::lux cells by insertinga spectinomycin-resistance cartridge (Spr ), creatinga gene-knockout library (Suzuki et al. 2000). Wescreened the members of this library for loss of coldinducibility of gene expression by monitoring luciferaseactivity at a low temperature. Only pdesB::lux/DHik33and pdesB::lux/DHik19, with disruption of the genesfor Hik33 and Hik19, respectively, exhibited reducedability to activate luciferase at low temperature,suggesting that Hik33 and Hik19 might be involvedin the perception and tranduction of cold signals.


DNA microarray analysis of Dhik33 cells indicatedthat Hik33 regulates the expression of 21 of 36 cold-inducible genes, with induction factors higher than 3.0(Fig. 3; Suzuki et al. 2001). These 21 genes includendhD2, hliA, hliB, hliC, feoB, crp, and genes for pro-teins of unknown function. By contrast, 15 of the 36cold-inducible genes were not regulated by Hik33.

Therefore, we can deduce that Synechocystis mighthave at least one other pathway for cold-signal trans-duction. DNA microarray analysis of the expression ofgenes in Dhik19 cells indicated that Hik19 is unlikely tobe a cold sensor.

To identify the Rre that is located downstream ofHik33, we screened an Rre-knockout library by RNAslot-blot hybridization using some of the cold-induciblegenes controlled by Hik33 as probes. We identifiedRre26 as 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 thatHik33 might negatively regulate the expression of a setof photosynthetic and high light-responsive genes. Theirhypothesis was based on changes in the global expres-sion of genes under normal conditions and the comple-mentation of the Dhik33 mutation in Synechocystis bythe homologous nblS gene from Synechococcus withrespect to the light-induced expression of hli genes, asmonitored by Northern blotting. However, because thecomplementation test did not examine the genome-wide expression of genes, it is possible that theirDHik33 mutant cells had, in addition to the mutationin hik33, a further mutation that might have producedchanges in gene expression under normal conditions.

To confirm our conclusion that Hik33 is a positiveregulator of signal transduction, we replaced the entireopen-reading frame of the hik33 gene with a spectino-mycin-resistance gene cassette that contained the Osequence, which is a strong terminator of transcriptionand inhibits the read-through of inserted genes on bothsides of the cassette (our unpublished work). The growthrate of cells with deletion of hik33 was similar to that ofthe wild-type and the insertion mutant of Hik33, contra-dicting the inferences made by Hsiao et al. (2004). DNAmicroarray analysis of the deletion mutant, comparingthe pattern of gene expression with that of the insertionmutant under normal conditions, confirmed the absenceof the major changes in gene expression reported byHsiao 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 hyperosmoticstress

We demonstrated previously that the perception ofhyperosmotic stress involves Hik33 and other unknowncomponents (Mikami et al. 2002). Later when we usedthe DNA microarray to investigate the impact of muta-tions in each Hik on changes in gene expression undersalt stress (Marin et al. 2003), we found that the

Cytoplasmic membrane

?

slr1927sll1611slr0955slr0236sll0494slr1436slr1974

Hik33

Rre26

ndhD2hliAhliBhliCfusycf39sigD

slr1747ssr2016slr0400sll1911slr0401sll0815sll1770

crhRrlpArbpAcbiOcbiQmutSdesBslr0082

feoBcrtPslr1544sll1483sll1541sll0086slr0616

Cold stress

Total: 21 Total: 15

A

htrA

Hik10?

?

Hyperosmotic stress

Hik34

Rre1

sll0939slr0967

Hik41

Hik33

fabGhliAhliB hliCgloAsigDsll1483slr1544ssr2016ssl3446sll1541

rlpArepAglpDsll1863sll1862sll1772slr0581

hspAclpB1sodBhtpGdnak2spkHgroESgroEL1groEL2dnaJ

Rre3Rre1 Rre17Rre31

sigBsll0528slr1119slr0852ssr3188

sll0846slr1963slr0959sll1884slr1603slr1915ssl2971slr1413

ssr1853slr0112sll0294slr0895slr1501sll0293sll0470

Hik2

Total: 11 Total: 19 Total: 5 Total: 2 Total: 1 Total: 14

Cytoplasmic membrane

B

Hik16

Fig. 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 3.0 are included in these schemes. Minor discrepan-

cies with respect to cold-inducible genes between this figure and Fig. 2

are 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 ofNaCl was significantly affected in DHik33-, DHik34-,DHik16-, and DHik41-mutant cells. In each of thesemutants, several genes were no longer induced by salt,or their inducibility by salt was markedly reduced.

Because hyperosmotic stress and salt stress mightaffect some aspects of the physiology of cyanobacterialcells similarly, we examined whether these Hiks mightalso regulate gene expression under hyperosmoticstress. DNA microarray analysis demonstrated thatthey are indeed involved in hyperosmotic-signal trans-duction. Fig. 3B shows that they regulate the expressionof 38 of 52 hyperosmotic stress-inducible genes(Paithoonrangsarid et al. 2004).

Screening our Rre-knockout library by RNA slot-blothybridization and with DNA microarray, we identifiedfour Rres, namely, Rre31, Rre1, Rre3, and Rre17, thatare 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 Rre1 and possibly Hik2, appeared tobe involved in the perception of hyperosmotic stress andtransduction of the signal (Paithoonrangsarid et al.2004). Fig. 3B shows a hypothetical model of the hyper-osmotic signal-transducing systems that involve theseHiks and Rres, including the hyperosmotic stress-inducible genes that are controlled by the individualHik-Rre systems.

The Hik33-Rre31 two-component system regulatesthe expression of 11 hyperosmotic stress-induciblegenes. Inactivation of either Hik33 or Rre31 resulted inthe elimination of or a marked reduction in the hyper-osmotic stress-inducible expression of these genes, indi-cating that Hik33 and Rre31 are tightly coupled in thesignal-transduction pathway. The Hik10-Rre3 two-component system regulates the hyperosmotic stress-inducible expression of only htrA, which encodes aserine protease.

The Hik16-Hik41-Rre17 system regulates the hyper-osmotic stress-inducible expression of sll0939 andslr0967. Inactivation of Hik16, of Hik41, or of Rre17eliminated the expression of these genes, suggesting thatHik16, Hik41, and Rre17 are all essential for the per-ception of hyperosmotic stress and for transduction ofthe corresponding signal. Hik41 probably acts down-stream of Hik16, because Hik41 is a hybrid-type Hikthat contains both a signal-receiver domain and a Hikdomain, whereas Hik16 is a typical Hik with a Hikdomain and potential sensory domain that, hypotheti-cally, spans the membrane seven times. It is also possi-ble that Hik16 and Hik41 might perceive hyperosmoticstress as a complex.

The Hik34-Rre1 system regulates the expression of 19hyperosmotic stress-inducible genes (for heat-shock pro-teins and for proteins of unknown function). The Hik2-Rre1 system regulates the expression of five genes thatinclude the sigB gene for a sigma factor. Rre1 mightperceive hyperosmotic signals from both Hik34 andHik2. However, the regulated genes are specific toeither His34 or Hik2 (Fig. 3B; Paithoonrangsarid et al.2004).

DNA microarray analysis revealed that expression of14 of the 52 hyperosmotic stress-inducible genes wasnot controlled by any of the five Hiks and four Rresdiscussed above (Fig. 3B). The signals, due to hyperos-motic stress, that induce the expression of these genesare probably perceived by unknown mechanisms thatdiffer 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 hyperosmoticstress. However, the genes controlled by the individualpathways are different (Shoumskaya et al. 2005), asdiscussed below.

His33 is a multifunctional regulator

As described above, Hik33 is involved in the perceptionand transduction of the cold signal and the hyperosmotic-stress signal (Fig. 3). However, the cold-responsivegenes controlled by Hik33 are not identical to thehyperosmotic stress-responsive genes controlled by thesame system. Fig. 4 shows that eight genes, includinghliA, hliB, hliC, and sigD, are induced by both kinds ofstress under the control of Hik33. However, Hik33 reg-ulates the expression of eight other genes, includingndhD2, fus, crtP, and feoB, in response to cold stressspecifically, and not to hyperosmotic stress, whereas itregulates the expression of three other genes, includingfabG 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 distinctsignals by a sensory system that includes Hik33. Inaddition, recent studies (Hsiao et al. 2004, Tu et al.2004) suggest that Hik33 might be involved in responseto light stress. Moreover, a homolog of Hik33 inSynechococcus is involved in sensing nutritional deficits(van Waasbergen et al. 2002).

Another level of compexity reflects the differentialinvolvement of Rres in signal transduction and geneexpression. Microarray analysis indicated that Rre26 is


involved in cold-signal tranduction (Fig. 3A), whereasRre31 is involved in hyperosmotic- and salt-signal trans-duction (Fig. 3B; Paithoonrangsarid et al. 2004,Shoumskaya et al. 2005). Moreover, eight genesrespond, in common, to cold stress and hyperosmoticstress via Rre26 and Rre31, respectively. Our observa-tions suggest that the mechanisms for perception of coldstress and hyperosmotic stress by Hik33 are complexand that as-yet-unidentified components exist that pro-vide the sensory systems with their respective activitiesfor induction of responses specific to each individualtype of stress.

Hik33 perceives the cold signal viarigidification of membrane lipids

The Hik33 sensory kinase includes a type-P linker, aleucine zipper, and a PAS domain (Fig. 5A; Los andMurata 1999, 2002, 2004, Mikami and Murata 2003).The type-P linker contains two helical regions in tandemthat are assumed to transduce stress signals via intramo-lecular structural changes that result from interactionsbetween the two helical regions and lead to intermole-cular dimerization of membrane proteins (Aravind et al.2003, Williams and Stewart 1999). In Hik33, cold stressmight promote a conformational change in the type-Plinker, with subsequent activation of Hik33 via 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, Mikamiand Murata 2003). Because it has been suggested that

changes in membrane fluidity might be involved in thesensing of temperature (Los and Murata 2000, Murata andLos 1997), it is likely that the transmembrane domains ofHik33 can recognize changes in membrane fluidity at lowtemperatures (Los and Murata 1999, 2000, 2004).

In 1996, we produced a series of mutants in whichthe extent of unsaturation of fatty acids is modified in astep-wise manner (Tasaka et al. 1996). In one suchmutant, the desA and desD genes for the D12 and D6fatty acid desaturases, respectively, are inactive as aresult of targeted mutagenesis. The desA–/desD–-doublemutant synthesize only a saturated C16 fatty acid and amono-unsaturated C18 fatty acid, regardless of growthtemperature, whereas wild-type cells synthesize di-unsaturated and tri-unsaturated C18 fatty acids in addi-tion to the mono-unsaturated C18 fatty acid (Tasakaet al. 1996). FTIR spectrometry revealed that the doublemutation of the desA and desD genes rigidified theplasma membrane of Synechocystis at physiologicaltemperatures (Szalontai et al. 2000).

ndhD2, fus, crtP, ycf39, feoB, slr1747, sll0086, slr0616 fabG,

gloA,ssl3044

8

3

8

1121

Cold; Hik33-Rre26Hyperosmotic;Hik33-Rre31

hliA, hliBhliC, sigDslr1544ssr2016, sll1483sll1541

Fig. 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 20 min after a

shift in growth temperature from 34� C to 22� C (cold stress) and after

addition of sorbitol at 0.5 M (hyperosmotic stress).

TM1 TM2Type-P

PAS His kinase

1 3248

201217

220270

310350

400 600 663

A

B

Type-P

PAS

His kinaseH H

H

LZ

LZ

Fig. 5. A hypothetical scheme for the structure of Hik33. (A) Domain

structure of Hik33. (B) A putative dimeric form of Hik33 and 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. TM1 and TM2, Transmembrane domains; LZ,

leucine zipper; Type-P, a type-P linker domain; and PAS, PAS domain

that contains amino acid motifs Per, Arnt, Sim, and phytochrome

(Taylor and Zhulin, 1999); H in circles, histidine residues that can be

phosphorylated in response to cold stress.


Using microarray, we examined the effects of theabove-described membrane rigidification on the expres-sion of genes at low temperature in Synechocystis(Inaba et al. 2003). We monitored changes in geneexpression in wild-type and desA–/desD– cells aftergrowth at 34� C and subsequent incubation at 22� Cfor 30 min. In wild-type cells, cold stress more thandoubled the levels of expression of 168 genes. IndesA–/desD– cells, in addition to the enhanced expres-sion of these same 168 cold-inducible genes, weobserved enhanced expression of 96 additional genes.Thus, rigidification of membrane lipids apparentlyenhanced the response of gene expression to low tem-perature in Synechocystis. By contrast, under isothermalconditions, the double mutation had no significanteffect on gene expression.

We divided cold-inducible genes into three groupsaccording to the effects of the double mutation (Inabaet al. 2003). The first group included genes that were notinduced by low temperature in wild-type cells but werestrongly induced by low temperature in desA–/desD–

cells. The second group included genes whose low-temperature inducibility was moderately enhanced bythe double mutation. The third group included geneswhose inducibility by low temperature was unaffectedby the double mutation. The response to low tempera-ture of the expression of the genes in these three groupsmight be regulated by different mechanisms withrespect to membrane rigidity. Induction of expressionof the genes in the first group might require greaterrigidification of membrane lipids than the low-temperatureresponses of genes in the second and third groups. Therigidification of membrane lipids did not enhance thecold inducibility of genes in the third group, perhapsbecause the rigidity of membranes in wild-type cells issufficient at low temperatures for the maximum inductionof expression of these genes.

To examine whether Hik33 might regulate the cold-responsive gene expression that depends on membranerigidity, we examined genome-wide gene expression indesA–/desD–/hik33– cells, in which the hik33 gene hadbeen mutated in addition to mutation of the desA anddesD genes. Mutation of Hik33 abolished or signifi-cantly reduced the inducibility by low temperature of10 of the 17 genes in the second group and of seven of25 genes in the third group. By contrast, mutation ofHik33 had no significant effect on the low-temperatureinducibility of genes in the first group. These resultsindicate that Hik33 regulates the expression of manygenes in the second and third groups. They also suggestthat the activity of Hik33 in the sensing of low tempera-ture depends on membrane rigidity and that there are atleast two other cold sensors, one of which depends on

membrane rigidification, while the other functions inde-pendently of membrane rigidity (Inaba et al. 2003).

Cold sensors and cold-signal transducers inother organisms

Aguilar et al. (2001) identified DesK of Bacillus subtilisas a cold-sensing Hik and DesR as the cognate Rre thatregulate the cold-inducible expression of the des genefor D5 desaturase. The desK and desR genes form anoperon on the genome of B. subtilis.

DesK is a membrane-bound protein with four trans-membrane domains and a Hik domain. However, incontrast to Hik33, DesK lacks the PAS and leucinezipper domains. DesK is a bifunctional enzyme withkinase and phosphatase activities. It has been suggestedthat DesK is involved in two signalling reactions: phos-phorylation in response to membrane rigidification anddephosphorylation in response to membrane fluidiza-tion (Mansilla et al. 2004). In fact, the carboxy-terminalportion of DesK (DesKC) acts as an autokinase as well asa phosphatase; the phosphoryl group of phosphorylatedDesKC is transferred to DesR. The resultant phosphory-lated DesR can be dephosphorylated in the presence ofDesKC in vitro. These findings suggest that DesK has theability to modify DesR through both its kinase and itsphosphatase activities, depending on the physical stateof the membrane. It is likely but, as yet, unproved thattransmembrane segments of DesK sense changes inmembrane fluidity due to changes in temperature(Albanesi et al. 2004).

DesR binds specifically to the promoter region of thedes for the D5 desaturase. Induction of expression of desin B. subtilis by the DesK-DesR two-component systemis inhibited by exogenous unsaturated fatty acids orisoleucine (Aguilar et al. 2001; Cybulski et al. 2002),suggesting the presence of a feedback loop between thefunction of the sensor and the extent of fatty acid unsa-turation. Cybulski et al. (2004) demonstrated that DesK,DesR, and the promoter region of the des gene interactdirectly with one another. The dephosphorylated formof DesR is unable to bind to a regulatory region of thedes gene. DesK phosphorylates dimeric DesR, whichbecomes a tetramer, and binds upstream of the promo-ter of the des gene in a sequence-specific manner,with activation of des through recruitment of RNApolymerase to the promoter. Thus, the DesK-DesRtwo-component system regulates the expression ofcold-inducible des, allowing the cell to optimize thefluidity of membrane phospholipids (Cybulski et al.2004, Mansilla and de Mendoza 2005).

The DesK-DesR system regulates the cold-inducibleexpression of the des gene but of no other genes. By


contrast, the cyanobacterial Hik33 sensor regulates theexpression of more than 50 cold-inducible genes(Mikami et al. 2002, Suzuki et al. 2001).

In plants, the discovery of the cold-regulation path-way that involves CBF/DREB led to further progress inthe characterization of cold-signal transduction (reviewsby Guy 1999, Thomashow 1999, Yamaguchi-Shinozakiand Shinozaki 2005, Xiong et al. 2002). Analysis of thetranscriptional control of two cold-inducible genes(rd29A and cor15a) in Arabidopsis thaliana led to theidentification of a cold-responsive element, the CRT/DRE [(C-repeat)/(dehydration responsive element)], intheir promoters (Shinozaki and Yamaguchi-Shinozaki2000). Members of a family of AP2-domain-transcriptionfactors, namely, DREB1 (DRE-binding protein) and CBF(CRT-binding factor), bind to the CRT/DRE element andactivate transcription. Expression of genes for these tran-scription factors is rapidly induced on cold treatment ofplants. Moreover, at normal temperatures, overexpres-sion of CBF1, CBF2, or CBF3 in transgenic A. thalianaenhanced the expression of 41 genes, 30 of which hadbeen identified as cold-inducible genes in wild-typeplants (Fowler and Thomashow 2003). Thus, CBF tran-scription factors might regulate the expression not onlyof cold-inducible genes but also of genes whose expres-sion is induced by other signals. By contrast, some cold-inducible genes do not appear to be controlled via theCBF pathway. Thus, other regulatory systems might existfor the cold-inducible regulation of approximately 60genes (Fowler and Thomashow 2003).

Despite the accumulation of important results aboutthe cold regulation of gene expression, little is knownabout the temperature sensors in plants. Sensors andtransducers of cold signals in plants remain to beidentified.

Conclusion

Genome-based systematic analysis is a powerful techni-que for the identification of Hiks and Rres that areinvolved in the perception and transduction of coldsignals and other kinds of stress signals. Using thisapproach, we showed, with relative ease, that Hik33regulates the expression of most of the cold-induciblegenes in Synechocystis, that membrane rigidification isintimately involved in the sensing of low temperature,and that Hik33 is also involved in the perception andtransduction of other types of stress. We must nowdetermine how the same Hik can perceive and trans-duce more than one kind of environmental signal andregulate the expression of different sets of genes (Losand Murata 2002, 2004, Mikami et al. 2002,Shoumskaya et al. 2005). Our findings cannot be

explained by the current model of two-component sys-tems, in which a Hik perceives a specific signal andregulates the expression of a particular set of genes viathe phosphorylation-dependent activation (or inactiva-tion) of a cognate Rre. It is likely that as-yet-unidentifiedcomponents are important in determining the specificityof the responses to individual types of stress. It is alsopossible that the sensors of environmental signals arehighly organized protein complexes, in which Hiks,Rres, and various unidentified components are some-how associated. To identify these components, weshall have to develop new techniques, which, mostprobably, will also exploit the information encoded inthe genomes of cyanobacteria and other organisms.

Acknowledgements – This work was supported by aGrant-in-Aid for Scientific Research on Priority Areas(no. 14086207) from the Ministry of Education,Science, Sports and Culture of Japan to N.M. It wasalso supported by grants from the Russian Foundationfor Basic Research (nos. 03-04-48581 and 05-04-50883)and by a grant from the ‘Molecular and Cell BiologyProgram’ of the Russian Academy of Sciences to D.A.L.

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Edited by C. Guy


Histidine kinase Hik33 is an important participant in cold-signal transduction in cyanobacteria

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