the histidine kinase hik33 perceives osmotic stress and cold stress in synechocystis sp. pcc 6803
TRANSCRIPT
Molecular Microbiology (2002)
46
(4), 905–915
© 2002 Blackwell Publishing Ltd
Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Science, 200246Original Article
Osmostress and cold sensor Hik33 in SynechocystisK. Mikami et al.
Accepted 5 August, 2002. *For correspondence. E-mail [email protected]; Tel. (
+
81) 564 55 7600; Fax (
+
81) 564 54 4866.
The histidine kinase Hik33 perceives osmotic stress and cold stress in
Synechocystis
sp. PCC 6803
Koji Mikami,
1
Yu Kanesaki,
1
Iwane Suzuki
1,2
and Norio Murata
1,2
*
1
Department of Regulation Biology, National Institute for Basic Biology, Okazaki, Japan.
2
Department of Molecular Biomechanics, Graduate University for Advanced Studies, Okazaki, Japan.
Summary
The stress imposed on living organisms by hyperos-motic conditions and low temperature appears to beperceived via changes in the physical state of mem-brane lipids. We compared genome-wide patterns oftranscription between wild-type
Synechocystis
sp.PCC 6803 and cells with a mutation in the histidinekinase Hik33 using a DNA microarray. Our resultsindicated that Hik33 regulated the expression of bothosmostress-inducible and cold-inducible genes. Therespective genes that were regulated by Hik33 underhyperosmotic and low-temperature conditions were,for the most part, different from one another. However,Hik33 also regulated the expression of a set of geneswhose expression was induced both by osmoticstress and by cold stress. These results indicate thatHik33 is involved in responses to osmotic stress andlow-temperature stress but that the mechanisms ofthe responses differ.
Introduction
Unicellular and multicellular organisms perceive changesin environmental conditions, such as osmolarity and tem-perature, and respond to these changes by regulating theexpression of genes that are crucial for growth and sur-vival under stress conditions (Kempf and Bremer, 1998;Wood, 1999). An increase in osmotic pressure has at leasttwo different effects on cells, namely, a decrease in cellvolume, which in turn deforms the cell membrane, and adecrease in the amount of water in the cell, whichincreases the concentrations of solutes in the cytoplasm(Kempf and Bremer, 1998; Wood, 1999). A downward shiftin temperature also modifies the physical state of mem-
brane lipids (Murata and Los, 1997; Vigh
et al
., 1998).Therefore, it is likely that individual cells perceive osmoticstress and low-temperature stress initially as changes inthe physical state of membrane lipids.
Considerable evidence indicates that pathways for thetransduction of osmotic and low-temperature signals areregulated by two-component systems (Perraud
et al
.,1999; Urao
et al
., 2000; Browse and Xin, 2001; Sakamotoand Murata, 2002), and histidine kinases have been iden-tified as osmosensors in both prokaryotic and eukaryoticcells. The proteins EnvZ and Sln1 have been studiedextensively as osmosensors in
Escherichia coli
and
Saccharomyces cerevisiae
respectively (Mizuno
et al
.,1982; Maeda
et al
., 1994; 1995). In
Dictyostelium discoi-deum,
DocA appears to play a central role in osmostresssignalling (Schuster
et al
., 1996). Moreover, a histidinekinase from
Arabidopsis thaliana
complemented the func-tion of Sln1 in yeast (Urao
et al
., 1999). In contrast, histi-dine kinases that act as cold sensors have been identifiedin prokaryotes, such as Hik33 (Sll0698) in
Synechocystis
sp. PCC 6803 (hereafter
Synechocystis
; Suzuki
et al
.,2000) and DesK in
Bacillus subtilis
(Aguilar
et al
., 2001).Recently, Ca
2
+
-permeable channels of the transient recep-tor potential (TRP) family were identified as cold sensorsin the mammalian nervous system (McKemy
et al
., 2002;Peier
et al
., 2002). The above-mentioned sensors ofosmotic stress and cold stress, with the exception ofDocA, are integral membrane proteins and, thus, it ispossible that they might recognize changes in the physicalstate of cell membranes that are induced by osmotic andlow-temperature stress. Indeed, it has been reported thatthe activities of the osmosensor KdpD and the heat sen-sor CpxA of
E. coli
, both of which are membrane-boundhistidine kinases, are influenced by the physical state ofthe cell membrane (Mileykovskay and Dowhan, 1997;Stallkamp
et al
., 1999).The close relationship between the physical state of the
cell membrane and the sensing of stress (for a review, seeLos and Murata, 2000) led us to ask whether the coldsensor Hik33 of
Synechocystis
might also perceiveosmotic stress. A DNA microarray, based on the completegenome of
Synechocystis,
provides a powerful tool for theanalysis of global gene expression (Hihara
et al
., 2001;Suzuki
et al
., 2001; Kanesaki
et al
., 2002). In the presentstudy, we used a DNA microarray to examine the contri-
906
K. Mikami, Y. Kanesaki, I. Suzuki and N. Murata
© 2002 Blackwell Publishing Ltd,
Molecular Microbiology
,
46
, 905–915
bution of Hik33 to the regulation by osmotic stress of geneexpression. Our results indicate that the cold sensor Hik33also acts as a sensor of osmotic stress in
Synechocystis
but that the mechanisms of its involvement in the sensingof cold stress and osmotic stress are not identical.
Results
Involvement of Hik33 in cell growth under hyperosmotic conditions
If Hik33 is involved in osmostress signalling, we wouldexpect that inactivation of the
hik33
gene would affect cellgrowth under hyperosmotic conditions. We observed thatcell growth in the presence of 0.5 M sorbitol was markedlyinhibited in
D
Hik33 cells, whereas sorbitol at the sameconcentration had a much smaller effect on the growth ofwild-type cells (data not shown). These results suggesteda role for Hik33 in the response of
Synechocystis
toosmotic stress.
Examination of the genome-wide expression of osmostress-inducible genes
Control experiments with DNA microarrays, in whichcDNAs labelled with Cy3 and Cy5 (fluorescent markers)were synthesized independently from the same sample ofRNA prepared from unstressed wild-type cells, indicatedthat the range of internal error corresponded to a differ-ence in levels of expression of up to twofold (Suzuki
et al
.,2001; Kanesaki
et al
., 2002). Therefore, genes whoselevels of expression were induced by osmotic stress toincrease to more than twice the uninduced levels weredefined as osmostress-inducible genes in our DNAmicroarray experiments. Figure 1 shows that an increasein osmolarity had a global effect on gene expression andthere appeared to be 257 osmostress-inducible genesin
Synechocystis
. Thus, approximately 7% of the genes in
Synechocystis
responded positively to osmotic stress. In
S. cerevisiae,
approximately 5% of all genes respond toosmotic stress (Rep
et al
., 2000).
Hik33 is an osmosensor in
Synechocystis
To identify genes whose expression is affected by Hik33upon exposure of cells to osmotic stress, we comparedgenome-wide patterns of transcription between wild-typeand
D
Hik33 cells. The results indicated that the expressionof approximately 80% of all osmostress-inducible geneswas affected to a greater or lesser extent by mutation ofHik33 (Fig. 1A and B; details can be accessed on theinternet at http://www.genome.ad.jp/kegg/kegg2.html).Moreover, as found previously to be the case for cold-
inducible genes (Suzuki
et al
., 2001), the genes that wereinduced by osmotic stress could be divided into threegroups: (i) group 1, genes that were fully regulated byHik33; (ii) group 2, genes that were partially regulatedby Hik33; and (iii) group 3, genes that were not regulatedby Hik33 (Table 1). These observations indicate thatHik33 is an osmosensor and that
Synechocystis
hasadditional osmosensors that regulate the genes in groups2 and 3.
Fig. 1.
Genome-wide patterns of transcription of osmostress-inducible genes.A. Gene expression in wild-type cells that had been exposed to 0.5 M sorbitol for 30 min was compared with that in unstressed cells.B. Gene expression in
D
Hik33 cells that had been exposed to 0.5 M sorbitol for 30 min was compared with that in unstressed cells. Red circles correspond to genes whose osmo-inducibility was depressed in
D
Hik33 cells, and blue circles correspond to genes whose osmo-inducibility was unaffected in
D
Hik33 cells. The assay was repeated three times in independent experiments and essentially the same results were obtained in every case. The data presented here are from one of the experiments. Dashed lines indicate the limit of exper-imental deviations. The number of osmostress-inducible genes in wild-type cells (257) was more than twice that (113) reported by Kanesaki
et al
. (2002). The difference might be due to the improve-ments in the quality of the DNA microarray and to differences in experimental conditions.
Osmostress and cold sensor Hik33 in
Synechocystis 907
© 2002 Blackwell Publishing Ltd,
Molecular Microbiology
,
46
, 905–915
Table 1.
Osmostress-inducible genes and effects of the inactivation of Hik33 on their induction by osmotic stress.
ORF Gene Product
Induction by 0.5 M sorbitol
Wild type
D
Hik33
Genes whose osmotic induction was strongly reduced in
D
Hik33 cells (-fold)sll0330
fabG
3-Ketoacyl-ACP reductase 29.9 3.9sll1483 Cell surface protein 23.3 2.1slr1516
sodB
Superoxide dismutase 12.9 3.9slr1884
trpS
Tryptophenyl-tRNA synthetase 8.1 2.4ssr2595
hliB
High light-inducible protein B 8.0 1.3slr1641
clpB
B subunit of Clp protease 7.1 2.1ssl2542
hliA
High light-inducible protein A 7.0 1.6slr0381
gloA
Lactoylglutathione lyase 5.2 1.3slr1748 Probable phosphoglycerate mutase 5.1 1.8ssl3044 Probable ferredoxin 4.5 1.0sll0416
groEL-2
HSP60 4.4 1.5ssl1633
hliC
High light-inducible protein C 3.5 1.0slr2076
groEL
HSP60 3.5 1.1slr0611
sds
Solanesyl diphosphate synthase 3.1 1.2sll0005 ABC1-like protein 3.0 1.1sll1852
ndkR
Nucleoside diphosphate kinase 3.0 0.6slr1291
ndhD2
NADH dehydrogenase subunit 4 2.8 0.7sll2012
sigD
RNA polymerase sigma factor 2.7 0.9slr0974
infC
Initiation factor IF-3 2.5 1.0
Genes whose osmotic induction was partially reduced in
D
Hik33 cells (-fold)slr1675
hypA
Hydrogenase expression protein 10.0 6.3sll0306
sigB
RNA polymerase sigma factor 9.0 4.1sll0170
dnaK
HSP70 5.3 2.5slr1285
hik34
Sensory histidine kinase 5.0 2.7slr1204
htrA
Serine protease 4.9 3.2sll0430
htpG
HSP90 4.6 2.5slr0753
P
P protein 4.2 2.1sll0680
pstS
Phosphate-binding protein precusor 4.0 1.9sll0789
copR
Response regulator 3.9 2.9slr0093
dnaJ
HSP40 3.6 1.5slr1991
cyaA
Adenylate cyclase 3.5 1.9sll0556 Na
+
/H
+
Antiporter 3.1 1.9slr0007 Sugar-phosphate nucleotidyltransferase 3.0 1.6sll1541 Lignostilbene-
a
,
b
-dioxygenase 2.9 1.7sll1797
ycf21
Unidentified protein 2.9 1.6slr1739
psbW
Photosystem II W protein 2.8 2.0slr2075
groES
HSP10 2.8 1.2slr0853
rimI
Alanine acetyltransferase 2.7 1.9ssl1911
gifA
Glutamine synthase-inactivating factor IF7 2.7 1.8sll0217 FMN-protein 2.7 1.8smr0011
rpl34
50S Ribosomal protein L34 2.7 1.6sll1733
ndhD3
NADH dehydrogenase subunit 4 2.6 1.9slr1192 Zinc-containing alcohol dehydrogenase 2.6 1.6slr1351
murF
Mur ligase 2.5 1.9slr0191
lytB
Amidase enhancer 2.5 1.7sll1041
cysA
Sulphate-transport ATP-binding protein 2.5 1.4slr0992 tRNA/rRNA methyltransferase 2.5 1.4sll0681
pstC
Phosphate-transport permease 2.5 1.2
Genes whose osmotic induction was unaffected in
D
Hik33 cells (-fold)sll1514
hspA
Small HSP 16.8 14.6slr0423
rlpA
Rare lipoprotein A precursor 6.7 9.8sll1566
ggpS
Glucosylglycerol-P synthase 4.2 4.9ssl3177
repA
Rare lipoprotein A precursor 3.6 4.3sll1085
glpD
Glycerol-3-P dehydrogenase 3.2 2.7sll0790
hik31
Sensory histidine kinase 2.7 2.2ssll0955 tRNA/rRNA methyltransferase 2.5 2.2sll1540
dpm1
Dolichyl-phosphate-mannose synthase 2.5 2.0
Cells, grown at 34
∞
C (to OD
730 nm
=
0.2), were incubated for 30 min in the presence of 0.5 M sorbitol or in its absence. Each value indicatesthe ratio of the level of expression in stressed cells to that in unstressed cells. The numbering of open reading frames (ORFs) corresponds tothat of Kaneko
et al
. (1996). This table lists genes whose transcripts were expressed at levels more than 2.5-fold higher in wild-type cells inresponse to 0.5 M sorbitol. In fact, 66% of Hik33-regulated genes encoded proteins of unknown function. The total list can be accessedat http://www.genome.ad.jp/kegg/kegg2.html.
908
K. Mikami, Y. Kanesaki, I. Suzuki and N. Murata
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 905–915
Genes whose osmo-inducibility was regulated by Hik33
DNA microarray analysis indicated that 59 and 151out of the 257 osmostress-inducible genes werefully and partially regulated by Hik33, respectively(see also http://www.gemone.ad.jp/kegg/kegg2.html).Thus, Hik33 fully and partially contributed to theosmo-inducibility of 23% and 58% of the total osmostress-inducible genes. Table 1 summarizes the osmostress-inducible genes that are annotated in the Cyanobase(http://www.kazusa.or.jp/cyano/) and the SynechocystisPCC6803 Gene Annotation Database in GenomeNetCommunity Databases (http://orf.genome.ad.jp/).
It appeared that the expression of particular sets ofgenes was regulated by Hik33. One set included genesfor proteins whose functions are related to the struc-tural maintenance and function of the cell wall andmembranes. It seems likely that Sll0330 (FabG; 3-ketoacyl-ACP reductase), which is required for theelongation of fatty acids, might be involved in the synthe-sis of fatty acids or lipopolysaccharides, whereas Sll1483(cell-surface lipoprotein) and Slr1351 (MurF; UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopimelate-D-alanyl-D-alanine ligase), required for the assembly ofpeptidoglycans, might be involved in the maintenanceof cell wall integrity. Moreover, expression of genes for thephosphate-transport system (phosphate-binding protein,Sll0679; PstS, Sll0680; PstC, Sll0681; PstA, Sll0682;and PstB, Sll0683) and ABC-like proteins of unknownfunction (Sll0005 and Slr0251) was also regulatedby Hik33.
Another set of genes encodes proteins that are locatedin the thylakoid membranes and are probably related tophotosynthesis. Proteins that are inducible by strong light(HliA, Ssl2542; HliB, Ssr2595; and HliC, Ssl1633) proba-bly protect and stabilize the photosynthetic machinery(He et al., 2001), whereas subunits D2 and D3 (NdhD2,Slr1291; and NdhD3, Sll1733) of NADH dehydrogenaseare involved in the cyclic flow of electrons in photosystemI and the uptake of CO2 respectively (Ohkawa et al.,2000a; b).
Another set of genes that was regulated by Hik33 con-sists of genes that are involved in the regulation of geneexpression. SigD (Sll2012) and SigB (Sll0306), whichare 70 kDa sigma factors (Goto-Seki et al., 1999), and aresponse regulator (Sll0789) are all transcription factors.Initiation factor IF-3 (InfC, Slr0974), 50S ribosomal pro-teins (Smr0011 and Ssr1604), a 30S ribosomal protein(Slr1356) and tryptophanyl-tRNA synthetase (TrpS, Slr1884) are, by contrast, involved in the control of transla-tion. Hik33 also regulated genes for proteins that areinvolved in signal-transduction pathways, such as Hik34(Slr1285) and adenylate cyclase CyaA (Slr1991). As the
genes for Hik31 (Sll0790) and the response regulatorSll0789 are included in the same operon, it is possible thatthese proteins might function cooperatively.
Genes for factors involved in the folding and turnover ofproteins form yet another set of Hik33-regulated genes.Hik33 regulates genes for protein chaperones such asDnaK (Sll0170), DnaJ (Slr0093), HptG (Sll0430), GroEL(Sll0416 and Slr 2076) and GroES (Slr 2075). In addition,Hik33 was also required for the osmostress-inducibleexpression of genes for proteases (HtrA, Slr1204; andClpB, Slr1641), which might be involved in proteinturnover.
Genes whose osmostress-inducible expression was not regulated by Hik33
Table 1 shows that the osmostress-inducible expressionof the hspA gene (sll1514), which encodes a small heat-shock protein of 16.6 kDa, was not regulated by Hik33.Moreover, Hik33 was also not involved in the osmostress-inducible expression of genes for precursors to rarelipoprotein RlpA (Slr0423) and RepA (Ssl3177). Thus,it appears that sets of osmostress-inducible genes forprotein turnover and cell wall maintenance are regulatednot only by Hik33 but also by other osmosensors inSynechocystis.
Synechocystis cells accumulate glucosylglycerol asa compatible solute under salt-stress conditions(Hagemann and Erdmann, 1994). The expression ofthe ggpS gene for glucosylglycerol-phosphate synthase(Sll1566) and the glpD gene for glycerol-3-phosphatedehydrogenese (Sll1085), which are involved in the syn-thesis of glucosylglycerol, was induced by the presenceof 0.5 M sorbitol (Table 1; also see Kanesaki et al., 2002).However, inactivation of the gene for Hik33 did not elimi-nate the induction by hyperosmotic stress of the expres-sion of these genes (Table 1). These observationssuggest that the osmostress-inducible expression of ggpSand glpD genes might be regulated in an unknownmanner by an as yet unidentified osmosensor.
Cold-inducible genes whose expression was regulated by Hik33
In a previous study, we examined the contribution of Hik33to cold-inducible gene expression using a DNA microarray(Suzuki et al., 2001). However, in view of the recent exten-sive improvements in the quality of Synechocystis-specificDNA microarrays, we re-examined the genome-wide pat-terns of transcription in wild-type and DHik33 cells upona downward shift in temperature, and we compared theeffects of the mutation in Hik33 on the cold-induciblegenes to the effects of this mutation on the osmostress-inducible genes.
Osmostress and cold sensor Hik33 in Synechocystis 909
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 905–915
We found that the expression of approximately 65%of cold-inducible genes was affected by the mutationin Hik33 (Fig. 2A and B; the data can be found athttp://www.genome.ad.jp/kegg/kegg2.html). Hik33 wasessential for the regulation of the expression of 16genes (36%) and was partially involved in the regulationof 12 genes (27%) out of a total of 45 cold-induciblegenes.
Table 2 summarizes the cold-inducible genes that areannotated in the Cyanobase (http://www.kazusa.or.jp/cyano/) and the Synechocystis PCC6803 Gene Annota-tion Database in GenomeNet Community Databases(http://orf.genome.ad.jp/). The Hik33-regulated genesencode proteins involved in gene expression (SigD,
Sll2012; Crh, Slr0083; and Fus, Slr1105), regulation ofphotosynthesis (NdhD2, Slr1291; NdhF, Slr 2009; HliA,Ssl2542; HliB, Ssr2595; HliC, Ssl1633; and CytM,Sll1245), responses to oxidative stress (XthA, Sll1854;and GshB, Sll1238) and membrane function (LivF, Slr1881; and DesB, Sll1441). These results are consistentwith previous reports of the cold-inducible expression ofgenes for w3 acyl-lipid desaturase (Los et al., 1997), cyto-chrome M (Malakhov et al., 1999) and three high light-inducible proteins (He et al., 2001). However, inactivationof the hik33 gene did not reduce the cold inducibility ofgenes for ribosomal proteins, an RNA-binding protein(Rbp1), the a subunit of RNA polymerase (RpoA), a pre-cursor to rare lipoprotein A (RlpA) and five ribosomalproteins (Rpl3, Rpl4, Rpl11, Rpl23 and Rps12). The infor-mation obtained with the new DNA microarray was basi-cally similar to that obtained in our previous study (Suzukiet al., 2001).
Discussion
Osmostress-inducible genes and their functions
In the present study, using a DNA microarray, we demon-strated that the histidine kinase Hik33 acts as an osmo-sensor. Osmotic stress induced the expression of 257genes in wild-type cells of Synechocystis cells (Fig. 1 andTable 1). The expression of these genes was controlledby Hik33 for the most part, and the various genes couldbe divided into several groups according to the functionsof their respective products (see Results). The first groupincludes genes for the synthesis and maintenance of cellwalls and membranes. Under osmotic stress due to 0.5 Msorbitol, water molecules leave cells via water channels,with the resultant shrinkage of cells that reduces the vol-ume of the cytoplasm to about half of the original volume(Allakhverdiev et al., 2000a). Such shrinkage of the cyto-plasm induces structural changes in the plasma mem-brane and the cell wall of Synechocystis cells. Theosmo-inducible expression of genes in this group mightbe related to the response of Synechocystis cells to theosmostress-induced changes in plasma membranes andcell walls.
The second group of genes includes genes for thephosphate transport system. The changes in the plasmamembrane due to the osmotic stress might havedepressed the activity of the phosphate transport system,which is located in the plasma membrane and is essentialfor the maintenance of cellular activity (Torriani, 1990;Wanner, 1993). The third group of genes includes a genefor the D2 subunit of NADH dehydrogenase (NdhD2,Slr1291) and genes for Hli proteins (Ssr2595, Ssl2542and Ssl1633). We demonstrated previously that photosys-tems I and II are inactivated under osmostress conditionsin Synechococcus sp. PCC 7942 (Allakhverdiev et al.,
Fig. 2. Genome-wide patterns of transcription of cold-inducible genes.A. Gene expression in wild-type cells that had been transferred from 34∞C to 22∞C and incubated for 20 min was compared with that in unstressed cells.B. Gene expression in DHik33 cells that had been transferred from 34∞C to 22∞C and incubated for 20 min was compared with that in unstressed cells. Red circles correspond to genes whose cold-inducibility was depressed in DHik33 cells and blue circles correspond to genes whose cold-inducibility was unaffected in DHik33 cells. The assay was repeated three times in independent experiments and essentially the same results were obtained in every case. Dashed lines indicate the limit of experimental deviations. The number of cold-inducible genes in wild-type cells (45) was almost twice that (24) reported by Suzuki et al. (2001). The difference might be due to improvements in the quality of the DNA microarray and to differences in experimental conditions.
910 K. Mikami, Y. Kanesaki, I. Suzuki and N. Murata
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 905–915
2000b), suggesting that osmotic stress might limit thesupply of ATP, which is mediated by the cyclic electrontransport of photosystem I. The osmostress-inducibleexpression of the nadhD2 gene, whose product is involvedin cyclic electron transport in photosystem I (Ohkawaet al., 2000a), may be important for the maintenance of asupply of ATP. It is possible that Hli proteins, which arelocated in thylakoid membranes (He et al., 2001), mightstabilize the photosystems against osmotic stress.
The fourth group of genes includes genes for transcrip-tion factors, such as a response regulator (Sll0789) andthe sigma factors SigB (Sll0306) and SigD (Sll 2012)(Goto-Seki et al., 1999), and translation factors, suchas IF-3 (Slr0974), 50S and 30S ribosomal proteins(Smr0011, Ssr1604 and Slr1356) and TrpS (Slr 1884). Itis probable that osmotic stress inhibits gene expressionat both the transcriptional and the translational level. Theexpression of the genes in the fourth group might be
important for compensatory enhancement of transcrip-tional and translational activity.
The last group is a set of heat-shock genes for chaper-ones and proteases that accelerate the turnover andrefolding of the damaged proteins that are generated byosmotic stress (Visick and Clarke, 1995). Expression ofthese genes might represent another strategy for copingwith the effects of osmotic stress on cellular activity. Forexample, HtrA, a serine protease in thylakoid membranes,degrades membrane-associated proteins, in particular,the damaged D1 protein of photosystem II (Itzhaki et al.,1998). This degradation probably accelerates reconstruc-tion of a new photosystem II complex and is supportedthe osmostress-inducible expression of the htrA (slr1204)gene (Table 1).
We also detected the osmostress-inducible expressionof genes for a small heat-shock protein (Sll1514) that isinvolved in refolding of proteins; for precursors to rare
Table 2. Cold-inducible genes and effects of the inactivation of Hik33 on their induction by low-temperature.
ORF Gene Product
Wild type DHik33
22∞C/34∞C 22∞C/34∞C
Genes whose cold inducibility was diminished in DHik33 cells (-fold)slr1291 ndhD2 NADH dehydrogenase subunit 4 6.6 1.3ssl2542 hliA High light-inducible protein A 4.8 1.0ssr2595 hliB High light-inducible protein B 3.7 1.0ssl1633 hliC High light-inducible protein C 2.9 0.8slr1105 fus Elongation factor EF-G 2.8 1.2slr0426 folE GTP cyclohydrolase I 2.7 1.0slr0399 ycf39 Protein of unknown function 2.5 1.1slr0611 sds Solanesyl diphosphate synthase 2.3 1.2sll2012 sigD RNA polymerase sigma factor 2.3 1.0sll1854 xthA Exodeoxyribonuclease III 2.1 1.1
Genes whose cold inducibility was partially reduced in DHik33 cells (-fold)slr0083 crh ATP-dependent RNA helicase 6.0 3.9slr2009 ndhF NADH dehydrogenase subunit F 2.5 1.4slr1881 livF Branched-chain amino acid transporter 2.4 1.3sll1441 desB w3 acyl-lipid desaturase 2.3 1.8slr1238 gshB Glutathione synthetase 2.2 1.3sll1245 cytM Cytochrome M 2.2 1.5
Genes whose cold inducibility was unaffected in DHik33 cells (-fold)sll0517 rbpA RNA-binding protein 8.0 5.2slr0423 rlpA Rare lipoprotein A precursor 2.5 2.5sll1799 rpl3 50S ribosomal protein L3 2.5 2.7slr0549 asd Asp b-semialdehyde dehydrogenase 2.4 2.0sll1743 rpl11 50S ribosomal protein L11 2.4 1.6sll1800 rpl4 50S ribosomal protein L4 2.3 1.9sll1801 rpl23 50S ribosomal protein L23 2.2 1.7sll1742 nusG Transcription antiterminator 2.2 1.7slr0550 dapA Dihydrodipicolinate synthase 2.2 2.1slr1093 folK 7,8-Dihydro-6-hydroxymethylpterin-pyrophosphokinase 2.1 1.7ssl3335 secE Secreting protein 2.0 1.8sll1818 rpoA RNA polymerase a subunit 2.0 1.7sll1096 rps12 30S ribosomal protein S12 2.0 1.8
Cells were grown at 34∞C for 16 h (34∞C cells) and then incubated at 22∞C for 20 min (22∞C cells). Each value indicates the ratio of the level ofthe mRNA from 22∞C cells to that from 34∞C cells. The data presented here are averages of results of three independent experiments. Genesthat gave ratios greater than 2.0 are listed. The numbering of open reading frames (ORFs) corresponds to that of Kaneko et al. (1996). Amongthe Hik33-regulated genes, 61% encoded proteins of unknown function. The total list can be accessed at http://www.genome.ad.jp/kegg/kegg2.html.
Osmostress and cold sensor Hik33 in Synechocystis 911
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 905–915
lipoproteins (RlpA, Slr0423; RepA, Ssl3177), which areassume to participate in maintenance of cell walls; andthe enzymes that are involved in the synthesis of gluco-sylglycerol (GgpS, Sll1566; GlpD, Sll1085). However, thegenes for these proteins were not regulated by Hik33(Fig. 3 and Table 1). Although Hik33 affects the expres-sion of a variety of groups of osmostress-inducible genes,other as yet unidentified osmosensors are needed forcomplete regulation of all the osmostress-inducible genes.The multiplicity and functional diversity of osmosensorshas been observed in E. coli (EnvZ and KdpD; Mizunoet al., 1982; Voelkner et al., 1993) and in yeast (Sln1 andSho1; Maeda et al., 1994; 1995).
Taken together, the various observations suggestthat the osmostress-inducible genes identified in thepresent study might play significant roles in the responseof Synechocystis to osmotic stress and that Hik33functions as an osmo-sensor that is important for theinduction of expression of these genes under hyperos-motic conditions.
Hik33 is a sensor of both osmotic and cold stress
Hik33 was identified as a cold sensor in Synechocystis(Suzuki et al., 2000; 2001). This conclusion was con-firmed in the present study (Table 2) and, thus, it appearsthat Hik33 functions both as an osmosensor and as a coldsensor. Figure 3 shows a schematic representation of therelationship between the genes induced by osmostressand those induced by cold stress. It is clear that Hik33
regulates distinct sets of genes under different stress con-ditions. In addition, Hik33 also regulates genes whoseexpression is induced both by osmostress and by coldstress. These genes include genes for proteins involvedin the regulation of photosynthesis (NdhD2, HliA, HliB andHliC) and gene expression (SigD and Crh), as well asgenes for solanesyl diphosphate synthase (Sds, Slr0611)and several unidentified proteins (Slr1544, Sll 1911,Sll1483, Sll1541, Slr0082 and Sll0668). Two other genes,encoding a precursor to rare lipoprotein (RlpA, Slr0423)and a protein of unknown function (Slr0955), wereinduced both by osmotic stress and by cold stress but theirexpression was not regulated by Hik33.
The results of our analysis are unexpected becauseboth osmotic stress and cold stress are assumed to influ-ence the physical state of membranes (Murata and Los,1997; Kempf and Bremer, 1998; Vigh et al., 1998; Wood,1999). Thus, we can postulate that Hik33 regulates similarsets of genes whose expression is induced under bothhyperosmotic and low-temperature conditions. Given thedifferential regulation of osmostress-inducible and cold-inducible genes via Hik33, it seems likely that osmoticstress and cold stress are recognized differently and theresultant signals are transduced differently by Hik33. How-ever, it is unclear how Hik33 might separately transducedifferent stress signals to cognate downstream targets,such as response regulators and transcription factors.Identification of response regulators and transcription fac-tors that act downstream of Hik33 and of other osmosen-sors involved in osmostress- and cold-signalling pathwayswill help us to clarify the cross-talk between the varioussignal transduction pathways.
Hik33 senses various kinds of stress
Hik33 was first identified as DspA, a chemical sensor ofdrugs such as inhibitors of photosynthesis (Bartsevichand Shestakov, 1995). Recently, van Waasbergen et al.(2002) identified NblS, a putative homologue of Hik33in Synechococcus elongatus PCC 7942, as a sensorof strong light and nutrient stress. The considerablehomology between NblS and Hik33 (58% at the aminoacid level) suggests that Hik33 might also play roles inresponses to strong light and nutrient stress in Syn-echocystis. As our analyses indicate that Hik33 is abifunctional sensor of osmotic and cold stress (Fig. 3,Tables 1 and 2), it is possible that Hik33 acts as asensor of several types of stress. However, Hik33 regu-lated different sets of genes under osmotic-stress andcold-stress conditions (Fig. 3, Tables 1 and 2). Theseobservations suggest that Hik33 might recognize thesedifferent stresses by different and as yet unknownmechanisms.
Fig. 3. Osmostress-inducible and cold-inducible genes that were regulated by Hik33 in wild-type cells. Large and small circles enclose genes whose expression was induced by osmotic stress and cold stress respectively. Rectangles in these circles enclose genes whose expression was regulated to a greater or lesser extent by Hik33 in cells under hyperosmotic stress and under cold stress. Genes outside rectangles appeared to be insensitive to the mutation in Hik33 in terms of their responses to the respective stresses. The rectangle in the overlapping region of the two circles encloses genes whose Hik33-regulated expression was observed under both kinds of stress.
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Structure of Hik33
As described previously (Suzuki et al., 2000), the struc-ture of Hik33 is typical of histidine kinases, with two trans-membrane regions at the amino terminus (aa 32–48 and201–217) and a transmitter domain at the carboxyl termi-nus (aa 420–625). Homology searches revealed threecharacteristic motifs (Fig. 4A), namely, a type-P linker(aa 217–269; Williams and Stewart, 1999; also known asa HAMP region; Aravind and Ponting, 1999), a leucinezipper domain (aa 270–309), and a PAS domain (Per,Arnt, Sim, and phytochrome; aa 300–419; Taylor andZhulin, 1999). As shown in Fig. 4A, the leucine zipperand PAS domains partially overlap one another.
The type-P linker seems to be the most importantmodule for signal transduction in Hik33 because mostmembrane-integrated osmosensors have a type-P linkerimmediately downstream of the second transmembraneregion (Fig. 4A). The type-P linker consists of two helicalregions in tandem (Fig. 4B). It has been proposed that ittransduces extracellular signals via intramolecular struc-tural changes that are due to interactions between the twohelical regions, which lead to the intermolecular dimeriza-tion of membrane proteins (Aravind and Ponting, 1999;Williams and Stewart, 1999). Such a conformationalchange, induced by osmotic stress, might generate adimeric form of Hik33, with the resultant activation of thehistidine kinase domain. The leucine zipper might alsoplay a role in stabilization of the dimerization of Hik33, asindicated in EnvZ (Yaku and Mizuno, 1997). The osmo-sensor Sln1 in yeast also has a putative type-P linker ina region that corresponds to the position of this domain
in Hik33 and EnvZ. However, there is a long sequencebetween the two helices (Fig. 4). It is possible that someaspects of the basic mechanism of transmembrane sig-nalling of osmotic stress are similar in prokaryotic andeukaryotic cells.
Perception of osmotic stress
Several lines of evidence suggest that a domain thatincludes the first transmembrane and periplasmic regionsis essential for perception of osmotic stress in EnvZ andSln1 (see, for example, Tokishita et al., 1992; Ostranderand Gorman, 1999; Waukau and Forst, 1999). However,there is no sequence homology between these two pro-teins. The stress-sensing domains in these proteins alsodiffer from the corresponding region in Hik33. Moreover,it is likely that the physical state of membrane lipids isimportant for the activity of membrane-bound histidinekinases, such as KdpD and the heat sensor CpxA in E.coli (Mileykovskaya and Dowhan, 1997; Stallkamp et al.,1999). The activity of KdpD depends on negativelycharged phospholipids that allow KdpD to communicatewith membranes via electrostatic interactions (Stallkampet al., 1999). CpxA is activated in mutant cells that lackphosphatidylethanolamine (Mileykovskaya and Dowhan,1997). The physical state of membrane lipids might alsobe affected by changes in turgor pressure that are pro-duced by osmotic imbalance. In yeast, Sln1 recognizeschanges in turgor pressure that result from osmotic stress(Tamas et al., 2000) but it remains unclear how changesin turgor pressure might alter the physical state of mem-brane lipids.
Fig. 4. Structural characteristics of membrane-bound osmosensors.A. Schematic diagrams of Hik33 from Syn-echocystis, EnvZ from Escherichia coli and Sln1 from Saccharomyces cerevisiae. Trans-membrane regions are indicated as black bars. Letters above the boxes refer to designations of motifs: P, type-P linker; L, leucine zipper domain; P, PAS domain; HK, histidine kinase domain; and RD, receiver domain.B. Comparison of the sequences of type-P linkers. Sequences of EnvZ and Sln1 were taken from Mizuno et al. (1982) and Ota and Varshavsky (1993) respectively. Strongly conserved proline, glutamic acid and hydrophobic residues are boxed.
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Transmembrane regions of sensors appear to be veryimportant for the perception of changes in the physicalstate of membranes. However, little is known about theway in which Hik33 distinguishes osmotic stress from coldstress at the perception step and the way in which Hik33senses these kinds of stress. Recently, Bogdanov et al.(2002) demonstrated that the membrane-bound lactosepermease of E. coli changes its conformation in responseto the phospholipid composition of membranes, suggest-ing that the physical state of membrane lipids might exertan effect on the conformation of membrane proteinsdirectly. Therefore, it is possible that Hik33 might senseosmotic stress and cold stress by two different mecha-nisms, in which Hik33 adopts two distinct conformationsaccording to the physical state of the membrane lipids,allowing Hik33 to interact with appropriate downstreamtargets in a stress-dependent manner. However, it is stillunclear whether Hik33 changes its conformation in a waythat reflects the altered physical state of membrane lipidsand, if so, what kind of changes in the physical state ofmembrane lipids affect the conformation of Hik33. Tounderstand the molecular mechanisms by which Hik33senses osmotic stress and cold stress differently, it will benecessary to identify the stress-sensing regions of Hik33and to characterize the interactions between Hik33 andmembrane lipids under different stress conditions. Suchefforts will help us to understand the mechanisms that areinvolved in the perception of abiotic stress, which might,in turn, provide new insights into the regulation of stressperception by bifunctional sensors, not only in photo-synthetic cyanobacteria but also in higher plants.
Experimental procedures
Cells and culture conditions
The wild-type strain of Synechocystis sp. PCC 6803 waskindly donated by J. G. K. Williams (Du Pont de Nemours,Wilmington, DE, USA). Wild-type cells were grown at 34∞Cin BG-11 medium (Stanier et al., 1971) buffered with 20 mMHepes-NaOH (pH 7.5) under continuous illumination fromincandescent lamps, as described previously (Wada andMurata, 1989). The DHik33 mutant, which had been gener-ated by Suzuki et al. (2000), was grown under the sameconditions, except that spectinomycin at 20 mg ml-1 wasadded to the medium for preculture. In this mutant, the wild-type gene for Hik33 is disrupted by insertional mutation witha spectinomycin-resistance gene cassette (Suzuki et al.,2000). Wild-type and mutant cells at the exponential phaseof growth (OD730 nm = 0.08) were used for all experiments.
Preparation of cDNAs for analysis with a DNA microarray
Cultures of cells that had been incubated with 0.5 M sorbitolwere combined with an equal volume of an ice-cold mixtureof phenol and ethanol (1:10, w/v) in an ice bath. Then total
RNA was prepared as described previously (Los et al., 1997)and treated with RNase-free DNase I (Nippon Gene, Tokyo,Japan) to remove any contaminating genomic DNA. Cy3- andCy5-labelled cDNAs were synthesized by reverse transcrip-tion with 20 mg of total RNA by incubation at 42∞C for 1 h with300 pmol random hexamer primers; 0.5 mM dATP, dGTP anddCTP respectively; 0.2 mM dTTP; 0.1 mM Cy3-dUTP or Cy5-dUTP (Amersham Pharmacia Biotech); 100 units of RNaseinhibitor (TaKaRa); and 50 units of reverse transcriptase XLfrom avian myeloblastosis virus (TaKaRa); in reaction bufferwhich had been provided by the manufacturer of the reversetranscriptase. Reaction mixtures were then supplementedwith an additional 50 units of reverse transcriptase XL andincubated further for 1 h at 42∞C. After termination of thereaction by gel filtration on a Centri-Sep spin column (AppliedBiosystems), the eluate that contained fluorescent dye-labelled cDNAs was purified by treatment with a mixture ofphenol, chloroform and isoamyl alcohol (25:24:1, v/v). ThencDNAs were precipitated in ethanol.
DNA microarray analysis
Genome-wide analysis of gene transcription was performedwith a DNA microarray, as described earlier (Suzuki et al.,2001; Kanesaki et al., 2002). We used the SynechocystisDNA microarray (CyanoCHIP ver. 1.1, lot no. 1003; TaKaRa),which included 3079 out of the 3169 genes of Synechocystis,for hybridization by incubating the array for 16 h at 65∞C withCy3- and Cy5-labelled cDNAs in 20 ml of 4¥ SSC (1¥ SSC iscomposed of 150 mM NaCl and 15 mM sodium citrate), 0.2%SDS, 5¥ Denhardt’s solution and 100 ng ml-1 of denaturedsalmon sperm DNA. After rinsing with 2¥ SSC at room tem-perature, the microarray was washed with 2¥ SSC at 60∞Cfor 10 min and 0.2¥ SSC that contained 0.1% SDS at 60∞Cfor 10 min. Then it was rinsed with distilled water at roomtemperature for 2 min. Moisture was removed completelywith an air spray before analysis with an array scanner(GMS418; Affimetrix). Signals were quantified with ImaGenever. 4.0 software (BioDiscovery) with normalization by refer-ence to the total intensity of signals from all genes with theexception of genes for rRNAs, which allowed us to calculatechanges in the level of the transcript of each gene relative tototal levels of mRNA.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Scien-tific Research (S) (no. 13854002) to N.M.; a Grant-in-Aid forScientific Research (C) (no. 14540606) to K.M.; and a Grant-in-Aid for Scientific Research on Priority Areas (C) (‘GenomeBiology’, no. 13206081) and a Grant-in-Aid for ExploratoryResearch (no. 14654169), both to I.S., from the Ministry ofEducation, Culture, Sports, Science and Technology ofJapan. It was also supported by a grant from the Salt ScienceResearch Foundation (no. 9942) to K.M.
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