Genomic analysis of the histidine kinase family in bacteria and archaea

16 Pages·2001·2.67 MB·English

Microbiology(2001),147,1197–1212 PrintedinGreatBritain
Genomic analysis of the histidine kinase family
in bacteria and archaea
Dong-jin Kim and Steven Forst
Authorforcorrespondence:StevenForst.Tel:›14142296373.Fax:›14142293926.
e-mail:sforst!uwm.edu
DepartmentofBiological Two-componentsignaltransductionsystems,consistingofhistidinekinase
Sciences,POBox413, (HK)sensorsandDNA-bindingresponseregulators,allowbacteriaandarchaea
UniversityofWisconsin,WI
torespondtodiverseenvironmentalstimuli.HKspossessaconserveddomain
53201,Milwaukee,USA
(H-boxregion)whichcontainsthesiteofphosphorylationandanATP-binding
kinasedomain.Inthisstudy,agenomicapproachwastakentoanalysetheHK
familyinbacteriaandarchaea.Basedonphylogeneticanalysis,differencesin
thesequenceandorganizationoftheH-boxandkinasedomains,andthe
predictedsecondarystructureoftheH-boxregion,fivemajorHKtypeswere
identified.Ofthe336HKsanalysed,92%couldbeassignedtooneofthefive
majorHKtypes.TheTypeIHKswerefoundpredominantlyinbacteriawhile
TypeIIHKswerenotprevalentinbacteriabutconstitutedthemajortype(13
of15HKs)inthearchaeonArchaeoglobusfulgidus.TypeIIIHKsweregenerally
moreprevalentinGram-positivebacteriaandwerethemajorHKtype(14of15
HKs)inthearchaeonMethanobacteriumthermoautotrophicum.TypeIVHKs
representedaminortypefoundinbacteria.ThefifthHKtypewascomposedof
thechemosensorHKs,CheA.SeveralbacterialgenomescontainedallfiveHK
types.Incontrast,archaealgenomeseithercontainedaspecificHKtypeor
lackedHKsaltogether.ThesefindingssuggestthatthedifferentHKtypes
originatedinbacteriaandthatspecificHKtypeswereacquiredinarchaeaby
horizontalgenetransfer.
Keywords: classificationscheme,phylogeneticanalysis,secondarystructureanalysis,
horizontalgenetransfer
INTRODUCTION RR.ModulationofthephosphorylatedstateoftheRR
controls either expression of target genes or cellular
In bacteria and archaea, two-component signal trans- behaviour,suchasswimmingmotility(Hoch&Silhavy,
ductionsystems(Kofoid&Parkinson,1988;Parkinson 1995). Environmental stimuli received by highly di-
& Kofoid, 1992; Forst & Roberts, 1994; Hoch & vergent sensory input domains provides specificity for
Silhavy,1995),alsoreferredtoasHis–Aspphosphorelay thesignaltransductionpathwayandcontrolsthelevelof
systems(Eggeretal.,1997),mediateadaptiveresponses thephosphorylatedstateoftheRR.Bacteriacanpossess
tochangesinenvironmentalconditions.Atypical two- more than 30 different two-component signal trans-
component signal transduction system consists of a ductionsystems(Mizuno,1997).Bacteriathatpossessa
membrane-bound sensor histidine kinase (HK) and a large number of HKs generally are able to adapt to a
cognate regulatory protein referred to as a response broad spectrum of environmental stimuli. While HKs
regulator(RR).HKsusuallyfunctionasdimericproteins have also been identified in fungi (Ota & Varshavsky,
thatundergotransautophosphorylationonaconserved 1993; Loomis et al., 1998), amoeba (Schuster et al.,
histidineresidueinresponsetospecificstimuli(Duttaet 1996;Changetal.,1998),Neurospora(Alexetal.,1996)
al.,1999).Thephosphorylgroupissubsequentlytrans- andArabidopsis(Changetal.,1993;Suzukietal.,1998),
ferred to an Asp residue in the receiver domain on the the HK content in eukaryotic genomes is much lower
than that found in bacterial genomes.
HKs consist ofan ATP-binding kinase domain and the
.................................................................................................................................................
Abbreviations:HK,histidinekinase;RR,responseregulator. H-box domain which includes the histidine site of
0002-4110#2001SGM 1197
D.-j. KIM and S. FORST
HK subtype HK H-box X-region +
(a)
Type IA
IB
IC
Type II
Type III
Type IV
CheA
Kinase type HK N G1 F G2 G3
(b)
Orthodox
Unorthodox
CheA
.................................................................................................................................................................................................................................................................................................................
Fig.1.Forlegendseefacingpage.
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Histidinekinasefamily
phosphorylation. The kinase domain consists of three type distribution differed markedly between bacteria
conserved consensus motifscalled theN-, G1- and G2- and archaea.
boxes,andafourth,morevariablesequence,theF-box
(Kofoid&Parkinson,1988;Stocketal.,1988,1995).In
METHODS
most HKs, the kinase domain is directly connected to
theC-terminalsideoftheH-boxdomain.Incontrast,in Analysis of bacterial and archaeal HK families. The HK
the chemosensor CheA, the H-box (P1 domain) resides familyofeachgenomewasassembledusingthegenetablesof
at the N terminus of the protein and is separated from thecompletedgenomeslistedinTIGR(http:}}www.tigr.org).
thekinasedomainbytheinterveningP2andP3modules Additionally, blast analysis using the transmitter domains
(Garzon&Parkinson,1996;Robinson&Stock,1999). (H-boxpluskinasedomain)ofEnvZ,CheA,NarX,YehUand
DcuS as the search sequences was performed. During this
ThestructuresoftheH-boxdomainoftheosmosensor, analysis ORFs were retrieved which lacked either H-box or
EnvZ (Tomomori et al., 1999) and the P1 domain of kinaseconsensusmotifs.Also,proteinssuchasHipAofE.coli
CheA(Zhou&Dalquihst,1997)havebeendetermined. and SpoIIAB of B. subtilis, which contained kinase domains
The H-box region of EnvZ consists of a four-helix butlackedanidentifiableH-boxdomainwereretrieved.Only
bundle structure formed by the dimeric association of thoseproteinsthatcontainedbothanidentifiableH-boxand
completekinasedomainwereincludedintheHKgenefamily.
two identical subunits while the P1 domain is a
monomericfour-helixbundlestructure.Whilethestruc- Alignment of H-box and kinase domains. The transmitter
tureofH-boxdomainsdiffer,thestructureofthekinase domains were initially aligned using the multi-sequence
domains of EnvZ of Escherichia coli (Tanaka et al., alignment program msa version 2.1. Refinement of the
1998)andCheAofThermatogamaritima(Bilwesetal., alignments was aided by blast search analysis and visual
1999)were showntobehomologous toeachotherand inspection.
totheATP-bindingdomainsofDNAgyraseBandHsp Phylogenetic analysis. A distance dendrogram of each HK
90. The phosphotransfer reaction can be reconstituted family was constructed using the unweighted pair-group
using liberated H-box and kinase domains (Garzon & method with arithmetic means (UPGMA) algorithm. Using
Parkinson, 1996; Park et al., 1998), indicating that the thismethod,fivedifferentHKtypeswereidentifiedinE.coli.
individual domains can be obtained as functionally Foreachgenomeanalysed,adataset,whichincludedtheHKs
intact modules. fromE.coli,wascreatedandsubsequentlyanalysedusingthe
UPMGAmethod.TheassignmentofHKtypesandsubtypes
Besidesthetypicaltwo-componentorganization,multi- within each genome analysed was accomplished using this
step His–Asp–His–Asp phosphorelay systems can be approach.
composedofindividualphosphotransferproteins.This
Secondary structure analysis. The PredictProtein server
modularorganizationhasbeenextensivelyinvestigated (http:}}www.embl-heidelberg.de}predictprotein}predict-
in the multi-step pathway controlling sporulation in protein.html) was used to predict the secondary structure of
Bacillus subtilis (Appleby et al., 1996; Fabret et al., theH-boxregionineachHKretrieved.Apredictedsecondary
1999; Hoch, 1995; Perraud et al., 1999). Multistep structurewasassignedtosequencesthatpossessedaliability
phosphotransferreactionscanalsooccurwithinasingle valueofgreaterthan7.
HK. These so-called hybrid HKs contain additional
phosphotransfermodulesreferredtoastheD1receiver
RESULTS
andtheHPtphosphotransferdomainsthatareattached
totheC-terminalsideofthekinasedomain(Applebyet CharacterizationoftheHKfamilyofE.coliK-12
al., 1996).
HKs were retrieved using the blast server within each
The number of HKs recognized has expanded enor- genomelistedintheTIGRmicrobialdatabase.Various
mouslywiththeadventofmicrobialgenomicsequencing transmitters,whichincludedboththeH-boxregionand
projects. The HK superfamily has been classified by kinase domain, were used as search probes. Retrieved
numerous criteria. Recently, Grebe & Stock (1999) ORFs containing both an H-box region and a kinase
separatedtheHKfamilyinto11differentsubtypesbased domain were included in the HK gene family. We
on cluster analysis of 348HKs. In the present study, initially analysed the genomes containing large HK
theHKfamiliesinthecompletedgenomesof22bacteria families. The HK family of the completed genomic
and 4 archaea was analysed. This genomic analysis sequence of E. coli K-12 (Blattner et al., 1997) was
divided the HK family into five major types. The HK analysed first since it has been extensively studied and
Fig.1.AlignmentoftheH-boxregionoftheHKsofE.coli.H-boxconsensussequencesareboxed.Boldcharactersdepict
invariable residues and characteristic conserved residues of a given subtype are represented by white letters. The
predictedhelix–loop–helixstructuresareunderlined.Thehelix–loop–helixstructureofEnvZisshownbythicklinesabove
theEnvZsequence.‘›’denotesthepositionoftheconservedpositiveaminoacidresidueattheendofhelix2.Proline
residuesintheputativeloopregionareshaded.(b)AlignmentofthekinasedomainoftheHKsofE.coli.Theconserved
motifsareenclosedinboxesandshaded.Boldcharactersdepictinvariableresiduesandcharacteristicconservedresidues
ofagivensubtypearerepresentedbywhiteletters.Thenumberofaminoacidresiduesbetweenmotifsarerepresented
by either a single dot (less than 10 residues), by double dots (10–25 residues) or double open circles (more than 25
residues).Dashesrepresentdeletedresidues.
1199
D.-j. KIM and S. FORST
BasS
YgiY
PhoR
YbcZ
BaeS
RstB
EnvZ
CpxA
IA KdpD
YedV
CreC
YfhK
BarA
RcsC
TorS
IB
ArcB
EvgS
AtoS
IC HydH
Type I
NtrB
PhoQ
Type II DcuS
DpiB
Che A
CheA
NarX
Type III NarQ
UhpB
Type IV YehU
B2380
0·1 changes
.................................................................................................................................................................................................................................................................................................................
Fig. 2. Phylogenetic analysis of the HKs of E. coli. Dendrogram of the 29HKs of E. coli analysed using the UPGMA
algorithm.ThealignmentsshowninFig.1(a)and(b)wereusedintheanalysis.
thefunctionof18ofitsHKsarepresentlyknown(Egger IIIandIVHKsoccupyseparatebranches.TypeIandII
et al., 1997). HKs both possess orthodox kinase domains, which
containtheN,G1,FandG2consensusmotifs.TypeIII
Twenty-nineHKswereretrievedfromthegenomeofE.
and IV HKs possess so-called unorthodox kinase
coli.Fig.1(a)showstheaminoacidsequencealignment
domains in which N1 of the N-box motif is either a
of the H-box and X regions and Fig. 1(b) shows the
glycine(TypeIII)oraproline(TypeIV)residue,theF-
alignment of the kinase domains. During the blast
box is absent and the G2 motif is truncated (Fig. 1b).
analysis, proteins which contained phosphorelay sub-
The conserved glycine residue identified on the C-
domainsbutlackedkinasedomainswereretrieved.For
terminal side of the G2 motif in almost all kinase
example, YojN contains an HPt domain but no
domainsisreferredtoastheG3site.TheTypeIgroup
identifiablekinasedomain.Thesetypesofproteinswere
containedthelargestnumberofmembers(72%).Within
not included in the HK gene family.
this group, three separate subtypes could be distin-
PhylogeneticanalysisseparatedtheHKgenefamilyinto guished. The Type IA group contained 12HKs, the
five major branches (Fig. 2). The analysis reveals that TypeIBgroupcontainedthehybridHKsandtheType
TypeIandIIHKsarerelatedtoeachotherwhileType IC group contained three HKs, including the nitrogen
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Histidinekinasefamily
Table1.CharacteristicfeaturesofHKtypes
HKtype H-boxconsensus* Kinasedomain MeanHtoN
distance(aa)
TypeI HEhR[P Orthodox 116
TypeII HE[[N[ Orthodox 96
TypeIII REhHD[h[[ Unorthodox 110
TypeIV PHFh[N[ Unorthodox 92
CheA HShKG[ Orthodox 325
*TheboldHrepresentstheinvariablehistidineresidueintheH-box.Conservedhydrophobicresidues
(I,L,V,M)aredesignatedby‘h’.
regulator,NtrB.WhilePhoQcontainedaTypeIH-box HKfamilyofPseudomonasaeruginosa
motifandanorthodoxkinasedomain,itdidnotbranch
TodeterminewhetherthefivemajorHKtypesfoundin
within the Type IA, IB and IC subgroups.
E. coli were present in other bacteria, a phylogenetic
EachofthefiveHKtypescontainedacharacteristicH- analysisofHKgenefamilyofPsd.aeruginosa(Stoveret
boxmotif(Fig.1a).TheH-boxofTypeIHKscontained al., 2000) using the UMPGA method was performed
the consensus motif HEhRTPh. Secondary structure (Fig.3).Psd.aeruginosapossesses63HKs.ThefiveHK
analysis predicted a helix–loop–helix structure (under- types found in E. coli were also identified in Psd.
linedinFig.1)intheH-boxregion.Thisstructurewas aeruginosa.Themajorityofthefamilymembers(86%)
shown to exist in the NMR solution structure of EnvZ were Type I HKs, containing typical orthodox kinase
(Tomomori et al., 1999). Proline residues were present domainsandH-boxmotifs.OneclusterwithintheType
in the loop region of numerous HKs and positively IAgroup(PA1396,1976,1992,3271and4936)contained
chargedresidueswerefoundattheendoftheX-region. orthodox kinase domains while the H-box motifs
The H-box motif of the Type II HKs possessed a contained a non-polar residue at position 4 and a
conservedasparagineresidueatposition5(theinvariant glutamine residue at position 5. This clade of HKs
H residue is defined as position 1) and lacked the formed a distinct branch within the Type IA group. In
positively charged residue and the proline at positions addition, a cluster of HKs in the Type IC group
4 and 6, respectively. These HKs lacked a predicted possessed the consensus H-box motif HDLNQPL in
helix–loop–helixstructure.TheH-boxmotifofTypeIII which the asparagine residue replaced the typical
HKswascharacterizedbyanR-E-LsequenceontheN- positively charged residue at position 4 and the glu-
terminalsideofthehistidinesiteofphosphorylationand tamineresidueatposition5washighlyconserved.Psd.
lacked the conserved positively charged and proline aeruginosa lacked Type II HKs and possessed four
residues.A helix–loop–helixstructurewas predicted in CheAs. Two HKs, PA3078 and PA4380, could not be
these molecules. The H-box motif of Type IV HKs assigned to the defined type, so were categorized as
contained a proline residue on the N-terminal side and unclassified.Helix–loop–helixstructureswerepredicted
theconservedsequenceFLFNALontheC-terminalside intheH-boxregionoftheTypeIandIIIHKsandtheH
of the histidine site of phosphorylation. Finally, the P1 toNdistancesforeachoftheHKtypesweresimilarto
domainofCheAcontainedtheconsensusmotifHSIKG thosefoundinthedifferentHKtypesofE.coli.Finally,
and exists at the N terminus of the protein. the majority of the HKs were found in operons with
cognate RRs.
Thedistancebetweentheconservedhistidineresidueof
the H-box and the conserved asparagine residue of the
kinase domain(Hto Ndistance)was characteristic for HKfamiliesinbacterialgenomes
the different HK subtypes. The mean H to N distance
The HK gene families of bacterial genomes listed as
wasapproximately116residuesand96residuesforthe
completed in the TIGR database were characterized
TypeIandIIHKs,respectively(Table1).ThemeanH
using the cluster analysis approach. We began the
to N distance was 110 residues and 92 residues for the
analysis with free-living micro-organisms containing
TypeIIIandIVHKs,respectively.Thekinasedomainof
relatively large genomes.
CheA was characterized by insertions between the N
and G1 boxes and the G1 and F boxes. The N-box of A total of 44HKs were identified in the genome of
CheA contained a histidine residue at the N1 position. Vibriocholerae(Heidelbergetal.,2000),whichincluded
ThelocalizationoftheH-boxattheNterminusofCheA three new proteins (VCA0705, VC0694, VCA0851) not
created an H to N distance of 325 residues (Table 1; previously listed in the TIGR gene table. V. cholerae
Kofoid & Parkinson, 1988). Finally, it has been shown possessed a large Type I group which included 7 Type
that26ofthe29HKsofE.coliareorganizedinoperons IA,9 TypeIBand 12TypeICmolecules (Table2). We
with cognate RRs (Mizuno, 1997). noted that a cluster of HKs within the Type IC group
1201
D.-j. KIM and S. FORST
BaeS
CpxA
PA0930
PA2687
PA3206
RstB
PA1158
PA1798
EnvZ
PA5199
PA3191
PA4102
KdpD
PA1636
PA5484
BasS
YgiY
PA4777
PA0757
PA2480 IA
PhoR
PA5361
YedV
PA4886
YbcZ
PA2810
PA1438
IA PA2524
CreC
PA0464
YfhK
PA1396
PA1976
PA1992
PA3271
PA4036
BarA
PA0928
PA4112
TorS
PA1611
PA3462
PA2824
PA4982
IB
PA2583
PA3044
PA3946
RcsC
ArcB
IB EvgS
PA4856
PA3974
PA3078
PA4380
AtoS
HydH
PA4725
PA4398
PA2571
PA2882
PA1336
PA5165
PA4293
PA5512 IC
PA4197
Type I PA4546
PA1243
PA2177
NtrB
PA5124
PA1098
IC PA4494
PA4117
PhoQ
PA1180
Type II PA2656
DcuS
DpiB
CheA
Che A PA0178
PA1458
PA0413
PA3704
NarX
PA3878
Type III
NarQ
UhpB
PA1979
PA0600
Type IV YehU
B2380
PA5262
0·1 changes
.................................................................................................................................................................................................................................................................................................................
Fig.3.PhylogeneticanalysisoftheHKsofPsd.aeruginosa.DendrogramshowingtheanalysisofacombineddatasetofE.
coliandPsd.aeruginosaHKs.HKsofPsd.aeruginosaarerepresentedbyboldtype.
possessed the H-box motif HDLNNP in which the possessed helix–loop–helix structures in the H-box
typical positively charged residue at position 4 was regionandameanHtoNdistanceof110residues.Type
substituted by an asparagine residue. The Type I HKs II, III, IV and CheA HKs were also present in V.
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Histidinekinasefamily
Table2.DistributionofHKtypesinbacteria
HKtype Escherichia Vibrio Pseudomonas Xylella Bacillus Synechocystis Deinococcus Thermotoga Aquifex
coli cholerae aeruginosa fastidiosa subtilis sp. radiodurans maritima aeolicus
TypeIA 12 7 24 4 6 11 13 4 –
TypeIB 5 9 11 1 0 19 2 – –
TypeIC 3 12 16 3 5 – – 1 –
TypeII 2 3 – – 4 – – – –
TypeIII 3 3 3 – 9 2 4 – –
TypeIV 2 2 1 1 3 – – – –
CheA 1 3 4 1 1 2 – 1 –
Unclassified – 4 2 1 8 4 1 2 3
TotalHKs 29* 44* 63* 12* 36 38 20 8 3
Total 4228 3885 5570 2904 4100 4594 3187 877 1512
proteins
Percentage 0–69 1–13 1–13 0–41 0–87 0–82 0–62 0–43 0–20
HKs
*IncludesPhoQ-likeHK.
cholerae.Additionally,fourHKsdidnotclusterwitha slr0331wasatypicalTypeIIIHK,slr1212containedan
definedgroupandwerethereforeplacedinanunclassi- atypicalH-boxsequence(HHRhKNNLQ)connectedto
fied category. Twenty-eight of the HKs were found on a typical unorthodox kinase domain. Type II and IV
thelargechromosomeofV.cholerae(2–96Mb)while16 HKswerenotpresentinSynechocystissp.Finally,only
were found on the small chromosome (1–07Mb). The 13 of the HKs were organized in operons with cognate
majority of the HKs existed in operons with cognate RRs (Mizuno et al., 1996).
RRs.
TheGram-positivebacteriumDeinococcusradiodurans
(White et al., 1999) contained 20HKs, 13 of which
Xylella fastidiosa (Simson, 2000), like E. coli and V.
cholerae, is a member of the c-subclass of the Proteo- belongedtotheTypeIAgroup.TheHKcontentofType
IIIHKs(4outof20)wasrelativelyhigh.Thegenomeof
bacteria.X.fastidiosacontainedpredominantlyTypeI
this bacterium possesses two chromosomes (2–6 and
HKs(Table2)andlackedTypeIIandIIIHKs.TheHKs
0–41Mb),amegaplasmid(0–18Mb)andasmallplasmid
inthis bacterium existed inoperonswith cognate RRs.
(45kb). Thirteen of the HKs were present on the large
The HK family of the Gram-positive bacterium B.
chromosome, three were located on the smaller
subtilis (Kunst, 1997) contained all five HK types. The
chromosomeandfourwerelocatedonthemegaplasmid.
TypeIgroup,containingsixTypeIA,fiveTypeICHKs
In the thermophilic bacterium Thermatoga maritima
and noTypeIBHKs, was not aslarge asthat found in
(Nelsonetal.,1999)themajorityoftheHKs(5outof8)
theGram-negativebacteriaanalysedabove,while25%
belongedtotheTypeIgroupwhiletwoHKsremained
of the HKs belonged to the Type III group. B. subtilis
unclassified. Seven of the eight HKs of T. maritima
contained a relatively large number of HKs which did
existed in operons with cognate RRs. Finally, the
not cluster within the five defined HK types. Three
hyperthermophilic bacterium, Aquifex aeolicus, which
unclassifiedHKs(YtsB,YvcQ,YxdK)clusteredtogether
is considered to be one of the earliest diverging
intoonecladewhilethreeotherunclassifiedHKs(YbdK,
eubacteria (Deckert et al., 1998), possessed three HKs,
YrkQ,YccG)formedaseparateclade(seeTable5).All
none of which could be classified. This organism is
oftheHKsofB.subtilisexistedinoperonswithcognate
motile and possesses polytrichous flagella but does not
RRwiththeexceptionoftheTypeICgroupwhichwere
contain an identifiable CheA protein.
orphans (Fabret et al., 1999).
Insummary,92%oftheHKsanalysedwereabletobe
A markedly different HK distributionwas found in the
assigned to one of the five major HK types. Several
cyanobacterium,Synechocystissp.(Kanekoetal.,1996).
bacteria contained all five HK types. The majority of
This bacterium possessed a large Type IB group but
HKs (63%) belonged to the Type I group while the
lackedTypeICHKs.SeveraloftheTypeIBHKsdidnot
distributionofthevarioussubtypesvariedconsiderably.
contain D1 or HPt modules. Whether these Type IB
In the bacteria, most of the HKs were organized in
moleculesrepresentHKstowhichadditionalphospho-
operons with cognate RRs, with the notable exception
relay modules had not been added or are hybrid types
of Synechocystis.
which lost the phosphorelay modules, remains to be
determined.Interestingly,thetwoCheAproteinsidenti-
HKfamiliesofhumanpathogens
fiedinSynechocystissp.containedadditionalphospho-
relay modules (Mizuno et al., 1996). Synechocystis sp. The size of the genomes of pathogenic bacteria is
contains two type III HKs, slr0331 and slr1212. While generallysmallerthanthatoffree-livingbacteria(Table
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D.-j. KIM and S. FORST
Table3.DistributionofHKtypesinpathogenicbacteria
HKtype Haemo- Neisseria Rickettsia Helico- Campylo- Chlamydo- Chlamydia Borrelia Treponema Myco-
philus meningi- prowazekii bacter bacter phila tracho- burgdorferi pallidum bacterium
influenzae tidis pylori jejuni pneumoniae matis tuberculosis
MC58 CWL029 serovarD
TypeIA 2 2 1 – – – – – – 9
TypeIB 1 – 2 – – – – 1 – –
TypeIC – 1 1 1 1 1 1 1 1 –
TypeII – – – – – – – – – –
TypeIII 1 1 – – – – – – – 4
TypeIV – – – – – – – – – –
CheA – – – 1 1 – – 2 1 –
Unclassified – 1 – 2 5 – – – – –
TotalHKs 4 5 4 4 7 1 1 4 2 13
Totalproteins 1730 2158 834 1590 1654 1073 894 1283 1041 3924
Percentage 0–23 0–23 0–48 0–25 0–42 0–09 0–11 0–31 0–19 0–33
HKs
HK subtype HK H-box
(a)
+
Type I
Type II
CheA
Kinase type HK N G1 F G2 G3
(b)
Orthodox
CheA
.................................................................................................................................................................................................................................................................................................................
Fig. 4. Alignment of the kinase and H-box regions of Arc. fulgidus. (a) Sequence alignment of the H-box regions.
Shadings and symbols are as in Fig. 1. Only AF1483 and CheA possessed predicted helix–loop–helix structures. (b)
Sequencealignmentofthekinasedomains.
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Histidinekinasefamily
BaeS
BasS
YgiY
PhoR
YbcZ
CpxA
RstB
EnvZ
IA
KdpD
YedV
CreC
YfhK
AtoS
IC
HydH
NtrB
BarA
RcsC
TorS
Type I
ArcB
IB
EvgS
AF1483
mth444
PhoQ
DcuS
DpiB
AF1721
AF1639
AF1515
Type II
AF0021
AF0208
AF0893
784830
AF0450
AF0770
AF1467
AF2109
AF1184
AF1452
CheA
AF1040
NarX
NarQ
UhpB
mth292
mth356
mth619
mth174
Type III mth468
mth446
mth560
mth823
mth985
mth123
mth1124
mth459
mth901
mth902
Type IV YehU
B2380
0·1 changes
.................................................................................................................................................................................................................................................................................................................
Fig. 5. Phylogenetic analysis of the HKs of Arc. fulgidus and Mbc. thermoautotrophicum. Dendrogram showing the
analysisofacombineddatasetofHKsofE.coli,Arc.fulgidusandMbc.thermoautotrophicum.HKsofArc.fulgidusand
Mbc.thermoautotrophicumarerepresentedbyboldtype.
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D.-j. KIM and S. FORST
Table4.DistributionofHKtypesinarchaea
HKtype Methanobacterium Archaeoglobus Pyrococcus Methanococcus Aeropyrum
thermoautotrophicum fulgidus horikoshii jannaschii pernix
TypeIA – – – – –
TypeIB 1 1 – – –
TypeIC – – – – –
TypeII – 13 – – –
TypeIII 14 – – – –
TypeIV – – – – –
CheA – 1 1 – –
TotalHKs 15 15 1 0 0
Totalproteins 1855 2436 2061 1738 2694
PercentageHKs 0–80 0–61 0–04 0 0
3). The mean HK content of the pathogenic bacteria motifHHRVKNNLQwhichwasidenticaltotheH-box
was 0–26%ascompared with 0–65%forthe free-living sequence found in Mycobacterium and Synechocystis.
bacteria. The human pathogenic bacteria contained HKs containing this sequence belong to the Type IIIB
predominantlyTypeIHKs(Table3).Interestingly,the group. Cluster analysis also showed that the HKs of
Gram-positive bacterium, Mycobacterium tuberculosis Mbc. thermoautotrophicum branched within the Type
(Daviesetal.,1998)containedarelativelyhighcontent III group forming a clade that was distinct from the E.
(4 of 13) of Type III HKs. One of the Type III HKs, coli group (Fig. 5). The mean H to N distance of the
Rv3220,containedatypicalunorthodoxkinasedomain Mbc. thermoautotrophicum HKs was 110 residues.
andtheatypicalH-boxsequenceHHRhKNNLQwhich Mbc. thermoautotrophicum possessed one orthodox
wassimilartotheH-boxofslr1212ofSynechocystissp. HKsubtypeanddidnotcontainsemi-orthodox,minor,
These proteins are referred to as Type IIIB HKs (see CheA, tripartite or hybrid HKs. Finally, most of the
Table 5). The majority of the HKs and RRs in these HKs were not organized in operons with cognate RRs.
bacteria were organized in operons with cognate RRs.
Methanococcus jannaschii (Bult et al., 1996) and
Helicobacter pylori and Campylobacter jejuni, both of Aeropyrum pernix (Kawarabayasi et al., 1999) were
which belong to the d-subclass of the Proteobacteria, previouslyfoundtolackHKs.Are-examinationofthese
containedseveralunclassifiedHKs.Thisfindingsuggests genomes confirmed that HKs were missing in these
that this group of bacteria may possess a unique HK organisms. The genome of Pyrococcus horikoshii has
typethatisnotyetidentifiedinthecurrentHKdataset. been completed recently (Kawarabayasi et al., 1998).
TheonlyHKfoundinthisgenomewasCheA(Table4).
TheHKandRRorganizationinarchaeawasmarkedly
AnalysisoftheHKfamilyofarchaealgenomes differentthanthatfoundinbacteria.MostoftheHKsin
Arc.fulgidusandMbc.thermoautotrophicumwerenot
The amino acid sequence alignment of the H-box
organizedinoperonswithacognateRR.OnlyAF0450
regions and kinase domains of the 15HKs of
ofArc.fulgidusandMTH0902andMTH0444ofMbc.
Archaeoglobusfulgidus(Klenketal.,1998)isshownin
thermoautotrophicum(Smithetal., 1997)were located
Fig. 4(a) and (b), respectively. Thirteen of the HKs
in operons with a cognate RR.
belongedtotheTypeIIsubtypewhileonlyonebelonged
totheTypeIgroup.TheH-boxmoduleofTypeIIHKs The genome sequence of the archaeon Halobacterium
contained the characteristic asparagine residue at sp. was recently completed (Ng et al., 2000). Of the 14
position 5. The H-box of the Type II HKs lacked a reportedHKs,weretrieved12HKs,8ofwhichformed
predictable secondary structure. Highly conserved glu- a clade within the Type I group but were distinct from
tamic acid and positively charged residues were the IA, IB and IC subtypes. These HKs appear to
identifieddownstreamoftheH-box(shadedinFig.4a). represent a subtype that is so far unique to Halo-
The kinase domains (Fig. 4b) possessed a conserved bacterium.ThreeTypeIIHKsandoneCheAwerealso
glycine residue in the F-box and the mean H to N identified. Finally, as found in Archaeoglobus, there
distancewas 96residues. Cluster analysis revealed that were more HKs (14) than RRs (6) in Halobacterium.
the Type II group of Archaeoglobus formed a separate
clade, designated the IIB subtype, within the Type II DISCUSSION
group of E. coli (Fig. 5). The 13HKs did not exist in
PhylogeneticanalysisofHKsinnumerousbacterialand
operonswiththenineRRsidentifiedinArchaeoglobus.
archaealgenomesledtotheidentification offive major
Earlier analysis of the Methanobacterium thermoauto- HK types. Of the 336 HKs analysed in this study 92%
trophicumgenomeidentified15HKs(Smithetal.,1997). couldbeassignedtooneofthefiveHKtypes.TypeIand
The H-box region of 14 of these HKs contained the IIHKspossessed orthodoxkinase domainswhile Type
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