Dissecting the protein architecture of DNA-binding transcription factors in bacteria and archaea

12 Pages·2017·4.73 MB·English

RESEARCH ARTICLE
Rivera-Gómezetal.,Microbiology2017;163:1167–1178
DOI10.1099/mic.0.000504
Dissecting the protein architecture of DNA-binding transcription
factors in bacteria and archaea
NancyRivera-Gómez,1,*MarioAlbertoMartínez-Núñez,2NinaPastor,3KatyaRodriguez-Vazquez4and
ErnestoPerez-Rueda5,6,*
Abstract
Gene regulation at the transcriptional level is a central process in all organisms where DNA-binding transcription factors
playafundamentalrole.ThisclassofproteinsbindsspecificallyatDNAsequences,activatingorrepressinggeneexpression
as a function of the cell’s metabolic status, operator context and ligand-binding status, among other factors, through the
DNA-binding domain (DBD). In addition, TFs may contain partner domains (PaDos), which are involved in ligand binding and
protein–protein interactions. In this work, we systematically evaluated the distribution, abundance and domain organization
of DNA-binding TFs in799 non-redundant bacterial and archaeal genomes. We found thatthe distributions of the DBDs and
their corresponding PaDos correlated with the size of the genome. We also identified specific combinations between the
DBDs and their corresponding PaDos. Within each class of DBDs there are differences in the actual angle formed at the
dimerization interface, responding to the presence/absence of ligands and/or crystallization conditions, setting the
orientation of the resulting helices and wings facing the DNA. Our results highlight the importance of PaDos as central
elements that enhance the diversity of regulatory functions in all bacterial and archaeal organisms, and our results also
demonstrate the role of PaDos in sensing diverse signal compounds. The highly specific interactions between DBDs and
PaDos observed in this work, together with our structural analysis highlighting the difficulty in predicting both inter-domain
geometry and quaternary structure, suggest that these systems appeared once and evolved with diverse duplication events
inalltheanalysedorganisms.
INTRODUCTION interactions. Today, diverse structures of bacterial and
archaeal TFs have been determined, and thousands of
Gene regulationat thetranscriptional levelisa central pro-
amino acid sequences are known for many more, allowing
cess in all organisms, and DNA-binding transcription fac-
forexhaustiveevaluationoftheseregulatoryproteinsindif-
tors (TFs) play a fundamental role. TFs specifically bind to
ferent organisms. In this context, the structural diversity
DNA,activatingorrepressinggeneexpressionasafunction
of the cell’s metabolic status, operator context and ligand and evolutionary distribution of TFs have been explored,
binding,amongotherfactors[1–3].Ingeneral,theTFscon- identifyingwingedhelix-turn-helix(wHTH)domainsasthe
sistofaDNA-bindingdomain(DBD),whichisinvolvedin most abundant DBDs in bacterial and archaeal TFs, fol-
specific contacts with DNA, and a partner domain (PaDo), lowed by homeodomain-like and lambda repressor-like
which is involved in ligand binding or protein–protein structures [4–6]. In contrast, structures such as ribbon-
Received15March2017;Accepted20June2017
Authoraffiliations:1CentrodeInvestigacionesenBiotecnología,UniversidadAutónomadelEstadodeMorelos,Cuernavaca,M(cid:19)exico;2Laboratoriode
EstudiosEcogenómicos,FacultaddeCiencias,UnidadAcad(cid:19)emicadeCienciasyTecnologíadeYucat(cid:19)an,UniversidadNacionalAutónomadeM(cid:19)exico,
M(cid:19)erida,Yucata(cid:19)n,M(cid:19)exico;3CentrodeInvestigaciónenDina(cid:19)micaCelular,IICBA.UniversidadAutónomadelEstadodeMorelosAv.Universidad1001,Col.
Chamilpa, Cuernavaca, Morelos 62209, M(cid:19)exico; 4Departamento de Ingeniería de Sistemas Computacionales y Automatización. Instituto de
Investigaciones en Matema(cid:19)ticas Aplicadas y en Sistemas. Ciudad Universitaria, Universidad Nacional Autónoma de M(cid:19)exico, M(cid:19)exico, D.F, M(cid:19)exico; 5
Instituto de Investigaciones en Matema(cid:19)ticas Aplicadas y en Sistemas, Universidad Nacional Autónoma de M(cid:19)exico, M(cid:19)erida, Yucat(cid:19)an, M(cid:19)exico; 6
DepartamentodeIngenieríaCelularyBiocata(cid:19)lisis,InstitutodeBiotecnología,UniversidadNacionalAutónomadeM(cid:19)exico,Cuernavaca,Morelos,M(cid:19)exico.
*Correspondence:NancyRivera-Gómez,[email protected];ErnestoPerez-Rueda,[email protected]
Keywords:transcriptionfactor;comparativegenomics;bacteriaandArchaea;proteinarchitecture;generegulation..
Abbreviations:Acyl-CoA(dCt),Acyl-CoAdehydrogenaseC-terminaldomain-like;Arginine,C-terminaldomainofargininerepressor;cAMP-bin,cAMP-
binding domain-like; C-Ttr, C-terminal domain of transcriptional repressor; DBD, DNA-binding domain; Dimeric, dimeric alpha+beta barrel; FDR,
false-discoveryrate;GAF,GMP-specificphosphodiesterases,adenylylcyclasesandFhlA;HMM,hiddenMarkovmodel;MetJ,methioninerepressor;
Methylat,methylatedDNA-proteincysteinemethyltransferasedomain;O6-MeG,O6-methylguanine;PaDo,partnerdomain;PBPII,periplasmic-binding
protein-like II; PLP-depend, pyridoxal phosphate-dependent transferase; Phoshotr, phoshotransferase/anion transport protein; pp(G)pp, guanosine
tetraphosphate; Rah, Ribbon helix-helix; Ribonucle, ribonuclease H-like; SAM, S-adenosylmethionine; SIS, sugar isomerase; Tex, Tex N-terminal
region-like;TF,transcriptionfactor;wHTH,wingedHelix-turn-helix.
SevensupplementarytablesandtwosupplementaryfiguresareavailablewiththeonlineSupplementaryMaterial.
000504ã2017TheAuthors
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helix-helix (Rhh), IHF-like and PhoU-like domains have identified by HMM profiles were scrutinized to assess their
been identified in lower proportions, and some of these domain organization by using the Superfamily database
havebeenassociatedwithspecificphyla,suchasRhh,which assignments[12].
isfoundalmostexclusivelyinProteobacteria[7].
Distribution of DBDsin bacterial and archaeal
In general, TFs contain structural domains involved in genomes
diverse functions, such as DNA and ligand binding; how-
In order to determine the abundance and distribution of
ever, the architectures of PaDos have only been systemati-
DBDs in the bacterial and archaeal genomes, the rate of
cally analysed for the wHTH regulatory proteins, whereas
occurrence of each structural superfamily of DBDs for the
integrative analyses to describe PaDos for proteins with
number of organisms per taxon was calculated, and from
otherDBDsarelacking[6].Therefore,inthisworkweeval-
this information a matrix was constructed. In this regard,
uatedtheproteinarchitecturesofDNA-bindingTFs,i.e.14
organisms were classified in terms of their corresponding
differentDBDsand298differentPaDos,in799non-redun-
phylum, according to the NCBI taxonomic classification
dant genomes, in terms of their diversity, abundance and
system, except for Firmicutes and Proteobacteria, which
distribution.WeconsideredTFsasthoseproteinsthatacti-
wereconsideredattheclasslevelbecauseofthelargenum-
vate or repress gene expression but do not belong to the
ber of organisms included. Finally, the matrix showing the
transcriptional basal machinery. From this analysis, we
presenceofeachsuperfamilypertaxonwasanalysedwitha
foundacorrelationbetweentheabundanceofDBDs,PaDos
hierarchical clustering approach considering a Manhattan
and genome size, and a highly specific association between
distance and complete linkage algorithm, with correlation
DBDsandPaDos.
uncentered as a similarity measure, as implemented in the
Mev4program[13].
METHODS
Enrichment analysisof DBDsand PaDos
Bacterial and archaeal genomes analysed
To evaluate the associations between the DBDs and their
The2786bacterialandarchaealgenomesusedinouranaly-
corresponding PaDos, an enrichment analysis using a one-
sisweredownloadedfromtheNCBIftpservergenomesec- tailed Fisher’s exact test was conducted. We set statistical
tion (http://ncbi.nlm.nih.gov). In this analysis, we only significance at a P-value of less than 0.05. Because several
considered open reading frames (ORFs) that encode pre- categoriesweretestedatthesametime,multiple-testingcor-
dicted protein sequences in all organisms. In order to rectionswereperformedusingtheBenjaminiandHochberg
exclude any bias associated with over-representation of step-upfalse-discoveryrate(FDR)-controllingprocedureto
organisms that have been completely sequenced and are calculate adjusted P-values. All analyses were performed
associated with particular divisions, non-redundant using the R software environment for statistical computing
genomes were considered from the study conducted by andgraphicsandpackagemulttest[14].
Martínez-Núñez et al. [8]. Briefly, the selection of the
organisms was carried out through the concatenation of 21 Structural analysis
conserved proteins across downloaded sequenced genomes. A total of 84 protein structures were downloaded from the
Next, a single data set for phylogenetic analysis was con- Protein Data Bank (www.rcsb.org) and manually inspected
structed, and we eliminated genomes located more closely with the Visual Molecular Dymanics software [15]. The
together on a phylogenetic tree, as previously reported, location of the DBD was determined from the domain
leaving a set of 710 bacterial and 89 archaeal genomes assignmentofthestructuralrecordandfromtheassociated
(Table S1, available with the online Supplementary reportofthestructure,whenavailable(TableS5).
Material).
RESULTS AND DISCUSSION
Identification of DNA-binding TFs and domain
assignations Content and diversity of DBDscombined with TFs
TFswereidentifiedbasedondiversesourcesofinformation, Based on HMM profiles constructed from known DBDs of
such as the DBD database assignments [9] and specialized bacterial TFs, a total of 122336 TFs were identified in 799
databases for model organisms, such as RegulonDB v6.0 non-redundant bacterial and archaeal genomes. Subse-
[10] and DBTBS v5.0 [11]. Therefore, hidden Markov quently, we determined the proportion of TFs for each
model (HMM) profiles of DBDs were constructed from organism, noting an increment in the total number of TFs
known TFs and from information in the RegulonDB and as a function of genome size, as has been previously
DBTBS databases to scan all the bacterial and archaeal described[8],i.e.smallgenomescontainasmallnumberof
genome sequences. The repertoire of TFs identified in the TFs, whereas large genomes contain a large number of TFs
model organisms was cross-checked to evaluate how effi- (Spearmancorrelation=0.9190052,P-value<2.2e–16)(Figs1
ciently the profiles identified possible TFs, i.e. the intersec- andS1).Inthiscontext,CandidatusCarsonellaruddiiPVis
tion between well-known TFs identified by HMM profiles. the organism with the smallest repertoire of TFs identified
The HMM profiles were used to identify potential DBDs sofar[16],withonlysixTFsidentifiedin182ORFs,repre-
by setting the E-value at (cid:20)10–4. Finally, those proteins senting 3.3% of its gene repertoire. On the other hand,
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0
0
8
s
ctor 600
a
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n
a
of tr
al 0
ot 0
T 2
0
8) 2) 9) 5) 9) 4) 9) 2) 7) 3) 6) 3) 9) 7) 2) 3) 3) 2) 2)
00 (4 00 (4 0 (10 0 (11 00 (8 00 (8 00 (6 00 (7 00 (5 00 (3 00 (1 00 (2 000 ( 00 (1 000 ( 500 ( 000 ( 500 ( 000 (
5 0 0 0 5 0 5 0 0 5 0 5 7 5 8 8 9 9 0
1 5 0 2 3 3 4 5 5 6 6 7 1
1 2
Genome size
Fig. 1.NumberofTFspergenomesize.Onthey-axisisthenumberofTFs,andonthex-axisarethegenomes,groupedatsizeinter-
valsof500ORFs.Thenumberoftotalgenomesperintervalisindicatedinbrackets.
Amycolatopsis mediterranei U32, with the largest genome MazE/MraZ-like superfamilies, among others (Table 1)
analysed in this work, also includes the highest number of [19].Subsequently,weexploredwhetherallthesuperfamily
TFs (814), which represents 10% of its genes that encode DBDsarehomogeneouslydistributedamongthe799bacte-
regulatoryproteins[17]. rial and archaeal genomes. We found that organisms with
reducedgenomesalsocontainedalowproportionofdiffer-
In order to evaluate how TFs vary within a species, 192
ent superfamily DBDs, with a slight increase in DBDs in
strains of E. coli, with ranges from 3708 (E. coli str. K-12
organismswithgenomesizesbetween1000and2000ORFs;
substr. MDS42) to 5611 (E. coli O26 H11 str. 11368), were
thenumberofDBDsremainedalmostconstantingenomes
chosen. Based on Supfam assignments we found a similar
withmorethan2000ORFs.Thus,anaverageof11different
result observed for the total of bacterial and archaeal
superfamily DBDs are associated with each organism.
genomes, suggesting that small genomes contain a small
Hence, each organism exhibits a low diversity of structural
repertoire of TFs versus large genomes with an increase of
DBDs, suggesting that these superfamilies have increased
its repertoire of TFs, and reinforcing the notion of regula-
mainlyviadiverseduplicationeventsfollowedbyevolution-
torydiversitybetweenandwithinspecies.
ary divergence. All these data reinforce the scenario pro-
To gain insights into the protein architecture of the DNA- posed by Itzkovitz et al. [20] for the evolution of TF
binding TFs, the complete set of regulatory proteins was superfamilies, where ‘simple’ organisms, which require few
analysed in terms of each protein’s number of structural TFs, might preferentially use certain superfamilies such as
domains (see Methods). From this structural dissection, we the lambda repressor-like and wHTH superfamiles. When
found that almost 40% of the TFs are mono-domain pro- these superfamilies reach the corresponding upper bound,
teins,50%oftheTFsareorganizedintwodomainsandthe new superfamilies are needed. At these points, organisms
remaining 10% exhibit three or more structural domains. shift their TF usage to novel superfamilies with greater
In addition, the DBDs of these regulatory proteins were degreesoffreedomandhighermaximalnumbers.Anexam-
classifiedinto14differentsuperfamiliesbasedonsuperfam- ple is the increased use of C2H2 zinc finger TFs in more
ilyassignments[18](Table1).Fromthisclassification,90% complexorganisms[20].
of DBDs were grouped into five large superfamilies: the
Distribution of DBDsis different in prokaryotic
wHTH, homeodomain, lambda repressor-like, C-terminal
genomes
effectordomainofbipartiteresponseregulators,andnucleic
acid-binding protein superfamilies. The 10% of TFs with In order to evaluate the distribution of DBDs across all the
three or more structural domains included the Rhh, TrpR- organismsstudied,24phylaandclassesofBacteria(Proteo-
like, flagellar transcriptional activator FlhD and AbrB/ bacteria and Firmicutes were divided into five and three
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Table1.DBDsanalysedinthiswork.Datainbracketsindicatetheabbreviationsusedinthemanuscript
DNA-bindingdomain Total Proportion Numberof PaDos Numberofsuperfamilies
families
WingedhelixDNA-bindingdomain(wing) 50534 0.413 38 34936 153
Homeodomain-like(home) 22135 0.181 6 14479 92
Lambdarepressor-likeDNA-bindingdomains(lamb) 13442 0.11 6 5591 107
C-terminaleffectordomainofthebipartiteresponseregulators(bipa) 12186 0.1 3 11763 62
Nucleicacid-bindingproteins(nucl) 11923 0.097 1 11893 42
PutativeDNA-bindingdomain(put) 4565 0.037 2 854 41
IHF-likeDNA-bindingproteins(IHFL) 1949 0.016 1 5 1
AbrB/MazE/MraZ-like(AbrB) 1790 0.015 3 123 7
Ribbon-helix-helix(Rhh) 1727 0.014 4 394 19
KorBDNA-bindingdomain-like(KorB) 1058 0.009 1 970 11
Glucocorticoidreceptor-like(DNA-bindingdomain)(gluc) 764 0.006 2 570 3
TrpR-like(TrpR) 228 0.002 1 15 3
FlagellartranscriptionalactivatorFlhD(FlhD) 82 7E-04 1 0 0
Haemolysin-expressionmodulatingproteinHHA(HHA) 50 4E-04 1 0 0
classes, respectively) and three phyla of Archaea were ana- superfamilies. These DBDs were identified in low propor-
lysed (Fig. 2). Subsequently, a cluster analysis, as described tions in diverse bacterial divisions, such as Nitrospirae,
in Methods, was performed. Four main groups of DBDs Verrucomicrobia, Acidobacteria, Chloroflexi, Thermi, Gem-
sharing similar taxonomic distributions were identified, matimonadetes, Deferribacteres and Synergistes, whereas
consideringadistancethresholdof9.79,whichcorresponds these DBDs were absent in Elusimicrobia, Fibrobacteres,
to the first plateau associated with the node-height plot. Dictyoglomi, Negativicutes, Fusobacteria and Nanoarch-
ThisanalysisisrelevanttoelucidatethoseDBDsuniversally aeota. The Rhh domain is a DBD consisting of four heli-
and specifically associated with bacterial and archaeal cell ces in an open array of two hairpins. Such domains have
divisions,suggestingcommonancestryand/orspecificline- been found in several bacterial and phage repressors,
ageassociations.Therefore,inthefollowing,wedescribethe including the E. coli K-12 methionine repressor (MetJ),
composition of each group. The first group included the which when combined with S-adenosylmethionine (SAM)
most abundant and widely distributed DBDs, such as the represses the expression of the methionine regulon and of
putativeDNA-bindingdomain,C-terminaleffectordomain enzymes involved in SAM synthesis [26]. This superfamily
of the bipartite response regulators, homeodomain, lambda represents 1.4% of the total DBDs identified in all the
repressor-like, nucleic acid-binding, wHTH and the IHF- genomes. The AbrB/MazE/MraZ-like superfamily includes
like superfamily proteins (Fig. 2). The wHTH, C-terminal transition-state regulators, putative regulators of cell wall
effector domain of the bipartite response regulators, and biosynthesis and regulators of phosphate uptake [27]. This
homeodomain-like DBDs are the most abundant superfa- superfamily is also widely distributed among prokaryotes,
milies identified in Bacteria and Archaea, and they also although in low proportions, but is not found in Nano-
have the greatest number of associated TF families identi- archaeota, Elusimicrobia and Gemmatimonadetes. The
fiedsofar(Table1),withalmostallfamiliesinvolvedinthe representative proteins associated with this group are not
regulationofprimarymetabolism,suchasLysR,Crp,ArgR, related at the sequence level; however, they share a com-
andAraC/XylS.Thelambdarepressor-likesuperfamilycon- mon fold [28]. MraZ and AbrB have been described as
sistsofsixfamiliesofregulatoryproteins,suchastheGalR/ two-domain proteins: one domain, which is in the N ter-
LacI family which is associated with nucleotide metabolism minus, interacts with DNA, whereas the other domain is
and carbon assimilation [21–23], both considered ancient located at the C terminus and plays a role in oligomeriza-
metabolic processes [24, 25]. Finally,the nucleic acid-bind- tion [27, 29, 30]. In this analysis, we found that 91% of
ing superfamily, which includes the cold-shock DNA-bind- the total proteins associated with this superfamily corre-
ing domain-like family, has been associated fundamentally sponded to monodomain proteins.
with temperature response regulation. An appealing
The third group included DBDs identified in low propor-
hypothesis is that the DBDs identified in both the bacteria
tions or absent in diverse taxonomic divisions, such as the
and archaea cellular domains have the same origin, as they
glucocorticoid receptor-like and the KorB superfamilies.
emergedbeforetheArchaea–Bacteriasplit.
The glucocorticoid receptor-like superfamily comprises a
The second group included DBDs identified in low pro- family of ligand-activated nuclear receptors that includes
portions in bacterial and archaeal cellular divisions, such the LIM domain and prokaryotic DksA/TraR C4-type zinc
as the ribbon-helix-helix and the AbrB/MazE/MraZ-like finger, which has been identified in bacterial and archaeal
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0 1.0
FlhDHHA TrpRGlucKorBAbrBRhhBipAPut IHFL Home Lamb NucWing
32.79
16.395
0.0
Crenarchaeota (27)
Euryarchaeota (61)
Nanoarchaeota (1)
Aquificae (7)
Thermotogae (8)
Dictyoglomi (2)
Chloroflexi (9)
Actinobacteria (89)
Planctomycetes (3)
Chlamydiae (8)
Verrucomicrobia (4)
Nitrospirae (1)
Acidobacteria (5)
Elusimicrobia (1)
Synergistetes (2)
Cyanobacteria (34)
Clostridia (48)
Bacilli (68)
Negativicutes (2)
Tenericutes (24)
Fibrobacteres (1)
Gemmatimonadetes (1)
Chlorobi (13)
Bacteroidetes (30)
Thermi (8)
Spirochaetes (16)
Fusobacteria (5)
Alphaproteobacteria (95)
Deltaproteobacteria (31)
Epsilonproteobacteria (20)
Gammaproteobacteria (120)
Betaproteobacteria (52)
Deferribacteres (3)
Fig. 2.DistributionofDBDsuperfamiliespertaxonomicaldivision.EachcolumndenotesaDBDsuperfamily,whereasrowsdenotetax-
onomicgroups.Thebar atthetopof thefigureindicatestherelativeabundanceofDBDsper group, witha valueof0 representing
absence(black), 0.01 to 0.3 representing variable distribution (blue), 0.31 to 0.9 representing moderateddistribution (green–yellow–
orange)and1(red)correspondingto100%presence.Thetotalnumberofgenomespertaxon isindicatedinbrackets.
genomes. DksA is a regulator that acts synergistically with which is mainly involved in the regulation of tryptophan
an alarmone, pp(G)pp (guanosine tetraphosphate, which biosyntheticgenes.
formsacomplexwithRNApolymeraseandregulatesamino
In summary, based on the distribution of the DBDs, four
acid metabolism-related genes) [31, 32]. KorB negatively
clusters wereidentified, suggestingthat, in general, thepat-
regulatesgenesoftheRK2korregulon[33].Theglucocorti-
tern of DBDs is influenced by the lifestyle of the organism.
coid receptor-like superfamily was mainly identified in
Therefore, the absence of some superfamilies, such as the
Proteobacteria,anditwasabsentinCyanobacteria,Synergis-
winged helix-turn-helix, homeodomain or lambda repres-
tetesandThermi,whereasKorBwasconstrainedtotheFir-
sor-like superfamilies, could be associated with events of
micutes,SynergistetesandThermibacterialdivisions.
gene loss as a consequence of the respective lifestyle, as
Finally,thefourthgroupincludedthreesmall-lengthsuper- found in Elusimicrobia, Fibrobacter and Nanoarchaeota,
families,mainlyidentifiedinEnterobacteriaandBurkholde- which are symbionts of insects, symbionts of ruminants,
ria,suchastheFlhDsuperfamily,involvedintheregulation and isolated in submarine hot vents, respectively [36–38],
of flagellum protein-encoding genes [34], HHA, a modula- whereas organisms with a large number of ORFs contain a
tor of the toxin alpha-hemolysin, involved in the response largediversityofDBDsinordertorespondtodiverseenvi-
toosmolarityandtemperaturechanges[35],andTrpR-like, ronmentalstimuli[8].
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Abundance of PaDos associated with TFs different PaDos per division, where Nanoarchaeota
(Archaea) and Tenericutes were the archaeal and bacterial
Inordertogaininsightintotheabundanceanddistribution
divisions with a minor proportion of PaDos, probably
of thePaDos associated with TFs, the PaDoswereanalysed
because these divisions include organisms with reduced
in the complete set of regulatory proteins. In total, 85331
genomes.Inthiscontext,smallgenomes,asinthearchaeon
PaDos were found to be associated with the repertoire of
Nanoarchaeum equitans Kin4-M [39], include 11 different
regulatory proteins and were classified into 298 different
PaDosassociatedwithtwodifferentDBDs.Onthecontrary,
superfamilies. In this context, the most abundant PaDos
theorganismwiththelargestgenomeanalysedinthiswork,
were associated with a large proportion of DBDs, such as
AmycolatopsismediterraneiU32(9,421ORFs),contains814
thewHTHDBD,whichcontains153differentPaDos(44%
TFs belonging to 11 different superfamilies and 613 PaDos
of the total), and the lambda repressor-like DBD, which
associated with 49 different superfamilies. Therefore, the
contains 107 different PaDos (26% of the total) (Table 1).
domain organization associated with the TFs, the relative
A correlation between DBDs and PaDos was observed
(Spearman correlation=0.97675, P-value <2.2e–16). For abundance of DBDs and their PaDos would contribute to
increasing the regulatory responses in Bacteria and
instance,wefoundthatthemostabundantPaDosarepref-
Archaea.
erentially associated with one DBD, such as the periplas-
mic-binding protein-like II (PBP II), which is intimately
Distribution of PaDos associated with DBDs
associated with the wHTH; CheY-like, which is mainly
In order to evaluate howthe PaDosare distributed in all the
associated with the C-terminal effector domain of the
organisms, an analysis based on their taxonomical classifica-
bipartite response regulators; and the Tetracycline repres-
tion was performed. This analysis is relevant under the
sor-like C-terminal domain, which is associated with the
hypothesis that a similar taxonomical distribution of PaDos
homeodomain-likesuperfamily.
andDBDssuggestsacommonancestryand/orspecificlineage
Inasubsequentstepandinordertoevaluatetheabundance associations. To do this, a hierarchical clustering approach
and distribution of PaDos described previously, we quanti- consideringa distancethresholdof18.04,whichcorresponds
fied the PaDos in all the bacterial and archaeal genomes. to the first plateau associated with the node-height plot,
From this analysis, we found that the number of PaDos revealed three main groups. The first group (Fig. 4a and
increased with genome size, similar to our findings with TableS2)included16PaDoswidelydistributedamongallthe
DBDs(Spearmancorrelation=0.8971116,P-value<2.2e–16) organisms, such as the GMP-specific phosphodiesterases,
(Figs 3 and S2). In addition, we found an average of 30 adenylyl cyclases and FhlA (GAF), and P-loop NTPase
0
0
6
0
0
5
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artner 300
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T
0
0
1
0
8) 2) 9) 5) 9) 4) 9) 2) 7) 3) 6) 3) 9) 7) 2) 3) 3) 2) 2)
00 (4 00 (4 0 (10 0 (11 00 (8 00 (8 00 (6 00 (7 00 (5 00 (3 00 (1 00 (2 000 ( 00 (1 000 ( 500 ( 000 ( 500 ( 000 (
5 0 0 0 5 0 5 0 0 5 0 5 7 5 8 8 9 9 0
1 5 0 2 3 3 4 5 5 6 6 7 1
1 2
Genome size
Fig. 3.NumberofPaDospergenomesize.Onthey-axisisthenumberofPaDos,andonthex-axisarethegenomes,groupedatsize
intervalsof500ORFs.Thetotalnumberofgenomesperintervalisindicatedinbrackets.
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(a) (b)
Iron 0PolynuclcAMP-bin LexA/Sig TetracycPBP-II Nucleic GAF Translat (SH3) P-loop KH-domain CheY-like Ribosomal (S5) Riho-PH ProkaryoNusA1.0 15.6120405 Rad51 DnaK0Pho N-t ParBArginine TPR GntC-TtrRossmannPBP-I NagBDimericPLP-depeChorismaRmlC-likAraC1Ribo-HL .0 RuvALexA 18.18475
7.8060503 9.092375
0 0
Crenarchaeota (27) Crenarchaeota (27)
Euryarchaeota (61) Euryarchaeota (61)
Nanoarchaeota (1) Nanoarchaeota (1)
Aquificae (7) Aquificae (7)
Thermotogae (8) Thermotogae (8)
Dictyoglomi (2) Dictyoglomi (2)
Chloroflexi (9) Chloroflexi (9)
Actinobacteria (89) Actinobacteria (89)
Planctomycetes (3) Planctomycetes (3)
Chlamydiae (8) Chlamydiae (8)
Verrucomicrobia (4) Verrucomicrobia (4)
Nitrospirae (1) Nitrospirae (1)
Acidobacteria (5) Acidobacteria (5)
Elusimicrobia (1) Elusimicrobia (1)
Synergistetes (2) Synergistetes (2)
Cyanobacteria(34) Cyanobacteria (34)
Clostridia (48) Clostridia (48)
Bacilli (68) Bacilli (68)
Negativicutes (2) Negativicutes (2)
Tenericutes (24) Tenericutes (24)
Fibrobacteres (1) Fibrobacteres (1)
Gemmatimonadetes (1) Gemmatimonadetes (1)
Chlorobi (13) Chlorobi (13)
Bacteroidetes (30) Bacteroidetes (30)
Thermi (8) Thermi (8)
Spirochaetes (16) Spirochaetes (16)
Fusobacteria (5)
Alphaproteobacteria (95) Fusobacteria (5)
Deltaproteobacteria (31) Alphaproteobacteria (95)
Epsilonproteobacteria (20) Deltaproteobacteria (31)
Gammaproteobacteria (120) Epsilonproteobacteria (20)
Betaproteobacteria (52) Gammaproteobacteria (120)
Deferribacteres (3) Betaproteobacteria (52)
Deferribacteres (3)
(c)
0 1.0
12.94243
6.471215
CCBNTFGCBTSFADEGBDCENATDCAPCVNAESeeihuhpull0lqccpyaaeeeeaiibylhrhheauapctonreseenrsticcanurtgilflllrmmrnsdhrttyiooaesaeoorrnoeuinnitaoiyoailcammlafiotempmrrrrosmbmlarbcooopartciroiortcpgbr pcriinraiiooocgabbyrvrid(hoaba obflcarciaucoi6p(cdhmtsicmlaoraapitieatcei8hoorcadthat itrt8eaticceotmexrc(eu eaayeiome)gt eaeeco(eo1)it(ttebsctoteer tt7r eaeeoe 4toreoe(tbiie3eer (iee o aboe9er)aro(snib81r((stsaosa)iti2s tae8boa2 a)atae a( ))ac b ((( a(4c(s 5b (i )(()d8(11cs 2at(2(1a 533 t()e)6a8 (et)6)( e2)c)())41e0(3rc1t93)r4ti7er))a)ti)e))ia) e))sa r (r i (5ai((3a19 2 (1)5(2) 1))02)0)
Fig. 4.DistributionandabundanceofPaDospercellulardivision.(a)ThemostabundantPaDos;(b)PaDoswithamoderatedistribu-
tion;(c)PaDoswithalowdistribution.EachcolumndenotesaPaDosuperfamily,whereasrowsdenotetaxonomicgroups.Thebarat
thetopofthefigureindicatestherelativeabundanceofeachPaDopergroup,withavalueof0representingabsence(black),0.01to
0.3representingvariabledistribution(blue),0.31to0.9representingmoderatedistribution(green–yellow–orange)and1(red)corre-
spondingto100%presence.Thetotalnumberofgenomespertaxonisindicatedinbrackets.
superfamilies. It is important to emphasizethat theP-loop is domain-like (cAMP bin) is associated with proteins devoted
involved in the hydrolysis of ATP or GTP and is an ancient to regulation of the catabolite repression mechanism. In this
domainidentifiedinalllifeforms [40].In addition,theGAF regard,thecAMPbinisinvolvedindimerizationofCrppro-
domain, which exhibits two main functions (cGMP binding teinandinteractionwiththeallostericeffectorcAMP[42,43].
anddimerization),isassociatedwithproteinsbelongingtothe PBP-IIisadomainprimarilyrelatedtotheLysRfamily,which
wHTHandtotheC-terminaleffectordomainofthebipartite belongs to the wHTH superfamily. Thus, PBP-II and cAMP
response regulators (bipartite), which is mainly identified in binareassociatedwithfamiliesthatregulatediversefunctions,
CrenarchaeotaandEuryarchaeota.TheGAFdomainwasalso such as basal metabolism and responses to stress. Therefore,
foundinFhlA(formatehydrogenlyasetranscriptionalactiva- these PaDos are intimately associated with their correspond-
tor), in the C-terminal domain of the heat-inducible ing DBDs, suggesting a probable common ancestor that was
transcription repressor HrcA and in the phytochrome chro- the origin of the other group members through duplication
mophore attachment domain [41]. The cAMP binding events.
1173
Rivera-Gómezetal.,Microbiology2017;163:1167–1178
Thesecondgroup(Fig.4bandTableS2)comprises19differ- addition, we found a large proportion of specific PaDos and
ent PaDos, including the C-terminal domain of arginine DBD associations, suggesting that those regulators increase
repressor (arginine), dimeric alpha+beta barrel (dimeric), the ability to sense signals, diversifying their number of spe-
PLP-dependent transferases (PLP-depend), ribonuclease H- cific domains and reducing the probable signal cross-talk in
like(Ribonucle)andTexN-terminalregion-like(Tex),among the cell. Another interesting observation is that some diverse
others.Inthisgroup,weidentifieddiversedomainsnotpres- TFs contain two different DBDs, such asthoseproteins con-
ent in all taxonomical divisions; indeed, some of them are taining the KorB and lambda repressor-like domains, AbrB
associated with specific regulators, such as the C-terminal and lambda repressor-like, or wHTH and bipartite DBDs.
familyArgR,whichfunctionsasatranscriptionalrepressorof Indeed,weidentifiednineproteinswithlambdarepressor-like
arginine metabolism. We speculate that the distribution of andAbrBDBDs.TheseproteinsaremembersoftheXrefam-
thesePaDossuggestsprobablegenelosseventsand/orlateral ily and regulate oxidative metabolic function-related genes,
genetransfer,butfurtherevidenceisrequired. suchasBzdR,whichisassociatedwiththeregulationofanaer-
obic catabolism of benzoate [47, 48]. Therefore, we suggest
Finally,thethird group (Fig. 4c and Table S2) includes261
that combinations between PaDos and DBDs significantly
PaDos, such as the methylated DNA-protein cysteine
increase the sensing of diverse signal compounds, decreasing
methyltransferase domain (Methylat), which is involved in
the cellular defence against the biological effects of O6- signalling cross-talk and making more efficient the response
methylguanine (O6-MeG) in DNA [44]. The phoshotrans- toenvironmentalstimuliinbacterialandarchaealorganisms.
ferase/anion transport protein (Phoshotr) domain, a struc-
Inaddition,theassociationofPaDosandDBDsintermsof
tural domain found in the cytoplasmic domain in certain
thegenomesizeswasevaluated(Fig.6andTableS7).Todo
anion transporter proteins [45], and the sugar isomerase
this,bacterialandarchaealgenomeswereclusteredininter-
(SIS)domain,describedasaphosphosugar-bindingdomain vals of 1000 ORFs and their corresponding PaDo–DBD
[46], have been identified in proteins that regulate the
associationswererepresentedandanalysedasnetworks[49,
expressionofgenesinvolvedinsynthesisofphosphosugars,
50].Fromthisanalysis,wefoundthatorganismswithsmall
possibly by binding to the end product of the pathway,
genome sizes not only contained a small number of DBDs
among other mechanisms. These domains show an erratic
butalsoalowdiversityofPaDosassociatedwiththem,cor-
pattern,wheremostarespecificallyassociatedwithparticu-
relating with the fact that organisms with small genome
lar DBDs and constrained to certain bacterial and archaeal
sizes are usually associated with stable and intracellular
taxonomicdivisions.
environmentsandmayhaveonly39PaDos(Fig.6a).Inthis
Insummary,thedistributionofPaDosinthedifferenttaxo- interval, the number of nodes and edges is the lowest
nomic groups reveals three main features: those that are observed so far (46 and 43),with an average path length of
mainly related to different superfamilies and present in all 2.87 (Table S4). In contrast, organisms with genome sizes
the taxonomical groups, such as the P-loop; those PaDos between 3000 and 4000 ORFs might contain 14 different
that combine with a single type of superfamily and specifi- DBDs associated with the largest diversity of PaDos, 191
cally with a type of DBD, such as C-terminal domain of (Fig. 6d). Based on this information, we suggest that the
transcriptional repressors (C-Ttr); and finally, those with a associations between DBDs and PaDos identified in organ-
heterogeneous distribution, such as Acyl-CoA dehydroge- ismsincludedinthisspanofgenomesizesrepresentaturn-
naseC-terminaldomain-like[Acyl-CoA(dCt)]. ingpoint,withthelargestnumberofnodes(212)andedges
(303),thelowestvalueofcentralizationandanaveragepath
Integrative analysisof the DBDsand their PaDos
length of 3.275. The largest genomes exhibit an increase in
In order to determine whether there are specific associations theaveragepathlengthandthenumberofnodesandedges
betweentheDBDsandtheircorrespondingPaDos,anenrich- diminishes, suggesting that the associations between these
ment analysis was performed. From this analysis, we found
classes of structural domains remain almost constant, not
1267 different combinations, forwhich614, or 48%, showed
onlyinnumberbutalsoindiversity.
statisticallysignificantassociations(TableS3).Fromthese,the
wHTH had the highest number of associated PaDos (314), Finally, we sought to determine whether structural analysis
whereasIHFcontainedonlyonePaDo,i.e.thelowestnumber could support the specific associations between DBDs and
of PaDos (Fig. 5 and Table S6). From this information, we PaDos.Todothis,88proteinswithstructuraldatawereevalu-
definedtheassociationsofthesestructuraldomainsaccording ated,36withtheDBDintheNterminus,ninewiththeDBD
totwomaingroups:(i)TFswheretheDBDsspecificallycom- intheCterminus,sevenwiththeDBDintheCterminusbut
bine with particular PaDos, such as the periplasmic binding associated with a two-component system (response regula-
protein-likeIIcombinedwiththewHTH DBDandtheperi- tors),25singledomains(withtheDBDlocatedinthemiddle,
plasmic binding protein-like I combined with the lambda flanked by small helices), and 11 single domains (with the
repressor-likeDBD;and(ii)promiscuousassociations,where DBD at one end) (Table S5). Many of the structures of the
PaDoscancombinewithdiverseDBDs,such as GAF, which TFs in this work correspond to dimers, although hexamers
isassociatedwiththewHTH,homeodomainandAbrBDBDs, andoctamersarealsopresent.Therewereinstancesofdomain
ortheP-loop,identifiedinregulatorswiththewHTH,homeo- swappingandalsoof‘side-by-side’dimers,andonecaseofa
domain, lambda repressor-like and bipartite domains. In tandem dimer (PDB ID 1P4X:SarS, with two HTH motifs in
1174
Rivera-Gómezetal.,Microbiology2017;163:1167–1178
ADP−ribUoDP−glucTTHA0068
PAilZraD/HWMDP40 PeptidCeYTH−likSuccinIsaotcitra
ToPrTDBe−PrpS−likeiInAnRodHieRnineintlEe−GlikrpENR−iMb(Aed−teaollilkxoYyprdC/leRcibtinS−ulp6e−rpoChxiaoCrsbaporhbna−TmnyaCptelp I−ICNY yeDmafunoMpaDlsiHceaStrt−anliskepos
Macro AbrBCaTrbIRa mdoomya
DnaK
TTHA1013
Lamb Cell gro Cobalami
PasrsBRNA vi TranspoKsorB Gluc Chaperon DNA clamTetrahyd
EPT/RTPC
PP2C−lik Methionimethylas
Sporulation
Putative CitratePrecorri
KorB
BRCT dom
Pentapep TipA−lik
LexA/Sig lambda PUT ferredox
oxidored
IHFL Myosin FMpNost−NAtAn Ahydrolases Winged
PRTase−l TPRHCP−like Ribbon
PFL−likeNucleoti
Sigma3
PUA doma Restrict Rhh Tetramer
YfbU−lik
L30RUe−hroolicdkaannEaDesssipmREiebrCoicfllaaDsvTsn raIoIDpNoomsLyo S−adenos PhosbpihpoaAlrRtitM Nitrite
DnaTBetrapyr IrPoTnS−regu PTSGlyceral ACP Resolvas RmlCS−ISl ikdoma His−Me
MM335Fic−likeMCP/YPpsBAPP−KI−Ilike MOPPhoshotrCBS−doma Glyoxala Nucl−cyc YVTN
AZciynlc− bCeoMtcAAadM1P−binBet v1−l Thioredo Homin4gFe−4 P−loop PYP ACT−like HistidKinelchH mDo−domai
AB1/MPN Actin−li AbrB Chemotax
HemCe−−TdPtreTTprSFk oAsryRmsuatbteredox Wing PhoU alpha/be Probable Bipa DTPoPlB6N N−CtheBHLeumxDR−−lliik
ADC−AliBkDeCN−Atr apnrimTM1602 HRDCMFDSinB/Yfi SMAD/FHNATF2−li TricorSnNigma2EAL
CarboxypSensorySAM ATPasTericorn2
TPryrpimto−pFphAoHlNCuhdoixrismaMetallo DnaJ GAF Quinopro Ribonucl GalactoCsheY−likTrpR
PhoCsrpehaGotintnAGtanlkuTycHrSoinAPcRMooTrPsSArGsmiBrmIABYCaA−N−nPcYniBLteRrGIPtoG1yib−gS2l−PoeTdICknNeHiGnp pdaelyroKcPmFeNDMraoAZNlNDCa−urlgcpdilPTenephiRoniesCoeieECsFF1tl−ae/BvTouLpIro TPhriooNlkaausrceyloTeriacnslat FAD/NNAagDCBalLciiguamnd Multihem Home HomeodAommdeat DalN reAAcetagmlyicdosid
Glycosyl
Nucl HomodimeAraCMethylaLtOeuxiAdorNedHTALTA−boAxLDH−lik
DNA−bind PA1324glutamin DNA brAealdolase
Rad51 post−HMHGMAPrphRydrogeYnWTDHPr−TlikrpeR−lik
KH−MdoicmroabiRAiaribfGosaTopemxFaoNmuDsA−LlyiksozymYfieA homeIF−D2H−PHaRolluyvnAucRlbpeotEa−lac TetracPychosphoDeN mu tran
Rho N−etIF2alph
Fig. 5.RelationshipsbetweenDBDsandPaDos.RedboxescorrespondtoDBDs;bluecirclesrepresentthepromiscuousPaDosassoci-
atedwithtwoormoresuperfamilies,suchastheGAFdomain,whichwasfoundtobeassociatedwiththewingedHelix-Turn-Helixand
homeodomainsuperfamilies.GreendiamondscorrespondtospecificPaDosrelatedtoonlyoneDBD,suchasParB,whichisassociated
withKorB.
tandem).Withineachclass,thereweredifferencesintheangle Theextentand nature oftheinterfaces between PaDos and
formed at thedimerizationinterface,respondingto thepres- DBDs are different in each class of TFs, and also within
ence/absence of ligands and/or crystallization conditions, each class. This isdue tothe hinges that connectthe DBDs
resulting in different orientations of the readout helices and and PaDos and the promotion of conformational changes
wingsfacingtheDNA.Thisvariabilityhighlightsthedifficulty upon ligand binding at the PaDos, altering the accessibility
in predicting both the interdomain geometry and quaternary of the DNA readout motif in the DBD (see, for example,
structure. CprK crystallized under different redox conditions, with
1175
Rivera-Gómezetal.,Microbiology2017;163:1167–1178
(a)
andwithoutligand,inFig.7a)[44].Intheabsenceofligand,
CprK without ligand: CprK with ligand putative regulator:
CprKdisplaysmodestinteractionsbetweentheDBDinthe 3E5Q 3E5U 1ZYB
C terminus and the PaDo in the N terminus, where the # #
linkerbetweenbothdomainsisdisorderedintheapo-struc-
ture. Another example corresponds to Crp of Bacteroides
thetaiotaomicron (PDB ID 1ZYB), which shows extensive
interactionsbetweenthe‘wings’oftheDBDsattheNandC
* *
* *
termini.
In proteins with the DBD located at the N terminus, the * *
DBD engages in DNA interactions through helices and the
wing, at different angles, contacting DNA in the major and FadR-DNA complex: 1HW2 YydK: 3BWG
(b)
(a) (b) ModE: 1B9M
AsnC: 2CG4
MntR-Mn complex: 10N1
180-1000 ORFs 1001-2000 ORFs
(c) (d)
(c)
Putative repressor: 1Y0U Sso10a: 1R7J SmtB: 1SMT Putative regulator: 1SFX
2001-3000 ORFs 3001-4000 ORFs
(e) (f)
Fig. 7. Structural analysis of DBDs and PaDos. Members of the
wingedhelix-turn-helixsuperfamilywereselectedbecausetheyrepre-
4001-5000 ORFs 5001-6000 ORFs sentthegeneralorganizationobservedinallTFs,i.e.theyformmulti-
plecombinationsbetweentheDBDsandPaDos.(a)Differentdegrees
(g) (h)
ofinteractionbetweenDBDsandotherdomains.Onesubunitisshown
asasolidcolour,andtheotherisshownasatransparentribbon.The
readinghelicesareindicatedbyasterisks.CprKdisplaysmodestinter-
actions between the DBD in the C terminus (orange) and the PaDo
(cyan).ProteinPDB ID 1ZYB showsextensiveinteractionbetweenthe
‘wings’oftheDBDs(darkandlightorange)attheNandCtermini.The
dimerizationhelixisshownincyan.Theputativereadinghelicesofthe
N-terminalDBDsaremarkedbyasterisksandthoseoftheC-terminal
6001-7000 ORFs 7001-10000 ORFs DBDareindicatedbyhashtags.(b)TheDBDattheNterminus(orange)
engagesinDNAinteractionsthroughhelicesandthewing,atdifferent
angles,contactingtheDNAinthemajorandminorgrooves.Onesub-
Fig. 6. Relationship between DBDs and PaDos as a function of unitisshowninasolidcolourandtheotherisshownasatransparent
genome size.In red are the DBDs, whereas PaDos are indicated in ribbon.TheDBDfromonechaincontactsthePaDo(cyan)oftheother
blue.(a)Organismswithgenomesizesbetween1and1000ORFs;(b) chain. (c) Single-domain DBDs (orange) with dimerization domains
between1001and2000ORFs;(c)between2001and3000ORFs;(d) (cyan)attheNterminus(PDBID1R7J),Cterminus(PDBID1Y0U)or
between3001and4000ORFs;(e)between4001and5000ORFs;(f) both(PDBIDs1SMTand1SFX).Onesubunitisshowninasolidcolour
between5001and6000ORFs;(g)between6001and7000ORFs;(h) andtheotherisshownasatransparentribbon.Allproteinsareasso-
between7001and10000ORFs. ciatedwiththewHTH.
1176