The TrmB family: a versatile group of transcriptional regulators in Archaea

12 Pages·2014·2.66 MB·English

Extremophiles(2014)18:925–936
DOI10.1007/s00792-014-0677-2
SPECIAL ISSUE: REVIEW 10th International Congress on Extremophiles
The TrmB family: a versatile group of transcriptional regulators
in Archaea
Antonia Gindner • Winfried Hausner •
Michael Thomm
Received:14March2014/Accepted:10July2014/Publishedonline:13August2014
(cid:2)TheAuthor(s)2014.ThisarticleispublishedwithopenaccessatSpringerlink.com
Abstract Microbesareorganismswhicharewelladapted and Wheelis in 1990 (Woese et al. 1990). This was con-
totheirhabitat.Theirsurvivaldependsontheregulationof firmed later by studies of the Archaeal biochemistry and
geneexpressionlevelsinresponsetoenvironmentalsignals. molecular biology. What distinguishes Archaea from the
The most important step in regulation of gene expression othertwodomainsisthefactthattheypossessbothbacterial
takes place at the transcriptional level. This regulation is and eukaryal properties. On the one hand, they have tran-
intriguing in Archaea because the eu-karyotic-like tran- scription, translation and DNA replication machineries
scription apparatus is modulated by bacterial-like tran- whicharesimilartothoseofeukaryoticorganisms(Keeling
scription regulators. The transcriptional regulator of mal and Doolittle 1995; Langer et al. 1995; Dennis 1997; Gra-
operon(TrmB)familyiswellknownasaverylargegroupof bowskiandKelman2003).Ontheotherhand,manygenes
regulatorsinArchaeawithmorethan250memberstodate. involved in metabolic processes are more similar to those
Onespecialfeatureoftheseregulatorsisthatsomeofthem encodedin bacterial genomes(Koonin et al. 1997).
can act as repressor, some as activator and others as both Alreadyinthelate1970sitwasdiscoveredthatArchaea
repressor and activator. This review gives a short updated have a multi-subunit RNA polymerase (RNAP) homolo-
overviewoftheTrmBfamilyandtheirregulatorypatternsin gous to the eukaryotic RNAP II (Zillig et al. 1979; Huet
differentArchaeaasalotofnewdatahavebeenpublishedon et al. 1983). In addition, the initiation factors TATA box
thistopicsincethelastreviewfrom2008. binding protein (TBP) and transcription factor B (TFB)
(Hausner et al. 1996; Ouhammouch et al. 2003), both
Keywords Archaea (cid:2) Transcriptional regulators (cid:2) Sugar related to their eukaryotic representatives, are important
metabolism (cid:2) TrmB family (cid:2) Transcription components of the Archaeal transcription machinery. An
Archaeal promoter consists of a transcription factor B rec-
ognitionelement(BRE)(Littlefieldetal.1999)comprising
Introduction
two adenines at -34/33 and a TATA box (Thomm and
Wich 1988) about -26/27 base pairs upstream of the
On the basis of 16S RNA analysis the third domain of life
transcriptionstartsite.Furthermore,aninitiatormotif(INR)
representingtheArchaeawasproposedbyWoese,Kandler
mostly consisting of a pyrimidine-purine di-nucleo-tide at
the transcription start site is essential for initiation (Reiter
CommunicatedbyM.W.W.Adams. et al. 1990; Hausner et al. 1991). Most DNA-binding pro-
teins with known gene-regulatory function resemble bac-
Thisarticleispartofaspecialissuebasedonthe10thInternational
terial activators or repressors (Kyrpides and Ouzounis
CongressonExtremophilesheldinSaintPetersburg,Russia,
1999). Just a small minority of these transcriptional regu-
September7–11,2014.
lators is homologous to eukaryotic proteins (Aravind and
A.Gindner(cid:2)W.Hausner(cid:2)M.Thomm(&) Koonin 1999; Bell and Jackson 2001). However, interac-
DepartmentofMicrobiologyandArchaeaCenter,Universityof
tions between bacterial-like regulators and the eukaryotic-
Regensburg,Universita¨tsstraße31,93053Regensburg,Germany
like basal transcription machinery are still a matter of
e-mail:[email protected];
[email protected] debate (Di Fioreet al. 2009).
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926 Extremophiles(2014)18:925–936
MostoftheDNA-bindingtranscriptionfactors(TFs)are 2007b). An overview of the distribution of TrmB family
repressors but also activators or proteins with both activi- proteins is given in Table 2. However, none of the TrmB
ties are known (Bell 2005; Geiduschek and Ouhammouch variantsispresentinallorganisms.Thisalignmentshowed
2005;GrohmannandWerner2011;Peeters etal.2013).A that P. furiosus contains four different TrmB-like proteins
family which contains all of the three possible TF combi- (Table 2). A closer look at their amino acid sequence
nations—repressors, activators or both—is the TrmB showed a 29 % sequence identity between TrmB and
family. Recently published data about the distribution of TrmBL1 (PF0124). Especially the N-terminus, which
proteins that possess a TrmB-like DNA–binding domain contains a HTH motif as DBD in TrmB, is highly con-
(DBD) (Pfam–ID: PF01978) and/or a TrmB-like effector- served (45 % sequence identity). In addition, a C-terminal
binding domain (EBD) (Pfam–ID: PF11495) show a EBD can be found in TrmBL1. TrmBL2 (PF0496) also
complete spreading all over the three domains of life possesses a high sequence identity to the N-terminus of
(Maruyama et al. 2011). The DBD belongs to the helix- TrmBindicatingDNAbindingbuttheC-terminalsequence
turn-helix (HTH) motifs which is prevalent in many Bac- is lacking 70 amino acids containing the sugar binding
teria and Archaea (Pe´rez-Rueda and Janga 2010). How- domain.ThesameistrueforTrmBL3(PF0661)(Leeetal.
ever, TrmB proteins that possess both a DBD and an EBD 2007b).
aremorecommoninArchaea.Mostofthemwerefoundin The presence of TrmB in Pyrococcus furiosus seems to
the phylum Euryarchaeota but some exist also in Cre- be a result of lateral gene transfer between Thermococcus
narchaeota as well as one in Thaum-, Nano- and Kor-ar- litoralis and P. furiosus (Imamura et al. 2004). The gene
chaeota, respectively (Maruyama et al. 2011). cluster containing TrmB and the ATP binding cassette
(ABC) transport system for maltose and trehalose (TM
Members of the TrmB family in Euryarchaeota operon)islocatedina16 kbregioninT.litoraliswhichis
flanked by one remaining IS element (DiRuggiero et al.
ProteinsoftheTrmBfamilycanbefoundin13 generainthe 2000). The cluster is composed of five genes, malE malF
Euryarchaeota.TheordersThermococcales,Halobacteriales malG treT trmB and malK forming a binding protein-
and Thermoplasmatales encode altogether 41 representa- dependent ABC transporter for maltose and trehalose with
tives in their genomes (Maruyama et al. 2011). In thermo- nearly identical sequence to the P. furiosus TM operon
philicArchaeaTFsoftheTrmBfamilyareinvolvedinthe (Fig. 1a)(Xavieretal.1996;Horlacheretal.1998;Greller
regulationofsugar metabolism,especially formaltose and etal.1999;Xavieretal.1999;DiRuggieroetal.2000;Lee
glucoseprocessing(vandeWerkenetal.2006;Kanaietal. et al. 2003; Qu et al. 2004).
2007; Lee et al. 2008). The gene loci which encode TrmB Pfu TrmB acts as transcriptional repressor for genes of
also flank genes that code for the maltose and/or trehalose the TM operon. A palindromic sequence—TACT-
transporters.Incontrast,thesegeneticlociarenotadjacentto NNNAGTA—wasdetectedasTrmBrecognitionsiteatthe
VNG1451C, the halobacterial version of TrmB in Halo- BRE/TATA box of the malE gene (Fig. 2a) (Lee et al.
bacterium salinarum (Schmid et al. 2009) or MreA, the 2003).Repressiontakesplacebypreventingtherecruitment
regulator of methanogenic pathways in Methanosarcina of the RNAP to the malE promoter (Fig. 3a). Maltose and
acetivorans(Reichlenetal.2012).Inrecentyears,moreand trehalose act as inducers by detaching TrmB from the rec-
moreTrmB-likeproteinsweredetectedindifferentArchaea ognition site (Fig. 3b) (Lee et al. 2003). These inducers,
andsofartheyarelikelytoplayanimportantroleindiverse however, show a difference in their binding affinity. Malt-
metabolicprocesses.TheresultsindicatethatTrmBarosein ose is bound to purified TrmB in a positive cooperative
oneoftheseorganismsandhassubsequentlybeendissemi- fashion whereas binding of trehalose shows no sigmoidal
natedafterwardsbyhorizontalgenetransfertootherArchaea binding behavior (binding of trehalose is at least 20-fold
orBacteria(DiRuggieroetal.2000).Thisreviewfocusseson lower than of maltose) (Lee et al. 2008). This could be
the best studied representatives of TrmB proteins in the explained in terms of their transport and metabolism.
EuryarchaeotaPyrococcusfuriosus,Thermococcuslitoralis, Studies in T. litoralis have shown that both maltose and
Thermococcuskodakarensis,Halobacteriumsalinarumand trehalose share an equal rate and a high affinity in trans-
Methanosarcinaacetivorans.Someofthecharacteristicsof portation but their metabolism is quite different. Whereas
theTrmBfamilyaresummarizedinTable 1. maltose is metabolized quickly, trehalose is just slowly
metabolized and accumulates to high internal concentra-
TrmBs in the Thermococcales tionswhenitispresentinthemedium(Lamosaetal.1998).
Nonetheless,PfuTrmBisalsoarepressorforgenesofa
A sequence alignment of Pyrococcus furiosus TrmB to separate maltodextrin ABC transporter (MD operon)
TrmB-like proteins in the order Thermococcales revealed (Fig. 1b). The MD operon is composed of the genes mdxE
five different clusters of TrmB-like proteins (Lee et al. mdxFmdxGpulAandmdxK(Koningetal.2002;Leeetal.
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Extremophiles(2014)18:925–936 927
Sulfolobales SulfolobusacidocaldariusMalR 349 40.3 DBD HTH Y51,N52 EBD G320,E326 Activator TworepeatsATAATACT Auto-regulationproposed (modifiedafteral.2013)
onet
Methano-sarcinalesMethanosarcinaacetivoransMreA 139 15.9 DBD HTH S66,L67 – – Repressor,activator maltodextrinoperal.2012;Wagner
Cren-archaeota Halobacteriales HalobacteriumsalinarumVNG1451C 360 39.4 wHTH Y61,D62 EBD G337,E343 Repressor,activator Glucoseglycerol TACTN7–8GAGTA Auto-regulation MDaltoseoperon,2009;Reichlenet
mal ose/metal.
Thermococcuskodakarensis Tk1769/Tgr/Tk0471/TrmBL1TrmBL2 341264 39.430.8 DBDDBD HTHHTH Y49,D50Y50,D51 EBD– G324,E330– Repressor,Repressor,activatorchromosoprotein Maltotriose CodingandTGM:intergenicTATCACN5GTGATAregions Auto-regulation TMelix-turn-helixmotif,trehalMaruyamaetal.2011;Schmid
h
cus 6 GTA nged)2007;
ThermococlitoralisTrmB 338 38.8 Dimer DBD wHTH Y50,D51 EBD G320,E32 Repressor Maltosetrehalose TACTNA3(TM) (w)HTH(wiKanaietal.
L3 ain,06;
TrmB 264 30.2 DBD Y50,E51 – – gdomal.20
al ndinnet
TrmBL2 264 30.6 DBD HTH Y50,D51 – – Putativechromosomprotein BDeffectorbivandeWerke
E3;
mBhomologs TrmBL1 341 39.4 Tetramer/Octamer DBD HTH Y49,D50 EBD G320,E326 Repressor,activator Maltosemaltotriosefructose TGM:TATCACN5GTGATA A-bindingdomain,8;Krugetal.201
Table1OverviewofthediscussedTr Euryarchaeota Thermococcales Pyrococcusfuriosus TrmB aa338 kDa38.8 structureDimer N-TerminusDBD wHTH DNA-bindingY50,D51aa C-TerminusEBD SugarbindingG320,E326aa ActingasRepressor InducersMaltosesucrosemaltotriosemaltodextrinstrehalose Co-repressorsGlucose(TM,MD)maltotriose(TM)maltose(MD) AGTA(TM)BindingmotifTACTN3TACT(MD) aakDaDBDaminoacids,kilodalton,DNLeeetal.2003,2005,2007a,2007b,200
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Table2 DistributionofTrmBfamilyproteinsintheThermococcales
TrmB TrmBL1 TrmBL2 TrmBL3 TrmBL4
P.furiosus PH1743 PF0124 PF0496 PF0661 -
T.litoralis Q7LYW4(100%) - - - -
T.kodakarensis - TK1769(67%) TK0471(82%) - -
P.horikoshii PH1034(71%) - PH0799(92%) - PH0751
P.abyssi - - PAB0838(91%) - -
ThevaluesinpercentshowtheidentityoftheaminoacidsequenceincomparisontothecorrespondingproteinofPyrococcusfuriosus.Aminus
signstatesthattheappropriateproteinismissingintheorganism.Theidentityamongthedifferentparalogousmemberswasbetween22and
30%(modifiedafterLeeetal.2007b)
Fig.1 The gene clusters of the trehalose/maltose (TM) and the promoterregionofmalE(green).(b)TheMDgeneclusterencoding
maltodextrin (MD) operon. a The TM gene cluster encoding the thebindingprotein-dependentABCtransporterformaltodextrinand
binding protein-dependent ABC transporter for trehalose/maltose, a anamylopullulanaseisshown.Aseparateregulatorismissinginthe
trehalose synthase and TrmB is shown. The red lines mark the MD operon. The red line tags the first half of the binding sequence
palindromic binding sequence of TrmB (TACTN AGTA) at the palindrome(TACT)(modifiedafterLeeetal.2008)
3
Fig.2 BindingsitesofPfuTrmBatthefirstgenesoftheTM(malE, palindrome.TheBrecognitionelement(BRE)andtheTATA-boxare
a) and MD (mdxE, b) operon. The binding sites are shown in bold showningreenboxes.TheThermococcalesglycolyticmotif(TGM,
letters.?1representsthetranscriptionstartsite.Thepromoterregion vandeWerkenetal.2006),justpresentinthemdxEpromoter,isina
ofthemalEgeneincludesaperfectpalindrome(underlined),whereas bluebox(modifiedafterLeeetal.2008)
the promoter region of mdxE just contains the first half of the
2003). This MD operon was identified with the same gene result of a duplication of the mdxK gene before the mal
arrangementinT.litoralisaswell.Accordingtoindividual geneclusterwastransferredfromT.litoralistoP.furiosus
genes the sequence identity varies from 51 to 83 % (Imamura et al. 2004).
between T. litoralis and P. furiosus (Imamura et al. 2004). IncontrasttothePfumalEpromoteroftheTMoperon,
DNA sequence comparison revealed that malK was the the Pfu mdxE promoter contains only the first half of the
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Extremophiles(2014)18:925–936 929
Fig.3 Models of the regulation mechanism of P.furiosus TrmB TrmBattheTMpromoterisTACT-N -AGTA,attheMDoperonof
3
(a and b) and TrmBL1 (a–d), T.littoralis TrmB (a and b), P. furiosus it is just the TACT sequence. Pfu TrmBL1 and Tk Tgr
T.kodakarensis Tgr (a–d) and H.salinarum VNG1451C (a–d) for bind to TATCAC-N -GTGATA (TGM), the binding motif in
5
genes where the proteins are acting asrepressor and/or as activator. H.salinarum is TACT-N -GAGTA. TBP, TATA binding protein;
7-8
All binding motifs are palindromic inverted repeats with cis- TFB, transcription factor II B; RNAP, RNA polymerase (modified
regulatory sequences in front of the BRE/TATA box or at the afterLeeetal.2008andKanaietal.2007)
transcriptionstartsite(?1).ThebindingmotifforPfuTrmBandTli
TrmB recognition sequence (TACT) at the transcription able to transport glucose but can metabolize it. Glucose
startsite(Fig. 2b).Thesecondhalfoftheinvertedrepeatis originatesfromthecytoplasmicdextrinmetabolismandits
missing and in vitro transcription experiments showed a presenceinexcessrepressesboththeTMandtheMDsys-
reduced binding affinity of TrmB to the MD operon (Lee tem and stops the uptake of glucose-producing sugars like
etal.2005).ThePfumalEgeneiscompletelyrepressedin maltose,trehaloseormaltodextrinseveninthepresenceof
the presence of 0.2 lM TrmB whereas Pfu mdxE is not inducers(Leeetal.2008).Inaddition,thepresenceofboth,
represseduntilanamountof1.6 lM.Furthermore,binding maltotrioseandmaltose,attheMDoperonatthesametime
of Pfu TrmB is controlled by differential sugar binding led to stronger repression showing also a co-repressor
activity. Maltose, sucrose, maltotriose, maltodextrins and activityofmaltosefortheMDsystem(Leeetal.2007a).
trehaloseareboundwithdecreasingorderofaffinity.Atthe TrmB of P. furiosus (PF1743) as well as of T. litoralis
TM promoter TrmB can be released using maltose and (Q7LYW4)consistsof338 aminoacids,formsaproteinof
trehalose as inducers. In contrast, bound TrmB at the MD 38.8 kDa and occurs at room temperature as a dimer
promotercanbereleasedonlyinthepresenceofmaltotriose, (Table 1)(Leeetal.2003).Hitherto,thePyrococcusTrmB
maltodextrinsandsucrose,butnotwithmaltoseortrehalose is the only one with a described crystal structure (Krug
(Lee et al. 2005). Later experiments showed that glucose etal.2006,2013).Astheconservedsequencesoftheother
acts as a co-repressor at the TM and MD operon, causing TrmB representatives argue for a similar structure we
strongerrepressionwhenmaltoseandmaltotrioseispresent, review the structural details of Pyrococcus TrmB in more
respectively (Lee et al. 2007a). Indeed, P. furiosus is not detail hereafter.
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The structure is split into an N-terminal DBD and a Da4-helix which is responsible for DNA recognition and
C-terminal EBD (Fig. 4). The N-terminal DBD contains a itscounterpartDa40 inthedimericprotein,TrmBisableto
winged-helix-turn-helix (wHTH) motif composed of four bind to two contiguous major grooves of duplex palin-
a-helices Da1–a4 and two b-strands Db1–b2. Via the dromic B-DNA (Krug et al. 2013). Especially tyrosine at
position 50 and to a lesser extent aspartic acid at position
51therebyseemtoplayanimportantroleconcerningDNA
binding (Fig. 5a) (Lee et al. 2007a). The wHTH motif is
followed by an amphipathic helix a5 containing a coiled-
coil (CC) motif which advances dimerization. Pairs of
hydrophobicaminoacidsinthisCCandacounterpartCC’,
Phe81/Ile910 and Phe84/Leu880 as well as their corre-
spondent part, provide a basis for interaction between two
monomer proteins to form a dimer (Fig. 5b) (Krug et al.
2013).
The C-terminus represents an EBD consisting of two
subdomains. The first subdomain is built up of an eight-
stranded sheet Eb1-8 flanked by two large helices Ea1–2
on the one side and one large helix Ea3 crossing the b-
sheet at the other side. The second subdomain, which is
connected to the first one by a short hinge, forms a strand
Eb9, a helix Ea4 and an irregular flattened seven-stranded
b-barrel with its axis parallel to the strands of the first
subdomain (Krug et al. 2006). The sugar recognition helix
Fig.4 The structure of Pyrococcus furiosus TrmB with bound
Ea3 is on the surface of the cleft between these two sub-
sucrose in yellow wireframe (ribbon presentation). The N-terminal
domains. Regardless of whether maltose or sucrose is
DNA-binding domain (DBD) consists of a winged helix-turn-helix
motif.Helixa4representstheDNArecognitionhelix.Thewingand bound, their nonreducing glucosyl moieties interact both
therecognitionhelixarecoloredyellow.Helices(a)andstrands(b) with the same six amino acids of the second C-terminal
areconsecutivelynumbered.Helixa5(CC)andashortlinkerconnect
subdomain—Asn , Gly , Met , Val , Ile and
the DBD to the sugar/effector binding domain (EBD) harboring 305 320 321 324 325
Glu (Fig. 6).Justtheorientationofmaltoseorsucrosein
sucrose(inyellow)(takenfromKrugetal.2013withpermissionfrom 326
theauthorsandthepublisher) the bound state is different. The reducing glucosyl moiety
Fig.5 aStructureofthePyrococcusfuriosusTrmBdimerinribbon helices as a hinge loop. The distances between the two recognition
presentation and bound sucrose in yellow wireframe. The structure helices (a4) are indicated. b Zooming of the coiled-coil formed by
represents the dimer created by -X, Y-X, 2/3-Z crystallographic two crystallographic counterparts of CC in ribbon presentation with
symmetryoperation.Onemonomeriscoloredgrey,theothermauve. side chains in stick representation. The hydrophobic residues Phe /
81
Theproteinpresumablybuildsadimerbyformingacoiledcoilofthe Ile ,Phe /Leu ,Leu /Phe andIle /Phe arerepresentedina
910 84 880 88 840 91 810
CChelicesofthetwomonomers.Thedimercanbeconsideredasa zipper-likearrangement(takenfromKrugetal.2013withpermission
result of domain swapping of the DBDs between two copies of an fromtheauthorsandthepublisher)
ancestral protein consisting of the EBD and the DBD with the CC
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Extremophiles(2014)18:925–936 931
glycolytic regulator), respectively, as the expected global
regulators with a 67 % amino acid identity (Table 2) (Lee
et al. 2007b).
TrmBL1 and Tgr are TGM-recognizing global sugar-
sensing transcription regulators of genes coding for gly-
colytic and gluconeogenic enzymes as well as for sugar
transport systems. TrmBL1 responds to maltose, maltotri-
ose and fructose which act as inducers to release DNA-
bound protein. Tgrjust responds to maltotriose as inducer.
Furthermore, both proteins play a binary regulatory role:
they are both activators for certain gluconeogenic genes
and repressors for glycolytic enzymes (Kanai et al. 2007;
Lee et al. 2008). If both proteins act as repressors at TGM
containing promoters, the TGM is situated downstream of
the BRE/TATA box, mostly overlapping the transcription
start site and interfering with the recruitment of RNAP
(Fig. 3a) (Kanai et al. 2007; Lee et al. 2007b) like it was
already shown for MDR1 of Archaeoglobus fulgidus (Bell
Fig.6 The sugar binding domain (EBD) of Pyrococcus furiosus
et al. 1999) or Phr of P. furiosus (Vierke et al. 2003).
TrmB in complex with sucrose. The TrmB residues which interact
withsucroseaswellasthedistancesofpotentialhydrogenbondsinA˚ This mechanism is also proposed for a potential glyco-
unitsareindicated.Theomitelectrondensitymapofsucroseisshown lytic regulon of P. furiosus including a phospho-sugar
atthe5rlevel(takenfromKrugetal.2013withpermissionfromthe
mutase (PF0588) and three a-amylase encoding genes
authorsandthepublisher)
(PF0272,PF0478andPF0477),asallofthecorresponding
promoters contain a TGM downstream of the BRE/TATA
of maltose contacts a seventh amino acid (Ser ), which box (van de Werken et al. 2006). In contrast, if TrmBL1
229
can be found in the sugar recognition helix Ea3, via andTgractasactivators,theTGMisfoundupstreamofthe
hydrogen bonding. However, it is not clear whether or not BRE/TATA box. Such an activation mechanism was
the fructosyl moiety of sucrose acts analogue (Krug et al. already shown for Ptr2 from Methanococcus jannaschii
2013). (Ouhammouch et al. 2003) which interacts with TBP or
In 2006 van de Werken et al. described a conserved TFB-RF1 (PF1088) from P. furiosus (Ochs et al. 2012)
sequence motif named TGM (Thermococcales glycolytic which interacts with TFB. A similar activation mode via
motif) at promoter sites of genes encoding glycolytic interaction with TBP, TFB, RNAP or even a yet unknown
enzymes as well as several other genes involved in sugar factor is also proposed for TrmBL1 (Fig. 3b) (Lee et al.
metabolism of P. furiosus and T. kodakarensis. The TGM 2008).
consists of a conserved inverted repeat interspaced by five In addition, an autoregulation mechanism was proposed
nucleotides (TATCAC-N -GTGATA). This putative cis- for Tgr in which maltotriose could be identified as a
5
acting element indicated a common transcriptional control potential physiological effector (Kanai et al. 2007). The
of the respective genes. The inverted repeat, however, is mechanismofautoregulationwasalreadycharacterizedfor
missing in Pyrococcus abyssi and Pyrococcus horikoshii Lrs14fromSulfolobussolfataricus(BellandJackson2000)
therebyindicatingthattheirreducedcataboliceffectiveness and an Lrp-like transcriptional regulator from P. furiosus
does not require such a regulatory system (van de Werken (Brinkman et al. 2000). However, Tgr controls the cur-
et al. 2006). rently largest regulon in Archaea, the Thermococcales
Since the genome of T. kodakarensis contains neither a glycolytic regulon with more than 30 genes (Kanai et al.
TM operon nor an ortholog of TrmB, it was hypothesized 2007).
that another global regulator capable of recognizing the Both, TrmBL1 andTgr,consist of341 amino acids and
TGM exists. Moreover, an ortholog of the MD operon have a molecular weight of 39.4 kDa (Table 1). TrmBL1
(TK1771-TK1775) as well as two paralogs to TrmB-like appears as tetramer and octamer in a balanced state but
genes (TK0471 and TK1769) can be found (Table 1) with additionofmaltoseormaltotrioseaswellasahighprotein
TK1769 being adjacent to the MD operon (Kanai et al. concentration shifts the equilibrium to the octameric form
2007). Sequence analysis of TrmB-like regulators which (Leeetal.2007b).AnalignmentoftheirN-terminalamino
may be involved in recognizing the TGM identified acid sequences revealed a highly conserved a-helix with
PF0124 from P. furiosus (TrmBL1, TrmB-like 1) and tyrosineatposition 49beingessentialforDNArecognition
TK1769 from T. kodakarensis (Tgr, Thermococcales (Fig. 7) (Lee et al. 2007b). Moreover, an alternative sugar
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Fig.7 Sequence alignment of the discussed TrmB homologs. The seven sugar binding amino acids of Pfu TrmB are shown in pink
boxes represent conserved protein domains. Dashes indicate gaps in letters. Organism abbreviations are as follows: Pfu, Pyrococcus
the alignment. Highly conserved amino acids are represented via furiosus; Tli, Thermococcus litoralis; Tk, Thermococcus kodakara-
asterisks.Dotsrepresentconservedaminoacids.Stronglyconserved ensis; Hs, Halobacterium salinarum; Ma, Methanosarcina acetivo-
aminoacidswithspecialfunctionarehighlightedinboldletters.The rans;Sa,Sulfolobusacidocaldarius
bindingaffinityandspecificityisproposedforTrmBL1and Moreover, a C-terminal EBD is missing in both proteins
Tgr in comparison to TrmB because just two out of seven (Fig. 7). Unlike Tgr, the Pfu TrmBL2 homolog TK0471 is
amino acids which are responsible for sugar binding in binding to both coding and intergenic regions thereby
TrmB are conserved at the C-terminus (Gly and Glu repressing transcription when bound to promoter regions
320 326
in P. furiosus, Gly and Glu in T. kodakarensis). (Maruyamaetal.2011).Furthermore,itseemstoplayamajor
324 330
However, the amino acids flanking the sugar contacting role as general chromosomal protein that is involved in
ones of TrmB are conserved in both indicating a similar agglutinatingDNA to thick fibrousstructures (Fig. 8) (Mar-
sugar binding pocket in all three proteins (Lee et al. uyamaetal.2011).RecentEMSAandChIP-Seqexperiments
2007b). in our laboratory revealed that TrmBL2 seems to have a
TrmBL2isthemostconservedproteinoftheTrmBfamily similarDNA-bindingbehaviorasTK0471ofT. kodakarensis
among the Thermococcales (Table 2). TK0471 of T. koda- (data not shown). In contrast, nothing is known about the
karensis shows an 82 % sequence identity to Pfu TrmBL2. functionofTrmBL2inP. horikoshii(PH0799)andP. abyssi
Furthermore, it also consists of 264 amino acids, but has a (PAB0838) so far, but the high identity of their amino acid
molecularweightof30.8 kDaincontrasttoPfuTrmBL2with sequences(92and91 %)toPfuTrmBL2indicatesasimilar
30.6 kDa (Table 1). A closer look at the highly conserved function.
N-terminalDBD of both proteinsreveals a HTH motif with Furthermore, there are two additional members of the
tyrosineatposition 50beingimportantforDNArecognition. Thermococcales TrmB family with unknown function,
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Extremophiles(2014)18:925–936 933
Fig.8 AFMimagesofa3-kbp
linearDNAofEscherichiacoli
(plasmidBluescriptIIlinearized
byHindIIIdigestion).Image
(a)showstheDNAwithout
protein.Image(b)illustratesthe
DNAincubatedwith
recombinantTK0471/TrmBL2
ataprotein-to-DNAratioof
10:1(wt/wt)(takenfrom
Maruyamaetal.2013with
permissionfromtheauthorsand
thepublisher)
TrmBL3(PF0661)inP. furiosusandTrmBL4(PH0751)in 2007; Lee et al. 2008; Schmid et al. 2009) and therefore
P. horikoshii. Sequence alignments argue for individual also seems to code for a putative sugar binding transcrip-
proteinswithintheseorganisms.InthecaseofTrmBL3the tional regulator. Interestingly, the gene locus coding for
N-terminalDBDispresentwhereastheC-terminalEBDis VNG1451C does not contain genes for the maltose and/or
missing (Fig. 7) (Lee et al. 2007b). trehalose ABC transporters like in thermophilic Archaea.
Nonetheless, VNG1451C seems to be a highly conserved
TrmB in Halobacterium salinarum regulator with a putative function related to sugar metab-
olism and the preservation ofredox balance (Schmidet al.
The haloarchaeal TrmB ortholog VNG1451C controls 2009). Phenotypic analysis of a VNG1451C knockout
approximately 113 promoters in the absence of glucose or mutant in absence of glucose revealed a vehement growth
glyceroltodirectlyregulategenesindiverseprocesseslike defect in the mutant, even in rich media. In addition, the
the central carbon and amino acid metabolism with bio- NAD?/NADH ratio was lower than in the wildtype strain.
synthesis of the cognate cofactors, vitamin and purine However, the growth defect as well as the NAD?/NADH
biosynthesisaswellassomemoreenzyme-codinggenesin ratio imbalance was reversed by the addition of glucose to
response to changes in carbon source availability. These the growth media whereas glycerol just partially had a
genes are either unique in Archaea or conserved across all complementational effect. Hence, this regulator seems to
domains of life (Schmid et al. 2009). In addition, have anutrientspecificitybecauseit reactson glucose and
VNG1451Cisabifunctionalregulatorwhichisinvolvedin glycerolbutnotonsugarslikegalactose,maltose,raffinose
the regulation of the redox and energy status of the cell in or sucrose as well as on pyruvate (Schmid et al. 2009).
response to nutrient availability. Surprisingly, no binding The binding motif of VNG1451C is a cis-regulatory
was observed in the presence of high glucose or glycerol element with the sequence TACT-N -GAGTA (Schmid
7-8
concentrations. Furthermore, VNG1451C bound to inter- et al. 2009) and is completely different from other char-
genic regions upstream of five transcription factors, acterized TrmB-binding sites like the TGM (van de Wer-
including its own promoter (Table 1), what is contributing ken et al. 2006). VNG1451C can act as both an activator,
to a differential regulation of genes which are not directly with its binding motif upstream of the promoter, and as a
influenced by the protein itself (Schmid et al. 2009). repressor with the binding motif downstream of the pro-
VNG1451C consists of 360 amino acids with a calcu- moter(Fig. 3a–d).ThismodelwasalreadyproposedforTk
lated molecular mass of 39.4 kDa. Its N-terminus contains Tgr and Pfu TrmBL1 (Kanai et al. 2007; Lee et al. 2008).
a winged HTH motif with tyrosine at position 61 possibly Recent data from Todor et al. (2013) revealed that the
beingessentialforDNAbindinglikeinPfuTrmB(Fig. 7). cobalamin biosynthesis pathway seems to be regulated by
Furthermore, VNG1451C shares a 21 % identity to the both VNG1451C and its dependent secondary regulators.
consensus sequence of the TrmB family signature (Pfam- In contrast, the purine biosynthesis pathway seems to be
ID: PF01978). ClustalW analysis showed that VNG1451C co-regulated by anon-VNG1451C-dependent transcription
has three active site residues (Gly , Glu and Asp ) factor. Taken together, the TrmB ortholog VNG1451C in
337 343 344
which are critical for sugar binding in the characterized H. salinarum plays an important role in recognizing the
TrmB orthologs (Fig. 7) (Krug et al. 2006; Kanai et al. nutrient availability and hence seems to directly regulate
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934 Extremophiles(2014)18:925–936
central metabolic enzyme-coding genes but also collabo- activator of the maltose regulon (Saci_1660-Saci_1666).
rates with other regulators to control peripheral metabolic It includes the ABC transporter malEFGK, an a-amylase
pathways (Todor et al. 2013). amyA and an a-glucosidase malA (Choi et al. 2013).
However, all other Sulfolobus species lack MalR homo-
TrmB in Methanosarcina acetivorans logs even though they retain a maltose transport regulon.
This most likely indicates that S. acidocaldarius obtained
The protein MreA (MA3302, Methanosarcina regulator of the regulator by horizontal gene transfer. MalR consists of
energy-convertingmetabolism)wasidentifiedbysequence 349 amino acids and has a molecular weight of 40.3 kDa.
analysis as a member of the TrmB family (Reichlen et al. Bioinformatic analysis of its amino acid sequence
2012). It consists of 139 amino acids with a calculated unveiled both a HTH domain with the conserved tyrosine
molecular mass of 15.9 kDa and therefore is the smallest at position 51 important for DNA binding and a TrmB
representative of the TrmB family so far (Table 1). The EBD which comprises two amino acids essential for sugar
DBDislocatedwithinthefirst111 N-terminalresiduesand binding in Pfu TrmBL1 (Gly and Glu ) (Fig. 7) (Lee
320 326
asequencealignmentwithTgrofT. kodakarensisindicates et al. 2007b). The amino acid sequence identity with
a similarity of 68 % (Reichlen et al. 2012). However, the P. furiosus TrmB and TrmBL1 is 24.1 and 22.7 %,
usuallyconservedtyrosineisreplacedbyaserine.Further- respectively. However, the expression of the genes of the
more,aregulatordomainattheC-terminusismissinglikein maltose regulon was just induced by Mal R with maltose
PfuTrmBL2andTrmBL3(Fig. 7)(Leeetal.2007b).MreA in the growth medium, indicating that MalR, in contrast
is a global regulator of distinct methanogenic pathways to its TrmB homologues, is an activator of the mal gene
(Reichlen et al. 2012). On the one hand, it represses genes cluster (Wagner et al. 2013).In addition, a MalR binding
which encode enzymes unique to pathways of methano- motif was detected upstream of the malE promoter with
genesisfrommethylotrophicsubstrates;ontheotherhand,it two 8-bp repeats (ATAATACT) located at -139 to -132
activates genes encoding enzymes which are idiomatic in and at -106 to -99, but binding of MalR to these repeats
methanogenesis from acetate. Transcriptional profiling of was independent of the addition of sugars like maltose,
wildtype versus mreA knockout strain revealed a diverse D-glucose or D-xylose in quantitative RT-PCR and
expression of 280 genes in acetate-grown cells. This indi- b-galactosidase activity assays. Furthermore, a feedback
catesakeyroleofMreAintheregulationofspecificgenes loop in which MalR regulates its own expression was
essential for growth with acetate. A certain binding motif proposed but unknown posttranscriptional processes or
liketheTGMofThermococcalescouldnotbeidentified. other regulators might also be involved in the regulation
of MalR activity. All in all, MalR seems to be an indi-
Members of the TrmB family in Crenarchaeota vidual regulator of maltose and maltodextrin transport
components and/or metabolism, while sucrose transport
Contrary to the Euryarchaeota, in the phylum Cre- and metabolism are not affected (Wagner et al. 2013).
narchaeotaonlythree organismscontainTrmB andTrmB-
like transcriptional regulators: Sulfolobus acidocaldarius,
Caldivirga maquilingensis and Thermophilum pendens. In Members of the TrmB family in Thaum-, Nano-
S. acidocaldarius the best studied regulator is MalR and Korarchaeota
(Saci_1161, mal regulon activator)—an activator of the
maltose regulon (Wagner et al. 2013). According to Mar- Members of the TrmB family were also discovered in the
uyama et al. (2011) also C. maquilingensis encodes two remaining Archaeal phyla Thaum-, Nano- and Kor-
different TrmBs in its genome—Cmaq_1188 (related to archaeota. Using phylogenetic sequence analysis one copy
MalR of S. acidocaldarius) and Cmaq_0601 (related to of TrmB could be detected in Nitrosopumilus maritimus,
TrmBL4 of P. horikoshii) (http://www.archaea.ucsc.edu). Nanoarchaeum equitans and Candidatus Korarchaeum
InT. pendensfourmembersoftheTrmBfamily withboth cryptofilum (Maruyama et al. 2011). Until now, none of
a DBD and an EBD domain similar to the TrmB domains these TrmBs is specified.
were identified via phylogenetic analysis by Maruyama
et al. 2011. However, the function of these proteins in
Caldivirga and Thermophilum is not known. Conclusions
TrmBs in Sulfolobus acidocaldarius TrmBsofthedifferentphylaintheArchaealdomainseem
to play an important role in regulation of transcription
MalR of S. acidocaldarius forms a separate group within of diverse metabolisms. Best studied are the TrmB pro-
the TrmB family because it is exclusively working as teins of the Thermococcales P. furiosus, T. litoralis and
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