The IclR-Type Transcriptional Repressor LtbR Regulates the Expression of Leucine and ...

14 Pages·2007·0.37 MB·English

JOURNALOFBACTERIOLOGY,Apr.2007,p.2720–2733 Vol.189,No.7
0021-9193/07/$08.00(cid:1)0 doi:10.1128/JB.01876-06
Copyright©2007,AmericanSocietyforMicrobiology.AllRightsReserved.
The IclR-Type Transcriptional Repressor LtbR Regulates the Expression
of Leucine and Tryptophan Biosynthesis Genes in the Amino Acid
(cid:1)
Producer Corynebacterium glutamicum
Iris Brune,1,2 Nina Jochmann,1,2 Karina Brinkrolf,1,3 Andrea T. Hu¨ser,1 Robert Gerstmeir,4†
Bernhard J. Eikmanns,4 Jo¨rn Kalinowski,1 Alfred Pu¨hler,2 and Andreas Tauch1*
Institutfu¨rGenomforschung,Centrumfu¨rBiotechnologie,Universita¨tBielefeld,Universita¨tsstraße25,D-33615Bielefeld,Germany1;
Lehrstuhlfu¨rGenetik,Fakulta¨tfu¨rBiologie,Universita¨tBielefeld,Universita¨tsstraße25,D-33615Bielefeld,Germany2;
InternationalNRWGraduateSchoolinBioinformaticsandGenomeResearch,Centrumfu¨rBiotechnologie,
Universita¨tBielefeld,Universita¨tsstraße25,D-33615Bielefeld,Germany3;andInstituteof D
MicrobiologyandBiotechnology,UniversityofUlm,D-89069Ulm,Germany4 o
w
Received13December2006/Accepted19January2007 n
lo
a
ThetranscriptionalregulatorCg1486ofCorynebacteriumglutamicumATCC13032isamemberoftheIclR d
e
proteinfamilyandbelongstotheconservedsetofregulatoryproteinsincorynebacteria.Adefineddeletionin d
thecg1486gene,nowdesignatedltbR(leucineandtryptophanbiosynthesisregulator),ledtothemutantstrain f
r
C. glutamicum IB1486. According to whole-genome expression analysis by DNA microarray hybridizations, o
m
transcriptionoftheleuBandleuCDgenesencodingenzymesoftheleucinebiosynthesispathwaywasenhanced

inC.glutamicumIB1486comparedwiththewild-typestrain.Moreover,thegenesofthetrpEGDCFBAoperon h
t
involved in tryptophan biosynthesis of C. glutamicum showed an enhanced expression in the cg1486 mutant t
p
strain.Bioinformaticspatternsearchesintheupstreamregionsofthedifferentiallyexpressedgenesrevealed :
/
the common 12-bp motif CA(T/C)ATAGTG(A/G)GA that is located downstream of the (cid:1)10 region of the /jb
mappedpromotersequences.DNAbandshiftassayswithastreptavidin-taggedLtbRproteindemonstratedthe .a
specificbindingofthepurifiedproteinto40-merscontainingthe12-bpmotiflocalizedinfrontofleuB,leuC, s
m
andtrpE,therebyconfirmingthedirectregulatoryroleofLtbRintheexpressionoftheleucineandtryptophan
.
biosynthesispathwaygenesofC.glutamicum.GeneshomologouswithltbRweredetectedupstreamoftheleuCD o
r
genesinalmostallsequencedgenomesofbacteriabelongingtothetaxonomicclassActinobacteria.TheltbR-like g
/
genes of Corynebacterium diphtheriae, Corynebacterium jeikeium, Mycobacterium bovis, and Bifidobacterium o
longum were cloned and shown to complement the deregulation of leuB, leuCD, and trpE gene expression in n

C.glutamicumIB1486. N
o
v
e
Corynebacteriumglutamicumisagram-positivesoilbacterium 2-IPMto3-IPM,whereasIPMdehydrogenasecatalyzestheoxi- m
that was discovered in the 1950s as a natural producer of L- dativedecarboxylationof3-IPMtoyield (cid:2)-ketoisocaproate(45, be
glutamicacid(65).Nowadays,itiswidelyusedfortheindustrial 46).Theaminationof(cid:2)-ketoisocaproatetoleucineiscarriedout r

2
productionofeconomicallyimportantaminoacids,especiallyL- by the branched-chain amino acid aminotransferase IlvE, en- 1
glutamicacidandL-lysine(22,32).Inadditiontothesetraditional codedbytheilvEgene.ThetransaminaseIlvEalsoparticipatesin ,
2
products, other amino acids such as L-leucine and L-tryptophan thesynthesisoftheaminoacidsisoleucine,valine,andphenylal- 0
can be produced by specially developed mutant strains of C. anine(40,35).Regulationoftheleucinebiosynthesispathwayis 18
glutamicum (64, 16). The biosynthesis pathways of both the apparently multivalent in C. glutamicum. The IPM synthase is b
branched-chainaminoacidleucineandthearomaticaminoacid subject to strong feedback inhibition by leucine, and the leuA y
g
tryptophan have been investigated in detail in recent years (27, gene is under negative transcriptional control, probably by an u
71).Thegenesinvolvedinleucinebiosynthesiswerelocalizedin alternativekindofattenuationthatisdirectedbytheLEUele- es
different regions of the C. glutamicum genome (29). The leuA mentintheupstreamregionofthegene(45,56).Incaseofthe t
geneencodesisopropylmalate(IPM)synthase,catalyzingthefirst
leuBgene,negativetranscriptionalregulationhasbeenproposed,
step in leucine biosynthesis by converting 2-oxoisovalerate to
sincetheactivityoftheleuBpromoterwassignificantlyreduced
2-IPM (45, 46). The two other enzymes that are unique to the
whenleucinewaspresentinthegrowthmedium,butanattenu-
leucine biosynthesis pathway are IPM dehydratase, encoded by
atorisabsentinfrontofthecodingregion(46).
theleuCDgenecluster,andIPMdehydrogenase,encodedbythe
Production of the aromatic amino acid tryptophan by fer-
leuB gene. IPM dehydratase is involved in the conversion of
mentation has made tremendous progress in recent years by
applyingmorerationalapproachesfortheimprovementofC.
glutamicum strains (26, 28). Biosynthesis of aromatic amino
*Correspondingauthor.Mailingaddress:Institutfu¨rGenomforschung,
Centrum fu¨r Biotechnologie, Universit¨at Bielefeld, Universit¨atsstraße 25, acids begins with the condensation of phosphoenolpyruvate
D-33615Bielefeld,Germany.Phone:[phone].Fax:[phone]. and erythrose 4-phosphate and proceeds to chorismate, from
E-mail:[email protected].
which the specific pathways to tryptophan, phenylalanine and
†Presentaddress:DegussaAG,ProjectHouseProFerm,D-33790
tyrosine branch. The genes of the tryptophan biosynthesis
Halle/Westfalen,Germany.
(cid:1)Publishedaheadofprinton26January2007. pathwayinC.glutamicumareorganizedasanoperon,consist-
2720
VOL.189,2007 THE LtbR REGULON 2721
TABLE 1. Plasmidsusedandconstructedinthisstudy
Plasmid Relevantcharacteristic(s) Sourceorreference
pK19mobsacB sacBlacZ(cid:2)Kmr;cloningvectorforallelicexchange 54
pK19(cid:7)cg1486 pK19mobsacBcarryingamodifiedcg1486generegion Thisstudy
pEC-K18mob2 lacZ(cid:2)Kmr;C.glutamicumcloningvector 31
pEC-XK99E P ;Kmr;C.glutamicumexpressionvector 31
trc
pASK-IBA3 P ;Apr;E.coliexpressionvector IBA,Go¨ttingen,Germany
tet
pNJ010 pEC-K18mob2carryingtheC.glutamicumcg1486gene Thisstudy
pNJ011 pEC-K18mob2carryingtheC.jeikeiumjk1222gene Thisstudy
pNJ012 pEC-K18mob2carryingtheC.diphtheriaedip1126gene Thisstudy
pNJ013 pEC-XK99EcarryingtheM.bovisMb3013gene Thisstudy
pNJ014 pEC-XK99EcarryingtheB.longumBL1261gene Thisstudy
pNJ015 pASK-IBA3carryingtheC.glutamicumcg1486gene Thisstudy
D
o
w
ing of six structural genes (trpE, trpG, trpD, trpCF, trpB, and leucineandtryptophanbiosynthesispathwaysofC.glutamicum n
trpA)thatencodeallenzymaticactivitiesrequiredforthecon- and that orthologs of this regulatory protein are highly con- lo
a
version of chorismate to tryptophan (39, 17). In addition to served in almost all sequenced bacterial species belonging to d
feedbackinhibitioncontrolofthreeenzymeswithinthispath- thetaxonomicclassActinobacteria. e
d
way (anthranilate synthase, anthranilate phosphoribosyltrans-
f
r
ferase,andtryptophansynthase),expressionofthetrpoperon o
MATERIALSANDMETHODS
m
isrepressedinthepresenceoftryptophan(57,28).Therefore,
Bacterialstrains,plasmids,andgrowthconditions.E.coliDH5(cid:2)MCR(15)
thestructureandregulatoryfunctionofthetrpoperoncontrol h
wasroutinelygrownat37°CinLuria-Bertanimedium(52)supplementedwith2g t
region have been studied extensively (53, 17). A small leader liter(cid:3)1glucoseandusedforstandardcloningprocedures.Selectionforthepresence tp
peptide,encodedbythetrpLgene,containsthreeconsecutive ofplasmidswasperformedwithkanamycin(50(cid:4)gml(cid:3)1forE.coliand25(cid:4)gml(cid:3)1 ://
tryptophan codons within the coding region, suggesting regu- forC.glutamicum)orampicillin(100(cid:4)gml(cid:3)1forE.coli).Isopropyl-(cid:5)-D-thiogalac- jb
lationofthetrpoperonexpressionbyclassicaltranscriptional topyranoside(IPTG)wasusedtoinducegeneexpressionbythePtrcpromoterof .a
pEC-XK99E(31).InductionofgeneexpressiononpASK-IBA3(IBA,Go¨ttingen, s
attenuation (53, 56). Mutations in the predicted attenuator Germany)wascarriedoutbyusing200(cid:4)gml(cid:3)1anhydrotetracycline.Thewild-type m
region were apparently responsible for high levels of read- strainC.glutamicumATCC13032(AmericanTypeCultureCollection,Manassas, .o
through transcription and a constitutive antitermination re- VA)andthemutantstrainC.glutamicumIB1486weregrownat30°CinLuria- rg
sponse of C. glutamicum (38, 21). Moreover, a putative oper- Bertanimedium(52)orinCGXIIminimalmediumcontaining30mgliter(cid:3)1pro- o/
tocatechuicacid(30).Growthofshake-flaskcultureswasmonitoredbymeasuring
ator sequence with similarity to the DNA binding site of the n
theopticaldensityat600nmwithanEppendorfBioPhotometer.Plasmidsusedand
tryptophan operon repressor TrpR from Escherichia coli was constructedinthisstudyarelistedinTable1. N
o
detected upstream of the (cid:3)10 promoter region of the trp DNAisolation,manipulation,andtransfer.PlasmidDNAwaspreparedfrom v
operon (53, 17), although a typical TrpR protein is not en- E.colicellsbyanalkalinelysistechniqueusingaQIAprepSpinMiniprepKit e
m
(QIAGEN,Hilden,Germany).ChromosomalDNAswerepreparedfromsmall
coded in the C. glutamicum genome sequence (5). However, aliquots of cells that were resuspended in HO and boiled for 5 min. The b
site-directed mutagenesis of the palindromic nucleotide se- supernatantswereclearedbycentrifugationat126,000(cid:6)gfor10minandfinally er
quence revealed a regulatory role in expression of the C. storedat(cid:3)20°C.ModificationofDNA,analysisbyagarosegelelectrophoresis, 2
glutamicumtrpoperon(17). andligationwereperformedbystandardprocedures(52).TransformationofE. 1
,
DNA-bindingtranscriptionalregulatorsrepresentkeycom- coliandC.glutamicumcellswasperformedbyelectroporation(58,59). 2
PCRtechniques.PCRexperimentswerecarriedoutwithaPTC-100thermo- 0
ponentsinthecontrolofbacterialgeneexpression.Theypos- cycler(MJResearch,Watertown,MA),PwoDNApolymerase(RocheDiagnos- 1
8
sess a DNA binding domain to recognize cognate operator tics,Mannheim,Germany),andchromosomalDNAastemplate.PCRproducts
b
sequences in front of their target genes and trigger a specific werepurifiedbyusingaPCRPurificationSpinKit(QIAGEN).Oligonucleotides y
transcriptionalresponseofthecelltochangingenvironmental used for PCR amplification were purchased from Operon Biotechnologies g
(Cologne,Germany). u
conditions (37). To understand transcriptional regulation of
Generationofadefinedcg1486mutantandgeneticcomplementation.The e
amino acid biosynthesis pathways in the wild-type strain C. geneSOEingmethod(25)wasappliedtoconstructapK19mobsacBderivative st
glutamicum ATCC 13032, we recently screened the complete thatissuitabletoperformanallelicexchangeofthecg1486geneinthechro-
genome sequence by different bioinformatics tools and char- mosomeofC.glutamicumATCC13032(54).TheamplifiedDNAfragmentwas
digestedwithEcoRIandclonedintothevectorpK19mobsacB(Table1).The
acterized the repertoire of potential regulators in this species
resultingplasmidpK19(cid:7)cg1486carriesamodifiedcg1486genethatisspecified
(5). In this study, the cg1486 gene, encoding a transcriptional
byadefineddeletionof134nucleotides.Genereplacementinthechromosome
regulatoroftheIclRproteinfamily,wasanalyzedbycompar- of C. glutamicum ATCC 13032 resulted in the mutant strain C. glutamicum
ativetranscriptomics,usingtheC.glutamicumwildtypeanda IB1486.ForgeneticcomplementationofC.glutamicumIB1486,thecomplete
defined cg1486 mutant strain. Therefore, a genome-wide ap- cg1486codingregionalongwiththenativepromoterregionwasamplifiedby
PCRwiththeprimerpaircompl_cg1486_aandcompl_cg1486_b(Table2).The
proachwitholigonucleotidemicroarrayhybridizationsincon-
PCRproductwasdigestedwithEcoRIandSalIandclonedintothecompatible
junction with bioinformatics predictions revealed the target sitesofthevectorpEC-K18mob2(Table1).TheresultingplasmidpNJ010was
genes of Cg1486 protein and potential DNA binding sites in transferredtoC.glutamicumIB1486byelectrotransformation.Forheterologous
the C. glutamicum genome. Verification of DNA binding was complementation of the cg1486 deletion in C. glutamicum IB1486, the or-
thologousgenesofCorynebacteriumjeikeiumK411(NationalCollectionofType
accomplishedbyDNAbandshiftassayswithpurifiedCg1486
Cultures,London,UnitedKingdom),CorynebacteriumdiphtheriaeDSM44123,
protein. The resulting data provided clear evidence that the
BifidobacteriumlongumDSM20219(DSMZ,Braunschweig,Germany),andMy-
Cg1486proteinisinvolvedintranscriptionalregulationofthe cobacteriumbovisBCGwereamplifiedbyPCRandclonedeitherintotheshuttle
2722 BRUNE ET AL. J.BACTERIOL.
TABLE 2. Primersusedforgeneamplificationinthisstudy
Primername Primersequencea
compl_cg1486_a..................................................................................................GATCTAGAATTCCGTTTCATGGGACAGTAT
compl_cg1486_b.................................................................................................GATCTAGTCGACAGCTCTTAAAGCAAAGCG
compl_jk1222_a..................................................................................................GATCTAGAATTCCTCCTCTGTGTCCGCTAT
compl_jk1222_b..................................................................................................GATCTAGGATCCTCGGGGAGTAGCTACTTC
compl_dip1126_a................................................................................................GATCTAGAATTCCGAATCACCTCTTCTACA
compl_dip1126_b................................................................................................GATCTAGTCGACGAGTAATGCCTAGAGTGA
compl_Mb3013_a...............................................................................................GATCTAGAATTCGTGAGACAGCATAGCGGT
compl_Mb3013_b...............................................................................................GATCTAGGATCCGTAGCACAACGCGGCTAG
compl_BL1261_a................................................................................................GATCTAGAATTCGCCATTGAACAACAGCTTGACC
compl_BL1261_b................................................................................................GATCTAGGATCCGTTGGGCTATCGGTTGGATTGAC
cg1486-strep1......................................................................................................ATGGTAGGTCTCAAATGGGACAGCAAGAAATTATCGAGGAC
cg1486-strep2......................................................................................................ATGGTAGGTCTCAGCGCTAAGCAAAGCGCCTAGGCGCTC
aRestrictionsitesusedforcloningofthePCRproductsareinbold. D
o
w
n
vectorpEC-K18mob2(inthecaseofthejk1222anddip1126genes)orintothe ofintensitieswascalculatedforeachspotaccordingtotheformulaMi(cid:9)log2(Ri/ lo
IPTG-inducibleexpressionvectorpEC-XK99E(inthecaseoftheMb3013and G).Inparticular,R (cid:9)I (cid:3)Bg andG (cid:9)I (cid:3)Bg ,whereI orI a
i i ch1i ch1i i ch2i ch2i ch1i ch2i
BL1261 genes). The respective primer sequences are listed in Table 2. The istheintensityofaspotinchannel1orchannel2andBg orBg isthe d
ch1i ch2i e
resultingplasmidspNJ011topNJ014(Table1)weretransferredtoC.glutami- backgroundintensityofaspotinchannel1orchannel2,respectively.The d
cumTotIaBl1R48N6AbpyreelpeactrraottiroannfsrfoomrmCat.iognlu.tamicumcultures.Forthepreparationof athveerfaogremiunltaenAsiit(cid:9)yinlobg2o(cid:1)th(cid:10)RchiGain(cid:11)n(e1l3s)w.Nasocramlcaulilzaatteidonfoarnedaechvaslupaottioanccoofrdthineghtyo- fro
totalRNA,C.glutamicumculturesweregrowninLuria-Bertanimedium(52). bridizationdatawereaccomplishedbytheEMMA2.2microarraydataanaly- m
Approximately5(cid:6)108cellswereharvestedfromexponentiallygrowingcultures sissoftware(11)(http://www.cebitec.uni-bielefeld.de/groups/brf/software/emma
h
(opticaldensityat600nmof2.5)bycentrifugationwith11,000(cid:6)gfor15s. _info/),usingasignalintensity(Avalue)cutoffof(cid:1)10.0andasignalintensity t
t
Subsequently,thesupernatantwasdecanted,andthepelletwastransferredinto ratio(Mvalue)cutoffof(cid:12)1,whichcorrespondstorelativeexpressionchanges p
:
liquid nitrogen. RNA isolation was performed with an RNeasy Mini Kit equaltoorgreaterthantwofold. //
(QIAGEN)insuchawaythatthefrozencellswereresuspendendin800(cid:4)lof BioinformaticstoolsforCg1486proteinbindingsitedetection.Theannotated jb
RLTbufferandimmediatelydisruptedbymeansoftheRiboLyserinstrument versionoftheC.glutamicumATCC13032genomesequence(29)wasusedto .a
(Hybaid,Heidelberg,Germany),usingFastProteinTubes(Qbiogene,Heidel- performagenome-widescreeningforputativeCg1486proteinbindingsites.The s
m
berg,Germany)andtwotimeintervalsof30satspeedlevel6.5withinterme- searchwasaccomplishedbyprofilehiddenMarkovmodel(HMM)analysisusing
.
diarycoolingoftheprobe.TheRNase-freeDNaseset(QIAGEN)wasusedfor the HMMER version 2.3.2 software package (http://hmmer.janelia.org/). An o
on-columndigestionofchromosomalDNA.AsecondDNaseIdigestionwas alignmentofpredictedCg1486bindingsitescompiledwiththeCLUSTALX rg
performed with DNase I (RNase free) from MBI Fermentas (St. Leon-Rot, program (62) was used to create a profile HMM by using the HMMBUILD /
o
Germany)tocompletelyremoveDNAfromthesample.AfterthesecondDNase module.ThecalculatedprofileHMM,inconjunctionwiththeHMMSEARCH
n
Itreatment,theRNAwaspurifiedaccordingtotheRNeasyMiniKitclean-up module, was applied to screen the C. glutamicum genome sequence for the
protocolandfinallystoredat(cid:3)80°C. presenceofCg1486proteinbindingsites.Thegenomicpositionsoftheresulting N
o
Detectionofdifferentialgeneexpressionbyreal-timeRT-PCR.Purifiedtotal hitswerecorrelatedwithcodingsequencesthatrevealeddifferentialexpression v
RNAofC.glutamicumcultureswasusedforreal-timereversetranscription-PCR inC.glutamicumIB1486comparedwiththewild-typestrainbyDNAmicroarray e
m
(RT-PCR) experiments with the LightCycler instrument (Roche Diagnostics) hybridization.Furthermore,theEMBOSSmotifsearchprogramfuzznuc(51)
and a Quanti-Tect SYBR Green RT-PCR Kit (QIAGEN). Oligonucleotides andthePredictRegulonserver(72)wereappliedtosearchforCg1486binding b
e
usedtomeasurerelativegeneexpressionwerepurchasedfromOperonBiotech- sites.TheidentifiedCg1486bindingsitesofC.glutamicumwerealignedwiththe r

nologies. Verification of RT-PCR products was performed by melting curve CLUSTALXprogramandusedtogenerateaprofileHMMforscreeningofthe 2
analysis. Differences in gene expression were determined by comparing the genome sequences of Corynebacterium efficiens (42), C. diphtheriae (8), C. 1
,
crossing points of two samples measured in duplicate. Crossing points were jeikeium(60),andCorynebacteriumurealyticum(61). 2
calculatedbytheLightCyclersoftware(RocheDiagnostics). Constructionandpurificationofastreptavidin-taggedCg1486protein.The 0
IdentificationoftranscriptionalstartsitesbytheRACE-PCRmethod.Forthe cg1486codingregionwasamplifiedbyPCRwiththeprimerpaircg1486-strep1 1
8
identificationoftranscriptionalstartsites,totalRNAwasisolatedfromC.glu- and cg1486-strep2 (Table 2) to generate a fusion protein of Cg1486 and a
b
tamicumculturesgrowninLuria-Bertanimedium(52).Rapidamplificationof carboxy-terminalstreptavidin(Strep)tag.ThePCRproductwasdigestedwith y
cDNAends(RACE)-PCRprimers(18-meroligonucleotides),binding200to300 BsaIandclonedintothevectorpASK-IBA3(Table1).Theresultingplasmid
g
nucleotidesdownstreamoftheannotatedtranslationalstartcodonofleuCand pNJ015wastransferredtoE.coliDH5(cid:2)MCRbyelectroporation.Toisolatethe u
cg1486,and1(cid:4)goftotalRNAwereusedforcDNAsynthesis.Theresulting Strep-taggedCg1486protein,E.coliDH5(cid:2)MCR(pNJ015)wasgrownfor16hat e
cDNAwasmodifiedandsubsequentlyamplifiedbytwoadditionalPCRsusinga 25°CinselectiveLuria-Bertanimediumcontaining200(cid:4)gml(cid:3)1anhydrotetra- st
5(cid:8)/3(cid:8)RACEKit,secondgeneration(RocheDiagnostics).PCRproductswere cylinetoinducetheproteinexpression.Approximately5(cid:6)1010E.colicellswere
clonedintothepCR2.1-TOPOvectorandtransferredintochemicallycompetent harvestedbycentrifugation,resuspendedinbufferW(1MTris-HCl,1.5MNaCl,
E. coli TOP10 cells (Invitrogen, Karlsruhe, Germany). DNA sequencing of 10mMEDTA,onetabletofRocheCompleteMiniproteaseinhibitors,pH8.0)
cloned RACE-PCR products was performed by IIT Biotech (Bielefeld, andtransferredintoaRiboLysertube(Hybaid).Celldisruptionbymeansofthe
Germany). RiboLyserinstrumentwascarriedoutwithaspeedrateof6.5andtwotime
Hybridizationofwhole-genomeDNAmicroarrays.Eightmicrogramsoftotal intervalsof30s.TheStrep-taggedCg1486proteinwaspurifiedfromtheprotein
RNAfromC.glutamicumcultureswasusedforcDNAsynthesis.Labelingof crudeextractwithaStrep-TactinSepharose-packedcolumn(IBA,Go¨ttingen,
probesandhybridizationoftheC.glutamicumwhole-genomeDNAmicroarray Germany)accordingtothemanufacturer’sinstructions.Aftertheproteincrude
were performed as described previously (6). Hybridization experiments were extractwasloaded,thecolumnwaswashedfivetimeswith1mlofbufferW.The
carried out in duplicate using label swapping. Since each DNA microarray Strep-tagged Cg1486 protein was eluted with 0.5 ml of buffer E (buffer W
containsfourreplicatesperC.glutamicumgene,atotalnumberofeightspotsper containing2.5mMdesthiobiotin).Theresultingeluatewasanalyzedbysodium
gene were available for calculating differential gene expression. During the dodecylsulfate-polyacrylamidegelelectrophoresis(52),furtherconcentratedby
imageacquisitionanddataanalysisprocess,meansignalandmeanlocalback- using5,000-molecular-weight-cutoffAmiconUltra-4centrifugalfilterunits(Mil-
groundintensitiesweredeterminedforeachspotofthemicroarrayimagesby lipore,Schwalbach,Germany),andfinallystoredat(cid:3)20°C.Theproteinconcen-
using the ImaGene 6.0 software for spot detection, image segmentation, and trationwasdeterminedwithaBio-Radproteinassaykit(Bio-RadLaboratories,
signalquantification(BioDiscovery,LosAngeles,CA).Aftersubtractionofthe Munich,Germany).ToverifythepurificationoftheStrep-taggedCg1486pro-
localbackgroundintensitiesfromthesignalintensities,thelog valueoftheratio tein,anin-geldigestionwithmodifiedtrypsin(Promega,Mannheim,Germany)
2
VOL.189,2007 THE LtbR REGULON 2723
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FIG. 1. Detailed genetic maps of the leuCD, leuB, and trpE regulatory regions of C. glutamicum. (A) Genetic organization of the leuCD /jb
upstreamregion.Themappedtranscriptionalstartsites((cid:1)1)andthededuced(cid:3)35and(cid:3)10hexamersofcorynebacterialpromotersarespecifically .
a
marked.Potentialribosome-bindingsitesinfrontofthecodingregionsareindicated;translationalstartcodonsareunderlined.Detected12-bp s
sequencesareshownasgrayboxes.ARipAbindingsiteinfrontoftheleuCgeneisboxed(69).(B)GeneticorganizationoftheleuBpromoter m
region.TheleuBpromoterwasmappedinanearlierstudy(46).The12-bpsequenceisshownasagraybox.(C)Geneticorganizationofthe .
o
regulatoryregionofthetryptophanoperon(17).The12-bpsequencedetectedinthisstudyismarked.ThetrpLlocuscodesforasmallleader r
g
peptideinfrontofthetrpEgeneandisapparentlyinvolvedintranscriptionalattenuation.Apredictedoperatorsequenceintheregulatoryregion /
ofthetryptophanoperonisboxed(17).RBS,ribosome-bindingsite. o
n

N
o
wascarriedout,andtheresultingpeptidemassesweredeterminedbymatrix- amino acids and a theoretical molecular mass of 24.8 kDa. v
e
assistedlaserdesorptionionization–time-of-flightmassspectrometry,applying Accordingtoproteinstructurepredictionsperformedwiththe m
anUltraflexmassspectrometer(BrukerDaltonics,Bremen,Germany)andthe SUPERFAMILY server (34), the Cg1486 protein contains a b
MASCOTsoftware(48)asdescribedpreviously(18). e
DNAbandshiftassayswithpurifiedCg1486protein.DNAbandshiftassays helix-turn-helix (HTH) motif of the winged-helix type within r
2
wereperformedwithCy3-labeled40-meroligonucleotides(OperonBiotechnol- itsamino-terminalregion.Moreover,Cg1486containsalarge 1
ogies)thatwereannealedwiththecorrespondingcomplementaryoligonucleo- domainwithsignificantstructuralsimilaritytoGAF-likeeffec- ,
tides to double-stranded DNA fragments by heating at 94°C for 5 min and 2
torbindingdomainsthatwereinitiallyidentifiedincyclicGMP 0
annealingonicefor15min.DuringDNAbandshiftassays,50pmolofStrep- 1
taggedCg1486proteinwasmixedwith0.05pmolofDNA,15%(vol/vol)glycerol, (cGMP)-specific phosphodiesterases, in several adenylyl cy- 8
and binding buffer (20 mM Na2HPO4, 50 mM NaCl, 5 mM MgCl2, 2 nM clases,andintheE.colitranscriptionfactorFhl(36).Dueto b
7d.i0th)iotothrgeeittoal,t1o0t%al v[voolul/mvoel]ogfly2c0er(cid:4)oll,.1T0h0e(cid:4)bginmdiln(cid:3)g1bbuofvfienrewsaesrummoadlibfiuemdiwn,heprHe these protein structure predictions and additional amino acid y g
sequencesimilaritiesoftheHTHdomainwiththeDNAbind- u
appropriatebyaddingeither5mMleucineor5mMtryptophan.Theassaywas
e
incubatedatroomtemperaturefor15minandthenseparatedat4°Cwitha2% ing domain of IclR-type regulators (14), the Cg1486 protein s
agarosegelpreparedingelbuffer(20mMNaHPO,pH7.0).Avoltageof80 canbeclassifiedintotheIclRfamilyoftranscriptionalregula- t
2 4
Vwassuppliedfor1.5h.TheagarosegelwasscannedwithaTyphoon8600 tors(5).TheIclRfamilycomprisesrepressors,activators,and
VariableModeImager(AmershamBiosciencesEurope,Freiburg,Germany).
regulatoryproteinswithdualfunctionthatcontrolamultiplic-
ityofcellularprocesses,suchastheglyoxylatebypassreactions
RESULTS
or the degradation of aromatic compounds (41). No member
MolecularfeaturesofthetranscriptionalregulatorCg1486 oftheIclRproteinfamilywashithertodescribedtobeinvolved
and genetic characterization of the mutant strain C. glutami- in transcriptional regulation of amino acid biosynthesis path-
cum IB1486. The leuCD operon (cg1487/cg1488) of C. glu- ways(41).
tamicum ATCC 13032 encodes the subunits of the IPM To elucidate a potential role of the regulatory protein
dehydratasethatcatalyzesthesecondstepoftheleucinebio- Cg1486inexpressionoftheleucinebiosynthesispathwayofC.
synthesispathway(29).Theregulatorygenecg1486waslocal- glutamicum,themutantstrainC.glutamicumIB1486,carrying
ized directly upstream of the leuCD operon on the opposite adefineddeletioninthecg1486codingregion,wasconstructed
DNAstrandoftheC.glutamicumgenomesequence(Fig.1A). by an allelic exchange procedure (54). Inactivation of the
The predicted regulatory protein Cg1486 has a length of 235 cg1486codingregionshouldinprincipleaffecttheexpression
2724 BRUNE ET AL. J.BACTERIOL.
of potential target genes of the transcriptional regulator alignmentwasperformedwiththeCLUSTALXtool(62).The
Cg1486inthemutantstrainandeventuallyexpressionofgenes DNAalignmentrevealedtheidentical12-bpsequenceCATA
involvedinleucinebiosynthesis.Oneexperimentalmethodto TAGTGAGAthatislocatedindifferentdistancestothetrans-
detectregulatoryinteractionsistomeasuredifferencesingene lationalstartcodonsoftheleuBandleuCgenes(Fig.1).Since
expression of potential target genes (23). Therefore, differen- thetranscriptionalstartsiteandthepromoteroftheleuBgene
tial expression of the leuA, leuB, leuCD, and ilvE genes was were determined previously (Fig. 1B) (46), we additionally
examined by real-time RT-PCR assays, comparing the gene mapped the promoters of the leuCD operon and the diver-
expressionintheC.glutamicumIB1486mutantwiththatofthe gently oriented cg1486 gene for a more precise functional
wild-type strain. For this purpose, total RNA was prepared analysis of the respective intergenic region (Fig. 1A). Total
fromtwoindependentlygrownC.glutamicumculturesofeach RNA was isolated from exponentially grown C. glutamicum
strain. Subsequent real-time RT-PCR assays demonstrated IB1486cellsandusedtoamplifyandsequencethe5(cid:8)endsof
thattheexpressionoftheleuCandleuDgeneswasenhanced theleuCDandcg1486transcriptsbytheRACE-PCRmethod.
approximately 100-fold in C. glutamicum IB1486, whereas ex- TheresultingDNAsequencesallowedtheidentificationofthe
pressionoftheleuBgenewasenhanced11.8-foldinthemutant transcriptionalstartsiteinfrontoftheleuCgenethatislocated D
strain (see Fig. 3). On the other hand, expression of the leuA 60nucleotidesupstreamofthetranslationalstartcodon(Fig. o
w
genewasmoderatelychangedandenhancedonly3.3-foldinC. 1A). In the case of the cg1486 gene, the mapped transcrip- n
glutamicum IB1486, whereas no significant difference in gene tional start site was identical with the adenine residue of the lo
a
expressionwasmeasuredinthecaseoftheilvEgene. annotated translational start codon (Fig. 1A), indicating the d
Thecg1486deletionmutantC.glutamicumIB1486wassub- expressionofaleaderlesstranscript(47). e
d
sequently complemented by transformation with plasmid The mapped transcriptional start sites were subsequently
f
pNJ010carryingtheclonedcg1486geneofthewild-typestrain. usedtodetermineappropriate(cid:3)35and(cid:3)10promoterregions ro
m
Todemonstratethiscomplementation,therelativeexpression accordingtothecharacteristicfeaturesofcorynebacterialpro-

oftheleuA,leuB,andleuCDgeneswasmeasuredbyreal-time moters (47). The deduced (cid:3)35 (GTGACA) and (cid:3)10 (TAT h
t
t
RT-PCR. C. glutamicum ATCC 13032 and C. glutamicum ACT)regionsoftheleuCDpromoterareseparatedbyaspacer p
:
IB1486,bothcarryingtheemptycloningvectorpEC-K18mob2 of18nucleotides(Fig.1A).Bothhexamersrevealedconsider- //
(Table 1), served as controls. As expected, expression of the ablesimilaritywiththe(cid:3)35(TTGCCA)and(cid:3)10[TA(C/T)A jb
.
a
leuA,leuB,andleuCDgeneswasenhancedalsointhevector- AT] consensus sequences of corynebacterial promoters (47).
s
carryingmutantstrainC.glutamicumIB1486(pEC-K18mob2) In the case of the cg1486 promoter, only the deduced (cid:3)10 m
comparedwiththerespectivewild-typecontrol,C.glutamicum region (TAAATT), which is located 7 bp upstream of the .o
r
ATCC 13032 (pEC-K18mob2). On the other hand, enhanced transcriptional and translational start site, revealed similarity g
expression of the leuA, leuB, and leuCD genes was no longer to the corynebacterial consensus promoter, whereas the (cid:3)35 o/
detectedinC.glutamicumIB1486(pNJ010),comparedwithC. region(ACCACG)islesswellconserved(Fig.1A).According n

glutamicumATCC13032(pEC-K18mob2),demonstratingthat to these mapping data, the common 12-bp sequence CATAT N
o
the cg1486 gene on pNJ10 complemented the cg1486 mutant AGTGAGAislocatedbetweenthe(cid:3)10promoterregionand v
e
(data not shown). The observed deregulation of leu gene ex- the translational start codon of the leuB and leuC genes (Fig.
m
pression in C. glutamicum IB1486 and C. glutamicum IB1486 1A and B). This location of the common 12-bp sequence in b
(pEC-K18mob2)canthereforebeattributedtothedeletionof frontofdifferentiallyexpressedgenessuggestsapotentialrole e
r
thecg1486gene. as a DNA binding site of a repressor protein (33), which is 2
1
Toelucidatetheroleofleucineindifferentialexpressionof consistentwiththeenhancedexpressionoftheleuBandleuCD
,

the leu genes of C. glutamicum, both the wild-type strain C. genesinthecg1486mutantC.glutamicumIB1486. 2
0
glutamicum ATCC 13032 and the cg1486 mutant C. glutami- Todetectidentical12-bpmotifsintheC.glutamicumATCC 1
8
cumIB1486weregrowninminimalmediumCGXII,andrel- 13032 genome sequence, bioinformatics analyses were per-

b
ativegeneexpressionwasmeasuredbyreal-timeRT-PCR.No formedwiththepatternrecognitiontoolfuzznuc(51)andthe y
differential expression of the leu genes was detected when C. PredictRegulonserver(72).Furthermore,aprofileHMMwas g
u
glutamicumIB1486wasgrowninminimalmediumcontaining built by means of an alignment of the identified 12-bp se-
e
5mMleucinecomparedwithgrowthinnonsupplementedmin- quencesandusedinamotifsearchagainsttheC.glutamicum s
t
imalmedium(datanotshown).Ontheotherhand,expression genomesequence.Thesebioinformaticssearchesrevealedthat
oftheleugenesdecreasedinthewild-typestrainwhenitwas the 12-bp sequence is solely present in front of the leuB and
cultivated in leucine-containing minimal medium, indicating leuCgenes.However,amultitudeofsimilarsequencestretches
thattheLtbRmutantlacksaleucine-dependentregulationof wasdetectedintheC.glutamicumgenomesequencewhenone
leugeneexpression.Thesedatafurthermoresuggestedthatthe to four mismatches within the motif sequence were included
Cg1486 protein is involved in negative transcriptional regula- (datanotshown).Forinstance,asimilar12-bpsequencewith
tion of the leu genes of C. glutamicum, either directly or fourmismatchesinthemotifsequencewaslocalizedupstream
indirectly. ofthemappedcg1486promoter(Fig.1A),butnosimilar12-bp
Detectionofpotentialoperatorsequencesbypromotermap- sequencesweredetectedbythedifferentcomputationalmeth-
ping and bioinformatics analysis. The real-time RT-PCR as- ods in the known promoter region of leuA (47) and in the
says demonstrated that the expression of the leu genes is upstream region of the ilvE gene. This result prompted us to
enhancedintheabsenceoftheregulatoryproteinCg1486.To examine the global gene expression of the cg1486 mutant C.
localize potential regulator binding sites in the upstream re- glutamicumIB1486bywhole-genometranscriptionalprofiling
gions of the differentially expressed genes, a DNA sequence withDNAmicroarrayhybridizationstosearchfordifferentially
VOL.189,2007 THE LtbR REGULON 2725
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
t
p
:
/
/
jb
.
a
FIG. 2. Ratio/intensity (M/A) plot deduced from DNA microarray hybridizations comparing the gene expression of the cg1486 mutant C.
s
glutamicumIB1486withthatofthewild-typestrainC.glutamicumATCC13032.TheanalyzedstrainswerecultivatedinLuria-Bertanimedium m
tomid-exponentialgrowthphase,revealingnoapparentdifferenceingrowthbehavior.TotalRNAwassubsequentlyisolatedfromtwobiological .
o
replicatesandappliedforDNAmicroarrayhybridizations,includinglabelswapping.GenesshowingenhancedexpressioninC.glutamicumIB1486 r
aremarkedbyblackdiamonds,geneswithdecreasedexpressionareindicatedbyblacktriangles,andgeneswithoutdifferentialexpressionpattern g
/
areshownbygrayspots.Selecteddifferentiallyexpressedgenesarenamedbytheiridentifiers.TheAvaluerepresentsthesignalintensity,andthe o
Mvaluerepresentsthesignalintensityratio,whichcorrespondstorelativeexpressionchanges.TheMvaluecutoffof(cid:12)1correspondstorelative n

expressionchangesequaltoorgreaterthantwofold. N
o
v
e
m
expressed genes that are specified by the presence of imper- geneswithenhancedexpression(Mvalueequaltoorgreater b
fectlyconserved12-bpsequences. than (cid:1)1) and eight genes with decreased expression (M e
r
DetectionofdifferentiallyexpressedgenesinC.glutamicum valueequaltoorlessthan(cid:3)1),comparedwiththewild-type 2
1
IB1486 by whole-genome DNA microarray hybridization. To strain.Theresultingdataaredepictedgraphicallyinaratio/ ,

detectgeneswithdifferentialexpressioninthecg1486mutant intensity (M/A) plot (Fig. 2). The respective genes showing 2
0
strain, whole-genome DNA microarray hybridizations were differentialexpressioninC.glutamicumIB1486arelistedin 1
8
carriedout,therebycomparingthegeneexpressionofC.glu- Table 3.
b
tamicumIB1486withthatofthewild-typestrainC.glutamicum Among the genes with significantly enhanced expression in y
ATCC 13032. Cell samples for RNA purification were har- C. glutamicum IB1486, the leuABCD genes involved in the g
u
vested from two independently grown C. glutamicum cultures
leucine biosynthesis pathway were detected. The genes with e
of each strain, and purified total RNAs were used for two s
the highest changes in transcription were leuC and leuD, t
DNA microarray hybridization assays, including label swap-
whereasleuBandleuAshowedsmallerchanges,whichiscon-
ping. The resulting hybridization data were evaluated and vi-
sistent with the initial measurements of differential gene ex-
sualizedwiththeImagenesoftware.Normalizationofthedata
pressioninthecg1486mutantbyreal-timeRT-PCRassays.In
by the LOWESS function and t test statistics were conducted
addition, almost all genes of the tryptophan biosynthesis
withtheEMMA2.2softwarepackage.Tominimizethenum-
operon(trpEGDCFBA)revealedanenhancedexpressioninC.
beroffalse-positivesignals,thedatawerestringentlyfilteredto
glutamicum IB1486, although differential expression of the
obtaingeneswithatleastsixstatisticallysignificantvaluesout
oftheeighttechnicalreplicatespresentonthetwohybridized trpB and trpA genes showed smaller changes, which may be
microarrays,alongwithanerrorprobabilityoflessthan5%for caused by the presence of an internal promoter in the trp
the t test. By applying the experimental threshold values of operon of C. glutamicum (43). Moreover, the genes of the
(cid:1)10forthesignalintensityAand(cid:12)1forthelog transformed putativerbsoperon(rbsRACBD)thatisapparentlyinvolvedin
2
signal intensity ratio M (corresponding to relative expression the uptake of ribose (68) were differentially expressed in C.
changesofatleasttwofold),asumtotalof50genesrevealed glutamicumIB1486,withtheexceptionofrbsD,whichisagain
differential expression in C. glutamicum IB1486, including 42 thelastgeneoftheputativepolycistronictranscript.
2726 BRUNE ET AL. J.BACTERIOL.
TABLE 3. Genesdifferentiallyexpressedinthecg1486mutantC.glutamicumIB1486
Geneidentifiera Gene Geneproductordeducedfunctionb Mvalue Avalue Pvalue
cg1488 leuD Isopropylmalatedehydratase,smallsubunit 4.58 12.58 7.87(cid:6)10(cid:3)8
cg1487 leuC Isopropylmalatedehydratase,largesubunit 4.35 11.93 5.25(cid:6)10(cid:3)8
cg1413 rbsB ABC-typeribosetransporter,substrate-bindingprotein 3.48 10.97 1.44(cid:6)10(cid:3)7
cg1412 rbsC ABC-typeribosetransporter,permeasesubunit 2.92 10.67 3.27(cid:6)10(cid:3)10
cg1658 PutativeMFS-typepermease 2.88 10.70 5.43(cid:6)10(cid:3)12
cg1453 leuB Isopropylmalatedehydrogenase 2.75 12.32 4.31(cid:6)10(cid:3)11
cg1411 rbsA ABC-typeribosetransporter,ATPasesubunit 2.70 10.50 1.24(cid:6)10(cid:3)8
cg1129 aroF Phospho-2-dehydro-3-deoxyheptonatealdolase 2.42 10.83 6.96(cid:6)10(cid:3)3
cg2893 PutativeMFS-typepermease 2.25 10.94 2.33(cid:6)10(cid:3)3
cg1410 rbsR ABC-typeribosetransporter,transcriptionalrepressor 2.18 10.16 5.67(cid:6)10(cid:3)9
cg2391 aroG 3-Deoxy-7-phosphoheptulonatesynthase 1.59 12.44 4.06(cid:6)10(cid:3)8
cg3359 trpE Anthranilatesynthase,subunitI 1.57 11.44 2.90(cid:6)10(cid:3)6
cg2894 Putativetranscriptionalregulator,TetRfamily 1.56 12.55 4.97(cid:6)10(cid:3)4
D
cg1612 Putativeacetyltransferase 1.50 11.50 4.47(cid:6)10(cid:3)3 o
cg3362 trpCF Phosphoribosylanthranilateisomerase 1.50 10.92 2.23(cid:6)10(cid:3)5 w
cg2565 Hypotheticalprotein 1.47 10.97 8.33(cid:6)10(cid:3)3 n
cg3361 trpD Anthranilatephosphoribosyltransferase 1.43 10.68 2.96(cid:6)10(cid:3)6 lo
cg1419 Putativesecondarytransporter 1.42 12.46 2.09(cid:6)10(cid:3)5 a
cg2610 PutativeABC-typetransporter,substrate-bindingprotein 1.40 12.21 4.28(cid:6)10(cid:3)4 de
cg3360 trpG Anthranilatesynthase,subunitII 1.36 10.64 2.87(cid:6)10(cid:3)6 d
cg2837 sucC Succinate-CoAligase(ADP-forming),betasubunit 1.34 13.44 2.76(cid:6)10(cid:3)3 f
cg2137 gluB ABC-typeglutamatetransporter,substrate-bindingprotein 1.30 14.43 4.51(cid:6)10(cid:3)9 ro
cg2836 sucD Succinate-CoAligase(ADP-forming),alphasubunit 1.30 12.93 2.98(cid:6)10(cid:3)4 m
cg0303 leuA Isopropylmalatesynthase 1.30 12.55 3.94(cid:6)10(cid:3)3 h
cg3047 ackA Acetatekinase 1.30 10.58 3.39(cid:6)10(cid:3)5 tt
cg2138 gluC ABC-typeglutamatetransporter,permeasesubunit 1.19 13.05 1.78(cid:6)10(cid:3)6 p
:
cg2559 aceB Malatesynthase 1.18 10.49 2.02(cid:6)10(cid:3)5 //
cg2136 gluA ABC-typeglutamatetransporter,ATPasesubunit 1.17 14.36 3.41(cid:6)10(cid:3)9 jb
cg0961 Putativehydrolase 1.17 11.05 3.83(cid:6)10(cid:3)4 .a
cg3096 Putativealdehydedehydrogenase 1.15 13.31 1.17(cid:6)10(cid:3)3 s
m
cg3022 Hypotheticalprotein 1.15 11.95 1.62(cid:6)10(cid:3)3
cg2636 catA Catechol1,2-dioxygenase 1.13 11.76 4.94(cid:6)10(cid:3)4 .o
cg2118 Putativetranscriptionalregulator,DeoRfamily 1.12 10.72 5.65(cid:6)10(cid:3)7 rg
cg2630 pcaG Protocatechuate3,4-dioxygenase,alphasubunit 1.11 11.14 1.20(cid:6)10(cid:3)3 /
cg1084 cgtR10 Two-componentsystem,responseregulator 1.11 11.04 2.27(cid:6)10(cid:3)7 on
cg1615 Putativepseudouridylatesynthase 1.10 11.61 4.13(cid:6)10(cid:3)3
N
cg1616 cmk Cytidylatekinase 1.09 12.03 1.52(cid:6)10(cid:3)3 o
cg2937 PutativeABC-typetransporter,substrate-bindingprotein 1.09 10.65 1.89(cid:6)10(cid:3)6 v
cg1617 PutativeGTPaseofunknownfunction 1.03 12.40 8.37(cid:6)10(cid:3)3 e
cg1085 Hypotheticalprotein 1.01 14.03 3.39(cid:6)10(cid:3)3 m
cg3329 Hypotheticalprotein 1.01 10.62 2.13(cid:6)10(cid:3)2 be
cg3363 trpB Tryptophansynthase,betachain 1.00 10.56 1.97(cid:6)10(cid:3)5 r
cg2102 sigB RNApolymerase,sigmafactorBsubunit (cid:3)1.02 12.76 8.39(cid:6)10(cid:3)9 2
cg2657 Putativemembraneprotein (cid:3)1.03 11.72 8.52(cid:6)10(cid:3)6 1
,
cg3309 Putativesecretedprotein (cid:3)1.07 11.09 1.54(cid:6)10(cid:3)5 2
cg2651 Hypotheticalprotein (cid:3)1.14 12.68 5.97(cid:6)10(cid:3)8 0
cg1121 PutativeMFS-typepermease (cid:3)1.15 12.29 3.37(cid:6)10(cid:3)10 18
cg0756 cstA CarbonstarvationproteinA (cid:3)2.57 11.08 2.49(cid:6)10(cid:3)6
b
cg2545 Putativesecretedormembraneprotein (cid:3)4.07 11.52 3.55(cid:6)10(cid:3)7 y
cg2546 PutativesecondaryC4-dicarboxylatetransporter (cid:3)4.46 12.08 5.56(cid:6)10(cid:3)9 g
u
aGenesspecificallylabeledinFig.2areinbold. e
bCoA,coenzymeA. s
t
IdentificationofdifferentiallyexpressedgenesofC.glutami- motifsinitiallylocalizedinfrontoftheleuBandleuCDgenes.
cumIB1486specifiedbythepresenceofa12-bpsequence.The Furthermore,animperfectlyconserved12-bpsequence,show-
results of the DNA microarray hybridization were subse- ing two mismatches, was detected upstream of the trpE gene
quently combined with data from bioinformatics pattern (Table4).Acloserinspectionoftherespectivegeneregionof
searches to determine the fraction of differentially expressed C. glutamicum revealed that the 12-bp sequence is located 16
genesofC.glutamicumIB1486thatarespecifiedbythepres- nucleotides in front of the trpL gene, thereby spanning the
ence of 12-bp sequences. Three computational methods were already mapped transcriptional start site of the tryptophan
appliedtosearchfor12-bpsequencesinthegenomesequence operon(Fig.1C)(17).ThetrpLlocuscodesforasmallleader
ofC.glutamicum,revealingthepresenceofsevenmotifsinthe peptideandisinvolvedintranscriptionalattenuationofthetrp
upstream region of genes or within coding regions that were operonexpression(38,21).Theother12-bpsequencesareless
detected as differentially expressed in C. glutamicum IB1486 wellconservedandcontainthreetofourmismatches(Table4).
(Table 4). This set of 12-bp sequences included the identical These sequence motifs are located (i) in front of the aroG
VOL.189,2007 THE LtbR REGULON 2727
TABLE 4. The12-bpsequenceslocalizedinfrontoforwithin 12-bp sequences. The respective DNA regions of the C. glu-
differentiallyexpressedgenesofC.glutamicumIB1486 tamicum genome represent appropriate candidate sequences
to prove a direct regulatory interaction by the transcriptional
Methodusedfor Distance
Gene
Gene 12-bpsequence 12-bpsequence (no.of regulatorCg1486.
identifier
predictiona nucleotides)b Direct interaction of the Cg1486 protein with 40-mer DNA
cg1487 leuC CATATAGTGAGA HMM PR PM (cid:3)31 sequences analyzed in vitro by DNA band shift assays. To
cg1453 leuB CATATAGTGAGA HMM PR PM (cid:3)16 demonstrateadirectinteractionofthetranscriptionalregula-
cg3359 trpE CACATAGTGGGA HMM PM (cid:3)269
torCg1486withthe12-bpregionsinfrontofthedifferentially
cg1617 CACCTAGTGGAA HMM PM (cid:3)24
cg2391 aroG CACACAGTTTGA HMM (cid:3)479 expressed genes of C. glutamicum IB1486, DNA band shift
cg1412 rbsC CACACAGTGCAA HMM (cid:1)91 assays were performed. For this purpose, a modified Cg1486
cg2545 CAAACAGTGGGA HMM (cid:1)87 protein was constructed by adding a carboxy-terminal Strep
tag. The complete coding region of cg1486 was amplified by
aPR,PredictRegulonserver;PM,patternmatchingwithfuzznuc.
b(cid:3), the 12-bp sequence is located in front of the coding region, and the PCR and cloned in E. coli into the anhydrotetracyline-induc-
distancefromthelastnucleotideofthe12-bpsequencetothefirstnucleotideof ibleexpressionvectorpASK-IBA3(Table1).TheStrep-tagged D
theannotatedstartcodonisindicated;(cid:1),the12-bpsequenceislocatedinthe o
Cg1486 protein was subsequently expressed in an induced E.
codingregion,andthedistancefromthefirstnucleotideoftheannotatedstart w
codontothefirstnucleotideofthe12-bpsequenceisindicated. colicultureandpurifiedtohomogeneitybyStrep-Tactinaffin- n
ity chromatography. Purification of the Cg1486 protein was lo
a
verifiedbymatrix-assistedlaserdesorptionionization–time-of- d
gene, encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate flightmassspectrometry(datanotshown). ed
synthase,whichisthefirstenzymeofthebiosynthesispathway DNA band shift assays were performed with the Strep- f
r
foraromaticaminoacids(9);(ii)withintherbsCgene,encod- tagged Cg1486 protein and double-stranded DNA fragments o
m
ing a permease subunit of a putative ABC-type ribose trans- containingthe12-bpsequences(Table4).Moreover,the12-bp

h
porter(68);(iii)upstreamofthecg1617gene;and(iv)within sequence located upstream of the cg1486 gene (Fig. 1A) was
t
t
thecg2545codingregionthatrevealedalowerexpressioninC. included in this assay. The selected 12-mers were equally ex- p
:
glutamicumIB1486(Table3). tended on both sides with the respective genome-specific se- //
jb
Considering potential operon structures of the C. glutami- quencestoyieldatotallengthof40nucleotides.Appropriate .
a
cumgenome(49),thedetected12-bpsequencesarerelatedto synthetic oligonucleotides were annealed to produce Cy3-la- s
seven transcription units comprising a sum total of 20 genes beled, double-stranded 40-mer fragments. Each DNA band m
.
(Fig. 3). Differential expression of these genes in C. glutami- shiftassaycontained0.05pmolofthelabeled40-merfragment o
r
cum IB1486 was verified by real-time RT-PCR assays, using and 50 pmol of the purified Cg1486 protein. As shown in g
/
the same experimental setup as applied for whole-genome Fig. 4A, three 40-mer fragments revealed a different electro- o
DNAmicroarrayhybridization.Theresultingdataaresumma- phoretic mobility when previously incubated with the Strep- n

N
rizedinFig.3,confirmingtheenhancedordecreasedexpres- tagged Cg1486 protein, comprising the probes of the leuC,
o
sionoftheselectedgenesinC.glutamicumIB1486.Thecom- leuB,andtrpEgenes.Nodifferencesinelectrophoreticmobil- v
e
binationofDNAmicroarrayhybridizationwithbioinformatics itywereobservedwhen40-mersequencespresentupstreamof m
patternsearchesthereforedefinedasetofgenesthatarenot thecg1412(rbsC),cg1486,cg1617,cg2391(aroG),andcg2545 b
e
only differentially expressed in the cg1486 mutant strain C. genes were used (Fig. 4B). The specificity of the DNA band
r

glutamicum IB1486 but are also specified by the presence of shift with the Strep-tagged Cg1486 protein was furthermore 2
1
,

2
0
1
8

b
y

g
u
e
s
t
FIG. 3. Relevantgenesandgeneclustersshowingdifferentialexpressioninthecg1486mutantC.glutamicumIB1486.Therespectivevaluesof
differentialgeneexpressionshownabovethearrowswerededucedfromreal-timeRT-PCRassays.Thevaluesaremeansoffourmeasurements.
PredictedRho-independenttranscriptionalterminatorsareindicatedbyhairpinstructures;12-bpsequencesdefinedbybioinformaticsanalysesare
shown as white boxes. The differentially expressed genes are involved in leucine and tryptophan biosynthesis, in ribose uptake, and in other
physiologicalprocesses.
2728 BRUNE ET AL. J.BACTERIOL.
D
o
w
n
lo
FIG. 4. Agarose gels of DNA band shift assays performed with the streptavidin-tagged Cg1486 protein. (A) DNA band shift assays with a
Cy3-labeled,double-stranded40-merscoveringthe12-bpsequencesinfrontoftheleuC,leuB,andtrpEgenes.DNAbandshiftswereperformed d
e
with0.05pmoloftheCy3-labeled40-merfragmentand50pmoloftheStrep-taggedCg1486protein.Assayswereseparatedina2%agarosegel d
andvisualizedbyfluorescenceimaging.Geneidentifiersareshownbelowtheagarosegels.Lane1,controlassaywithoutCg1486protein;lane2, f
DNAbandshiftassaywithCg1486protein.(B)DNAbandshiftassayswithCy3-labeled,double-stranded40-merscoveringthe12-bpsequences ro
ofthecg1412(rbsC),cg1486,cg1617,cg2391(aroG),andcg2545genes.(C)DNAcompetitionexperimentwitha40-merfragmentcoveringthe m
12-bpsequenceinfrontoftheleuCgene.Atotalof50pmoloftheStrep-taggedCg1486proteinand0.05pmolofaCy3-labeled40-meralongwith
h
increasing concentrations of the same nonlabeled 40-mer fragment (40mer) were added to the assay. (D) DNA band shift assays with the t
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Cy3-labeled40-merfragmentrepresentingtheupstreamregionoftheleuCgeneandmodifiedbindingbuffercontainingeither5mMleucine(Leu) p
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or5mMtryptophan(Trp). //
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.
a
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examined by competition assays. When an excess of nonla- tified in front of the leuB and leuCD genes of these species m
beled 40-mer was added to the assay, the DNA band shift of (datanotshown).Asimilar12-bpsequencewas,furthermore, .o
r
the leuC probe was prevented more and more (Fig. 4C), ap- detectedinfrontofthetrpLlocusoftheC.efficienstrpoperon g
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parently by competition between labeled and increasing (datanotshown).ThepresenceofltbR-likeregulatorygenesin o
amounts of nonlabeled 40-mers. Moreover, the DNA band the leuCD gene region was therefore examined in 42 se- n

shiftassaywasperformedwithamodifiedbindingbuffercon- quenced genomes of gram-positive bacteria belonging to the N
o
taining either leucine or tryptophan as potential effector sub- taxonomicclassActinobacteria.Theanalysiswasaccomplished v
e
stancesoftheregulatoryproteinCg1486(Fig.4D).DNAband by means of the SEED software environment (44) and addi-
m
shiftsweredetectedintheabsenceandpresenceofleucineand tionalproteinsimilaritysearchesthatwereperformedwiththe b
tryptophan, indicating that both amino acids are dispensable BLASTPprogram(1).Thesecomputationalmethodsrevealed e
r
for the in vitro interaction of Cg1486 with the 40-mer frag- that the genomic organization of the ltbR and leuCD gene 2
1
ments and not involved in alleviating the DNA binding prop- regionisconservedin38ofthehithertosequencedactinobac-
,

ertiesoftheCg1486protein.Nevertheless,theDNAbandshift terial genomes, including all sequenced species of the genera 2
0
assaysconfirmedinvitrothedirectbindingoftheStrep-tagged Acidothermus, Arthrobacter, Bifidobacterium, Brevibacterium, 1
8
Cg1486proteintothreespecific40-merDNAfragmentscon- Corynebacterium,Frankia,Janibacter,Kineococcus,Mycobacte-

b
taining the 12-bp sequences that were localized in the up- rium,Nocardia,Nocardioides,Propionibacterium,Rhodococcus, y
stream regions of the leuC, leuB, and trpE genes, thereby Rubrobacter,Salinispora,Streptomyces,andThermobifida(data g
u
demonstratingthattheCg1486proteinisinvolvedintranscrip- not shown). Exceptions are Leifsonia xyli subsp. xyli, the se-
e
tional regulation of the leucine and tryptophan biosynthesis quenced strains of Tropheryma whipplei, and the unclassified s
t
pathways of C. glutamicum. Due to the apparent direct regu- actinobacterial species Symbiobacterium thermophilum. The
latory role of the Cg1486 protein in leucine and tryptophan conserved genomic organization suggests that genes homolo-
biosynthesisofC.glutamicum,thecg1486genewasnamedltbR gouswithltbRofC.glutamicummayhaveasimilarregulatory
(leucineandtryptophanbiosynthesisregulator). function in gene expression of other species belonging to the
Detection of ltbR orthologs in genome sequences of Acti- taxonomicclassActinobacteria.
nobacteria. A recent classification of genes encoding DNA- Heterologous complementation of C. glutamicum IB1486
bindingtranscriptionalregulatorsinsixsequencedcorynebac- withltbR-likegenesfromCorynebacterium,Mycobacterium,and
terial genomes revealed that the LtbR (Cg1486) protein Bifidobacteriumspecies.Toassesswhethertheactinobacterial
belongstothecommonsetofregulatorsinthesespecies(4,5). ltbR-like genes are functionally equivalent in transcriptional
The homologous genes of C. efficiens (ce1426), C. diphtheriae regulationofgeneexpression,heterologouscomplementation
(dip1126),C.jeikeium(jk1222),andC.urealyticum(cu09_0562 assays were conducted with the ltbR mutant C. glutamicum
andcu11_1897)arelocatedupstreamoftheleuCDgenesina IB1486 and the ltbR-like genes of C. diphtheriae DSM44123
divergent orientation as well. In addition, DNA motifs with (dip1126),C.jeikeiumK411(jk1222),M.bovisBCG(Mb3013),
similarity to the 12-bp sequence of C. glutamicum were iden- and B. longum DSM20219 (BL1261). The selected ltbR-like
VOL.189,2007 THE LtbR REGULON 2729
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FIG. 5. HeterologouscomplementationassaysofC.glutamicumIB1486withltbR-likegenesfromC.diphtheriae(CD),C.jeikeium(CJ),M. .a
bovis(MB),andB.longum(BL).RelativeexpressionoftheleuCD,leuB,andtrpEgeneswasmeasuredbyreal-timeRT-PCRandnormalizedby s
m
usingthevaluesdeterminedintheplasmid-carryingcontrolstrainsC.glutamicumATCC13032(pEC-K18mob2)andC.glutamicumATCC13032
(pEC-XK99E),abbreviatedC1andC2,respectively.Thevaluesaremeansoffourmeasurements;standarddeviationsareindicated. .o
r
g
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geneswereamplifiedbyPCRandclonedinE.coliintoeither sayswiththeltbRmutantC.glutamicumIB1486demonstrated n

theshuttlevectorpEC-K18mob2(dip1126andjk1222)orthe thattheltbR-likegenesofC.diphtheriae,C.jeikeium,M.bovis, N
o
shuttleexpressionvectorpEC-XK99E(Mb3013andBL1261). andB.longumnotonlyarelocatedinaconservedpositionin v
e
The resulting plasmids pNJ011 to pNJ014 (Table 1) were fi- the actinobacterial genomes but also can functionally replace
m
nallytransferredintotheltbRmutantC.glutamicumIB1486by thetranscriptionalregulatorLtbRofC.glutamicum. b
electrotransformation.RelativeexpressionoftheleuCD,leuB, e
r
andtrpEgenesintheplasmid-carryingmutantC.glutamicum 2
DISCUSSION 1
IB1486 was subsequently measured by real-time RT-PCR
,

assays and compared with that of the wild-type strain C. glu- TheleucinebiosynthesisgenesleuBandleuCDaresubjectto 2
0
tamicum ATCC 13032 carrying the empty cloning vectors. transcriptionalcontrolbytheIclR-typerepressorLtbR.Inthe 1
8
Therefore, C. glutamicum IB1486 was also transformed with present study, the regulon of the transcriptional regulator

b
pEC-K18mob2 or pEC-XK99E and served as control. As ex- LtbRofC.glutamicumwasexamined,andthetopologyofits y
pected, the expression of leuCD, leuB, and trpE was signifi- gene regulatory network was deduced from DNA microarray g
u
cantly enhanced in the control strains C. glutamicum IB1486 hybridizations, bioinformatics analysis, and DNA band shift
e
(pEC-K18mob2) and C. glutamicum IB1486 (pEC-XK99E) assays. The ltbR gene is located on the opposite DNA strand s
t
comparedtotherespectivegeneexpressionlevelsoftheplas- upstreamoftheleuCDgenes,encodingIPMdehydratase(29).
mid-carrying wild-type controls (Fig. 5). On the other hand, IPM dehydratase as well as IPM synthase and IPM dehydro-
expression of the leuCD, leuB, and trpE genes significantly genase, encoded by the leuA and leuB genes, represent the
decreased in C. glutamicum IB1486 harboring plasmids enzymesuniquetotheleucinebiosynthesispathwayofC.glu-
pNJ011 to pNJ014, indicating that the cloned ltbR-like genes tamicum (45, 46). Expression of the three enzymes is coordi-
fromC.diphtheriae,C.jeikeium,M.bovis,andB.longumcom- nately down-regulated when appropriate amounts of leucine
plementedthederegulationoftheleuCD,leuB,andtrpEgene are present in the growth medium, suggesting negative tran-
expressioninthemutantstrain(Fig.5).Anexceptionwasthe scriptionalregulationoftherespectivegenes(45).However,a
ltbR-like gene BL1261 from B. longum that reduced the ex- previous comparison of the regulatory DNA regions of the
pressionoftheleuCDgenesinC.glutamicumIB1486(pNJ014) leuAandleuBgenesrevealednonucleotidesequencesimilar-
to only 30% of the values measured in the control strain C. ities, already indicating different mechanisms for transcrip-
glutamicumIB1486(pEC-XK99E),indicatingthatonlypartial tionalregulationofleugeneexpressioninC.glutamicum(46).
complementation by the BL1261 gene occurred in this assay A small gene coding for a potential leader peptide with four
(Fig. 5). Nevertheless, the heterologous complementation as- consecutiveleucineresidueswasdetectedupstreamoftheleuA