Molecular characterization of lysR-lysXE, gcdR-gcdHG and amaR-amaAB operons for lysine ...

13 Pages·2016·2.69 MB·English

Microbiology(2016),162,876–888 DOI10.1099/mic.0.000277
Molecular characterization of lysR-lysXE,
gcdR-gcdHG and amaR-amaAB operons for
lysine export and catabolism: a comprehensive
lysine catabolic network in Pseudomonas
aeruginosa PAO1
Sai Madhuri Indurthi,1 Han-Ting Chou1 and Chung-Dar Lu1,2
Correspondence 1Department ofBiology,GeorgiaState University, Atlanta,GA30303,USA
Chung-DarLu 2Department ofClinical LaboratoryandNutritional Sciences,UMassLowell, Lowell,
[email protected] MA01854,USA
AmongmultipleinterconnectedpathwaysforL-Lysinecatabolisminpseudomonads,ithas
beenreportedthatPseudomonasaeruginosaPAO1employsthedecarboxylaseandthe
transaminasepathways.However,upuntilnow,knowledgeofseveralgenesinvolvedin
operationandregulationofthesepathwayswasstillmissing.Transcriptomeanalysescoupled
withpromoteractivitymeasurementsandgrowthphenotypeanalysesledustoidentifynew
membersinL-LysandD-Lyscatabolismandregulation,includinggcdR-gcdHGforglutarate
utilization,dpkA,amaR-amaABandPA2035forD-Lyscatabolism,lysR-lysXEforputativeL-Lys
effluxandlysPforputativeL-Lysuptake.ThegcdHGoperonencodesanacyl-CoAtransferase
(gcdG)andglutaryl-CoAdehydrogenase(gcdH)andisunderthecontrolofthetranscriptional
activatorGcdR.GrowthonL-LyswasenhancedinthemutantsoflysXandlysE,supportingthe
operationofL-Lysefflux.ThetranscriptionalactivatorLysRisresponsibleforL-Lysspecific
inductionoflysXEandthePA4181-82operonofunknownfunction.Theputativeoperatorsites
ofGcdRandLysRwerededucedfromserialdeletionsandcomparativegenomicsequence
analyses,andtheformationofnucleoproteincomplexeswasdemonstratedwithpurified
His-taggedGcdRandLysR.TheamaABoperonencodestwoenzymestoconvertpipecolate
to2-aminoadipate.InductionoftheamaABoperonbyL-Lys,D-Lysandpipecolaterequiresa
functionalAmaR,supportingconvergenceofLyscatabolicpathwaystopipecolate.Growthon
Received 19November2015 pipecolatewasretardedinthegcdGandgcdHmutants,suggestingtheimportanceofglutarate
Revised 9March2016 inpipecolateand2-aminoadipateutilization.Furthermore,thisstudyindicatedlinksinthecontrol
Accepted 10March2016 ofinterconnectednetworksoflysineandargininecatabolisminP.aeruginosa.
INTRODUCTION making this initial step in the proposed pathway a bottle-
neck for L-lysine catabolism in this organism. Cadaverine,
Multiple and interconnected catabolic pathways exist for
the product of LdcA, is further degraded to 5-aminovale-
the utilization of lysine in microbes. As shown in Fig. 1,
L-lysine is catabolized via the monooxygenase pathway in rate through the c-glutamylation pathway for polyamine
Pseudomonas putida, while in Pseudomonas aeruginosa it catabolism that is controlled by PauR (Chou et al., 2013;
is mainly mediated via the decarboxylase pathway (Chou Yao et al., 2011). Both monooxygenase and decarboxylase
et al., 2010; Revelles et al., 2004, 2005). Expression of the pathways converge at 5-aminovalerate, which is then con-
lysine decarboxylase LdcA was found to be inducible by vertedtoglutaratebyapairoftransaminaseandsemialde-
the arginine regulator ArgR and L-arginine but not hyde dehydrogenase encoded by the gabDT operon (Chou
L-lysine in P. aeruginosa PAO1 (Chou et al., 2010), et al., 2013; Revelles et al., 2004). Glutarate is proposed to
produce acetyl-CoA that can enter the Krebs cycle (Numa
et al., 1964), and glutaryl-CoA dehydrogenase enzyme
Abbreviations: AMV, 5-amino valerate; CAD, cadaverine; EMSA,
electrophoreticmobilityshiftassay;GA,glutaricacid;L-Pip,L-pipecolate. (an enzyme in glutarate catabolism) activity has been
reported in crude extracts of P. aeruginosa (Fothergill &
The GenBank/EMBL/DDBJ (GEO) accession number for the Gene
ChipdatausedinthisstudyisGSE75502. Guest, 1977). However, none of the genes for glutarate
876 000277G2016TheAuthors PrintedinGreatBritain
LysinecatabolicpathwaysinP.aeruginosa
L-LYS D-LYS into glutarate (Fothergill & Guest, 1977). In P. aeruginosa,
PP3722 (alr)
ArgR IdcA aruH amaC dauA DauR tFhAeDa-ldphepaeanmdeinnot Dg-raomupinoofaDc-iLdydsechayndbroegdeenaamseinDaateudAb(yLith&e
CAD KAH Lu,2009a).ItwasnotclearhowD-Lyscatabolismwasoper-
pauA1
AruSR ated in this organism due to very limiting information
A4A5 davB ΔP2C
aboutpotentialplayersinthecatabolicpathway.
pauB1 dpkA
spuC
Many of the intermediate compounds in the proposed
PauR L-PIP
lysine catabolic pathways in P. aeruginosa PAO1 can
pauC
amaB
serve as better sources of carbon and nitrogen than lysine
davA ΔP6C
pauD2 itself. It is very likely that lysine catabolism is composed
AmaR
of multiple modules of biochemical reactions, and the
AMV 2-AAS
expressionofeachmoduleiscontrolledbyaspecificinter-
gabT davT amaA
mediate compound and its corresponding regulators.
2-AAP To gain a better understanding on the interconnections
gabD davD
of L-Lys and D-Lys catabolism in P. aeruginosa, we con-
GA
2-KAP ducted transcriptome analysis to identify several possible
gcdG PA2035 (PAO1)
Krebs missing links and new players, and these thus identified
GcdR Glutaryl-CoA α-KG cycle members were subjected to further characterization to tie
gcdH their physiological functions and regulatory mechanisms
Crotonyl-CoA Acetyl-CoA into an integrated metabolic network.
Fig.1.PictorialrepresentationoflysinecatabolisminPseudomo-
METHODS
nas aeruginosa PAO1 and Pseudomonas putida KT2440. Path-
waysinP.aeruginosaPAO1andP.putidaKT2440areindicated Strainsandgrowthconditions.Bacterialstrainsusedinthisstudy
by solid black arrows and dashed grey arrows, respectively. are listed in Table 1. Luria–Bertani (LB) medium was used for bac-
Genes that are subjected to control by transcriptional regulators terialgrowthwiththefollowingsupplements asrequired:ampicillin
ArgR, PauR, GcdR, DauR and AmaR are labelled in different at100mgml21andcarbenicillinat100mgml21forbothEscherichia
colours. LYS, lysine; CAD, cadaverine; AMV, d-aminovalerate; coli and P. aeruginosa. Minimal medium P (MMP) supplemented
GA, glutaric acid; KAH, a-keto-e-aminohexanoate (keto-lysine); with specific carbon sources and nitrogen sources was used for the
DP2C,D1-piperideine-2-carboxylate;L-PIP,L-pipecolate;DP6C, growthofP.aeruginosa(Haasetal.,1977).Unlessspecified,mutant
D1-piperideine-6-carboxylate; 2-AAS, 2-aminoadipate semiade- strainswithasinglelesionbytransposoninsertionusedinthisstudy
(Table 2) were obtained from the stock centre at University of
hyde; 2-AAP, 2-aminoadipate; 2-KAP, 2-ketoadipate; a-KG,
Washington,Seattle,USA(Jacobsetal.,2003).
a-ketoglutarate.
Transcriptome analysis. Two independent sets of P. aeruginosa
PAO1 cultures were grown aerobically in MMP media with 10mM
L-Gluwith10mML-Lys,cadaverine(CAD),5-aminovalerate(AMV),
catabolism and its regulation have been characterized in
glutaricacid(GA),D-Lysor5mML-pipecolate(L-Pip).Cellsinthe
Pseudomonas. exponential growth phase were harvested and RNA samples were
extractedfromcellswithanRNeasyPlusMinikit(Qiagen).Following
The presence of a lysine racemase has been reported in
the protocol of Affymetrix, cDNA was synthesized, fragmented and
P. putida to convert L-Lys to D-Lys (Radkov & Moe, labelled. Labelled cDNA was used for GeneChip Microarrays with a
2013; Revelles et al., 2005). The catabolic pathway for P. aeruginosa Genome Array (Affymetrix). Data were collected and
D-Lys catabolism in this organism (Fig. 1) has been pro- analysedbycomparinggeneexpressionundereachtestconditionto
posed to degrade D-Lys to 2-aminoadipate, which is then thatincellsgrowninL-Glufollowingthesameparametersasdescribed
further degraded to a-ketoglutarate to enter the Krebs previously(Chouetal.,2008).DatawereprocessedbyMicroarraySuite
5.0software,normalizingtheabsoluteexpressionsignalvaluesofall
cycle. Several genes encoding enzymes of this proposed
chips to a target intensity of 500. Only genes showing consistent
pathway in P. putida KT2440 have been identified, includ-
expressionprofilesinduplicateswereselectedforfurtheranalysis.
ingPP3590forD-Lystransaminase,PP3596forD-Lysdehy-
drogenase, dpkA for D1-piperideine-2-carboxylate
Construction of gene promoter::lacZ fusions. For the con-
reductase (Muramatsu et al., 2005; Revelles et al., 2005,
struction of lacZ fusions, the regulatory regions of the genes were
2007),andamaBandamaAinconversionofpipecolate to amplifiedfromthegenomicDNAofP.aeruginosaPAO1byPCRwith
2-aminoadipate (Revelles et al., 2005). P. aeruginosa does specific primer pairs as follows: GCD1-F and GCD1-R for pGCD1;
notpossessthelysineracemase;however,L-Lyscatabolism LYS1-F and LYS1-R for pLYS1; AMA1-F and AMA1-R for pAMA1;
can potentially be connected to the D-Lys pathway via an and PA4181-F and PA4181-R for pPA4181. Purified PCR products
weredigestedwithspecificrestrictionenzymesandligatedtopQF50
arginine-pyruvate transaminase AruH (Chou et al., 2010;
(Farinha&Kropinski,1990)digestedwiththesameenzymesbefore
Yang&Lu,2007a).Oneearlyreportsuggestedthepresence theyweretransformedintocellsofE.coliDH5a.Thepositiveclones
of another route through L-lysine 6-amino transferase to wereselectedonLBplatescontainingampicillin,andthenucleotide
make L-pipecolate, which again could possibly convert sequence of the inserts was confirmed by DNA sequencing.
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S.M.Indurthi,H.-T.ChouandC.-D.Lu
Table1.Bacterialstrains,plasmidsandprimersusedinthisstudy
Strainorplasmid Genotypeordescription Sourceorreference
Escherichiacoli
DH5a Hoststrainforgenecloning BRL
TOP10 HoststrainforthepBADvector Invitrogen
P.aeruginosa
PAO1 Wild-type Gallegosetal.(1997)
PW1815 gcdG-F01::ISlacZ/hah Jacobsetal.(2003)
PW1817 gcdH-A02::ISphoA/hah Jacobsetal.(2003)
PW1819 gcdR-C02::ISphoA/hah Jacobsetal.(2003)
PW2858 PA1026-G12::ISphoA/hah Jacobsetal.(2003)
PW2859 amaA-E05::ISphoA/hah Jacobsetal.(2003)
PW2862 amaB-B10::ISlacZ/hah Jacobsetal.(2003)
PW3259 dpkA-D05::ISphoA/hah Jacobsetal.(2003)
PW4475 dhcR-G02::ISlacZ/hah Jacobsetal.(2003)
PW4478 atoB::ISlacZ/hah Jacobsetal.(2003)
PW4480 PA2002-D03::ISlacZ/hah Jacobsetal.(2003)
PW4481 bdhA-H08::ISphoA/hah Jacobsetal.(2003)
PW4520 PA2035-D05::ISphoA/hah Jacobsetal.(2003)
PW8087 PA4181-F03::ISlacZ/hah Jacobsetal.(2003)
PW8089 PA4182-B01::ISlacZ/hah Jacobsetal.(2003)
PW8366 lysR-F10::ISphoA/hah Jacobsetal.(2003)
PW8368 lysX-G02::ISlacZ/hah Jacobsetal.(2003)
PW8370 lysE-A07::ISphoA/hah Jacobsetal.(2003)
PW9274 amaR-E05::ISlacZ/hah Jacobsetal.(2003)
PW9379 PA4979-G11::ISphoA/hah Jacobsetal.(2003)
PW9381 PA4980-B07::ISphoA/hah Jacobsetal.(2003)
PW9382 PA4981-C03::ISphoA/hah Jacobsetal.(2003)
PAO5503 aruH::Tcr Yang&Lu(2007b)
MJ27 ParentalstrainofMJ99 Wargo&Hogan(2009)
MJ99 DdhcAB Wargo&Hogan(2009)
Plasmids
pQF50 blalacZtranscriptionalfusionvector Farinha&Kropinski(1990)
pLYS1-5 lysX::lacZfusionofpQF50 Thisstudy
pGCD1-5 gcdH::lacZfusionofpQF50 Thisstudy
pAMA amaA::lacZfusionofpQF50 Thisstudy
pPA4181 PA4181::lacZfusionofpQF50 Thisstudy
pBAD-HisE VectormodifiedfrompBAD-HisC Li&Lu(2009b)
pGCDR-HisC 6His-taggedGcdRproteinexpressionvector Thisstudy
pLYSR-HisC 6His-taggedLysRproteinexpressionvector Thisstudy
Primers
GCD1-F 59-ATGCGGATCCGGGTCAGGGACAACTCCTCC-39 Thisstudy
GCD1-R 59-ATGCAAGCTTCCGGTCTCGCCCATCTCGCG-39 Thisstudy
LYS1-F 59-ATGCGGATCCGCAGGCGTTCCGGGGCACC-39 Thisstudy
LYS1-R 59-ATGCAAGCTTCCGTGGCGAAGGCGGCGGTCAG-39 Thisstudy
AMA1-F 59-ATGCGGATCCGTCGGCGTTCGCAGGGTG-39 Thisstudy
AMA1-R 59-ATGCGGATCCGCCCCTCCAGTCGCACGCA-39 Thisstudy
PA4181-F 59-ATGCGGATCCGCGCAGGGACTCCAGCAGGT-39 Thisstudy
PA4181-R 59-ATGCAAGCTTGCGCTCTACGGCGCGGCCG-39 Thisstudy
LysR-F 59-ATCTCATGATTTTGTTCGACTACAAGTTGCTCGCCGC-39 Thisstudy
LysR-R 59-GGGGCCTGACACCCGCACCAAGCC-39 Thisstudy
GcdR-F 59-ATCTCATGAGACGCAAGATCCCTTCCACCG-39 Thisstudy
GcdR-R 59-GGGGCCCAGCCCGTTGGCCTCGC-39 Thisstudy
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LysinecatabolicpathwaysinP.aeruginosa
Table2.GrowthphenotypeofvariousP.aeruginosaknockoutmutants
CellsfromtwoindependentcoloniesweregrowninMMPwiththecompoundsindicatedasthesolesourceofcarbonandnitrogen(20mM)exceptfor
GA(10mM)where0.5%NH Clwasaddedasthenitrogensource.Aerobicgrowthat378Cwasfollowedfor4daysandrecordedasfollowsundereach
4
growthcondition:+++,bettergrowththanWTPAO1;++,samegrowthasWTPAO1;+,weakergrowththanWTPAO1;+/2,verypoorgrowth
comparedwithWTPAO1;2,nogrowthevenafter4days.NT,Nottested;NA,notapplicable.Significantchangesonthegrowthphenotypewere
markedingrey.ThePWstrainsoftransposoninsertion mutantswereobtainedfromtheUniversity ofWashingtonPseudomonasstockcentre.
MJ27andMJ99weregiftedbyDrM.J.Wargo(UniversityofVermont).
Growthwith:
PAID* Strain Genotype TransposondirectionD L-Arg L-Lys CAD AMV GA L-Pip
PAO1 WT NA ++ ++ ++ ++ ++ ++
PAO446 PW1815 gcdG R ++ 2 2 2 2 2
PAO447 PW1817 gcdH R ++ 2 2 2 2 2
PAO448 PW1819 gcdR F ++ 2 2 2 2 NT
PA1026 PW2858 R ++ ++ ++ ++ ++ ++
PA1027 PW2859 amaA R ++ + ++ ++ ++ 2
PA1028 PW2862 amaB F ++ + ++ ++ ++ 2
PA1252 PW3259 dpkA R ++ ++ ++ ++ ++ NT
PA1998 PW4475 dhcR R ++ ++ ++ ++ ++ ++
PA2001 PW4478 atoB F ++ ++ ++ ++ ++ ++
PA2002 PW4480 R ++ ++ ++ ++ ++ ++
PA2003 PW4481 bdhA R ++ ++ ++ ++ ++ ++
PA2035 PW4520 R ++ + ++ ++ ++ 2
PA4181 PW8087 F ++ ++ ++ ++ ++ NT
PA4182 PW8089 R ++ ++ ++ ++ ++ NT
PA4363 PW8366 lysR F ++ +++ ++ ++ ++ NT
PA4364 PW8368 lysX F ++ +++ ++ ++ ++ NT
PA4365 PW8370 lysE F ++ +++ ++ ++ ++ NT
PA4914 PW9274 amaR F ++ + ++ ++ ++ 2
PA4976 PAO5503 aruH ++ ++ ++ ++ ++ NT
PA4979 PW9379 F ++ ++ ++ ++ ++ NT
PA4980 PW9381 R ++ ++ ++ ++ ++ NT
PA4981 PW9382 lysP R ++ +/2 ++ ++ ++ NT
MJ27d WT ++ ++ ++ ++ ++ ++
PA1999-2000 MJ99d dhcAB ++ ++ ++ ++ ++ ++
*PAIDnumberswerefromthePAO1genomeannotationproject(http://www.pseudomonas.com).
DTransposondirectionreferstotheabsolutedirectionofthetransposonandtranscriptionaldirectionofastronginternalpromotercarriedbythe
transposon in the genome. F, forward; R, reverse. Forward is oriented parallel to increasing nucleotide base number and PA gene annotation
number.
dMJ27istheparentalstrainofMJ99andthecomparisonismadebetweenthesetwoandnottheWTPAO1above.
Plasmids with serial deletions from the full-length inserts were con- 0.2%L-arabinose(w/v)wasaddedtotheculturewhentheOD600was
structedbythesameapproachwithspecificprimers. approximately 0.5–0.6. After 4h of vigorous shaking at 18uC, cells
wereharvestedbycentrifugationandstoredat280uCuntiluse.
Cloning, expression and purification of hexa-histidine-tagged The cell pellets were suspended in phosphate buffer A (20mM
LysRandGcdR.ThestructuralgeneslysRandgcdRwereamplifiedby Na2HPO4, 0.5M NaCl, 20mM imidazole, pH7.5) with PMSF
PCR from the genomic DNA of P. aeruginosa PAO1 using the fol- (1mM)asaproteaseinhibitorandthecellswererupturedbyFrench
lowing primer pairs: LysR-F and LysR-R for lysR; and GcdR-F and press. Cell debris was removed by centrifugation at 30000g for
GcdR-R for gcdR. The resulting PCR products were digested with 30min. The supernatant was applied to a HisTrap HP column (GE
specific restriction enzymes and cloned into the expression vector Healthcare),theHis-taggedLysRwaselutedat20–60%bufferBand
pBAD-HisE(Li&Lu,2009b)sothattheCterminusofLysRandGcdR His-taggedGcdRwaselutedat40–70%bufferB(20mMNa2HPO4,
wasfusedin-framewiththeHis6-tagwhiletheNterminuswaspre- 0.5M NaCl, 1M imidazole, pH7.5). The target fractions were ana-
ceded by a ribosome-binding site and an arabinose-inducible pro- lysed by SDS-PAGE, pooled and concentrated using an Amicon
moter in the plasmid. The resulting plasmids, pLYSR and pGCDR, Ultra-15 centrifugal filter unit (molecular mass cut-off, 30kDa;
wereintroducedintoE.coliTop10(Invitrogen).Foroverexpressionof Millipore)tochangethebufferto20mMTris/HCl(pH7.5).Protein
LysRandGcdRproteins,therecombinantstrainsofE.coliweregrown concentration was determined by the method of Bradford (Kruger,
in LB medium containing ampicillin at 37uC. For over-expression, 1994).
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S.M.Indurthi,H.-T.ChouandC.-D.Lu
Electrophoreticmobilityshiftassays(EMSA).The DNA probes that displayed over threefold induction with significant
(2ng)wereallowedtointeractwithdifferentconcentrationsofGcdR signallevelsinduplicateswereselectedforfurtheranalyses.
orLysRinthereactionbuffercontaining50mMTris/HCl(pH7.5or
pH8.5, respectively), 50mM KCl, 1mM EDTA, 5% (v/v) glycerol, ListedinTable3isaselectedgroupofgenesthatweresub-
4mM DTT and 200mg acetylated BSA ml21. After incubation for jectedtomoredetailedcharacterizationinthisstudy.Some
10min at room temperature, 20ml of each reaction mixture was genesthatpassedtheinitialscreeningparametershavebeen
loaded on a polyacrylamide gel (6%) in Tris/borate/EDTA buffer characterized in our previous studies as members of the
(pH8.7).ThegelswerestainedwithSYBRGoldsolution(Invitrogen) c-glutamylationpathwayforCADandpolyaminecatabolism
for20min,washedtwicewithdeionizedH O,andscanned.
2 (Chouetal.,2013;Yaoetal.,2011),andhencewereinten-
tionally omitted from the list. Therefore, in this table we
RESULTS mainly focused on genes that are either new members or
less characterized members in the Lys metabolic network.
Transcriptome profiling In particular, this study characterized three operons
(lysXE-R, gcdHG-R and amaAB-R) that play significant
Many of the intermediate compounds in the proposed
roles in Lys utilization in PAO1, apart from some other
lysine catabolic pathways in P. aeruginosa PAO1 can
serve as better sources of carbon and nitrogen than lysine genes that are activated by L-lysine.
itself. We hypothesized that lysine catabolism is composed
of multiple modules of biochemical reactions, and each
Growth phenotypes
module is controlled by a specific intermediate compound
anditscorrespondingregulatorymechanism atthegenetic Mutants of various genes under study were obtained from
level. To get a more detailed understanding of lysine cata- the transposon mutant library stock centre (University of
bolism and regulation, DNA microarray experiments were Washington). These mutants were checked for growth on
conducted to get a snapshot of gene expression during L-Lys, CAD, AMV, GA, L-Pip or L-Arg as the sole source
exponential growth phase in the WT strain PAO1 grown of carbon and nitrogen on minimal medium plates and
in glutamate (L-Glu) minimal medium supplemented the growth was recorded as in Table 2. In the case of glu-
with L-Lys, CAD, AMV, GA, D-Lys or L-Pip. The tarate, which can only serve as carbon source, ammonium
expression profiling under each test condition was com- chloride (0.5%) was included in the growth medium as
pared with that of cells grown in L-Glu, and the genes nitrogen source.
Table3.SelectedgenesfromDNAmicroarrayanalysisforL-Lyscatabolism
Signal intensityD
PA ID* Gene Name Glu Lys CAD AMV GA D-Lys Pip Description
PAO446 gcdG 266 1263 1698 2294 11738 615 2256 Acyl-CoA transferase
PAO447 gcdH 242 9746 7201 9183 19218 1572 4215 Glutaryl-CoA dehydrogenase
PAO448 gcdR 115 83 69 91 156 56 58 Transcriptional regulator
PA1027 amaA 139 1230 197 238 81 4858 6105 NAD-dependent aldehyde dehydrogenase
PA1028 amaB 75 483 231 144 136 2460 3521 FAD-dependent oxidoreductase
PA1252 dpkA 113 205 465 279 105 285 252 L-Malate dehydrogenase
PA1998 dchR 126 284 351 322 165 221 238 Transcriptional regulator
PA1999 dchA 54 72 215 371 7081 161 153 Dehydrocarnitine CoA transferase
PA2000 dchB 63 156 380 624 5767 157 165 Dehydrocarnitine CoA transferase
PA2001 atoB 101 165 175 259 3763 95 73 Acetyl-CoA acetyltransferase
PA2002 38 165 185 136 542 95 103 Conserved hypothetical protein
PA2003 bdhA 137 402 411 375 1387 315 257 3-Hydroxybutyrate dehydrogenase
PA2035 51 198 167 35 82 1659 2974 Probable decarboxylase
PA4181 169 5721 432 385 191 344 416 Hypothetical protein
PA4182 234 8589 423 457 282 276 250 Transcriptional factor
PA4363 lysR 54 59 35 104 45 65 28 Inhibitor of chromosome initiation IciA
PA4364 lysX 38 2448 125 30 60 182 162 Hypothetical protein
PA4365 lysE 33 2304 181 111 37 89 115 Lysine efflux permease
PA4914 amaR 187 321 299 337 137 239 142 Transcriptional regulator
PA4976 aruH 130 630 130 53 111 180 149 Arginine:pyruvate transaminase
PA4981 lysP 38 5855 189 39 61 100 93 Amino acid permease
*PAIDnumberswerefromthePAO1genomeannotationproject(http://www.pseudomonas.com).
DGeneChiprawdataweremeanvaluesoftwoindependentsetsofcultures.Significantchangesonthesignalintensityweremarkedinboldtype.
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LysinecatabolicpathwaysinP.aeruginosa
ItwasfoundthatmutantsofgcdG,gcdHorgcdRwerecom- support the route for converting 2-aminoadipate into glu-
pletelydefective forgrowth onL-Lys, CAD,AMV,GA and tarate, but not a-ketoglutarate as has been suggested by
L-Pip but grew normally on L-Arg. Based on the genome early reports (Perfetti et al., 1972; Revelles et al., 2005).
annotation, the divergent gcdR-gcdHG operon (Fig. 2a)
encodesatranscriptionalregulator,theglutaryl-CoAdehy- Intheproposedpathway,conversionofL-Pipinto2-amino-
drogenaseandaputativeacyl-CoAtransferase,respectively. adipaterequirestwosequentialreactionscatalysedbyAmaB
Theresultsofgrowth phenotypeanalysessupportthepro- andAmaA.IndeedtheamaAandamaBmutantswerecom-
posed functions of these genes in glutarate utilization. pletelydefectiveforgrowthonL-Pip,supportingtheirrolein
Surprisingly, it was found that these mutants could not L-Pip and hence D-Lys catabolism. Furthermore, these two
grow on L-Pip, which is the intermediate compound in mutationsalsoaffectedgrowthonL-Lys,suggestingthepre-
D-lysine catabolism and was proposed to be converted sence of a metabolic flow that is branched out from L-Lys
into 2-aminoadipate (Fig. 1). The results would therefore catabolism(Fig.1). Asimilarpatternofgrowthphenotypes
(a) (b)
gcdR gcdH gcdG SA (nmole OD min–1)
600 GcdR
L-Glu L-Glu+GA
pGCD1 (508bp) lacZ 71 914 +
pGCD2 (363bp) lacZ 88 1155 +
GTG
pGCD2 (317bp) lacZ 86 1196 +
CAC
pGCD4 (317bp) lacZ 51 53 –
pGCD5 (288bp) lacZ 63 44 –
(c)
(d)
Fig.2.IdentificationofGcdRbindingsiteonthegcdHpromoterregion.(a)SchematicpresentationsofthedivergentgcdR-
gcdGH operons and constructs of the gcdH::lacZ fusions. Five plasmids with serial deletions in the regulatory regions
werelabelledwithplasmidnames.(b)Measurementsofb-galactosidaseactivityfromfusionplasmidsandsummaryofGcdR
bindingactivitywiththegcdHregulatoryregioncarriedbytheseplasmids.BindingofGcdRtotheregionisindicatedby‘+’
andnobindingisindicatedby‘2’;SA,specificactivity.(c)NucleotidesequenceofthegcdHregulatoryregion.Locationsof
59endsforpGCD2,pGCD3,pGCD4andpGCD5andATGinitiationcodonsforgcdHandgcdRareindicatedwitharrow-
heads or arrows and labels.The 210and 235 sequences for the putative promoter are also labelled. The proposed GcdR
operator site is in bold italicized letters with double underlining. S.D., Shine–Dalgarno sequence for ribosomal binding.
(d)MultiplesequencealignmentsfortheputativeGcdRoperatorsinthegcdHregulatoryregionsofrepresentativespeciesof
Pseudomonas in the following order: P. aeruginosa (PAO1), P. denitrificans (ATCC13867), P. mendocina (NK01),
P.fluorescens(Pf01),P.entomophila(L48),P.stutzeri(CCUG29243),P.fulva(12-X)andP.putida(KT2440).
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S.M.Indurthi,H.-T.ChouandC.-D.Lu
wasalsoobservedforthemutantofamaRencodingaputa- constructed and introduced into WT PAO1 as described
tivetranscriptionalregulatoroftheamaABoperon. in Methods. The ability of various compounds to activate
the gcdH promoter was tested. Consistent with the results
Among genes that were induced specifically by L-lysine,
ofGeneChipanalysis(Table3),asimilarpatternofpromo-
PA4981 (lysP) encodes a putative amino acid permease,
ter induction by L-Lys, CAD, AMV, GA, D-Lys and L-Pip
and the lesion in this gene conferred a strong growth
was observed in PAO1 harbouring pGCD1 by measure-
defect on L-Lys. We also compared the growth phenotype ments of b-galactosidase activities (Fig. 3).
of the lysP mutant and the WT strain PAO1 on glucose
minimal medium with different nitrogen sources, Amongthecompoundstested,exogenousglutarateexerted
including ammonium, L-Arg, D-Arg, L-Lys, and D-Lys. thestrongesteffectonpromoterinduction,suggestingglu-
AnapparentgrowthdefectofthelysPmutantwasobserved tarate as the potential effector compound for promoter
withD-LysandL-LysbutnotD-ArgandL-Arg.Itsuggested activation. To test this hypothesis, the levels of gcdH
the significant role of LysP as a potential Lys transporter. promoter activation by glutarate were also measured in
the gcdG and gcdR mutants harbouring pGCD1.
Onthecontrary,amongallmutantstested,itwasfoundthat
IncomparisonwiththelevelintheWTPAO1,thispromo-
thelysR,lysXandlysEmutantsgrewbetterthantheparental
ter was activated more than fivefold in the gcdG mutant,
strain on L-Lys. The lysX gene codes for a protein of
which was expected to accumulate glutarate inside the
unknown functionwhilethelysEgenecodesforaputative
cell without the acyl-CoA transferase activity. On the con-
amino acid exporter of the LysE/ArgO family, showing
trary, this promoter showed no sign of induction in the
45% sequence identity to the arginine exporter ArgO and
gcdR mutant (Fig. 3). These results support the hypothesis
thelysineexporterLysOofE.coli(Nandineni&Gowrishan-
of GcdR as transcriptional activator of the gcdHG operon
kar,2004;Pathania&Sardesai,2015).UpstreamofthelysXE
in response to glutarate as the signal molecule.
operon,thedivergentlysRgenewasannotatedasiciA(Tho¨ny
etal.,1991)bythegenomeproject,andthetranscriptional
regulator encoded exhibited 41% sequence identity to
DNA binding activity of GcdR
ArgPofE.coliincontrolofArgOexpression.Assupported
by other lines of evidence described below, the enhanced The results of growth phenotype analysis and promoter
growth on L-Lys of the lysR mutant was very likely due to measurements indicated the potential function of GcdR
itsactivationeffectonlysXEexpression. asthetranscriptionalregulatorofthegcdHGoperoninglu-
tarate catabolism. Recombinant GcdR with a hexa-histi-
dine tag at its carboxyl terminus was expressed and
Induction of the gcdHG promoter
purified from a strain of E. coli as described in Methods.
To study regulation of the gcdHG operon, a gcdH::lacZ ThisrecombinantGcdRwasusedtodemonstrateitsinter-
transcriptional fusion plasmid (pGCD1; Fig. 2a) was actionswiththegcdHpromoterregionbyEMSA.Asshown
4854
5000
4500
1)
–
yn
vitmi
c actiD 600 1500 1258
SpecifiD/O420 1000 914 933
O
(
500 330 396
246 225 240
165
71 39 33 77 62 74 75 92 81
0
u s D V A s p u A u A u A s p u A s p
Gl Ly A M G Ly Pi Gl G Gl G Gl G Ly Pi Gl G Ly Pi
-L -L C A -D -L -L -L -L -D -L -L -D -L
PAO1 PW1815 PW1819 PW3259 PW9274
(WT) (ΔgcdG) (ΔgcdR) (ΔdpkA) (ΔamaR)
Fig.3.ExpressionprofileofthegcdHpromoterinvivo.ThegcdHpromoteractivitywasmonitoredbymeasuringb-galactosi-
dase activity in theWT PAO1 andits mutants harbouring the pGCD1 plasmid.Cells were grown in L-Glu (10mM)minimal
medium in the presence of various compounds as indicated (10mM). Specific activity values represent the mean¡SD of
threemeasurementsforeachgrowthcondition.
882 Microbiology162
LysinecatabolicpathwaysinP.aeruginosa
None GA None GA None GA with the restriction enzyme EcoRI, which is located in
(GcdR) 0 12 24 12 24 0 12 24 12 24 0 12 24 12 24 the middle of the second conserved sequence motif (data
(nM) not shown). Site-directed mutagenesis of the first three
bases in the proposed GcdR operator from GTG in
C pGCD3 to ACA in pGCD4 resulted in complete loss of
GcdR binding in vitro (Fig. 4) and loss of promoter
F
activation in vivo (Fig. 2).
pGCD1 pGCD3 pGCD4 Induction of lysXE by L-Lys
Asdescribedabove,lysEwasproposedtoencodetheputa-
Fig.4.DemonstrationofGcdR–DNAinteractions.ThegcdHpro- tiveL-Lysexporter,anditformsaputativeoperonwiththe
moterregionsasinpGCD1,pGCD3orpGCD4wereusedasthe upstreamlysXofunknownfunction.Totesttheexpression
probe.PurifiedGcdR–His(6)oftheconcentrationsindicatedwas profilingoflysXE,alysX::lacZtranscriptionalfusionplas-
used with or without 5mM GA as indicated in each reaction. mid (pLYS1) was constructed and introduced into WT
F,freeprobe;C,nucleoproteincomplexes.Locationsoffreeprobes PAO1. The ability of various compounds to activate this
andmajornucleoproteincomplexesarealsomarkedwitharrows. promoter was tested. As shown in Fig. 5, it was found
that this promoter was induced by L-Lys only, consistent
with the results of the DNA microarray experiments
in Fig. 4, GcdR forms nucleoprotein complexes with DNA (Table 3).
fragments covering the gcdHG promoter region carried by
The activity of the lysX promoter was also measured in
pGCD1. Although the presence of glutarate in the binding
various mutants in order to understand the regulatory
reactiondidnotcauseanychangeinaffinityofGcdRtothe
mechanism of the lysXE operon. It was found that lysine-
probe DNA, it seemed to favour nucleoprotein complexes
dependent induction of the lysX promoter was completely
that migrate slower on the gel in comparison with those
abolished in strain PW8366 carrying a lesion in lysR
without glutarate.
(Fig.5).Onthecontrary,thelevelofinductionbyL-Lysin
In order to further narrow down the GcdR binding site, the lysX or lysE mutant was even higher than that in the
shorter probes carried by pGCD2, pGCD3, pGCD4 and WT strain PAO1. These results supported the proposed
pGCD5(Fig.2a)weregeneratedandtestedforinteractions functions of LysE and LysR as L-Lys exporter and lysine-
with GcdR (Fig. 4). The result of serial deletions led us to responsive transcriptional activator, and suggested a link
identifyaregionof30bpthatmaycarrytheGcdRoperator ofLysXtothelysineexportsystem.
siteasdescribedbelow.Itwasalsosupportedbythepromo-
teractivitymeasurements(Fig.2b)fromplasmidscarrying
the same series of promoter fragments as in the EMSA. DNA binding activity of LysR
Site-directed mutagenesis of the proposed GcdR operator
in pGCD4 or absence of the proposed GcdR operator in LysR was overexpressed and purified with a hexa-histidine
pGCD5 resulted in complete abolishment of glutarate- tag at its carboxyl terminus as described in Methods. This
dependent induction of the gcdH promoter activity. recombinant LysR was used to demonstrate its interaction
with the lysX promoter region by EMSA. LysR alone was
abletobindtoaDNAfragmentcarryingthelysXpromoter
Multiple sequence alignment of gcdHG regioninpLYS1toformnucleoproteincomplexes(C1and
promoters in Pseudomonas C2, Fig. 6). We also tested the potential effects of two
amino acids, L-Lys and L-Arg, on LysR–DNA interactions.
Information from comparative genomics indicated that
However,onlyminimaleffectsbythepresenceofthesetwo
gcdR-gcdHG genes are highly conserved among various
amino acids could be detected in vitro, which cannot
species of Pseudomonas (http://www.pseudomonas.com).
explain the observed effects in vivo.
Multiple sequence alignment was conducted for the
gcdHG regulatory regions from representative species, In an attempt to identify the LysR operator sites, multiple
which revealed a highly conserved sequence motif. sequencealignmentswereconductedwiththelysXpromo-
In accordance with the results of promoter analysis and ter sequences from PAO1 and representative strains from
gel shift assays shown in Figs 2 and 4, it appeared that different gene clusters. As shown in Fig. 7(c), a conserved
the sequence motif (59-GTGAGAAAATAGCAC-39) sequence motif was found in this regulatory region that
109bp upstream of the gcdH initiation codon might be a could potentially be the Lys operator site. This finding is
potential candidate for the GcdR operator. A second con- further supported by the loss of LysR binding in EMSA
served sequence motif was found downstream of the pro- with lysX promoter DNA fragments lacking this motif in
posed GcdR operator site (Fig. 2d). However, this site pLYS4 and pLYS5 (Fig. 6b) and also by the lack of tran-
was excluded as the GcdR operator site as binding of scriptionalactivationinthesetwoplasmidsinthepresence
GcdR to the pGCD1 probe was retained after digestion of L-Lys in the WT PAO1 in Fig. 7.
http://mic.microbiologyresearch.org 883
S.M.Indurthi,H.-T.ChouandC.-D.Lu
2500
2038
2000
1)
–
yn
c activitD mi600 1500
SpecifiD/O420 1000 942
O
( 456
500
9 10 5 8 9 11 25 12 8
0
u s D V A g u s u s u s
Gl Ly A M G Ar Gl Ly Gl Ly Gl Ly
-L -L C A -L -L -L -L -L -L -L
PAO1 PW8366 PW8368 PW8370
(WT) (ΔlysR) (ΔlysX) (ΔlysE)
Fig. 5.Expression profile of the lysX promoter in vivo.lysXE promoter activity was monitored by measuring b-galactosidase
activity in WT and mutant PAO1 harbouring the pLYS1 plasmid. Cells were grown in L-Glu alone (20mM) or L-Glu+L-Lys
(10mM each) minimal medium. Specific activity values represent the mean¡SD of three measurements for each growth
condition.
Induction of the amaAB promoter
From transcriptome analysis, amaAB of P. aeruginosa
(a) PAO1 was found to be inducible to a comparable level
(LysR) None 5 mM L-Lys 5 mM L-Arg None 5 mM L-Lys by D-Lys and L-Pip. This operon was also induced by
(nM) 0 8 32 0 8 32 0 8 32 0 8 32 0 8 32 L-Lysbuttoarelativelylowlevel.Tosubstantiatethisfind-
ing, the amaA::lacZ transcriptional fusion plasmid
C2
(pAMA1) was constructed and introduced into the WT
C1
strainPAO1toanalysetheexpressionprofilingofthispro-
moter.AsshowninFig.8,theamaApromoterwasinduced
F byL-Lys,D-LysandL-Pip,consistentwiththeDNAmicro-
array experiments (see Table 3).
DivergentfromtheamaABoperon,thePA1026genecodes
pLYS1 p4181 for a putative transcriptional elongation factor of the
(b) GreA/GreB family (http://www.pseudomonas.com). How-
pLYS2 pLYS3 pLYS4 pLYS5 ever, the knockout mutant of this gene grew normally on
(LysR)
(nM) 0 8 32 0 8 32 0 8 32 0 8 32 allcompoundstestedinthisstudy(Table2),anditdidnot
affecttheexpressionprofilingoftheamaApromoter(data
not shown). These results led us to exclude PA1026 as the
regulatorygeneofamaAB.InthecaseofP.putidaKT2440,
the PP5259 gene encoding a transcriptional regulator of
the LysR family was also divergently transcribed from the
amaAB operon in this bacterium (Revelles et al., 2005).
On the basis of sequence similarity, we found PA4914 of
PAO1 as the possible orthologue of PP5259. As shown in
Fig. 6. Demonstration of LysR–DNA interactions. (a) Promoter Fig.8,activationoftheamaABpromoterbyL-LysorD-Lys
was lost completely in the PA4914 knockout mutant
DNA as in pLYS1 or pPA4181 was used as the probe. Purified
(PW9274)suggestingthatPA4914isthetranscriptionalacti-
LysR–His(6) of the concentrations indicated was used with or
vatoroftheamaABoperon.WethereforedesignatedPA4914
without 5mM L-Lys or L-Arg in the reaction. (b) Promoter DNA
as in pLYS2, pLYS3, pLYS4 or pLYS5 was used as the probe. asamaR.ConsistentwithitsroleinactivationofamaAB,the
All reactions were carried out in the presence of 5mM L-Lys. amaRmutantexhibitedthesamepatternofgrowthdefects
F,freeprobe;C1andC2,nucleoproteincomplexes. onL-LysandL-PipastheamaAandamaBmutants(Table2).
884 Microbiology162
LysinecatabolicpathwaysinP.aeruginosa
(a) (b)
IysR IysX IysE SA (nmole OD min–1)
600 LysR
L-Glu L-Glu+L-Lys
pLYS1 (661 bp) IacZ 9 456 +
pLYS2 (473 bp) IacZ 39 572 +
pLYS3 (407 bp) IacZ 36 443 +
pLYS4 (371 bp) IacZ 56 31 –
pLYS5 (339 bp) IacZ 75 58 –
(c)
(d)
Fig. 7. Identification of LysR operator on the lysXE promoter region. (a) Schematic presentations of the divergent lysR-
lysXE operons and constructs of the lysX::lacZ fusions. Five plasmids of serial deletions in the regulatory regions were
labelledwithplasmidnames. (b)Measurements ofb-galactosidase activity fromfusionplasmids andsummary ofLysR bind-
ingactivitywiththelysXEregulatoryregioncarriedbytheseplasmids.BindingofLysRtotheregionisindicatedby‘+’and
no binding is indicated by ‘2’. (c) Nucleotide sequence of the lysXE regulatory region. Locations of 59 ends for pLYS3,
pLYS4 and pLYS5 and ATG initiation codons for gcdH and gcdR are indicated with arrowheads or arrows and labels. The
210 sequence for the putative promoter is also labelled. The proposed LysR operator site is in bold italicized letters with
double underlining. (d) Multiple sequence alignments for the putative LysR operators in the lysXE regulatory regions of
representative species of Pseudomonas in the following order: P. aeruginosa (PAO1), P. denitrificans (ATCC13867),
P. mendocina (NK01), P. fluorescens (Pf01), P. entomophila (L48), P. stutzeri (CCUG29243), P. fulva (12-X) and
P. putida (KT2440). The proposed LysR site was marked with divergent arrows. The ATG initiation codon (CAT on the
strand shown here) of lysR was marked with double lines for PAO1 and a single line for other species. The dashed line
denotes the ribosomal binding site. Also shown on the right side of this panel are the two types of gene organization for
lysineexporterandtranscriptionalregulatorinthesespeciesofPseudomonas.
Induction of the amaAB operon by L-Lys was 2-carboxylate to 2-aminoadipate. One missing link in the
abolished in the PA1252 mutant converged pathway is the enzyme converting D9-piper-
AlthoughL-LyscatabolisminP.aeruginosaPAO1ismainly idine-2-carboxylate to L-Pip. One potential candidate for
thisenzymewasencodedbyPA1252,basedonitssequence
through the decarboxylase pathway, L-Lys can also be
similarity to DpkA (PP3591) of P. putida KT2440 that has
channelled into the D-Lys pathway via the transaminase
pathway (Chou et al., 2010). As shown in Fig. 1, L-Lys been previously shown to catalyse this reaction in D-Lys
andD-Lyscatabolismmayconvergeata-keto-e-aminohex- and D-Pro catabolism (Muramatsu et al., 2005; Revelles
anoate(akaketo-lysine),whichthenspontaneouslycyclizes et al., 2005). From the results of transcriptome analysis
toformD9-piperidine-2-carboxylate.Thisconvergencewas (Table 3), the PA1252 gene was found not to be inducible
further supported by our findings that L-Lys is able to byL-Lys, D-Lys orL-Pip. However,activation oftheamaA
inducetheamaABoperon(Table3,Fig.8),whichencodes promoter by L-Lys and D-Lys was completely lost in the
enzymes for the subsequent degradation of D9-piperidine- PA1252 mutant (Fig. 8), supporting its role in the
http://mic.microbiologyresearch.org 885