Transcriptome Analysis of Pseudomonas putida KT2440 ... · Transcriptome Analysis of Pseudomonas putida KT2440 Harboring the ... mid-free, spontaneous restriction-deﬁcient derivative

JOURNAL OF BACTERIOLOGY, Oct. 2007, p. 6849–6860 Vol. 189, No. 190021-9193/07/$08.00�0 doi:10.1128/JB.00684-07Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transcriptome Analysis of Pseudomonas putida KT2440 Harboring theCompletely Sequenced IncP-7 Plasmid pCAR1�†

Masatoshi Miyakoshi,1‡ Masaki Shintani,1 Tsuguno Terabayashi,1 Satoshi Kai,1Hisakazu Yamane,1 and Hideaki Nojiri1,2*

Biotechnology Research Center1 and Professional Programme for Agricultural Bioinformatics,2

The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received 1 May 2007/Accepted 19 July 2007

The IncP-7 plasmid pCAR1 of Pseudomonas resinovorans CA10 confers the ability to degrade carbazole upontransfer to the recipient strain P. putida KT2440. We designed a customized whole-genome oligonucleotidemicroarray to study the coordinated expression of pCAR1 and the chromosome in the transconjugant strainKT2440(pCAR1). First, the transcriptome of KT2440(pCAR1) during growth with carbazole as the sole carbonsource was compared to that during growth with succinate. The carbazole catabolic car and ant operons wereinduced, along with the chromosomal cat and pca genes involved in the catechol branch of the �-ketoadipatepathway. Additionally, the regulatory gene antR encoding the AraC/XylS family transcriptional activatorspecific for car and ant operons was upregulated. The characterization of the antR promoter revealed that antRis transcribed from an RpoN-dependent promoter, suggesting that the successful expression of the carbazolecatabolic operons depends on whether the chromosome contains the specific RpoN-dependent activator. Next,to analyze whether the horizontal transfer of a plasmid alters the transcription network of its host chromo-some, we compared the chromosomal transcriptomes of KT2440(pCAR1) and KT2440 under the same growthconditions. Only subtle changes were caused by the transfer of pCAR1, except for the significant induction ofthe hypothetical gene PP3700, designated parI, which encodes a putative ParA-like ATPase with an N-terminalXre-type DNA-binding motif. Further transcriptional analyses showed that the parI promoter was positivelyregulated by ParI itself and the pCAR1-encoded protein ParA.

Many catabolic plasmids can be transferred horizontally be-tween different bacteria and play an important role in thedistribution of the ability to degrade and utilize recalcitrantchemical compounds (15, 90). Several catabolic plasmidswithin members of the genus Pseudomonas have been identi-fied and have been classified mostly into incompatibility groupsIncP-1, IncP-2, IncP-7, and IncP-9. Recently, the completegenome sequences of several IncP-1, IncP-7, and IncP-9 cata-bolic plasmids were determined (16, 28, 44, 45, 47, 76, 77, 83,86, 93). The 199,035-bp catabolic plasmid pCAR1 was origi-nally discovered in Pseudomonas resinovorans CA10, which isable to utilize carbazole as its sole source of carbon, nitrogen,and energy (53, 56), and was the first IncP-7 plasmid to becompletely sequenced (45).

pCAR1 carries the car and ant operons, which encode theupper and meta pathway enzymes and the anthranilate 1,2-dioxygenase, respectively (see Fig. 1A). The operons are tran-scribed from two identical anthranilate-inducible promoters,Pant, under the control of the AraC/XylS family activator AntR(85), and the constitutive promoter PcarAa also originates thetranscription of the car operon (50). Carbazole catabolism

begins with the upper pathway to yield anthranilate and 2-hy-droxypenta-2,4-dienoate, and then 2-hydroxypenta-2,4-dieno-ate is mineralized into pyruvate and acetyl coenzyme A (acetyl-CoA) by the meta pathway enzymes (53) whereas anthranilateis converted into catechol by the anthranilate 1,2-dioxygenase(see Fig. 1B). Catechol, one of the central intermediates of thearomatic catabolic pathway, is degraded into acetyl-CoA andsuccinyl-CoA via the chromosomally encoded �-ketoadipatepathway (36). Thus, the induction of pCAR1-borne catabolicoperons confers the ability to grow with carbazole and anthra-nilate upon the recipient strain.

pCAR1 is a self-transmissible plasmid that has been conju-gally transferred into P. putida KT2440 (75). KT2440 is a plas-mid-free, spontaneous restriction-deficient derivative of P.putida mt-2 (2, 64), which was originally isolated in Japan (51).Because of a defect in its normal system of restriction againstDNA uptake, KT2440 is thought to be an ideal host for ex-panding the range of growth substrates via the recruitment ofcatabolic plasmids. Because the complete genomic sequence ofP. putida KT2440 is known (52) and the DNA sequence of thecatabolic plasmid pCAR1 is also known (45), we were able todesign a customized high-density oligonucleotide microarrayfor P. putida KT2440 containing pCAR1 that allowed us toinvestigate the expression profile of the entire genome.

In this study, using our customized microarray, we analyzedthe range of expression of pCAR1-borne genes and the coor-dinated response of the host chromosome in cells of thetransconjugant strain KT2440(pCAR1) grown with carbazoleas the carbon source. For comparison, succinate was used as analternative carbon source since it is generally regarded as a

* Corresponding author. Mailing address: Biotechnology ResearchCenter, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: 81(3)5841-3064. Fax: 81(3)5841-8030. E-mail:[email protected].

† Supplemental material for this article may be found at http://jb.asm.org/.

‡ Present address: Department of Environmental Life Sciences,Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan.

� Published ahead of print on 3 August 2007.

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good carbon source for KT2440 and has been used in severalprevious transcriptome studies (87, 88) and proteomic studies(8, 38, 42, 69, 94). The operons required for carbazole catab-olism, both on pCAR1 and on the chromosome, were stronglyinduced, and the regulatory gene antR was also upregulatedduring growth with carbazole. Next, to examine whether thehorizontal transfer of a plasmid alters the transcription net-work of its host chromosome, the transcriptional changes inthe host chromosome of KT2440(pCAR1) relative to that of itsparental strain KT2440 under the same growth conditions werefurther analyzed. Only subtle changes were observed, exceptfor the chromosomal hypothetical gene PP3700, which wassignificantly induced by the carriage of pCAR1.

MATERIALS AND METHODS

Bacterial strains and growth conditions. Bacterial strains used in this study arelisted in Table 1. The Escherichia coli strains were grown at 37°C in Luria broth(68). The Pseudomonas strains were grown in Luria broth and in nitrogen-plusmineral medium 4 (NMM-4) with 1.0 mg of carbazole or succinate ml�1 as thesole carbon source (73). The following antibiotic was added to the medium:ampicillin (100 �g ml�1), kanamycin (50 �g ml�1), or gentamicin (15 �g ml�1).For plate cultures, the above-described media were solidified with 1.6% agar(wt/vol).

RNA extraction. To obtain initial optical densities at 600 nm (OD600) of 0.05,100 ml of NMM-4 supplemented with 1.0 mg of carbazole or sodium succinateml�1 was inoculated with cells of the Pseudomonas strains from an overnightculture in Luria broth. The cells were incubated at 30°C on a rotary shaker at 120rpm and were monitored by measuring the OD600 or the number of CFU permilliliter.

TABLE 1. Bacterial strains and plasmids used in this study

Bacterial strain or plasmid Relevant characteristicsa Source orreference

StrainsE. coli

DH5� F� �80dlacZ�M15 �(lacZYA-argF)U169 endA1 recA1 hsdR17(rK� mK

�) deoR thi-1 supE44� gyrA96 relA1

Toyobo

S17-1 �pir recA thi pro hsdR; RP4-2 integrated into the chromosome (kan::Tn7 ter::Mu) �pir 14

P. putidaKT2440 Wild-type strain 2KT2440(pCAR1) KT2440 harboring pCAR1 75KT2440�rpoN(pCAR1) rpoN::Gmr mutant of KT2440(pCAR1) This studyKT2440(pCAR1�antR) antR::Gmr mutant of KT2440(pCAR1) This study

P. resinovoransCA10 Wild-type strain 56CA10dm4 Derivative of CA10 from which pCAR1 was removed 74

PlasmidspBBR1MCS-2 Kmr lacZ� mob 41pBRKantR pBBR1MCS-2 with SalI-BamHI fragment containing antR This studypBRKrpoN pBBR1MCS-2 with SalI-BamHI fragment containing rpoN This studypBRKparI pBBR1MCS-2 with SalI-BamHI fragment containing parI This studypBRKparA pBBR1MCS-2 with HindIIII-BglII fragment containing parA This studypBRKparB pBBR1MCS-2 with BglII-SpeI fragment containing parB This studypBRKpmr pBBR1MCS-2 with KpnI-XbaI fragment containing pmr This studypK18mobsacB Kmr lacZ� sacB 71pK19mobsacB Kmr lacZ� sacB 71pKrpoN::Gmr pK18mobsacB with EcoRI-HindIII fragment of pUCrpoN::Gmr This studypKantR::Gmr pK19mobsacB with SalI-BamHI fragment of pTORF23::Gmr This studypMEGluc Gmr; promoterless luc�NF 50pMCantA253 pMEGluc containing the region from �200 to �53 relative to antA transcription start point This studypMEGparI-200 pMEGluc containing the region from �200 to �20 relative to parI transcription start point This studypMEGparI-100 pMEGluc containing the region from �100 to �20 relative to parI transcription start point This studypMEGparI-50 pMEGluc containing the region from �50 to �20 relative to parI transcription start point This studypMEGparI-50C39T pMEGparI-50 with C-to-T mutation at �39 This studypMEGparI-50C38T pMEGparI-50 with C-to-T mutation at �38 This studypMEGparI-40 pMEGluc containing the region from �40 to �20 relative to parI transcription start point This studypSJ12 pBluescript II KS(�) with 0.7-kb SmaI fragment containing a nonpolar Gmr cassette 35pT7Blue T-vector Apr lacZa NovagenpTrpoN pT7Blue with 1.5-kb PCR fragment containing rpoN This studypTORF23 pT7Blue with 1.0-kb PCR fragment containing antR This studypTORF23::Gmr pTORF23 with 0.7-kb SmaI fragment containing Gmr cassette inserted into EcoRV site

within antRThis study

pT7PP3700int pT7Blue with PCR fragment containing PP3701-PP3700 intergenic region This studypUC19 Apr lacZa 92pUCA741 pUC19 with 8.9-kb SalI insert of pCAR1 DNA 53pUCrpoN pUC19 with EcoRI-HindIII fragment containing rpoN This studypUCrpoN::Gmr pUCrpoN with 0.7-kb SmaI fragment containing Gmr cassette inserted into EcoRV-HincII

site within rpoNThis study

a Apr, Gmr, and Kmr indicate resistance to ampicillin, gentamicin, and kanamycin, respectively.

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A total of 109 cells at early exponential phase were mixed with RNA ProtectBacteria reagent as recommended by the manufacturer (QIAGEN, Valencia,CA). Total RNA was extracted using the RNeasy midi kit (QIAGEN). Theeluted RNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI)at 37°C for 1 h. After the inactivation of the DNase at 65°C for 15 min with thestop reagent supplied by the manufacturer, RNA samples were again purifiedwith the RNeasy mini column according to the cleanup protocol of the manu-facturer (QIAGEN).

Microarray analysis. The genome sequences and open reading frame (ORF)predictions for P. putida KT2440 and pCAR1 were obtained from GenBankaccession numbers NC_002947 and AB088420, respectively. The 6,181,863-bpchromosome of KT2440 contains 5,350 predicted ORFs and 21 rRNAs, and the199,035-bp pCAR1 contains 190 ORFs. A customized high-density oligonucle-otide array (NimbleGen Systems Inc., Madison, WI) designed for the KT2440and pCAR1 genomes contained 178,230 different 24-mer probes paired withperfect-match (PM) oligonucleotides and their corresponding mismatch oligo-nucleotides with substitutions by transversion at positions 6 and 12 from the 5end. Fifteen adequate probe pairs were selected for each ORF, and probe pairsassigned to pCAR1 were produced in triplicate.

The cDNA synthesis, hybridization, and scanning were performed by Nimble-Gen Systems Inc. Microarray data analysis was performed using the robustmultiarray average method (34) based on the log2 values of the absolute signalintensities for PM probes only. The hybridization signals assigned to pCAR1could not be compared because of the lack of pCAR1 in plasmid-free KT2440but were used for normalization together with those assigned to the KT2440chromosome. Student’s t test for each PM probe and each biological replicate,followed by the Bonferroni correction, was used to identify genes showing dif-ferential expression patterns (P 0.05). The data presented are the averages ofresults from two independent experiments.

Quantitative reverse transcription-PCR (qRT-PCR). Reverse transcriptionwas performed with 30-�l reaction mixtures containing 6 �g of total RNA, 375ng of random primers (Invitrogen, Carlsbad, CA), 750 U of SuperScript II(Invitrogen), 30 U of RNase OUT (Invitrogen), 1� First Strand buffer (Invitro-gen), 10 mM dithiothreitol, and 0.5 mM deoxynucleoside triphosphates (ToyoboCo. Ltd., Tokyo, Japan). After the RNA and random primers had been dena-tured at 70°C for 10 min and annealed at 25°C for 10 min, the remaining reagentswere added and the mixture was incubated at 25°C for 10 min, 37°C for 60 min,and 42°C for 60 min and held at 70°C for 10 min to inactivate the enzymes. Todegrade the RNA, 10 �l of 1 N NaOH was added, and the reaction mixture washeated at 65°C for 30 min; the mixture was neutralized with 10 �l of 1 N HCl.

The cDNA quantification was performed using the ABI 7300 real-time PCRsystem (Applied Biosystems, Foster City, CA). The primers used for qRT-PCRwere designed using the Primer3 program (67). Detailed information on theprimer sequences used in this study is available upon request. All of the productswere between 100 and 150 bp in length. The univ16S-F and univ16S-R primer setused to measure the transcription of 16S rRNA was designed based on the 16SrRNA sequences from P. putida KT2440 (GenBank accession no. NC_002947)and P. resinovorans CA10 (GenBank accession no. AB047273). Each 20-�l re-action mixture contained 10 �l of Power SYBR green PCR master mix (AppliedBiosystems), 200 nM concentrations of each specific primer, and the cDNA. Thereaction conditions were as follows: 95°C for 10 min for enzyme activation and40 cycles of 95°C for 5 s, 60°C for 20 s, and 72°C for 30 s. A melting-curve analysiswas performed to verify the amplification specificity. To quantify the transcrip-tion of each gene, the copy number was determined by generating a standardcurve using a series of 10-fold dilutions (from 100 pM to 1 fM) of the target PCRproduct inserted into the pT7Blue T-vector (Novagen, Madison, WI). For sam-ple normalization, 16S rRNA was used as an internal standard. All of thereactions were performed at least in triplicate, and the data were normalizedusing the average for the internal standard.

Primer extension. An IRD800-labeled primer, ANTR-R or PP3700-10R(Aloka, Ltd., Tokyo, Japan), which anneals to the coding region from �57 to�76 of antR or �353 to �372 of parI relative to the annotated translation startpoint, was used. Primer extension was performed with 20 �l of 1� first-strandbuffer containing 10 �g of total RNA, 2 pmol of the IRD800-labeled primer, 200U of SuperScript III (Invitrogen), 80 U of RNase OUT (Invitrogen), 10 mMdithiothreitol, and 0.5 mM deoxynucleoside triphosphates (Toyobo Co. Ltd.).After the denaturation of the RNA and the IRD800-labeled primer at 65°C for5 min, the remaining reagents were added and the mixture was incubated at 50°Cfor 30 min. The extended product was purified by phenol-chloroform extractionand ethanol precipitation and then dissolved in 2 �l of H2O and 1 �l of IR2 stopsolution (LI-COR Inc., Lincoln, NE). The solution was then denatured at 95°Cfor 2 min and subjected to electrophoresis using a LI-COR model 4200L-2auto-DNA sequencer. A sequence ladder was obtained using the same primer

and the template plasmid pUCA741 (53) or pT7PP3700int, containing the in-tergenic region from PP3701 to PP3700 amplified from the total DNA of KT2440by PCR using the primer pair PP3700int-F and PP3700int-R.

Reporter assay. To obtain the Pant transcriptional fusion, the 253-bp BamHI-HindIII fragment of pBRCantA253 (85), which contains the region from �200to �53 relative to the transcription initiation site of antA, was inserted into thereporter vector pMEGluc (50) to yield pMCantA253.

Transcriptional fusions of the parI promoter were prepared by amplifyingappropriate DNA fragments upstream of parI from pT7PP3700int by usingadequately designed primer sets. The EcoRI restriction site was introduced intothe forward primers PP3700F-200, PP3700F-100, PP3700F-50, and PP3700F-40,and the NcoI restriction site CCATGG was introduced into the reverse primerPP3700R�18 to match its CAT nucleotides to the start codon of parI. Forwardprimers PP3700F-50C39T and PP3700F-50C38T were designed to introduceartificial mutations at �39 and �38 relative to the transcription start point,respectively. The amplified fragments were sequenced to confirm the nucleotidesequences and then inserted into the reporter vector pMEGluc (50), which wasdigested with EcoRI and NcoI restriction enzymes, to yield the pMEGparI series(Table 1).

To overexpress the candidate activators for the parI promoter, the codingregion of parI (PP3700), parA, parB, or pmr (orf70) was amplified from the totalDNA of KT2440(pCAR1) by using the primer pair PP3700-F and PP3700-R,ParApCAR1-F and ParApCAR1-R, ParBpCAR1-F and ParBpCAR1-R, or70exp-F and 70exp-R, respectively. Each amplified fragment was sequenced toconfirm the nucleotide sequence and then inserted into the broad-host-rangevector pBBR1MCS-2 (41), which was digested with the appropriate restrictionenzymes to yield pBRKparI, pBRKparA, pBRKparB, and pBRKpmr (Table 1).

Electrocompetent cells of Pseudomonas strains were prepared as described byChoi et al. (12). Electrotransformation was performed using Gene Pulser II(Bio-Rad, Hercules, CA) as described previously (85). Overnight cultures of theresultant transformants were inoculated into 5 ml of Luria broth or NMM-4supplemented with 1.0 mg of succinate ml�1 with or without 0.2 mg of sodiumanthranilate ml�1 to yield an initial OD600 of 0.05, and the cells were grown tothe mid-exponential phase. In a 96-well microtiter plate, 50 �l of the culture andan equal volume of Picagene LT2.0 (Toyo Ink Co. Ltd., Tokyo, Japan) weremixed and shaken for 20 s, and the relative light units (RLU) were measured for10 s by using Centro LB960 (Berthold Technologies GmbH & Co. KG, BadWildbad, Germany). Each sample was assayed independently at least threetimes.

Disruption and complementation of the rpoN and antR genes. The rpoN andantR genes were amplified from the genomic DNA of KT2440(pCAR1) using theprimers rpoNF-EcoRI with rpoNR-HindIII and ORF23-F with ORF23-R andinserted into the pT7Blue T-vector to yield pTrpoN and pTORF23, respectively.The EcoRI-HindIII fragment of pTrpoN was subcloned into pUC19 to yieldpUCrpoN. To disrupt the rpoN and antR genes, a SmaI fragment containing thenonpolar Gmr cassette of pSJ12 (35) was inserted into the EcoRV-HincII sites ofrpoN in pUCrpoN and the EcoRV site of antR in pTORF23 in the samedirection; the resulting plasmids were designated pUCrpoN::Gmr andpTORF23::Gmr, respectively. Subsequently, the EcoRI-HindIII fragment ofpUCrpoN::Gmr or the SalI-BamHI fragment of pTORF23::Gmr was insertedinto pK18mobsacB and pK19mobsacB (71) to obtain pKrpoN::Gmr andpKantR::Gmr, respectively. As described previously (59), pKrpoN::Gmr orpKantR::Gmr was introduced into KT2440(pCAR1) by filter mating with E. coliS17-1 �pir transformants, and the double-crossover recombinants were screened.To complement rpoN and antR, the SalI-BamHI fragments of pTrpoN andpTORF23 were inserted into the broad-host-range vector pBBR1MCS-2 (41) toobtain pBRKrpoN and pBRKantR, respectively, and then these plasmids wereintroduced into the P. putida strains by electroporation (85).

Transcriptome accession number. The data discussed in this publication havebeen deposited in the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through Gene Expression Omnibus series ac-cession number GSE7650.

RESULTS

The KT2440(pCAR1) transcriptome during growth withcarbazole. P. putida KT2440 cannot utilize anthranilate as itssole source of carbon and energy (36); however, the conjuga-tive transfer of pCAR1 from P. resinovorans CA10 enabledKT2440 to utilize either carbazole or anthranilate as its solesource of carbon and nitrogen. In this study, we first analyzed

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the transcriptome of KT2440(pCAR1) during growth with car-bazole or succinate. The rate of KT2440(pCAR1) growth withcarbazole was significantly lower than that of growth with suc-cinate (data not shown).

Microarray analysis showed that the levels of transcription of38 genes assigned to pCAR1 differed more than twofold (P 0.05) during growth with carbazole (Table 2). As expected,among the 30 upregulated genes, the 17 structural genes con-

stituting the pCAR1-borne car and ant operons (Fig. 1A),which encode the upper and meta carbazole pathway enzymesand the anthranilate 1,2-dioxygenase, respectively, were themost significantly induced during the growth of KT2440(pCAR1) with carbazole. Microarray analysis showed that orf9and antA, both of which are transcribed from Pant (85), wereinduced by 39- and 43-fold, respectively. These values werecalculated from 10 PM probes that were specific for each geneand 5 PM probes common to orf9 and antA, the 5 regions ofwhich are identical because of the transposition of ISPre1 (53).In addition to the structural genes, the regulatory gene antRwas upregulated by 4.1-fold during growth with carbazole.Among the rest of the upregulated genes in KT2440(pCAR1),the transposase gene tnpAc of the 72.8-kb class II carbazolecatabolic transposon Tn4676 (74) was induced by 2.9-fold dur-ing growth with carbazole (Table 2). This finding suggests thatthe transposition of Tn4676 is enhanced during growth withcarbazole. All eight downregulated genes (Table 2) corre-sponded to a gene cluster from orf100 to orf108 and wereoriented in the same direction, although orf103 was signifi-cantly downregulated by only 1.8-fold (P � 7.0E�07) as cal-culated by the microarray analysis.

A total of 139 chromosomal genes showed significantchanges of more than twofold (P 0.05); 63 were upregulatedand 76 were downregulated during growth with carbazole (seeTables S2 and S3 in the supplemental material). The majorityof the upregulated chromosomal genes were hypothetical (seeTable S2 in the supplemental material). However, we detectedsignificant upregulation of the algT, rpoH, and rpoS genes,which encode alternative sigma factors (48). Similarly, themucA and algN genes, which encode anti-sigma proteins forAlgT and are located downstream of algT, were also upregu-lated. Conversely, a large number of genes associated withprotein synthesis and energy metabolism were downregulatedduring growth with carbazole (see Table S3 in the supplemen-tal material). This finding may be attributable to the decreasedrate of KT2440(pCAR1) growth with carbazole. In E. coli,ribosomal protein operons are regulated by the growth rate (9,29, 37). Interestingly, the downregulated cyoABCD genes en-code the cytochrome o ubiquinol oxidase, which is involved incatabolite repression in addition to its role in the electrontransfer chain (17, 58). Therefore, it is possible that cataboliterepression is relieved during growth with carbazole.

Catechol is produced from anthranilate by the pCAR1-en-coded anthranilate 1,2-dioxygenase, and the chromosomallyencoded �-ketoadipate pathway enzymes catalyze further cat-echol degradation (Fig. 1B). We found that the catC and catAgenes of the KT2440 chromosome were significantly inducedduring growth with carbazole (see Table S2 in the supplemen-tal material). Although catB was excluded by the criteria of themicroarray analysis because of its relatively high P value afterthe Bonferroni correction (P � 0.07), qRT-PCR analysisshowed that growth with carbazole induced the transcription ofcatB by 160-fold (Table 3). This result is consistent with thefindings of a previous study of P. putida PRS2000, which re-vealed that the catBCA genes are activated by the LysR familytranscriptional regulator CatR in response to cis,cis-muconate(30), and those of a study of the original host strain P. resino-vorans CA10 (54). Additionally, genomic analysis of theKT2440 chromosome has revealed that another catA homo-

TABLE 2. Change in expression of pCAR1 genes during growthwith carbazole

Genea Change(n-fold)b P valuec Product or functiona

antC 18.1 4.5E�24 Reductase of anthranilate1,2-dioxygenase

antB 31.8 1.6E�14 Small subunit of oxygenase ofanthranilate 1,2-dioxygenase

antA 43.1 3.3E�23 Large subunit of oxygenase ofanthranilate 1,2-dioxygenase

antR 4.1 5.3E�14 Transcriptional regulatororf9 39.3 2.2E�23 Fusion gene productcarAa 43.5 9.7E�29 Oxygenase of carbazole

1,9a-dioxygenasecarAa 38.0 3.3E�28 Oxygenase of carbazole

1,9a-dioxygenasecarBa 47.9 2.9E�32 Subunit of meta cleavage enzymecarBb 22.9 1.9E�26 Subunit of meta cleavage enzymecarC 41.9 5.5E�33 meta cleavage compound hydrolasecarAc 37.4 6.7E�34 Ferredoxin of carbazole

1,9a-dioxygenaseorf7 30.4 4.5E�30 Hypothetical proteincarAd 26.8 1.6E�20 Ferredoxin reductase of carbazole

1,9a-dioxygenasecarD 19.3 1.6E�33 2-Hydroxypenta-2,4-dienoate

hydrataseorf33 9.0 2.1E�17 Hypothetical proteinorf34 13.1 3.5E�21 Hypothetical proteincarF 17.4 1.9E�22 Acetaldehyde dehydrogenasecarE 10.0 1.3E�25 4-Hydroxy-2-oxovalerate aldolaseorf40 3.3 1.7E�06 Hypothetical proteintnpRa 3.3 1.8E�11 Resolvaseorf150 2.1 3.4E�05 Hypothetical proteinorf159 4.4 7.6E�14 Hypothetical proteinorf161 2.4 5.9E�09 Hypothetical proteinorf162 2.4 1.0E�09 Hypothetical proteinorf165 2.3 4.8E�09 Hypothetical proteinorf167 2.0 1.3E�06 Probable helicaseorf170 2.5 2.3E�05 Methyl-accepting chemotaxis

proteinorf173 2.2 1.7E�06 Hypothetical proteinorf174 2.4 2.1E�09 Hypothetical proteintnpAc 2.9 6.0E�09 Transposaseorf100 �3.0 7.6E�16 Hypothetical proteinorf101 �4.1 3.9E�17 Putative cobalamin biosynthesis

proteinorf102 �2.6 6.8E�14 Putative cobalamin biosynthesis

proteinorf104 �3.9 1.1E�19 Hypothetical proteinorf105 �2.2 2.4E�06 Hypothetical proteinorf106 �4.0 3.8E�16 Hypothetical proteinorf107 �2.6 1.2E�10 Hypothetical proteinorf108 �3.7 9.3E�09 Hypothetical protein

a From the complete nucleotide sequence of pCAR1, in which the carAa geneis duplicated (45).

b The values indicate the mean levels of upregulation (positive values) anddownregulation (negative values) of gene expression during growth with carba-zole compared to that during growth with succinate.

c P values after the Bonferroni correction.



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logue known as catA2 (PP3166) is located within the ben genecluster (36). qRT-PCR analysis indicated a low level of catA2transcription even during growth with carbazole, althoughcatA2 was induced 5.9-fold (Table 3). This finding suggests thatCatA2 is not involved in catechol oxygenation, at least duringcarbazole utilization, but it is still possible that catA2 transcrip-tion is induced along with that of the ben genes under thecontrol of BenR in response to benzoate (13). The enzymesPcaD, PcaF, and PcaIJ, which are encoded by discrete geneclusters pcaRKFTBDCP and pcaIJ, constitute the downstream

portion of the �-ketoadipate pathway (36). In P. putidaPRS2000, the IclR family transcriptional regulator PcaR isrequired for the induction of pcaK, pcaF, pcaBCD, and pcaIJ inresponse to �-ketoadipate (30). Although our microarray anal-ysis showed only a 3.3-fold induction of pcaF during growthwith carbazole because of our strict criteria (see Table S2 inthe supplemental material), qRT-PCR analysis verified thatpcaF and pcaIJ were induced during growth with carbazole,although the induction of pcaD was relatively weak (Table3). This result indicates that the chromosomal �-ketoadi-pate pathway is integrated into the overall carbazole cata-bolic pathway.

Transcriptional regulation of the pCAR1-borne regulatorygene antR. It has been suggested previously that an additionalantR homologue is located on the CA10 chromosome (85),similar to the homologues found near the chromosomalantABC genes on several Pseudomonas chromosomes, includ-ing antR of P. fluorescens MB214 (65) and orfAN of P. putidaP111 (GenBank accession no. AY026914), and also those oncompletely sequenced chromosomes, such as PFL_0759 of P.fluorescens Pf-5, PA2511 of P. aeruginosa PAO1, andPA14_32160 of P. aeruginosa PA14 (http://www.pseudomonas.com/). In contrast, KT2440 cannot utilize anthranilate as itssole carbon source, and its chromosome lacks antR andantABC. However, there have been several reports of cross-regulation among the AraC/XylS family members, such as thechromosome-encoded BenR and the pWW0-encoded XylS

FIG. 1. (A) Genetic organization of the car and ant gene clusters on pCAR1. Black, gray, and white pentagons represent regulatory genes,structural genes, and unknown ORFs, respectively. The ORF numbers are indicated below. The gray box at the 5 region of orf9 represents thetransposed portion of antA (53). Arrows with solid and dotted lines indicate the induced and constitutive transcripts originating from Pant andPcarAa, respectively (50, 85). (B) Carbazole catabolic pathway in P. putida KT2440 harboring pCAR1. The enzymes constituting the upper and metapathways shown in the rounded box are encoded on pCAR1 (53), whereas the �-ketoadipate pathway enzymes are encoded on the KT2440chromosome (36). CarAaAcAd, carbazole 1,9a-dioxygenase; CarBaBb, 2-aminobiphenyl-2,3-diol 1,2-dioxygenase; CarC, 2-hydroxy-6-oxo-6-(2-aminophenyl)-hexa-2,4-dienoic acid hydrolase; CarD, 2-hydroxypenta-2,4-dienoate hydratase; CarE, 4-hydroxy-2-oxovalerate aldolase; CarF,acetaldehyde dehydrogenase (acylating); AntABC, anthranilate 1,2-dioxygenase; CatA, catechol 1,2-dioxygenase; CatB, cis,cis-muconate-lacton-izing enzyme; CatC, muconolactone isomerase; PcaD, �-ketoadipate enol-lactone hydrolase; PcaIJ, �-ketoadipate succinyl-CoA transferase; PcaF,�-ketoadipyl-CoA thiolase.

TABLE 3. Quantitative RT-PCR analysis of �-ketoadipatepathway genes

Gene

Relative quantity of mRNA duringgrowth witha: Induction

(n-fold)Succinate Carbazole

catB (PP3715) 0.0015 0.0001 0.24 0.02 160catA2 (PP3166) 0.00021 0.00002 0.0013 0.0001 5.9pcaF (PP1377) 0.00086 0.00011 0.036 0.005 41pcaD (PP1380) 0.0015 0.0001 0.014 0.001 8.7pcaI (PP3951) 0.0024 0.0004 0.10 0.01 42pcaJ (PP3952) 0.0024 0.0003 0.042 0.004 18

a cDNA was synthesized from total RNA of P. putida KT2440(pCAR1) duringgrowth with succinate or carbazole by a standard method for microarray analysis.The mRNA level for each gene was divided by the 16S rRNA level. Values areaverages standard deviations of results from at least five independent exper-iments.



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(13, 18), because of the conserved binding sequence of thegenes (5-TGCA-N6-GGNTA-3) (26), and a total of 40 regu-latory genes belonging to the AraC/XylS family are present onthe KT2440 chromosome (19). Reporter analysis using a Pant

transcriptional fusion showed that the promoter activity inKT2440(pBBR1MCS-2)(pMCantA253) was not significantlychanged by supplementation with anthranilate (181 25RLU/OD600 unit without anthranilate and 243 68 RLU/OD600 unit with anthranilate) but that the promoter activityin KT2440(pBRKantR)(pMCantA253), which expresses theAntR protein, was significantly increased (517 122 RLU/OD600 unit without anthranilate and 272,500 38,000 RLU/OD600 unit with anthranilate). Therefore, it was concludedthat AntR is the only pathway-specific activator of Pant inKT2440(pCAR1) in response to anthranilate.

Microarray analysis showed that the regulatory gene antRwas upregulated during growth with carbazole. Because thetranscriptional regulation of antR has not yet been investigated,primer extension analysis was performed to map the antR tran-scription start site. A single product was detected 55 nucleotides(nt) upstream of the translation start site of antR, and the signalintensity of the antR transcript was significantly higher in carba-zole-grown KT2440(pCAR1) cells than in succinate-grown cells(Fig. 2A). As well, the same transcription start site of antR wasdetected in P. resinovorans CA10, the original host strain ofpCAR1 (Fig. 2A). Upstream of the transcription start site ofantR, at positions �24 and �12, we found conserved GG and GCdoublets (Fig. 2B), which are characteristic binding motifs for thesigma factor RpoN. The sequence flanking the core promoterwas similar to the extended consensus promoter sequence 5-YTGGCACGNNNNTTGCW-3 (where Y is T or C and W isA or T) (5), and the GenomeMatScan program (11) assignedit a score of 14.56.

To confirm that the antR promoter depends on RpoN RNApolymerase, the rpoN gene in KT2440 harboring pCAR1 wasdisrupted. The rpoN-disrupted strain KT2440�rpoN(pCAR1)could not grow with anthranilate and carbazole as its sources ofcarbon and energy, consistent with the findings in a previousreport (39), its growth rate in Luria broth was lower than thatof the wild-type strain KT2440(pCAR1). The antR-disrupted

strain KT2440(pCAR1�antR) was also unable to grow withanthranilate and carbazole as carbon and energy sources, butits growth rate in Luria broth was identical to that of thewild-type strain. The expression of rpoN in trans allowedKT2440�rpoN(pCAR1) alone to utilize anthranilate, whereasthe expression of antR in trans complemented bothKT2440(pCAR1�antR) and KT2440�rpoN(pCAR1). Theseresults indicate that antR transcription is RpoN dependent.

Chromosomal genes affected by the carriage of pCAR1.Next, to analyze the changes in the expression of the KT2440chromosome in response to the carriage of pCAR1, the tran-scriptome of KT2440(pCAR1) was compared with that of theisogenic plasmid-free strain KT2440 growing under the sameconditions. The growth curves for growth with succinate weresimilar, but a lower growth rate of KT2440(pCAR1) wasobserved (Fig. 3); the doubling times of KT2440 andKT2440(pCAR1) at early exponential phase were estimated tobe 43 and 51 min, respectively. Microarray analysis showed

FIG. 2. Identification of the antR promoter. (A) Primer extension was performed using equal amounts of total RNA from KT2440(pCAR1)cells grown with succinate (lane 1) and carbazole (lane 2) and from CA10 cells grown with succinate (lane 3) and carbazole (lane 4). Lanes G, A,T, and C correspond to the sequence ladder obtained using the same primer. (B) Nucleotide sequence of the antR promoter region. Thetranscription start site (�1) is indicated by an arrow, and the �12 and �24 duplexes of the antR promoter are underlined. The extended consensuspromoter sequence (5) is aligned below (Y is T or C and W is A or T), and asterisks indicate identical nucleotides. The start codon of antR is boxed.

FIG. 3. Growth curves of P. putida KT2440 and KT2440(pCAR1)in NMM-4 with 1.0 mg of sodium succinate ml�1. Samples for mi-croarray analysis were collected at an OD600 of �0.2.



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that, compared to the genes in KT2440, only 10 chromosomalgenes in KT2440(pCAR1) were differentially expressed at leasttwofold (P 0.05) (Table 4). Unexpectedly, a hypotheticalgene, PP3700, showed an exceptional 41.8-fold induction in thepresence of pCAR1.

A conserved-domain search of the NCBI conserved-domaindatabase (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml/)showed that the protein encoded by PP3700 had modular or-ganization. At its N terminus, the translational start point ofwhich was different from that annotated in the databases (seebelow), an HTH DNA-binding motif of the Xre family oftranscriptional regulators was conserved (Fig. 4A). The Xrefamily is a large family of proteins with an HTH motif similar

to that of the � repressor and the Cro protein of � bacterio-phage (62, 66, 70) and is also represented by the Bacillussubtilis prophage PBSX repressor Xre (91), the B. subtilis de-velopment regulator SinR (21), and the restriction-modifica-tion system control proteins such as C.PvuII and C.AhdI (49,78). As well as the HTH motif, the PP3700 protein possessedthe Walker-type ATPase motif, which is characteristic of theParA family ATPases involved in the active partitioning oflow-copy-number plasmids and the segregation of chromo-somes upon cell division (23), composed of the A box kGGxxK(ST), the A box gx[rk]uuuudxDp, and the B box duuUuD(Fig. 4B), where x represents any amino acid, u represents abulky hydrophobic amino acid, lowercase letters indicate atleast 80% conservation, and uppercase letters indicate 100%conservation (40). The KT2440 chromosome carries a total offive parA homologues, which were categorized into clusters ofthe orthologous COG1192 group (http://v2.pseudomonas.com/), consisting of parA in the vicinity of oriC (55) and fourorphan parA homologues that lack the cognate parB genes.The Walker motifs were highly conserved among the fiveCOG1192 proteins (Fig. 4B). However, completely sequencedPseudomonas chromosomes commonly carry at least threeCOG1192 genes in a conserved locus (http://v2.pseudomonas.com/), and PP3700 is specifically found in the KT2440 chro-mosome. Because PP3700 was identified as an inducible or-phan parA homologue, PP3700 is hereafter designated parI(inducible parA homologue).

Activation of the promoter of the chromosomal parI gene inthe presence of pCAR1. Primer extension was performed toidentify the transcription start point of parI. A single signal wasdetected from the total RNA of KT2440(pCAR1) but not inthe absence of pCAR1 (Fig. 5A). The extension product waspositioned at a guanine base 104 nt downstream of the anno-

TABLE 4. Differential expression of P. putida KT2440chromosomal genes affected by pCAR1

ORFa Genenamea

Change(n-fold)b P valuec Description of producta

PP0457 rplB 2.1 5.3E�03 Ribosomal protein L2PP0789 ampD 2.5 9.6E�03 N-acetyl-anhydromuramyl-

L-alanine amidase AmpDPP1149 5.4 3.7E�07 Hypothetical proteinPP2161 3.0 4.2E�05 Hypothetical proteinPP3700 parI 41.8 7.1E�05 Hypothetical proteinPP4870 5.0 2.1E�02 AzurinPP1560 �2.0 3.5E�02 Hypothetical proteinPP3921 �3.2 5.0E�03 Hypothetical proteinPP3991 �5.0 2.1E�02 Hypothetical proteinPP4117 �3.6 2.4E�02 Hypothetical protein

a From the annotated genome (52) as indicated by The Institute for GenomicResearch (http://www.tigr.org).

b The values indicate the mean levels of upregulation (positive values) anddownregulation (negative values) of gene expression in KT2440(pCAR1) com-pared to that in KT2440.

c P values after the Bonferroni correction.

FIG. 4. Multiple-sequence alignment of the N-terminal HTH motif (A) and the Walker-type ATPase motif (B) of the PP3700 protein (ParI).The sequences used are as follows: CI, NCBI GenPept accession no. AAA72530; Cro, GenPept accession no. AAA32245; C.AhdI, GenPeptaccession no. AAP78483; C.PvuII, GenPept accession no. AAA96335; SinR, GenPept accession no. BAA12542; Xre, GenPept accession no.CAA84042; PP0002 protein, GenPept accession no. AAN65636; PP2412 protein, GenPept accession no. AAN68024; PP4334 protein, GenPeptaccession no. AAN69913; PP5070 protein, GenPept accession no. AAN70635; and PP3700 protein, GenPept accession no. AAN69297. TheN-terminal 40 amino acids of the PP3700 protein are truncated according to the result of primer extension analysis (Fig. 5B). Asterisks, colons,and periods indicate the conserved, strongly similar, and weakly similar residues, respectively. Bars above the alignments represent the HTH motif(A) and the Walker A, A, and B motifs. Residue numbers of terminal amino acids are indicated on both sides. Numbers of interval amino acidsbetween Walker A and B motifs are indicated within the sequences.



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tated translation start point of PP3700, however (Fig. 5B).Accordingly, such an annotated translation start point is un-likely, and the ATG trinucleotide located 123 nt downstreamof the annotated translation start point is preferable for thestart codon; this codon is preceded by an AG-rich ribosomalbinding site at a 5-nt distance (Fig. 5B). Putative �35 and �10hexamers, 5-TGTTTT-N17-TACGCT-3, were found up-stream of the transcription start point.

To characterize the promoter of parI (parIp), reporter anal-ysis was performed using a series of deletions in the regionupstream of parI. Luciferase expression from the 220-bp pro-moter region from �200 to �20 relative to the transcriptionstart point was significantly increased in KT2440(pCAR1) cellsbut was negligible in KT2440 cells (Fig. 5C). Furthermore,deletion of the promoter region to �40 did not change thebasal promoter activity but significantly reduced the inductionrate in the presence of pCAR1. A 13-bp palindromic sequence,5-GGCACTCTGTGCC-3, from �50 to �38 relative to thetranscription start point was found (Fig. 5B). Even in thepresence of pCAR1, each replacement of cytosines by thy-mines at �39 and �38 reduced the activity of the 70-bp pro-moter (from �50 to �20) by 25% 8% and 40% 12%,respectively, although the basal promoter activity remained atthe same level as that in the absence of pCAR1. These resultssuggest that the region up to at least �50 is essential for theactivation of parIp in response to the carriage of pCAR1 andthat the palindromic sequence in cis plays an important role inthe activation of parIp.

Transcriptional regulation of parI. Because proteins of theXre family appear to bind to short palindromic sequences (3, 4,

7, 24, 57, 60), it is possible that ParI binds the 13-bp palin-dromic sequence just upstream of parIp to mediate its owntranscription. To investigate whether ParI autoregulates itstranscription, luciferase expression from the 220-bp region ofparIp was measured in KT2440 cells harboring pBRKparI so asto express ParI under the control of the tac promoter in thepBBR1MCS-2 vector. Compared to that in the negative control,KT2440(pBBR1MCS-2), luciferase expression in KT2440(pBRKparI) increased by 26-fold (Fig. 6A), indicating thatParI positively regulates its own transcription.

Because the truncation of the possible cis-acting regulatorysite resulted in the remarkable repression of parIp activity evenin the presence of pCAR1 rather than causing activation (Fig.5C), parIp was thought to require an activator encoded onpCAR1. The first candidate for this activator was the pCAR1-encoded MvaT family transcriptional regulator (79), desig-nated Pmr (plasmid-encoded MvaT-like regulator), becausethe disruption of pmr in KT2440(pCAR1) resulted in the sig-nificant downregulation of parI (T. Terabayashi, M. Miyakoshi,and H. Nojiri, unpublished data). However, parIp activity wasonly slightly repressed in KT2440 harboring pBRKpmr, whichoverexpressed the Pmr protein (Fig. 6A). This result suggeststhat Pmr indirectly upregulates the transcription of parI. Thenext candidates for the activator of parIp were pCAR1-en-coded ParA (ParApCAR1) and ParBpCAR1, whose genes werealso significantly downregulated in the pmr-deficient mutant(Terabayashi et al., unpublished). When ParApCAR1 was ex-pressed in KT2440 by pBRKparA, luciferase expression fromthe promoter increased by 90-fold compared to that in thenegative control (Fig. 6A). In contrast, the expression of

FIG. 5. Identification of the parI promoter. (A) Mapping of the parI transcription start point. Primer extension was preformed using equalamounts of total RNA from KT2440 (lane 1) and KT2440(pCAR1) (lane2). Lanes G, A, T, and C correspond to the sequence ladder obtainedusing the same primer. (B) Nucleotide sequence of the parI promoter region. The arrow indicates the transcription start site (�1). The �35 and�10 hexamers are underlined. The 13-bp palindromic sequence is shown in bold type. The annotated start codon of PP3700 and the modified startcodon in this study are boxed in dashed and solid lines, respectively. (C) Deletion analysis of the parIp promoter region. The DNA regions fusedwith the reporter gene in pMEGparI plasmids are shown to the left. Luciferase activities in KT2440 and KT2440(pCAR1) are shown. Values anderror bars correspond to averages and standard deviations of results from at least three independent experiments.



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ParBpCAR1 by pBRKparB did not affect parIp activity. There-fore, we concluded that the plasmid-encoded protein ParApCAR1

triggered the transcriptional activation of parI in KT2440(pCAR1).

Additionally, the activation mechanism of the 220-bp parIpregion was investigated in the original host of pCAR1, P.resinovorans CA10. However, luciferase expression from parIpin CA10 cells was similar to that in the isogenic strainCA10dm4 lacking pCAR1 (Fig. 6B). Furthermore, the over-expression of ParApCAR1 in CA10dm4 failed to activate parIp.This result suggests that ParApCAR1 indirectly activates thetranscription of parI in KT2440. In contrast, the overexpressionof ParI activated parIp, even in the heterogeneous CA10dm4cells (Fig. 6B). Together with the results obtained in theKT2440 cell environment (Fig. 6A), these results suggest thatthe direct transcriptional activator of parI is ParI itself.

DISCUSSION

We first analyzed the transcriptome of P. putida KT2440(pCAR1) during growth with carbazole, the recalcitrant aro-matic compound not used as the sole carbon source by KT2440without pCAR1. Microarray and qRT-PCR analyses showedthat the pCAR1-borne car and ant genes required for theupstream catabolic pathway and the chromosomal cat and pcagenes involved in the downstream catabolic pathway werestrongly induced. The car and ant operons are activated byAntR in response to anthranilate (85), whereas the cat and pcagenes are activated by CatR in response to cis,cis-muconateand PcaR in response to �-ketoadipate, respectively (30). Inaddition to the car and ant structural genes, the regulatorygene antR was upregulated during growth with carbazole. Theidentification of the transcription start site of antR revealedthat the antR promoter depends on RpoN in KT2440(pCAR1)as well as in CA10, the original host strain of pCAR1 (Fig. 2A),indicating that antR transcription is similarly regulated in bothhost strains. Generally, transcription from RpoN-dependentpromoters requires additional transcriptional activators (10).Therefore, the absence of RpoN-dependent regulatory genesin pCAR1 (45) implies that antR transcription requires an

unidentified RpoN-dependent activator whose gene should oc-cur on the host chromosome, at least on the KT2440 and CA10chromosomes. Thus, we concluded that the successful expres-sion of the carbazole catabolic operons on pCAR1 is highlydependent on the host chromosome. By using the availablecomplete genome sequence of KT2440, the identification ofthe activator for antR among the 22 RpoN-dependent activa-tors encoded by the KT2440 chromosome (11) will reveal theentire transcriptional cascade that occurs between pCAR1 andits host chromosome for carbazole catabolism. Furthermore, itwill be necessary to investigate whether and how the transcrip-tomes of pCAR1 differ among several heterogenic host strains.

In addition, various other responses were exerted duringgrowth with carbazole, starting from the alternative sigma fac-tor genes algT, rpoH, and rpoS at the top of the hierarchicbacterial transcriptional networks (48). AlgT is the counterpartof AlgU from P. aeruginosa PAO1, which is required for thetranscription of genes involved in exopolysaccharide alginatebiosynthesis and the coordinated transcription of rpoH (27,72). Although the role of AlgT in P. putida has not beenelucidated (48), it has been reported previously that rpoH istranscribed from an AlgT-dependent promoter in P. putida (1,46). RpoH is the major sigma factor involved in the heat shockresponse. In KT2440(pWW0), toluene-like aromatic com-pounds induce a large number of genes involved in the heatshock response (18), although the amount of RpoH is regu-lated at the posttranslational level rather than at the transcrip-tional level (46). Of the heat shock response genes (18), onlyibpA was found to be upregulated during growth with carba-zole (see Table S2 in the supplemental material), probably dueto the strict criteria of the microarray analysis. In contrast, thestationary phase-specific sigma factor RpoS plays a generalrole in stress responses. The transcription of rpoS in Pseudo-monas is under the control of several global regulators thatrespond to cell density and other as-yet-unknown signals(89). Given that rpoS transcription is elevated during thestationary phase (95), the upregulation of rpoS in KT2440(pCAR1) during growth with carbazole may have been causedby the decreased growth rate. It has been reported that, along

FIG. 6. Luciferase activities of the parI promoters in P. putida KT2440 cells (A) and P. resinovorans CA10dm4 cells (B). The effector plasmidsintroduced into the cells along with the reporter plasmid pMEGparI-200 are indicated on the left. P. resinovorans CA10dm4 harboring pCAR1represents P. resinovorans CA10. Values and error bars correspond to averages and standard deviations of results from at least three independentexperiments.



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with the integration host factor and the repressor TnpC, RpoSregulates the transcription of the transposase gene of Tn4652and that, therefore, the transposition frequency of Tn4652increases with the time of carbon starvation (31, 32, 33).Tn4652 is a deletion derivative of the toluene catabolic trans-poson Tn4651 that belongs to the class II transposons (22, 84).Thus, it is possible that tnpAc of the class II transposonTn4676, whose level of expression was higher during growthwith carbazole (Table 2), is also under the control of RpoS.

Next, we analyzed the transcriptome of the KT2440 chro-mosome in the presence of pCAR1 compared to that in theabsence of pCAR1 under the same growth conditions. Ourmost notable finding was the orphan parA homologue of P.putida KT2440, designated parI, that was specifically tran-scribed in the presence of pCAR1. The ParA family ATPases,in concert with the cognate ParB proteins and centromere-likeparS sites, are required for the active partitioning mechanismsof low-copy-number plasmids and are also involved in thesegregation of chromosomes upon cell division (20). The chro-mosomal parAB loci are found in close vicinity to the oriCregions of a wide range of bacteria, not including E. coli andother members of the Enterobacteriaceae family (6). The chro-mosomal parAB genes identified in the oriC region of KT2440(55) are particularly important for chromosome partitioning inspecific physiological states when cells are undergoing a reduc-tion in growth rate (25, 43). In contrast, orphan parA homo-logues that lack their cognate parB genes downstream arecommonly found in bacterial chromosomes. Knowledge of thefunctions of orphan ParA homologues is still limited, but sev-eral have been reported very recently. PpfA of Rhodobactersphaeroides, which is encoded within the chemotaxis locus (61),regulates the number and positions of cytoplasmic chemotaxisprotein clusters (82). In Caulobacter crescentus, MipZ affectsthe assembly and positioning of the FtsZ cytokinetic ring andis thereby thought to synchronize cytokinesis with chromosomesegregation (80). Interestingly, ParB of C. crescentus interactsboth with ParA, mediating chromosome segregation, and withMipZ, determining the site of FtsZ ring formation. By analogy,it is possible that ParI interacts with the chromosome- andplasmid-encoded ParA and ParB proteins.

In addition to the positive regulation by the ParI proteinitself, the pCAR1-encoded ParA protein activated the tran-scription of parI in KT2440 (Fig. 6A). However, in the heter-ologous strain CA10, parIp was activated by ParI but not byParApCAR1 (Fig. 6B), indicating that ParApCAR1 indirectly ac-tivates parIp in KT2440. Therefore, the regulation of parIpdepends on a KT2440 chromosomally encoded protein, whoseactivation is probably triggered by the ParApCAR1 protein.Since ParI itself activates parIp even in the heterologous strainand parI is not found among completely sequenced Pseudomo-nas chromosomes except for the KT2440 chromosome, it ismost likely that the direct regulator of parIp is ParI itself.Further studies will be needed to clarify whether the HTHmotif of ParI binds to the 13-bp palindromic sequence and howParApCAR1 triggers the activation of parIp. Since the partition-ing system of narrow-host-range IncP-7 plasmids is distinctfrom that of other plasmids (81), the high levels of similarity ofParA proteins among IncP-7 plasmids raise the possibility thatparI is also induced when another IncP-7 plasmid is introducedinto KT2440.

This study shows that an alien IncP-7 plasmid can be inte-grated into the KT2440 host chromosome to endow the strainwith the ability to degrade recalcitrant aromatic compounds,while exerting an unexpected effect on the transcription of thechromosomal gene. It is noteworthy that KT2440 is a derivativeof P. putida mt-2 from which the toluene-xylene catabolicIncP-9 plasmid pWW0 was removed (51, 64). The expressionof the catabolic operons on pWW0 involves the participationof plasmid-encoded pathway-specific regulators XylR and XylSand a number of host accessory regulatory elements that arenot exclusive to this catabolic pathway (63). Recently, the tran-scriptome of the original strain KT2440(pWW0) as affected inresponse to the toluene-like aromatic compounds has beenstudied using the whole genomic microarray (18). However, itremains unclear how the carriage of pWW0 affects the chro-mosomal transcriptome (namely, how the removal of pWW0changed the transcriptome of the original host). Further com-prehensive studies using a variety of the incompatibility groupsof plasmids and the genera of the hosts will provide new in-sights into the regulatory networks constructed during the hor-izontal transfer of mobile genetic elements.

ACKNOWLEDGMENTS

This work was supported by the Program for Promotion of BasicResearch Activities for Innovative Biosciences (PROBRAIN) and agrant-in-aid (hazardous chemicals) from the Ministry of Agriculture,Forestry and Fisheries of Japan (HC-07-2325-1) to H.N. M.M. andM.S. were supported by research fellowships from the Japan Societyfor the Promotion of Science for Young Scientists.

We are grateful to GeneFrontier Inc., Tokyo, Japan, for assistancewith microarray experiments.

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Transcriptome Analysis of Pseudomonas putida KT2440 ... · Transcriptome Analysis of Pseudomonas putida KT2440 Harboring the ... mid-free, spontaneous restriction-deﬁcient derivative