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Journal of Bacteriology, October 2007, p. 6849-6860, Vol. 189, No. 19
0021-9193/07/$08.00+0     doi:10.1128/JB.00684-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transcriptome Analysis of Pseudomonas putida KT2440 Harboring the Completely Sequenced IncP-7 Plasmid pCAR1{triangledown} ,{dagger}

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

Biotechnology Research Center,1 Professional Programme for Agricultural Bioinformatics, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan2

Received 1 May 2007/ Accepted 19 July 2007


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ABSTRACT
 
The IncP-7 plasmid pCAR1 of Pseudomonas resinovorans CA10 confers the ability to degrade carbazole upon transfer to the recipient strain P. putida KT2440. We designed a customized whole-genome oligonucleotide microarray to study the coordinated expression of pCAR1 and the chromosome in the transconjugant strain KT2440(pCAR1). First, the transcriptome of KT2440(pCAR1) during growth with carbazole as the sole carbon source was compared to that during growth with succinate. The carbazole catabolic car and ant operons were induced, along with the chromosomal cat and pca genes involved in the catechol branch of the ß-ketoadipate pathway. Additionally, the regulatory gene antR encoding the AraC/XylS family transcriptional activator specific for car and ant operons was upregulated. The characterization of the antR promoter revealed that antR is transcribed from an RpoN-dependent promoter, suggesting that the successful expression of the carbazole catabolic 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 chromosome, we compared the chromosomal transcriptomes of KT2440(pCAR1) and KT2440 under the same growth conditions. Only subtle changes were caused by the transfer of pCAR1, except for the significant induction of the hypothetical gene PP3700, designated parI, which encodes a putative ParA-like ATPase with an N-terminal Xre-type DNA-binding motif. Further transcriptional analyses showed that the parI promoter was positively regulated by ParI itself and the pCAR1-encoded protein ParA.


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INTRODUCTION
 
Many catabolic plasmids can be transferred horizontally between different bacteria and play an important role in the distribution of the ability to degrade and utilize recalcitrant chemical compounds (15, 90). Several catabolic plasmids within members of the genus Pseudomonas have been identified and have been classified mostly into incompatibility groups IncP-1, IncP-2, IncP-7, and IncP-9. Recently, the complete genome sequences of several IncP-1, IncP-7, and IncP-9 catabolic plasmids were determined (16, 28, 44, 45, 47, 76, 77, 83, 86, 93). The 199,035-bp catabolic plasmid pCAR1 was originally discovered in Pseudomonas resinovorans CA10, which is able to utilize carbazole as its sole source of carbon, nitrogen, and energy (53, 56), and was the first IncP-7 plasmid to be completely sequenced (45).

pCAR1 carries the car and ant operons, which encode the upper and meta pathway enzymes and the anthranilate 1,2-dioxygenase, respectively (see Fig. 1A). The operons are transcribed 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 the transcription of the car operon (50). Carbazole catabolism begins with the upper pathway to yield anthranilate and 2-hydroxypenta-2,4-dienoate, and then 2-hydroxypenta-2,4-dienoate is mineralized into pyruvate and acetyl coenzyme A (acetyl-CoA) by the meta pathway enzymes (53) whereas anthranilate is converted into catechol by the anthranilate 1,2-dioxygenase (see Fig. 1B). Catechol, one of the central intermediates of the aromatic catabolic pathway, is degraded into acetyl-CoA and succinyl-CoA via the chromosomally encoded ß-ketoadipate pathway (36). Thus, the induction of pCAR1-borne catabolic operons confers the ability to grow with carbazole and anthranilate upon the recipient strain.


Figure 1
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  		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 the transposed portion of antA (53). Arrows with solid and dotted lines indicate the induced and constitutive transcripts originating from Pant and PcarAa, respectively (50, 85). (B) Carbazole catabolic pathway in P. putida KT2440 harboring pCAR1. The enzymes constituting the upper and meta pathways shown in the rounded box are encoded on pCAR1 (53), whereas the ß-ketoadipate pathway enzymes are encoded on the KT2440 chromosome (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-lactonizing enzyme; CatC, muconolactone isomerase; PcaD, ß-ketoadipate enol-lactone hydrolase; PcaIJ, ß-ketoadipate succinyl-CoA transferase; PcaF, ß-ketoadipyl-CoA thiolase.

pCAR1 is a self-transmissible plasmid that has been conjugally transferred into P. putida KT2440 (75). KT2440 is a plasmid-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 against DNA uptake, KT2440 is thought to be an ideal host for expanding the range of growth substrates via the recruitment of catabolic plasmids. Because the complete genomic sequence of P. putida KT2440 is known (52) and the DNA sequence of the catabolic plasmid pCAR1 is also known (45), we were able to design a customized high-density oligonucleotide microarray for P. putida KT2440 containing pCAR1 that allowed us to investigate the expression profile of the entire genome.

In this study, using our customized microarray, we analyzed the range of expression of pCAR1-borne genes and the coordinated response of the host chromosome in cells of the transconjugant strain KT2440(pCAR1) grown with carbazole as the carbon source. For comparison, succinate was used as an alternative carbon source since it is generally regarded as a good carbon source for KT2440 and has been used in several previous transcriptome studies (87, 88) and proteomic studies (8, 38, 42, 69, 94). The operons required for carbazole catabolism, both on pCAR1 and on the chromosome, were strongly induced, and the regulatory gene antR was also upregulated during growth with carbazole. Next, to examine whether the horizontal transfer of a plasmid alters the transcription network of its host chromosome, the transcriptional changes in the host chromosome of KT2440(pCAR1) relative to that of its parental strain KT2440 under the same growth conditions were further analyzed. Only subtle changes were observed, except for the chromosomal hypothetical gene PP3700, which was significantly induced by the carriage of pCAR1.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. Bacterial strains used in this study are listed 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-plus mineral medium 4 (NMM-4) with 1.0 mg of carbazole or succinate ml–1 as the sole carbon source (73). The following antibiotic was added to the medium: ampicillin (100 µg ml–1), kanamycin (50 µg ml–1), or gentamicin (15 µg ml–1). For plate cultures, the above-described media were solidified with 1.6% agar (wt/vol).


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  		TABLE 1. Bacterial strains and plasmids used in this study

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 succinate ml–1 was inoculated with cells of the Pseudomonas strains from an overnight culture in Luria broth. The cells were incubated at 30°C on a rotary shaker at 120 rpm and were monitored by measuring the OD600 or the number of CFU per milliliter.

A total of 109 cells at early exponential phase were mixed with RNA Protect Bacteria reagent as recommended by the manufacturer (QIAGEN, Valencia, CA). Total RNA was extracted using the RNeasy midi kit (QIAGEN). The eluted 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 the stop reagent supplied by the manufacturer, RNA samples were again purified with the RNeasy mini column according to the cleanup protocol of the manufacturer (QIAGEN).

Microarray analysis. The genome sequences and open reading frame (ORF) predictions for P. putida KT2440 and pCAR1 were obtained from GenBank accession numbers NC_002947 and AB088420, respectively. The 6,181,863-bp chromosome of KT2440 contains 5,350 predicted ORFs and 21 rRNAs, and the 199,035-bp pCAR1 contains 190 ORFs. A customized high-density oligonucleotide array (NimbleGen Systems Inc., Madison, WI) designed for the KT2440 and pCAR1 genomes contained 178,230 different 24-mer probes paired with perfect-match (PM) oligonucleotides and their corresponding mismatch oligonucleotides with substitutions by transversion at positions 6 and 12 from the 5' end. Fifteen adequate probe pairs were selected for each ORF, and probe pairs assigned to pCAR1 were produced in triplicate.

The cDNA synthesis, hybridization, and scanning were performed by NimbleGen Systems Inc. Microarray data analysis was performed using the robust multiarray average method (34) based on the log2 values of the absolute signal intensities for PM probes only. The hybridization signals assigned to pCAR1 could not be compared because of the lack of pCAR1 in plasmid-free KT2440 but were used for normalization together with those assigned to the KT2440 chromosome. Student's t test for each PM probe and each biological replicate, followed by the Bonferroni correction, was used to identify genes showing differential expression patterns (P < 0.05). The data presented are the averages of results from two independent experiments.

Quantitative reverse transcription-PCR (qRT-PCR). Reverse transcription was performed with 30-µl reaction mixtures containing 6 µg of total RNA, 375 ng of random primers (Invitrogen, Carlsbad, CA), 750 U of SuperScript II (Invitrogen), 30 U of RNase OUT (Invitrogen), 1x First Strand buffer (Invitrogen), 10 mM dithiothreitol, and 0.5 mM deoxynucleoside triphosphates (Toyobo Co. Ltd., Tokyo, Japan). After the RNA and random primers had been denatured at 70°C for 10 min and annealed at 25°C for 10 min, the remaining reagents were 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. To degrade the RNA, 10 µl of 1 N NaOH was added, and the reaction mixture was heated at 65°C for 30 min; the mixture was neutralized with 10 µl of 1 N HCl.

The cDNA quantification was performed using the ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA). The primers used for qRT-PCR were designed using the Primer3 program (67). Detailed information on the primer sequences used in this study is available upon request. All of the products were between 100 and 150 bp in length. The univ16S-F and univ16S-R primer set used to measure the transcription of 16S rRNA was designed based on the 16S rRNA sequences from P. putida KT2440 (GenBank accession no. NC_002947) and P. resinovorans CA10 (GenBank accession no. AB047273). Each 20-µl reaction mixture contained 10 µl of Power SYBR green PCR master mix (Applied Biosystems), 200 nM concentrations of each specific primer, and the cDNA. The reaction conditions were as follows: 95°C for 10 min for enzyme activation and 40 cycles of 95°C for 5 s, 60°C for 20 s, and 72°C for 30 s. A melting-curve analysis was performed to verify the amplification specificity. To quantify the transcription of each gene, the copy number was determined by generating a standard curve using a series of 10-fold dilutions (from 100 pM to 1 fM) of the target PCR product inserted into the pT7Blue T-vector (Novagen, Madison, WI). For sample normalization, 16S rRNA was used as an internal standard. All of the reactions were performed at least in triplicate, and the data were normalized using 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 start point, was used. Primer extension was performed with 20 µl of 1x first-strand buffer containing 10 µg of total RNA, 2 pmol of the IRD800-labeled primer, 200 U of SuperScript III (Invitrogen), 80 U of RNase OUT (Invitrogen), 10 mM dithiothreitol, and 0.5 mM deoxynucleoside triphosphates (Toyobo Co. Ltd.). After the denaturation of the RNA and the IRD800-labeled primer at 65°C for 5 min, the remaining reagents were added and the mixture was incubated at 50°C for 30 min. The extended product was purified by phenol-chloroform extraction and ethanol precipitation and then dissolved in 2 µl of H2O and 1 µl of IR2 stop solution (LI-COR Inc., Lincoln, NE). The solution was then denatured at 95°C for 2 min and subjected to electrophoresis using a LI-COR model 4200L-2 auto-DNA sequencer. A sequence ladder was obtained using the same primer and the template plasmid pUCA741 (53) or pT7PP3700int, containing the intergenic region from PP3701 to PP3700 amplified from the total DNA of KT2440 by 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 –200 to +53 relative to the transcription initiation site of antA, was inserted into the reporter vector pMEGluc (50) to yield pMCantA253.

Transcriptional fusions of the parI promoter were prepared by amplifying appropriate DNA fragments upstream of parI from pT7PP3700int by using adequately designed primer sets. The EcoRI restriction site was introduced into the forward primers PP3700F-200, PP3700F-100, PP3700F-50, and PP3700F-40, and the NcoI restriction site CCATGG was introduced into the reverse primer PP3700R+18 to match its CAT nucleotides to the start codon of parI. Forward primers PP3700F-50C39T and PP3700F-50C38T were designed to introduce artificial mutations at –39 and –38 relative to the transcription start point, respectively. The amplified fragments were sequenced to confirm the nucleotide sequences and then inserted into the reporter vector pMEGluc (50), which was digested with EcoRI and NcoI restriction enzymes, to yield the pMEGparI series (Table 1).

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

Electrocompetent cells of Pseudomonas strains were prepared as described by Choi et al. (12). Electrotransformation was performed using Gene Pulser II (Bio-Rad, Hercules, CA) as described previously (85). Overnight cultures of the resultant transformants were inoculated into 5 ml of Luria broth or NMM-4 supplemented with 1.0 mg of succinate ml–1 with or without 0.2 mg of sodium anthranilate ml–1 to yield an initial OD600 of 0.05, and the cells were grown to the mid-exponential phase. In a 96-well microtiter plate, 50 µl of the culture and an equal volume of Picagene LT2.0 (Toyo Ink Co. Ltd., Tokyo, Japan) were mixed and shaken for 20 s, and the relative light units (RLU) were measured for 10 s by using Centro LB960 (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). Each sample was assayed independently at least three times.

Disruption and complementation of the rpoN and antR genes. The rpoN and antR genes were amplified from the genomic DNA of KT2440(pCAR1) using the primers rpoNF-EcoRI with rpoNR-HindIII and ORF23-F with ORF23-R and inserted into the pT7Blue T-vector to yield pTrpoN and pTORF23, respectively. The EcoRI-HindIII fragment of pTrpoN was subcloned into pUC19 to yield pUCrpoN. To disrupt the rpoN and antR genes, a SmaI fragment containing the nonpolar Gmr cassette of pSJ12 (35) was inserted into the EcoRV-HincII sites of rpoN in pUCrpoN and the EcoRV site of antR in pTORF23 in the same direction; the resulting plasmids were designated pUCrpoN::Gmr and pTORF23::Gmr, respectively. Subsequently, the EcoRI-HindIII fragment of pUCrpoN::Gmr or the SalI-BamHI fragment of pTORF23::Gmr was inserted into pK18mobsacB and pK19mobsacB (71) to obtain pKrpoN::Gmr and pKantR::Gmr, respectively. As described previously (59), pKrpoN::Gmr or pKantR::Gmr was introduced into KT2440(pCAR1) by filter mating with E. coli S17-1 {lambda}pir transformants, and the double-crossover recombinants were screened. To complement rpoN and antR, the SalI-BamHI fragments of pTrpoN and pTORF23 were inserted into the broad-host-range vector pBBR1MCS-2 (41) to obtain pBRKrpoN and pBRKantR, respectively, and then these plasmids were introduced into the P. putida strains by electroporation (85).

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


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RESULTS
 
The KT2440(pCAR1) transcriptome during growth with carbazole. P. putida KT2440 cannot utilize anthranilate as its sole source of carbon and energy (36); however, the conjugative transfer of pCAR1 from P. resinovorans CA10 enabled KT2440 to utilize either carbazole or anthranilate as its sole source of carbon and nitrogen. In this study, we first analyzed the transcriptome of KT2440(pCAR1) during growth with carbazole or succinate. The rate of KT2440(pCAR1) growth with carbazole was significantly lower than that of growth with succinate (data not shown).

Microarray analysis showed that the levels of transcription of 38 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 constituting the pCAR1-borne car and ant operons (Fig. 1A), which encode the upper and meta carbazole pathway enzymes and the anthranilate 1,2-dioxygenase, respectively, were the most significantly induced during the growth of KT2440 (pCAR1) with carbazole. Microarray analysis showed that orf9 and antA, both of which are transcribed from Pant (85), were induced by 39- and 43-fold, respectively. These values were calculated from 10 PM probes that were specific for each gene and 5 PM probes common to orf9 and antA, the 5' regions of which are identical because of the transposition of ISPre1 (53). In addition to the structural genes, the regulatory gene antR was 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 carbazole catabolic transposon Tn4676 (74) was induced by 2.9-fold during growth with carbazole (Table 2). This finding suggests that the transposition of Tn4676 is enhanced during growth with carbazole. All eight downregulated genes (Table 2) corresponded to a gene cluster from orf100 to orf108 and were oriented in the same direction, although orf103 was significantly downregulated by only 1.8-fold (P = 7.0E–07) as calculated by the microarray analysis.


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  		TABLE 2. Change in expression of pCAR1 genes during growth with carbazole

A total of 139 chromosomal genes showed significant changes of more than twofold (P < 0.05); 63 were upregulated and 76 were downregulated during growth with carbazole (see Tables S2 and S3 in the supplemental material). The majority of the upregulated chromosomal genes were hypothetical (see Table S2 in the supplemental material). However, we detected significant upregulation of the algT, rpoH, and rpoS genes, which encode alternative sigma factors (48). Similarly, the mucA and algN genes, which encode anti-sigma proteins for AlgT and are located downstream of algT, were also upregulated. Conversely, a large number of genes associated with protein synthesis and energy metabolism were downregulated during growth with carbazole (see Table S3 in the supplemental material). This finding may be attributable to the decreased rate 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 encode the cytochrome o ubiquinol oxidase, which is involved in catabolite repression in addition to its role in the electron transfer chain (17, 58). Therefore, it is possible that catabolite repression is relieved during growth with carbazole.

Catechol is produced from anthranilate by the pCAR1-encoded anthranilate 1,2-dioxygenase, and the chromosomally encoded ß-ketoadipate pathway enzymes catalyze further catechol degradation (Fig. 1B). We found that the catC and catA genes of the KT2440 chromosome were significantly induced during growth with carbazole (see Table S2 in the supplemental material). Although catB was excluded by the criteria of the microarray analysis because of its relatively high P value after the Bonferroni correction (P = 0.07), qRT-PCR analysis showed that growth with carbazole induced the transcription of catB by 160-fold (Table 3). This result is consistent with the findings of a previous study of P. putida PRS2000, which revealed that the catBCA genes are activated by the LysR family transcriptional regulator CatR in response to cis,cis-muconate (30), and those of a study of the original host strain P. resinovorans CA10 (54). Additionally, genomic analysis of the KT2440 chromosome has revealed that another catA homologue known as catA2 (PP3166) is located within the ben gene cluster (36). qRT-PCR analysis indicated a low level of catA2 transcription even during growth with carbazole, although catA2 was induced 5.9-fold (Table 3). This finding suggests that CatA2 is not involved in catechol oxygenation, at least during carbazole utilization, but it is still possible that catA2 transcription is induced along with that of the ben genes under the control of BenR in response to benzoate (13). The enzymes PcaD, PcaF, and PcaIJ, which are encoded by discrete gene clusters pcaRKFTBDCP and pcaIJ, constitute the downstream portion of the ß-ketoadipate pathway (36). In P. putida PRS2000, the IclR family transcriptional regulator PcaR is required for the induction of pcaK, pcaF, pcaBCD, and pcaIJ in response to ß-ketoadipate (30). Although our microarray analysis showed only a 3.3-fold induction of pcaF during growth with carbazole because of our strict criteria (see Table S2 in the supplemental material), qRT-PCR analysis verified that pcaF and pcaIJ were induced during growth with carbazole, although the induction of pcaD was relatively weak (Table 3). This result indicates that the chromosomal ß-ketoadipate pathway is integrated into the overall carbazole catabolic pathway.


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  		TABLE 3. Quantitative RT-PCR analysis of ß-ketoadipate pathway genes

Transcriptional regulation of the pCAR1-borne regulatory gene antR. It has been suggested previously that an additional antR homologue is located on the CA10 chromosome (85), similar to the homologues found near the chromosomal antABC genes on several Pseudomonas chromosomes, including antR of P. fluorescens MB214 (65) and orfAN of P. putida P111 (GenBank accession no. AY026914), and also those on completely sequenced chromosomes, such as PFL_0759 of P. fluorescens Pf-5, PA2511 of P. aeruginosa PAO1, and PA14_32160 of P. aeruginosa PA14 (http://www.pseudomonas.com/). In contrast, KT2440 cannot utilize anthranilate as its sole carbon source, and its chromosome lacks antR and antABC. However, there have been several reports of cross-regulation among the AraC/XylS family members, such as the chromosome-encoded BenR and the pWW0-encoded XylS (13, 18), because of the conserved binding sequence of the genes (5'-TGCA-N6-GGNTA-3') (26), and a total of 40 regulatory genes belonging to the AraC/XylS family are present on the KT2440 chromosome (19). Reporter analysis using a Pant transcriptional fusion showed that the promoter activity in KT2440(pBBR1MCS-2)(pMCantA253) was not significantly changed by supplementation with anthranilate (181 ± 25 RLU/OD600 unit without anthranilate and 243 ± 68 RLU/OD600 unit with anthranilate) but that the promoter activity in KT2440(pBRKantR)(pMCantA253), which expresses the AntR protein, was significantly increased (517 ± 122 RLU/OD600 unit without anthranilate and 272,500 ± 38,000 RLU/OD600 unit with anthranilate). Therefore, it was concluded that AntR is the only pathway-specific activator of Pant in KT2440(pCAR1) in response to anthranilate.

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


Figure 2
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  		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. The transcription start site (+1) is indicated by an arrow, and the –12 and –24 duplexes of the antR promoter are underlined. The extended consensus promoter 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.

To confirm that the antR promoter depends on RpoN RNA polymerase, the rpoN gene in KT2440 harboring pCAR1 was disrupted. The rpoN-disrupted strain KT2440{Delta}rpoN(pCAR1) could not grow with anthranilate and carbazole as its sources of carbon and energy, consistent with the findings in a previous report (39), its growth rate in Luria broth was lower than that of the wild-type strain KT2440(pCAR1). The antR-disrupted strain KT2440(pCAR1{Delta}antR) was also unable to grow with anthranilate and carbazole as carbon and energy sources, but its growth rate in Luria broth was identical to that of the wild-type strain. The expression of rpoN in trans allowed KT2440{Delta}rpoN(pCAR1) alone to utilize anthranilate, whereas the expression of antR in trans complemented both KT2440(pCAR1{Delta}antR) and KT2440{Delta}rpoN(pCAR1). These results 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 KT2440 chromosome in response to the carriage of pCAR1, the transcriptome of KT2440(pCAR1) was compared with that of the isogenic plasmid-free strain KT2440 growing under the same conditions. The growth curves for growth with succinate were similar, but a lower growth rate of KT2440(pCAR1) was observed (Fig. 3); the doubling times of KT2440 and KT2440(pCAR1) at early exponential phase were estimated to be 43 and 51 min, respectively. Microarray analysis showed that, compared to the genes in KT2440, only 10 chromosomal genes in KT2440(pCAR1) were differentially expressed at least twofold (P < 0.05) (Table 4). Unexpectedly, a hypothetical gene, PP3700, showed an exceptional 41.8-fold induction in the presence of pCAR1.


Figure 3
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  		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 microarray analysis were collected at an OD600 of ~0.2.


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  		TABLE 4. Differential expression of P. putida KT2440 chromosomal genes affected by pCAR1

A conserved-domain search of the NCBI conserved-domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml/) showed that the protein encoded by PP3700 had modular organization. At its N terminus, the translational start point of which was different from that annotated in the databases (see below), an HTH DNA-binding motif of the Xre family of transcriptional regulators was conserved (Fig. 4A). The Xre family is a large family of proteins with an HTH motif similar to that of the {lambda} repressor and the Cro protein of {lambda} bacteriophage (62, 66, 70) and is also represented by the Bacillus subtilis prophage PBSX repressor Xre (91), the B. subtilis development regulator SinR (21), and the restriction-modification system control proteins such as C.PvuII and C.AhdI (49, 78). As well as the HTH motif, the PP3700 protein possessed the Walker-type ATPase motif, which is characteristic of the ParA family ATPases involved in the active partitioning of low-copy-number plasmids and the segregation of chromosomes 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 a bulky hydrophobic amino acid, lowercase letters indicate at least 80% conservation, and uppercase letters indicate 100% conservation (40). The KT2440 chromosome carries a total of five parA homologues, which were categorized into clusters of the orthologous COG1192 group (http://v2.pseudomonas.com/), consisting of parA in the vicinity of oriC (55) and four orphan parA homologues that lack the cognate parB genes. The Walker motifs were highly conserved among the five COG1192 proteins (Fig. 4B). However, completely sequenced Pseudomonas chromosomes commonly carry at least three COG1192 genes in a conserved locus (http://v2.pseudomonas.com/), and PP3700 is specifically found in the KT2440 chromosome. Because PP3700 was identified as an inducible orphan parA homologue, PP3700 is hereafter designated parI (inducible parA homologue).


Figure 4
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  		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, GenPept accession 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, GenPept accession no. AAN69913; PP5070 protein, GenPept accession no. AAN70635; and PP3700 protein, GenPept accession no. AAN69297. The N-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 acids between Walker A' and B motifs are indicated within the sequences.

Activation of the promoter of the chromosomal parI gene in the presence of pCAR1. Primer extension was performed to identify the transcription start point of parI. A single signal was detected from the total RNA of KT2440(pCAR1) but not in the absence of pCAR1 (Fig. 5A). The extension product was positioned at a guanine base 104 nt downstream of the annotated translation start point of PP3700, however (Fig. 5B). Accordingly, such an annotated translation start point is unlikely, and the ATG trinucleotide located 123 nt downstream of the annotated translation start point is preferable for the start codon; this codon is preceded by an AG-rich ribosomal binding site at a 5-nt distance (Fig. 5B). Putative –35 and –10 hexamers, 5'-TGTTTT-N17-TACGCT-3', were found upstream of the transcription start point.


Figure 5
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  		FIG. 5. Identification of the parI promoter. (A) Mapping of the parI transcription start point. Primer extension was preformed using equal amounts of total RNA from KT2440 (lane 1) and KT2440(pCAR1) (lane2). Lanes G, A, T, and C correspond to the sequence ladder obtained using 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 start codon in this study are boxed in dashed and solid lines, respectively. (C) Deletion analysis of the parIp promoter region. The DNA regions fused with the reporter gene in pMEGparI plasmids are shown to the left. Luciferase activities in KT2440 and KT2440(pCAR1) are shown. Values and error bars correspond to averages and standard deviations of results from at least three independent experiments.

To characterize the promoter of parI (parIp), reporter analysis was performed using a series of deletions in the region upstream of parI. Luciferase expression from the 220-bp promoter region from –200 to +20 relative to the transcription start point was significantly increased in KT2440(pCAR1) cells but was negligible in KT2440 cells (Fig. 5C). Furthermore, deletion of the promoter region to –40 did not change the basal promoter activity but significantly reduced the induction rate in the presence of pCAR1. A 13-bp palindromic sequence, 5'-GGCACTCTGTGCC-3', from –50 to –38 relative to the transcription start point was found (Fig. 5B). Even in the presence of pCAR1, each replacement of cytosines by thymines at –39 and –38 reduced the activity of the 70-bp promoter (from –50 to +20) by 25% ± 8% and 40% ± 12%, respectively, although the basal promoter activity remained at the same level as that in the absence of pCAR1. These results suggest that the region up to at least –50 is essential for the activation of parIp in response to the carriage of pCAR1 and that the palindromic sequence in cis plays an important role in the activation of parIp.

Transcriptional regulation of parI. Because proteins of the Xre family appear to bind to short palindromic sequences (3, 4, 7, 24, 57, 60), it is possible that ParI binds the 13-bp palindromic sequence just upstream of parIp to mediate its own transcription. To investigate whether ParI autoregulates its transcription, luciferase expression from the 220-bp region of parIp was measured in KT2440 cells harboring pBRKparI so as to express ParI under the control of the tac promoter in the pBBR1MCS-2 vector. Compared to that in the negative control, KT2440(pBBR1MCS-2), luciferase expression in KT2440 (pBRKparI) increased by 26-fold (Fig. 6A), indicating that ParI positively regulates its own transcription.


Figure 6
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  		FIG. 6. Luciferase activities of the parI promoters in P. putida KT2440 cells (A) and P. resinovorans CA10dm4 cells (B). The effector plasmids introduced into the cells along with the reporter plasmid pMEGparI-200 are indicated on the left. P. resinovorans CA10dm4 harboring pCAR1 represents P. resinovorans CA10. Values and error bars correspond to averages and standard deviations of results from at least three independent experiments.

Because the truncation of the possible cis-acting regulatory site resulted in the remarkable repression of parIp activity even in the presence of pCAR1 rather than causing activation (Fig. 5C), parIp was thought to require an activator encoded on pCAR1. The first candidate for this activator was the pCAR1-encoded MvaT family transcriptional regulator (79), designated Pmr (plasmid-encoded MvaT-like regulator), because the disruption of pmr in KT2440(pCAR1) resulted in the significant downregulation of parI (T. Terabayashi, M. Miyakoshi, and H. Nojiri, unpublished data). However, parIp activity was only slightly repressed in KT2440 harboring pBRKpmr, which overexpressed the Pmr protein (Fig. 6A). This result suggests that Pmr indirectly upregulates the transcription of parI. The next candidates for the activator of parIp were pCAR1-encoded ParA (ParApCAR1) and ParBpCAR1, whose genes were also significantly downregulated in the pmr-deficient mutant (Terabayashi et al., unpublished). When ParApCAR1 was expressed in KT2440 by pBRKparA, luciferase expression from the promoter increased by 90-fold compared to that in the negative control (Fig. 6A). In contrast, the expression of ParBpCAR1 by pBRKparB did not affect parIp activity. Therefore, 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 parIp region was investigated in the original host of pCAR1, P. resinovorans CA10. However, luciferase expression from parIp in CA10 cells was similar to that in the isogenic strain CA10dm4 lacking pCAR1 (Fig. 6B). Furthermore, the overexpression of ParApCAR1 in CA10dm4 failed to activate parIp. This result suggests that ParApCAR1 indirectly activates the transcription of parI in KT2440. In contrast, the overexpression of ParI activated parIp, even in the heterogeneous CA10dm4 cells (Fig. 6B). Together with the results obtained in the KT2440 cell environment (Fig. 6A), these results suggest that the direct transcriptional activator of parI is ParI itself.


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DISCUSSION
 
We first analyzed the transcriptome of P. putida KT2440 (pCAR1) during growth with carbazole, the recalcitrant aromatic compound not used as the sole carbon source by KT2440 without pCAR1. Microarray and qRT-PCR analyses showed that the pCAR1-borne car and ant genes required for the upstream catabolic pathway and the chromosomal cat and pca genes involved in the downstream catabolic pathway were strongly induced. The car and ant operons are activated by AntR in response to anthranilate (85), whereas the cat and pca genes are activated by CatR in response to cis,cis-muconate and PcaR in response to ß-ketoadipate, respectively (30). In addition to the car and ant structural genes, the regulatory gene antR was upregulated during growth with carbazole. The identification of the transcription start site of antR revealed that 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 both host strains. Generally, transcription from RpoN-dependent promoters requires additional transcriptional activators (10). Therefore, the absence of RpoN-dependent regulatory genes in pCAR1 (45) implies that antR transcription requires an unidentified RpoN-dependent activator whose gene should occur on the host chromosome, at least on the KT2440 and CA10 chromosomes. Thus, we concluded that the successful expression of the carbazole catabolic operons on pCAR1 is highly dependent on the host chromosome. By using the available complete genome sequence of KT2440, the identification of the activator for antR among the 22 RpoN-dependent activators encoded by the KT2440 chromosome (11) will reveal the entire transcriptional cascade that occurs between pCAR1 and its host chromosome for carbazole catabolism. Furthermore, it will be necessary to investigate whether and how the transcriptomes of pCAR1 differ among several heterogenic host strains.

In addition, various other responses were exerted during growth with carbazole, starting from the alternative sigma factor genes algT, rpoH, and rpoS at the top of the hierarchic bacterial transcriptional networks (48). AlgT is the counterpart of AlgU from P. aeruginosa PAO1, which is required for the transcription of genes involved in exopolysaccharide alginate biosynthesis and the coordinated transcription of rpoH (27, 72). Although the role of AlgT in P. putida has not been elucidated (48), it has been reported previously that rpoH is transcribed from an AlgT-dependent promoter in P. putida (1, 46). RpoH is the major sigma factor involved in the heat shock response. In KT2440(pWW0), toluene-like aromatic compounds induce a large number of genes involved in the heat shock response (18), although the amount of RpoH is regulated at the posttranslational level rather than at the transcriptional level (46). Of the heat shock response genes (18), only ibpA was found to be upregulated during growth with carbazole (see Table S2 in the supplemental material), probably due to the strict criteria of the microarray analysis. In contrast, the stationary phase-specific sigma factor RpoS plays a general role in stress responses. The transcription of rpoS in Pseudomonas is under the control of several global regulators that respond to cell density and other as-yet-unknown signals (89). Given that rpoS transcription is elevated during the stationary phase (95), the upregulation of rpoS in KT2440 (pCAR1) during growth with carbazole may have been caused by the decreased growth rate. It has been reported that, along with the integration host factor and the repressor TnpC, RpoS regulates the transcription of the transposase gene of Tn4652 and that, therefore, the transposition frequency of Tn4652 increases with the time of carbon starvation (31, 32, 33). Tn4652 is a deletion derivative of the toluene catabolic transposon Tn4651 that belongs to the class II transposons (22, 84). Thus, it is possible that tnpAc of the class II transposon Tn4676, whose level of expression was higher during growth with carbazole (Table 2), is also under the control of RpoS.

Next, we analyzed the transcriptome of the KT2440 chromosome in the presence of pCAR1 compared to that in the absence of pCAR1 under the same growth conditions. Our most notable finding was the orphan parA homologue of P. putida KT2440, designated parI, that was specifically transcribed in the presence of pCAR1. The ParA family ATPases, in concert with the cognate ParB proteins and centromere-like parS sites, are required for the active partitioning mechanisms of low-copy-number plasmids and are also involved in the segregation of chromosomes upon cell division (20). The chromosomal parAB loci are found in close vicinity to the oriC regions of a wide range of bacteria, not including E. coli and other members of the Enterobacteriaceae family (6). The chromosomal parAB genes identified in the oriC region of KT2440 (55) are particularly important for chromosome partitioning in specific physiological states when cells are undergoing a reduction in growth rate (25, 43). In contrast, orphan parA homologues that lack their cognate parB genes downstream are commonly found in bacterial chromosomes. Knowledge of the functions of orphan ParA homologues is still limited, but several have been reported very recently. PpfA of Rhodobacter sphaeroides, which is encoded within the chemotaxis locus (61), regulates the number and positions of cytoplasmic chemotaxis protein clusters (82). In Caulobacter crescentus, MipZ affects the assembly and positioning of the FtsZ cytokinetic ring and is thereby thought to synchronize cytokinesis with chromosome segregation (80). Interestingly, ParB of C. crescentus interacts both with ParA, mediating chromosome segregation, and with MipZ, determining the site of FtsZ ring formation. By analogy, it is possible that ParI interacts with the chromosome- and plasmid-encoded ParA and ParB proteins.

In addition to the positive regulation by the ParI protein itself, the pCAR1-encoded ParA protein activated the transcription of parI in KT2440 (Fig. 6A). However, in the heterologous strain CA10, parIp was activated by ParI but not by ParApCAR1 (Fig. 6B), indicating that ParApCAR1 indirectly activates parIp in KT2440. Therefore, the regulation of parIp depends on a KT2440 chromosomally encoded protein, whose activation is probably triggered by the ParApCAR1 protein. Since ParI itself activates parIp even in the heterologous strain and parI is not found among completely sequenced Pseudomonas chromosomes except for the KT2440 chromosome, it is most likely that the direct regulator of parIp is ParI itself. Further studies will be needed to clarify whether the HTH motif of ParI binds to the 13-bp palindromic sequence and how ParApCAR1 triggers the activation of parIp. Since the partitioning system of narrow-host-range IncP-7 plasmids is distinct from that of other plasmids (81), the high levels of similarity of ParA proteins among IncP-7 plasmids raise the possibility that parI is also induced when another IncP-7 plasmid is introduced into KT2440.

This study shows that an alien IncP-7 plasmid can be integrated into the KT2440 host chromosome to endow the strain with the ability to degrade recalcitrant aromatic compounds, while exerting an unexpected effect on the transcription of the chromosomal gene. It is noteworthy that KT2440 is a derivative of P. putida mt-2 from which the toluene-xylene catabolic IncP-9 plasmid pWW0 was removed (51, 64). The expression of the catabolic operons on pWW0 involves the participation of plasmid-encoded pathway-specific regulators XylR and XylS and a number of host accessory regulatory elements that are not exclusive to this catabolic pathway (63). Recently, the transcriptome of the original strain KT2440(pWW0) as affected in response to the toluene-like aromatic compounds has been studied using the whole genomic microarray (18). However, it remains unclear how the carriage of pWW0 affects the chromosomal transcriptome (namely, how the removal of pWW0 changed the transcriptome of the original host). Further comprehensive studies using a variety of the incompatibility groups of plasmids and the genera of the hosts will provide new insights into the regulatory networks constructed during the horizontal transfer of mobile genetic elements.


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

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


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FOOTNOTES
 
* Corresponding author. Mailing address: Biotechnology Research Center, 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: anojiri{at}mail.ecc.u-tokyo.ac.jp Back

{triangledown} Published ahead of print on 3 August 2007. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back

{ddagger} Present address: Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan. Back


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Journal of Bacteriology, October 2007, p. 6849-6860, Vol. 189, No. 19
0021-9193/07/$08.00+0     doi:10.1128/JB.00684-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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