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Biosci. Biotechnol. Biochem., 73 (3), 744–746, 2009 Note The Complete Nucleotide Sequence of pCAR2: pCAR2 and pCAR1 Were Structurally Identical IncP-7 Carbazole Degradative Plasmids y Yurika TAKAHASHI, Masaki SHINTANI, Hisakazu YAMANE, and Hideaki NOJIRI Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received September 22, 2008; Accepted November 6, 2008; Online Publication, March 7, 2009 [doi:10.1271/bbb.80665] pCAR1 and pCAR2 are IncP-7 self-transmissible determined by shotgun sequencing, followed by manual carbazole degradative plasmids. Their respective hosts gap closing. P. resinovorans CA10dm4D2,6) a P. resin- showed clearly different conjugative host ranges. Their ovorans strain CA10 harboring pCAR2 instead of complete nucleotide sequences were virtually the same, pCAR1, was cultivated in Luria broth7) for 15 h. The and can be regarded as structurally the same plasmid, full-growth culture (100 ml) was used for extraction of indicating that the difference in the conjugative host pCAR2. The preparation of pCAR2 from the cells was range was caused by host cell backgrounds. carried out using a Large-Construct Kit (Qiagen, Santa Clarita, CA) according to the protocol recommended by Key words: carbazole; host range; IncP-7 plasmid; the manufacturer. Shotgun sequencing was performed pCAR1; Pseudomonas by Hitachi High-Technologies (Tokyo). After sequenc- ing, the two assembled contigs were aligned with a pCAR1 is a carbazole degradative plasmid isolated whole sequence of pCAR1 using the MAVID program from Pseudomonas resinovorans CA10.1,2) The entire (http://baboon.math.berkeley.edu/mavid/).8) Possible two 199,035-bp nucleotide sequence of the plasmid3) and gap regions, which corresponded to 13,000–13,077 and incompatibility testing4) indicated that pCAR1 belongs 168,560–168,683 in pCAR1, sequenced in 2003,3) were to the IncP-7 group. On the other hand, another PCR-amplified from pCAR2 using two primer sets for carbazole degradative plasmid, pCAR2, was isolated each gap: GapA-1F [50-CAAATGTATCGCCTGGC- from Pseudomonas putida HS01, and had a genetic TTG-30] and GapA-1R [50-GAACAACTGCCCTCC- structure similar to that of pCAR1.5) Mating analyses AACAC-30], GapA-2F [50-TGGGAGCAAGGGTCTT- showed that both pCAR1 and pCAR2 are self- ACAC-30] and GapA-2R [50-GCCATGAGCACATCT- transmissible plasmids, and that the conjugative host ACTGG-30], GapB-1F [50-GCGTCTCTCTGCACAAA- (recipient) range of pCAR2 from HS01 (P. chlorora- CCT-30] and GapB-1R [50-GTCGGTAGTGCTTTCGC- phis, P. fluorescens, P. putida, P. resinovorans, and TTT-30], and GapB-2F [50-GATGTATGCAGAAGCG- P. stutzeri) was broader than that of pCAR1 from TGGA-30] and GapB-2R [50-ATAGCTGCGTGCCT- CA10 (P. putida and P. resinovorans).6) When pCAR1 GAAGAT-30]. The reaction mixture (25 ml) contained and pCAR2 were transferred individually from the same 1 mg of isolated pCAR2, 25 mmol of each primer, 5 mmol cell background, the range and frequency of conjugation of each dNTP, 1Â Ex Taq Buffer (Takara Bio, Shiga, were similar.6) Shintani et al. assumed the two reasons Japan), and 0.625 units of Ex Taq DNA polymerase for this differences as follows: (i) although the genetic (Takara Bio). The reaction conditions were as follows: structures of the two plasmids are similar,5) it is possible 30 s (94 C) to denature DNA and 30 cycles of 30 s that the conjugative functions are different because of (94 C), 30 s (62 C), and 1 min (72 C). The 269-bp, somewhat nucleotide alterations; and/or (ii) if the 534-bp, 309-bp, and 710-bp resulting PCR products conjugative genes in both strains are at least functional, were cloned into pT7Blue T-vector (Invitrogen, Carls- the differences in the host factors supplied by the donor bad, CA), and the nucleotide sequences of the inserts cell can cause the change in the conjugative host range. were determined using either an ABI Prism 310 DNA Previous transcriptional analyses showing that tra/trh analyzer (Applied Biosystems, Warrington, UK) or a genes on pCAR1 and pCAR2 were similarly transcribed model 4200L-2 auto-DNA sequencer (LI-COR, Lincoln, in CA10 and HS01 respectively suggested the possibil- NE), as described previously.5) Each gap region was ities that the conjugative machinery of both plasmids sequenced at least 3 times using independent clones. As functioned similarly, and that unknown host factors a result, the pCAR2 was established as a 200,231-bp affected the conjugative recipient range and/or fre- circular plasmid. There were only two differences quency of conjugation. In this study, to attain a definite between pCAR2 and pCAR13) (Fig. 1). One was a conclusion as to whether the conjugative host range is single nucleotide substitution, and the other was the affected by the plasmid or the host chromosome, we additional copy of insertion sequence (IS), ISPre1. A determined the complete nucleotide sequence of pCAR2 single base substitution was found in the tnpA1 trans- to elucidate the differences between pCAR2 and pCAR1 posase gene: the 25,757th thymine in the pCAR1 more precisely. sequence published in 2003 was replaced by cytosine The entire nucleotide sequence of pCAR2 was in pCAR2. Nonetheless, the substitution was at the third y To whom correspondence should be addressed. Fax: +81-3-5841-8030; E-mail: [email protected] Abbreviations: DR, direct repeat; IR, inverted repeat; IS, insertion sequence; ORF, open reading frame Complete Nucleotide Sequence of pCAR2 745 Single base base of the codon and did not affect the amino acid substitution sequence of the encoded protein. On pCAR1 from CA10, we found four homologous ISs (three copies of ISPre1 and a single copy of ISPre2), which located ant around the car and ant operons (Fig. 1).3) In the car nucleotide sequence of pCAR2, we found an additional pCAR1 copy of ISPre1 in the intergenic region between the recT 199,035 bp gene and ORF117 (Figs. 1 and 2). We named the transposase gene in the additional copy of ISPre1, tnpA5. The ISPre1 found in this study was identical to ISPre1 containing tnpA4 and ISPre1 containing tnpA1, and nearly identical to ISPre1 containing tnpA2, with pCAR2 one base pair mismatch (in the 1,192-bp nucleotide Additional sequence between the direct repeats). 200,231 bp copy of ISPre1 Previously, Southern hybridization analysis indicated that pCAR1 or the chromosome of strain CA10 (the Fig. 1. Schematic Representation of pCAR2 Compared with pCAR1. original host of pCAR1) contained an additional copy of The circle represents pCAR1. Each pentagon represents one open 2) reading frame (ORF). The black boxes with white pentagons and the ISPre1, although pCAR1 just after the isolation of one with the dotted pentagon indicate ISPre1 and ISPre2 respec- P. resinovorans CA10, which was sequenced in 2003, tively. The gray arrows indicate the car or ant operons. A single had three copies of ISPre1.3) As shown in Fig. 3, the base substitution was found in the tnpA1 transposase gene of ISPre1: restriction fragment length predicted from physical the 25,757th thymine in the pCAR1 sequence was replaced by mapping around ISPre1 of pCAR2 was in agreement cytosine in pCAR2. An additional copy of ISPre1 was found in the intergenic region between the recT gene and ORF117. In the with that of the DNA fragments from the previously 2) DDBJ/EMBL/GenBank DNA databases, pCAR2 was renamed as unidentified ISPre1. Hence we speculate that pCAR1, pCAR1.2 (AB474758). used in the previous analysis of 2001,2) has an additional Sequenced pCAR1 recT ORF117 pCAR2 recT tnpA5 ORF117 Sequenced pCAR1 tnpA4 tnpA1 tnpA2 ISPre1 Fig. 2. Insertion Positions of an Additional Copy of ISPre1 on pCAR2 and Its Alignment with the Other Copies of ISPre1 on pCAR1. Bold lines indicates the nucleotide sequences, and the pentagons indicate genes with transcriptional directions. The inverted repeats (IRs) of ISPre1 and direct repeats (DRs) formed by the transposition are indicated in boxes and underlining respectively. Each number corresponds to the nucleotide number in the pCAR1 sequence (DDBJ/EMBL/GenBank accession no. AB088420) published in 2003. ABRV Eco I RI+ I RI dIII I I I RV RI I RV I RI dIII dIII dIII Eco Sma Kpn Hin Eco (kb) Sma Kpn Eco Eco Hin Pvu Hin Eco Kpn Hin Eco Sma 19.3 EcoRI+EcoRV 7.7 SmaI 6.2 4.3 KpnI 3.5 2.7 HindIII 5 kb EcoRI 1.9 1.5 Fig. 3. Comparison of Restriction Enzyme Sites around ISPre1 Containing the tnpA5 Gene with Previous Southern Hybridization Results.2) A, Southern hybridization results2) for digested total DNA of CA10 using the 332-bp PvuI-HindIII fragment of ISPre1 as a probe. Restriction endonucleases used in digestion are shown in the panel. In each lane, the bands that cannot be explained from the pCAR1 sequence determined in 20033) are indicated by arrowheads. B, Physical map and restriction fragments around ISPre1, containing the tnpA5 gene found between recT and ORF117 on pCAR2. The pentagons on the physical map indicate the sizes, locations, and directions of transcription of the ORFs. The black box indicates ISPre1 containing the tnpA5 gene. The solid lines indicate the locations of the restriction fragments using the enzymes shown at the left. The shaded region corresponds to the 332-bp PvuI-HindIII fragment used in the previous hybridization analysis. 746 Y. TAKAHASHI et al. copy of ISPre1 in the same region as pCAR2. This result Acknowledgment suggests that ISPre1 transposed to the additional region of pCAR1, which is between the recT gene and This study was supported by the Program for ORF117, since its isolation in 19931) following a Promotion of Basic Research Activities for Innovative previous Southern hybridization study published in Biosciences (PROBRAIN) of Japan.

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