Recipient Range of IncP-7 Conjugative Plasmid pCAR2 from Pseudomonas putida HS01 is Broader than from Other Pseudomonas Strains

The carbazole-degradative plasmid pCAR2 was isolated from Pseudomonas putida and had a genetic structure similar to that of pCAR1, the IncP-7 archetype plasmid. Mating analyses of pCAR2 with various recipient strains showed that it could transfer from HS01 to Pseudomonas recipients: P. chlororaphis, P. fluorescens, P. putida, P. resinovorans and P. stutzeri. The range of recipients changed when different hosts were used as a donor of pCAR2. The range of the plasmid from strain HS01 was broader than that using P. resinovorans CA10dm4 or P. putida KT2440. When pCAR1 or pCAR2 was transferred from the same cell background, the range and frequency of conjugation were now similar. Quantitative RT-PCR analyses indicated that tra/trh genes on both plasmids were similarly transcribed in each donor strain suggesting that the conjugative machinery of both plasmids may function similarly, and that other host factors are affecting the recipient range and frequency of conjugation.     

Behavior of the IncP-7 carbazole-degradative plasmid pCAR1 in artificial environmental samples

In artificial environmental samples, the behavior of the IncP-7 conjugative plasmid pCAR1, which is involved in the catabolism of carbazole, was monitored. Sterile soil and water samples supplemented with carbazole were prepared. After inoculation with Pseudomonas putida harboring pCAR1, seven species of the genus Pseudomonas, and three other bacterial species, were monitored for carbazole degradation, bacterial survival, and conjugative transfer of pCAR1. In artificial soils, more than 90% of the carbazole was degraded in samples with high water content, suggesting that the water content is a key factor in carbazole degradation in artificial soils. In three of the artificial environmental water samples, more than 95% of the carbazole was degraded. Transconjugants were detected in some artificial water samples, but not in the artificial soil samples, suggesting that pCAR1 is preferably transferred in aqueous environments. Composition analysis of the artificial water samples and examination of conjugative transfer indicated that the presence of the divalent cations Ca(2+) and Mg(2+) promoted the plasmid transfer. The presence of carbazole also increases in incidence of transconjugants, probably by enhancing their growth. In contrast, humic acids in the liquid layer of artificial soil samples appeared to prevent conjugative transfer.     

Complete nucleotide sequence of carbazole/dioxin-degrading plasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and the presence of large catabolic transposon Tn4676

The car and ant operons originally isolated from Pseudomonas resinovorans strain CA10 contain the genes encoding the carbazole/dioxin-degrading enzymes and anthranilate 1,2-dioxygenase, respectively, and are located on the plasmid pCAR1. The entire nucleotide sequence of pCAR1 was determined to elucidate the mechanism by which the car operon may have been assembled and distributed in nature. pCAR1 is a 199,035-bp circular plasmid, and carries 190 open reading frames. Although the incompatibility group of pCAR1 is unclear, its potential origin for replication, OriP, and Rep and Par proteins appeared to be closely related to those of plasmid pL6.5 isolated from Pseudomonas fluorescens. The potential tellurite-resistance klaABC genes identified in the neighboring region of repA gene were also related to those in IncP plasmid originally identified from pseudomonads. On the other hand, we found genes encoding proteins that showed low but significant homology (20-45% identity) with Trh and Tra proteins from Enterobacteriaceae, which are potentially involved in conjugative transfer of plasmids or genomic island, suggesting that pCAR1 is also a conjugative plasmid. In pCAR1, we found tnpAcCST genes that encoded the proteins showing >70% length-wise identities with those are encoded by the toluene/xylene-degrading transposon Tn4651 of TOL plasmid pWW0. Both car and ant degradative operons were found within a 72.8-kb Tn4676 sequence defined by flanking tnpAcC and tnpST genes and bordered by a 46-bp inverted repeat (IR). Within Tn4676 and its flanking region, we found the remnants of numerous mobile genetic elements, such as the duplicated transposase genes that are highly homologous to tnpR of Tn4653 and the multiple candidates of IRs for Tn4676 and Tn4653-like element. We also found distinct regions with high and low G+C contents within Tn4676, which contain an ant operon and car operon, respectively. These results suggested that multiple step assembly could have taken place before the current structure of Tn4676 had been captured.     

Large plasmid pCAR2 and class II transposon Tn4676 are functional mobile genetic elements to distribute the carbazole/dioxin-degradative car gene cluster in different bacteria

The carbazole-catabolic plasmid pCAR1 isolated from Pseudomonas resinovorans strain CA10 was sequenced in its entirety; and it was found that pCAR1 carries the class II transposon Tn4676 containing carbazole-degradative genes. In this study, a new plasmid designated pCAR2 was isolated from P. putida strain HS01 that was a transconjugant from mating between the carbazole-degrader Pseudomonas sp. strain K23 and P. putida strain DS1. Southern hybridization and nucleotide sequence analysis of pCAR1 and pCAR2 revealed that the whole backbone structure was very similar in each. Plasmid pCAR2 was self-transmissible, because it was transferred from strain HS01 to P. fluorescens strain IAM12022 at the frequency of 2×10^–7 per recipient cell. After the serial transfer of strain HS01 on rich medium, we detected the transposition of Tn4676 from pCAR2 to the HS01 chromosome. The chromosome-located copy of Tn4676 was flanked by a 6-bp target duplication, 5-AACATC-3. These results experimentally demonstrated the transferability of pCAR2 and the functionality of Tn4676 on pCAR2. It was clearly shown that plasmid pCAR2 and transposon Tn4676 are active mobile genetic elements that can mediate the horizontal transfer of genes for the catabolism of carbazole.     

Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities

The transferability of plasmids pCAR1, pB10, R388, and NAH7 was compared using the same donor-recipient system at different cell density combinations in liquid or on a solid surface. pCAR1 was efficiently transferred in liquid, whereas the other plasmids were preferentially transferred on a solid surface. Difference of liquid or solid affected the transfer frequency especially at lower cell densities.     

Characterization of the Replication, Maintenance, and Transfer Features of the IncP-7 Plasmid pCAR1, Which Carries Genes Involved in Carbazole and Dioxin Degradation

Isolated from Pseudomonas resinovorans CA10, pCAR1 is a 199-kb plasmid that carries genes involved in the degradation of carbazole and dioxin. The nucleotide sequence of pCAR1 has been determined previously. In this study, we characterized pCAR1 in terms of its replication, maintenance, and conjugation. By constructing miniplasmids of pCAR1 and testing their establishment in Pseudomonas putida DS1, we show that pCAR1 replication is due to the repA gene and its upstream DNA region. The repA gene and putative oriV region could be separated in P. putida DS1, and the oriV region was determined to be located within the 345-bp region between the repA and parW genes. Incompatibility testing using the minireplicon of pCAR1 and IncP plasmids indicated that pCAR1 belongs to the IncP-7 group. Monitoring of the maintenance properties of serial miniplasmids in nonselective medium, and mutation and complementation analyses of the parWABC genes, showed that the stability of pCAR1 is attributable to the products of the parWAB genes. In mating assays, the transfer of pCAR1 from CA10 was detected in a CA10 derivative that was cured of pCAR1 (CA10dm4) and in P. putida KT2440 at frequencies of 3 × 10⁻¹ and 3 × 10⁻³ per donor strain, respectively. This is the first report of the characterization of this completely sequenced IncP-7 plasmid.     

Characterization of the replication, maintenance, and transfer features of the IncP-7 plasmid pCAR1, which carries genes involved in carbazole and dioxin degradation

Isolated from Pseudomonas resinovorans CA10, pCAR1 is a 199-kb plasmid that carries genes involved in the degradation of carbazole and dioxin. The nucleotide sequence of pCAR1 has been determined previously. In this study, we characterized pCAR1 in terms of its replication, maintenance, and conjugation. By constructing miniplasmids of pCAR1 and testing their establishment in Pseudomonas putida DS1, we show that pCAR1 replication is due to the repA gene and its upstream DNA region. The repA gene and putative oriV region could be separated in P. putida DS1, and the oriV region was determined to be located within the 345-bp region between the repA and parW genes. Incompatibility testing using the minireplicon of pCAR1 and IncP plasmids indicated that pCAR1 belongs to the IncP-7 group. Monitoring of the maintenance properties of serial miniplasmids in nonselective medium, and mutation and complementation analyses of the parWABC genes, showed that the stability of pCAR1 is attributable to the products of the parWAB genes. In mating assays, the transfer of pCAR1 from CA10 was detected in a CA10 derivative that was cured of pCAR1 (CA10dm4) and in P. putida KT2440 at frequencies of 3 x 10(-1) and 3 x 10(-3) per donor strain, respectively. This is the first report of the characterization of this completely sequenced IncP-7 plasmid.     

Conjugative transfer of the IncP-7 carbazole degradative plasmid, pCAR1, in river water samples

The transfer of the IncP-7 carbazole degradative plasmid pCAR1 from Pseudomonas putida SM1443 (derived from strain KT2440) into bacteria of river water samples was monitored using a reporter gene encoding red fluorescent protein (RFP). The number of transconjugants drastically increased in the presence of carbazole, and most appeared to belong to the genus Pseudomonas. The results suggest that the presence of carbazole benefits the appearance of transconjugants belonging to the genus Pseudomonas. Intriguingly, we also detected the transfer of pCAR1 into non-Pseudomonas, Stenotrophomonas-like bacteria.