Carbazole-Degradative IncP-7 Plasmid pCAR1.2 Is Structurally Unstable in Pseudomonas fluorescens Pf0-1, Which Accumulates Catechol, the Intermediate of the Carbazole Degradation Pathway

Abstract

We determined the effect of the host on the function and structure of the nearly identical IncP-7 carbazoledegradative plasmids pCAR1.1 and pCAR1.2. We constructed Pseudomonas aeruginosa PAO1(pCAR1.2) and P. fluorescens Pf0-1Km(pCAR1.2) and compared their growth on carbazole-and succinate-containing media with that of P. putida KT2440(pCAR1.1). We also assessed the stability of the genetic structures of the plasmids in each of the three hosts. Pf0-1Km(pCAR1.2) showed dramatically delayed growth when carbazole was supplied as the sole carbon source, while the three strains grew at nearly the same rate on succinate. Among the carbazole-grown Pf0-1Km(pCAR1.2) cells, two types of deficient strains appeared and dominated the population; such dominance was not observed in the other two strains or for succinate-grown Pf0-1Km(pCAR1.2). Genetic analysis showed that the two deficient strains possessed pCAR1.2 derivatives in which the carbazoledegradative car operon was deleted or its regulatory gene, antR, was deleted by homologous recombination between insertion sequences. From genomic information and quantitative reverse transcription-PCR analyses of the genes involved in carbazole mineralization by Pf0-1Km(pCAR1.2), we found that the cat genes on the chromosome of Pf0-1Km, which are necessary for the degradation of catechol (a toxic intermediate in the carbazole catabolic pathway), were not induced in the presence of carbazole. The resulting accumulation of catechol may have enabled the strain that lost its carbazole-degrading ability to have overall higher fitness than the wild-type strain. These results suggest that the functions of the chromosomal genes contributed to the selection of plasmid derivatives with altered structures.Horizontal gene transfer plays an important role in the evolution and adaptation of prokaryotes (12,40). Plasmids are extrachromosomal elements that are replicated autonomously and provide beneficial functions to their hosts, such as resistance to antibiotics and degradation of recalcitrant aromatic compounds. The essential functions encoded on plasmid backbones, e.g., replication initiation, active partitioning, and conjugative transfer, are partially dependent on host factors (1). For example, De Gelder et al. (5) showed that the host range of the multiresistance plasmid pB10 was influenced by donor strains within an activated-sludge microbial community. Accessory genes are also regulated by host factors, and there are reports showing that catabolic operons on plasmids require host regulatory elements (21, 28). Although these results suggest the importance of the host in imparting plasmid function, additional investigations are necessary to understand the mechanisms of plasmid survival and to determine how plasmid dynamics are affected by hosts with different types of genomic organization.The carbazole-catabolic plasmid pCAR1 was discovered in Pseudomonas resinovorans CA10 (27) and was the first IncP-7 plasmid to be completely sequenced (20,26,36). Carbazole is a structural analog of dioxin...

Behavior of Various Hosts of the IncP-7 Carbazole-Degradative Plasmid pCAR1 in Artificial Microcosms

“…intermediate compound of the carbazole degradation pathway, and accumulation of catechol is toxic to the cells), 16) (ii) they may have been alive and may have adhered to the particles in the soil microcosms, as is confirmed by the fact that Pf0-1 was isolated as a strain adhesive to the soil particles. 24) Carbazole degraders in the other soil microcosms inoculated with CA10L(pCAR1::rfp) or IAM1511L(pCAR1::rfp) decreased similarly, although the rates of the latter were slower than those of the former (Fig.…”

“…2). Considering that Pf0-1L(pCAR1::rfp) did not degrade carbazole effectively, 16) it is not surprising that the carbazole degraders decreased and that we did not detect carbazole degradation in MRA (Fig. 2B).…”

“…For instance, we found a difference in host ability to metabolize carbazole as between P. putida KT2440(pCAR1) and P. fluorescens Pf0-1(pCAR1), and that the growth rate of Pf0-1(pCAR1) is much slower than that of KT2440(pCAR1). 16) This difference in the growth rate is due to distinct regulation mechanisms of gene transcription encoding catechol degradation systems, catechol being an intermediate compound in the carbazole degradation pathway. 16) In this study, we constructed three new host strains of pCAR1::rfp (P. fluorescens, P. resinovorans, and P. chlororaphis), and monitored their behaviors in artificial microcosms.…”

“…16) This difference in the growth rate is due to distinct regulation mechanisms of gene transcription encoding catechol degradation systems, catechol being an intermediate compound in the carbazole degradation pathway. 16) In this study, we constructed three new host strains of pCAR1::rfp (P. fluorescens, P. resinovorans, and P. chlororaphis), and monitored their behaviors in artificial microcosms. A comparison of behavior among different host strains gave us insight into important aspects enabling us to predict their fates in the natural environment.…”

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

“…In the case of pCAR1-containing Pf0-1, the growth defect on medium supplemented with carbazole as sole carbon and energy source stemmed from differential regulation of the transcription of catechol-degradative cat genes in Pf0-1 and other hosts. 92) Two sets of cat genes encoding catechol ortho-cleavage pathway enzymes in Pf0-1 are present in benzene-degradative ben operons. No expression of these was induced by metabolic intermediate compounds of the catechol degradative pathway, but it was induced by benzoate, 92) whereas those in the other hosts were induced by cis,cis-muconate, a metabolic intermediate of catechol.…”