Identification and characterization of the conjugal transfer region of the pCg1 plasmid from naphthalene-degrading Pseudomonas putida Cg1

Woojun Park, Che O. Jeon, Amy M. Hohnstock-Ashe, Stephen C. Winans, Gerben J. Zylstra, Eugene L. Madsen

Research output: Contribution to journalArticle

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Abstract

Hybridization and restriction fragment length polymorphism data (K. G. Stuart-Keil, A. M. Hohnstock, K. P. Drees, J. B. Herrick, and E. L. Madsen, Appl. Environ. Microbiol. 64:3633-3640, 1998) have shown that pCg1, a naphthalene catabolic plasmid carried by Pseudomonas putida Cg1, is homologous to the archetypal naphthalene catabolic plasmid, pDTG1, in P. putida NCIB 9816-4. Sequencing of the latter plasmid allowed PCR primers to be designed for amplifying and sequencing the conjugal transfer region in pCg1. The mating pair formation (mpf) gene, mpfA encoding the putative precursor of the conjugative pilin subunit from pCg1, was identified along with other trb-like mpf genes. Sequence comparison revealed that the 10 mpf genes in pCg1 and pDTG1 are closely related (61 to 84% identity) in sequence and operon structure to the putative mpf genes of catabolic plasmid pWW0 (TOL plasmid of P. putida) and pM3 (antibiotic resistance plasmid of Pseudomonas. spp). A polar mutation caused by insertional inactivation in mpfA of pCg1 and reverse transcriptase PCR analysis of mRNA showed that this mpf region was involved in conjugation and was transcribed from a promoter located upstream of an open reading frame adjacent to mpfA. lacZ transcriptional fusions revealed that mpf genes of pCg1 were expressed constitutively both in liquid and on solid media. This expression did not respond to host exposure to naphthalene. Conjugation frequency on semisolid media was consistently 10- to 100-fold higher than that in liquid media. Thus, conjugation of pCg1 in P. putida Cg1 was enhanced by expression of genes in the mpf region and by surfaces where conditions fostering stable, high-density cell-to-cell contact are manifest.

Original languageEnglish
Pages (from-to)3263-3271
Number of pages9
JournalApplied and Environmental Microbiology
Volume69
Issue number6
Publication statusPublished - 2003 Jun 1
Externally publishedYes

Fingerprint

Pseudomonas putida
naphthalene
plasmid
plasmids
Plasmids
gene
Genes
genes
Fimbriae Proteins
liquid
Foster Home Care
liquids
antibiotic resistance
operon
Operon
Microbial Drug Resistance
Pseudomonas
Reverse Transcriptase Polymerase Chain Reaction
Restriction Fragment Length Polymorphisms
Open Reading Frames

ASJC Scopus subject areas

  • Biotechnology
  • Environmental Science(all)
  • Microbiology

Cite this

Identification and characterization of the conjugal transfer region of the pCg1 plasmid from naphthalene-degrading Pseudomonas putida Cg1. / Park, Woojun; Jeon, Che O.; Hohnstock-Ashe, Amy M.; Winans, Stephen C.; Zylstra, Gerben J.; Madsen, Eugene L.

In: Applied and Environmental Microbiology, Vol. 69, No. 6, 01.06.2003, p. 3263-3271.

Research output: Contribution to journalArticle

Park, Woojun ; Jeon, Che O. ; Hohnstock-Ashe, Amy M. ; Winans, Stephen C. ; Zylstra, Gerben J. ; Madsen, Eugene L. / Identification and characterization of the conjugal transfer region of the pCg1 plasmid from naphthalene-degrading Pseudomonas putida Cg1. In: Applied and Environmental Microbiology. 2003 ; Vol. 69, No. 6. pp. 3263-3271.
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AU - Park, Woojun

AU - Jeon, Che O.

AU - Hohnstock-Ashe, Amy M.

AU - Winans, Stephen C.

AU - Zylstra, Gerben J.

AU - Madsen, Eugene L.

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