Naphthalene and Donor Cell Density Influence Field Conjugation of Naphthalene Catabolism Plasmids

Abstract

We examined transfer of naphthalene-catabolic genes from donor microorganisms native to a contaminated site to site-derived, rifampin-resistant recipient bacteria unable to grow on naphthalene. Horizontal gene transfer (HGT) was demonstrated in filter matings using groundwater microorganisms as donors. Two distinct but similar plasmid types, closely related to pDTG1, were retrieved. In laboratory-incubated sediment matings, the addition of naphthalene stimulated HGT. However, recipient bacteria deployed in recoverable vessels in the field site (in situ) did not retrieve plasmids from native donors. Only when plasmid-containing donor cells and naphthalene were added to the in situ mating experiments did HGT occur.

nahR, encoding a LysR-type transcriptional regulator, is highly conserved among naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site and in extracted community DNA b bThe GenBank accession number for the sequences of the tnpA-like gene, nahG and nahR of P. putida NCIB 9816-4 is AF491307. The GenBank accession numbers for the sequences of the nahR–nahG intergenic region and the nahR homologue genes of strains Cg1, Cg2, Cg5, Cg7, Cg9, Cg11, Hg8 and N1 are AF491308–AF491315, respect

“…The degenerate primers designed from these nahR sequences successfully amplified the entire nahR-like genes (921 bp ; using primers onahR-F and onahR-R) and internal fragments (315 bp ; using onahR-F and inahR-R) from five wild-type strains, Cg1, Cg2, Cg5, Cg7, Cg9, (Fig. 2, lanes 9, 10 ; 13, 14 ; 17, 18 ; 21, 22 ; and 25, 26 ; respectively) from our coal tar waste study site in Glens Falls, NY (Herrick, 1995 ;Herrick et al, 1997 ;Hohnstock et al, 2000). P. putida NCIB 9816-4 and P. putida G7 (isolated originally from soil from Bangor, Wales and Berkeley, CA, respectively) were used as positive controls (Fig.…”

“…Bacterial isolates (Cg1, Pseudomonas putida Cg1 ; Cg2, Pseudomonas fluorescens Cg2 ; Cg5, P. fluorescens Cg5 ; Cg7, Pseudomonas strain Cg 7 ; Cg9, P. fluorescens Cg9 ; Cg11, Pseudomonas mendocina Cg11 ; N1, P. fluorescens N1 ; and Hg8, Burkholderia gladioli Hg8) and their plasmids from the coal-tarcontaminated site have been described (Herrick, 1995 ;Herrick et al, 1997 ;Hohnstock et al, 2000 ;Stuart-Keil et al, 1998). These and other bacterial strains, vectors and plasmids used in this study are listed in Table 1.…”

“…Naphthalene-degrading bacteria, and plasmid-cured and other strains derived therefrom were grown at 22 mC (room temperature) and maintained on mineral salts basal medium (MSB ; Stanier, 1966) with either naphthalene (vapour or a suspension of crystals ; MSB-N) or glucose (MSB-G) as sole carbon source. Other culture media were prepared according to the procedures described by Herrick et al (1997) and Hohnstock et al (2000). All Escherichia coli strains were grown with shaking (60 r.p.m.)…”

“…Sediment samples were obtained in South Glens Fall, NY (Herrick, 1995 ;Herrick et al, 1997 ;Hohnstock et al, 2000). Sediments representing a broad spectrum of locations and contaminant exposure histories were examined.…”

Gentisate 1,2-dioxygenase, in the third naphthalene catabolic gene cluster of Polaromonas naphthalenivorans CJ2, has a role in naphthalene degradation

“…Then, to construct the nagI knockout mutants, pK19mobsacBgnagIx was mobilized from E. coli S17-1/lpir cells containing pK19mobsacBDnagIx (~5.0610 8 -1.0610 9 , grown overnight in LB broth with 20 mg kanamycin ml 21 ) into rifampicin-resistant P. naphthalenivorans CJ2 cells (~1.0610 9 to 2.0610 9 , grown overnight in MSB-P broth with 200 mg rifampicin ml 21 ) through mating on R2A agar medium at 25 uC for 8-16 h as described by Hohnstock et al (2000) and Park et al (2007a). Three transconjugants (strains CJ2DnagIxkm r ) were selected from each of the R2A plates containing kanamycin (20 mg ml 21 ) and rifampicin (200 mg ml 21 ) at 25 uC.…”