Plasmids Responsible for Horizontal Transfer of Naphthalene Catabolism Genes between Bacteria at a Coal Tar-Contaminated Site Are Homologous to pDTG1 from Pseudomonas putida NCIB 9816-4

Abstract

The presence of a highly conserved nahAc allele among phylogenetically diverse bacteria carrying naphthalene-catabolic plasmids provided evidence for in situ horizontal gene transfer at a coal tar-contaminated site (J. B. Herrick, K. G. Stuart-Keil, W. C. Ghiorse, and E. L. Madsen, Appl. Environ. Microbiol. 63:2330–2337, 1997). The objective of the present study was to identify and characterize the different-sized naphthalene-catabolic plasmids in order to determine the probable mechanism of horizontal transfer of the nahAc gene in situ. Filter matings between naphthalene-degrading bacterial isolates and their cured progeny revealed that the naphthalene-catabolic plasmids were self-transmissible. Limited interstrain transfer was also found. Analysis of the restriction fragment length polymorphism (RFLP) patterns indicated that catabolic plasmids from 12 site-derived isolates were closely related to each other and to the naphthalene-catabolic plasmid (pDTG1) of Pseudomonas putidaNCIB 9816-4, which was isolated decades ago in Bangor, Wales. The similarity among all site-derived naphthalene-catabolic plasmids and pDTG1 was confirmed by using the entire pDTG1 plasmid as a probe in Southern hybridizations. Two distinct but similar naphthalene-catabolic plasmids were retrieved directly from the microbial community indigenous to the contaminated site in a filter mating by using a cured, rifampin-resistant site-derived isolate as the recipient. RFLP patterns and Southern hybridization showed that both of these newly retrieved plasmids, like the isolate-derived plasmids, were closely related to pDTG1. These data indicate that a pDTG1-like plasmid is the mobile genetic element responsible for transferring naphthalene-catabolic genes among bacteria in situ. The pervasiveness and persistence of this naphthalene-catabolic plasmid suggest that it may have played a role in the adaptation of this microbial community to the coal tar contamination at our study site.

Raman‐FISH: combining stable‐isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function

“…Therefore, using the Raman‐FISH approach, we could directly assign and quantify the metabolism of the labelled compound in situ to a key group which we knew to possess the capability from independent measures (selective isolation and molecular characterization of the strains). Such whole cell findings confirm previous reports on the naphthalene degrading capability of members of the genus Pseudomonas (Cane and Williams, 1982; Yen and Serdar, 1988; Simon et al ., 1993; Stuart‐Keil et al ., 1998; Dennis and Zylstra, 2004; Izmalkova et al ., 2006) . More specifically, pseudomonads have previously been implicated as important members of the in situ community during large scale remediation strategies at the site from which the samples were derived (Ferguson et al.…”

Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool

“…The main advantages of this method are its direct selection for transferable plasmids and its independence from culturing plasmid hosts. This approach has been successfully used by several groups to isolate a wide diversity of plasmids that encode Hg R or antibiotic resistance, or the ability to degrade a specific organic compound, from various environments (Bale et al, 1987(Bale et al, , 1988Fry & Day, 1990;Top et al, 1995;Lilley et al, 1996;Dahlberg et al, 1997;Drnen et al, 1998Drnen et al, , 1999Smit et al, 1998;Stuart-Keil et al, 1998;Dröge et al, 2000;Schneiker et al, 2001;Heuer et al, 2002;van Overbeek et al, 2002;Smalla et al, 2006;Ono et al, 2007). All of the six plasmids from WWTP described in detail in this review, and pKJK5 from barley rhizosphere (Bahl et al, 2007), were obtained using this method with selection on antibiotic resistance.…”

Coselection for resistance to multiple late-generation human therapeutic antibiotics encoded on tetracycline resistance plasmids captured from uncultivated stream and soil bacteria

“…Transconjugants were verified as the recipient host strain (Ps. putida SM1315) by rep-PCR fingerprinting using BOX primers (Rademaker et al 1998;Stuart-Keil et al 1998). Cells were lysed, and PCR amplification carried out essentially as described previously for ERIC PCR fingerprinting (Herrick et al 1997), except with the single BOX primer (Rademaker et al 1998) and using the Failsafe PCR system with buffer G (Epicentre Technologies, Inc., Madison WI) according to manufacturer's instructions.…”

“…This method allows for the capture of plasmids that are transmissible and actively transferring, even from bacteria that have not been cultured. It has been used to isolate mercury resistance plasmids (Bale et al 1988;Lilley et al 1996;Dahlberg et al 1997), pollutant catabolic plasmids (Stuart-Keil et al 1998;Hohnstock et al 2000), mobilizing plasmids (which can mobilize other, nonself-transmissible plasmids) (Dronen et al 1998;Van Elsas et al 1998) and transmissible antibiotic resistance plasmids from manure and other habitats (Smalla et al 2000;Heuer et al 2011). A phenotype-independent plasmid capture method has been used to isolate plasmids from the human gut microflora (Jones and Marchesi 2007;Jones et al 2010).…”