Physical mapping and characterization of a catabolic plasmid from the deep-subsurface bacterium Sphingomonas sp. strain F199

Abstract

A supercoiled 180-kb plasmid, pNL1, has been isolated from the deep-subsurface, chemoheterotrophic Sphingomonas sp. strain F199, and a physical map was generated. Analysis of a pNL1-derived cosmid library indicated that catechol 2,3-dioxygenase activity was linked to two distinct regions of the plasmid. Thus, the genes for aromatic catabolism in this Sphingomonas strain are, at least in part, plasmid encoded.Surface soil and aquatic bacteria capable of using aromatic hydrocarbons as carbon and energy sources are relatively common and make in situ bioremediation of petroleum hydrocarbon-contaminated soils and shallow sediments using indigenous microorganisms feasible. Although many of these catabolic pathways have been described for bacteria residing in soil and near-surface environments (23), relatively little is known about the physiology of bacteria in the deep subsurface.A subsurface organism, strain F199, isolated from uncontaminated sediments 407 m below the soil surface at the Department of Energy Savannah River Site has the ability to use toluene, all xylene isomers, p-cresol, naphthalene, salicylate, and benzoate as sole carbon and energy sources (7). This isolate has been classified in the genus Sphingomonas on the basis of 16S rRNA gene sequence and fatty acid analysis (8). Although the members of the genus Sphingomonas are known to have extensive abilities to degrade xenobiotic compounds (15,19,25), little is known about the genes involved or their organization.Strain F199 was previously shown to harbor two large plasmids of over 100 kb (7). Because catabolic genes are often found on plasmids in aerobic, gram-negative bacteria (22,26,27), the plasmids in F199 were of particular interest.Megaplasmid isolation. A number of approaches (1, 9, 11, 24) were used in attempts to purify F199 plasmid DNA, with minimal success, possibly because of copurification of polysaccharides produced by this microorganism. Better yields of plasmid were obtained by the method of Casse et al. (3); for this, F199 cells were grown in King's medium B (13) at 30ЊC in shaking cultures. To remove small fragments of DNA, the precipitated DNA resulting from alkaline lysis purification was dissolved in 400 l of 10 mM Tris-HCl-1 mM EDTA (pH 7.5) (TE) and further purified by centrifugation (2 h at 100,000 ϫ g) through a sucrose gradient consisting of 2.5-ml layers of 40, 30, 20, and 10% (wt/vol) sucrose in TE. Fractions containing pure plasmid were pooled and concentrated by use of Centricon 100 (Amicon Inc., Beverly, Mass.) spin filters or by ethanol precipitation. These procedures resulted in an essentially pure preparation of a single large plasmid termed pNL1.Previous data (7) suggested that F199 contained two megaplasmids. While working out plasmid isolation procedures, it became apparent that the two bands seen earlier were different forms of pNL1. Nonetheless, recent results (data not shown) indicate that F199 indeed has a second, even larger plasmid, but this replicon could not be isolated by the methods described above.Megapla...

Degradation of phenanthrene via meta-cleavage of 2-hydroxy-1-naphthoic acid by Ochrobactrum sp. strain PWTJD

“…3c), indicating the presence of catechol 2,3‐dioxygenase activity. This was also confirmed by the immediate appearance of a yellow‐colored product when catechol was sprayed on colonies in a Luria–Bertani agar plate (Stillwell et al , 1995) induced with phenanthrene, 2‐hydroxy‐1‐naphthoic acid or salicylic acid. However, none of these activities could be detected in the cell‐free extract obtained from succinate‐grown cells.…”

The Sphingomonas Plasmid pCAR3 Is Involved in Complete Mineralization of Carbazole

“…Although it is thought that these plasmids are important for the degradative abilities of these organisms, few studies have reported on whole sequences of plasmids isolated from sphingomonads. The only example is seen in pNL1 identified in a naphthalene-and biphenyl-degrading bacterium, Novosphingobium aromaticivorans F199 (58), in which the entire sequence was determined (48). Thus, it is necessary to investigate other sphingomonad plasmids to better understand their function, diversity, and molecular evolution.…”

Taxonomic Study of Aromatic-Degrading Bacteria from Deep-Terrestrial-Subsurface Sediments and Description of Sphingomonas aromaticivorans sp. nov., Sphingomonas subterranea sp. nov., and Sphingomonas stygia sp. nov.

“…One such organism, strain F199' (T = type strain), was among the isolates obtained from deep Atlantic coastal plain sediments mentioned above and was found to degrade a broad range of aromatic compounds, including xylene, toluene, and naphthalene (18). The ability to degrade these compounds is encoded on a 180-kb plasmid in F199T (52), which may have evolved because sedimentary organic material is the principal source of energy in this organism's environment (18). Strain F199' and several other subsurface bacterial isolates that can degrade various aromatic compounds were subsequently shown to be members; of the genus Sphingomonas (16).…”