Dumont et al. 2006 - The Department of Ecology and Evolutionary ...

1246 M. G. Dumont et al.the soil clone and therefore the position of the mptG geneon this clone from an uncultivated Methylocystis sp. isunusual. Within the genome of Methylococcus capsulatus(Bath) (AE017282), the mptG gene is also positioned inclose proximity to a pmoCAB operon, but is separated byfour genes, fhcC, fhcD, fhcA and fhcB, that encode subunitsof the formyltransferase/hydrolase complex for C 1transfer.In summary, the retrieval of SIP-generated 13 C-DNAfrom environmental samples provides access to thegenomes of bacteria in the environment that were involvedin specific metabolic processes. As shown in this study,the genes encoding metabolic pathways are often linkedon chromosomes and therefore it is possible by combiningDNA-SIP and metagenomics to retrieve targeted geneticinformation with minimal sequencing effort. With continuedadvances in genomics and DNA sequence analysis,it may soon become more feasible to reconstruct thecomplete genomes of microbial populations and consortiadirectly from the environment (Venter et al., 2004). Untilnow, metagenomic studies have been largely restricted tothe most abundant organisms in the community, but DNA-SIP offers a breakthrough means by which ecologicallyrelevant, and potentially subdominant, community membersmay be characterized and their metabolic functionsrevealed.Experimental proceduresSoil sampling and SIPThe characteristics of the Gisburn forest soil were describedpreviously (Radajewski et al., 2002). Soil was collected inAugust 2002 and SIP experiments were performed essentiallyas described previously (Radajewski et al., 2002).Briefly, a soil slurry was made by adding 10 ml of ANMSmedium (NMS medium (Whittenbury et al., 1970) containing0.5 g NH 4 Cl, 0.5 g KNO 3 and buffered with 4 mM phosphate,pH 3.5) to 5 g of soil. The slurry was incubated in a 125 mlserum vial sealed with a butyl stopper on a rotary shaker(∼100 rpm) at room temperature (20–25°C) and in the dark.The soil DNA was harvested after consumption of a total of50 ml of 13 CH 4 (Linde) added in 10 ml aliquots to the soilslurry microcosm (Radajewski et al., 2002). The headspacewas flushed with air between injections to limit theaccumulation of 13 CO 2 from complete oxidation of 13 CH 4 bymethanotrophs.DNA extraction from soilThe protocol for DNA extraction from soil was based on themethod previously described (Zhou et al., 1996), with severalmodifications. The soil slurry was placed in a 35 ml Oakridgetube and centrifuged at 6000 g in a JA20 rotor. The soil pelletwas suspended in 13.5 ml of extraction buffer [100 mM Tris-HCl (pH 8.0), 100 mM sodium EDTA (pH 8.0), 100 mMsodium phosphate (pH 8.0), 1.5 M NaCl, 1% (w/v) CTAB] towhich 100 µl of fresh Proteinase K (10 mg ml −1 ) was added.The tube was placed horizontally on a 200 rpm shaker at37°C for 30 min. 1.5 ml of 20% (w/v) SDS was added andthe tube was placed at 65°C for 2 h and mixed by inversionevery 15 min. The supernatant was decanted into a cleantube after centrifugation for 10 min at 6000 g at 25°C. Thesoil pellet was again suspended in 4.5 ml of extraction bufferand 0.5 ml of 20% (w/v) SDS, incubated at 65°C, centrifugedas before and the supernatant added to the first aliquot. Thecrude extract (∼20 ml) was gently extracted with chloroform[containing 4% (v/v) isoamyl alcohol to minimize foaming]and centrifuged at 16 000 g for 10 min at 25°C. The aqueousphase was transferred to a clean tube with a wide bore 5 mlpipette tip, made by cutting ∼5 mm from the tip end. Care wastaken to leave the interface undisturbed. The DNA was precipitatedfrom the aqueous phase by adding 0.6 vol.(∼10.5 ml) of 2-propanol, mixing gently and incubating for 1 hat room temperature. The DNA was pelleted by centrifugationat 16 000 g for 20 min at 20°C. The DNA pellet was rinsedwith 5 ml of 70% (v/v) ethanol and air-dried for 20 min.UltracentrifugationTotal DNA from the extraction was dissolved in 20 ml of TEbuffer (Sambrook and Russell, 2001) at 4°C for 16 h. Thevolume of DNA solution was measured and exactly 1 g ml −1CsCl was dissolved by gentle mixing. Five hundred microlitresof ethidium bromide (10 mg ml −1 ) was added and thesolution transferred to a 25 mm × 89 mm polyallomer Quick-Seal ultracentrifuge tube (Beckman). Unfilled tube volumewas filled by adding 1 g ml −1 CsCl in TE buffer. The tube wascentrifuged at 180 000 g (45 000 rpm) for 20 h. If the positionof the DNA in the gradient could not be seen in visible light,the tube was exposed to long wavelength UV (365 nm) for aminimum time (< 2 s); it was found that indiscriminate UVexposure subsequently made it impossible to clone the DNA.The portion of gradient containing the DNA was collectedusing a 16-gauge needle and 2.5 ml syringe (Sambrook andRussell, 2001). Another 250 µl of ethidium bromide(10 mg ml −1 ) was added to the DNA solution, which wastransferred to a new 25 mm × 89 mm Quick-Seal tube andcentrifuged as before. DNA bands were collected with aneedle and syringe as before. The DNA was then transferredto a 13 mm × 51 mm polyallomer Quick-Seal tube and centrifugedat 265 000 g (55 000 rpm) in a VTi65 rotor at 20°C.The bands were collected independently ( 13 C-DNA followedby 12 C-DNA) and each placed in a new 13 mm × 51 mmQuick-Seal tube and centrifuged as before. The repeatedcentrifugations were performed to ensure that the 13 C-DNAwas purified from contaminating 12 C-DNA and coextractedsoil organics. Ethidium bromide was removed from DNApreparations by repeated extractions with water-saturated 1-butanol (Sambrook and Russell, 2001). The CsCl wasremoved by dialysis against TE buffer and the DNA stored at4°C.The desalted DNA was purified by electrophoresis on a 1%(w/v) low melting point agarose gel in 1× TAE buffer withoutadded ethidium bromide. Low melting point agarose (Gibco)was used because it has a large pore size which allows therestriction enzyme to penetrate the agarose plug (Sambrookand Russell, 2001). To minimize DNA shearing, 3 mm was© 2006 The AuthorsJournal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 1240–1250