, 2005; Nadalig et al, 2011) In this study, we examined

, 2005; Nadalig et al., 2011). In this study, we examined selleck chemicals cmuA sequences obtained from seawater samples, and methyl halide enrichment

cultures, from the Arabian Sea and English Channel to determine the presence and diversity of marine methyl halide-degrading bacteria that utilise the methyl halide degradation pathway involving the enzyme CmuA. Stand-alone pumps (SAPs; Challenger mark 2 SAP; Challenger Oceanic, UK) were used to obtain large-volume samples from the deep-chlorophyll maximum at stations of the NERC AMBITION research cruise in the Arabian Sea on board the RRS Charles Darwin in 2001 (Cruise CD132; Fig. 1). SAPs were left in place varying times, and the sample volume through the 293-mm-diameter, 0.2-μm pore-size filters was calculated using time and flow rate (Table 1). DNA extraction was achieved by rinsing SAP filters in 5 mL

filtered seawater, and then the filtrate was taken up in 1 mL RNALater (Ambion) and stored at 4 °C. An amount of 0.5 mL of this filtrate was centrifuged (14 000  g ) and DNA isolated from the resulting pellet using a Qiagen DNA extraction kit with the DNA eluted in 100 μL sterile deionised water (M. Wyman, pers. commun.). One microlitre of this DNA extract, or of a 1 : 10 diluted extract (typically 5–50 ng of DNA), was used as a template for PCR amplification of cmuA. PCR mixtures were 2.5 mM Dinaciclib datasheet MgCl2, 200 μM each dNTP, 25 pmol of primers cmuAF802/cmuAR1609 (Miller et al., 2004), 1.3 M betaine, 1.3% (vol/vol) DMSO, in 1× Invitrogen Taq DNA Polymerase buffer and 2.5 U of Taq DNA Polymerase (Invitrogen, Paisley, UK) in a total volume of 50 μL, made up with sterile deionised water. Thermal cycling was carried out on a Hybaid Touchdown thermal cycler with initial denaturation at 95 °C for 5 min, whereupon the Taq DNA Polymerase was added as a hot start, followed by 35 cycles of 1 min at 95 °C, 1 min at 55 °C and 1 min at Decitabine price 72 °C, followed by a final extension step of 72 °C for 10 min. Genomic DNA from Hyphomicrobium chloromethanicum strain CM2 was used as a positive control. Enrichment cultures were

set up with seawater on a range of substrates during a research cruise on board the RRS Charles Darwin in 2001 (Cruise CD132). Water samples were taken at eleven stations (Fig. 1a) using a SeaBird rosette sampler equipped with 24 × 30 L Niskin bottles and conductivity, temperature and depth (CTD) devices. The exact system configuration can be found in the AMBITION Cruise report, from the Biological Oceanographic Data Centre website (http://www.bodc.ac.uk/projects/uk/mfmb/fieldwork_programme/documents/cd132_cruise_report.pdf). The Niskin bottles were subsampled using their integral taps and a short length of Tygon tubing into 2-L polycarbonate bottles rinsed three times with seawater sample. Two litres of water from 5 m depth (surface) and the chlorophyll maximum for each station (as determined by the CTD profile) were vacuum-filtered through 47-mm, 0.

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