Where YE was omitted, the media contained either the normal concentration of thiosulfate or 5.33 mM arsenite (or 2.67 mM for those strains
sensitive to arsenite) as an electron donor. In the case of arsenite-amended media, pre-CT99021 mw cultures Selleck PD0332991 were grown in the presence of 2.67 mM arsenite. To determine autotrophic growth yield as a product of As(III) oxidised, triplicate cultures were grown in liquid MCSM without YE or thiosulfate containing either 0.66 or 1.33 mM As(III), at 25°C in static conditions. To test concentrations greater than 1.33 mM, initial cultures containing 1.33 mM As(III) were inoculated. As soon as the As(III) had been oxidised, more As(III) was added from a concentrated (0.13 M) stock solution to a final concentration of 1.33 mM. Once this had been oxidised, the process was repeated until the desired total quantity of As(III) had been added. The oxidation of As(III) to As(V) was analysed as described by Battaglia-Brunet et al. [31]. The pH was adjusted to pH 6.0 using a sterile NaOH solution before each As(III) addition. Once all of the As(III) had been oxidised, each culture was centrifuged at 10 kg for 15 min and the pellet resuspended in 10 mL MCSM. The total organic
carbon concentration of this suspension was analysed using an OI ANALYTICAL 1010 apparatus according to the AFNOR NF EN 1484 method. The influence of As(III) on final cell concentration in the presence of an organic substrate was determined with strains 3As and T. arsenivorans
in MCSM complemented with 0.1 or 0.2 g L-1 yeast extract. LDN-193189 purchase Final cell concentration was determined by measuring optical density at 620 nm. Strain motility was assessed using growth media supplemented with 0.3% agar as described previously [36]. Three separate cell cultures of each strain were analysed in triplicate. Differential protein expression analysis T. arsenivorans and Thiomonas sp. 3As strains were grown in MCSM and m126, respectively, with or without 2.7 mM As(III). Cells were harvested by centrifugation (7 K g, 10 min, 4°C). Cell lysis was performed as described previously [37]. Proteins were precipitated using the 2-D Clean-up kit (Amersham Biosciences) and resuspended in rehydratation 4��8C buffer (364 g L-1 thiourea, 1000 g L-1 urea, 25 g L-1 CHAPS, 0.6% (v/v) IPG buffer Pharmalyte, 10 g L-1 DTT and 0.01% (w/v) bromophenol blue). Protein concentration was determined using the 2-D Quant kit (Amersham Biosciences). Three hundred μg of this extract were loaded onto an 18 cm pH 4–7 IPG strip using the cup-loading technique (manifold, GE Healthcare Biosciences, Australia). IEF was conducted using the IPGPhor system (10 min at 150 V, 10 min at 500 V, 10 min at 1,000 V, 1.5 h at 4,000 V, and 4 to 5 h at 8,000 V, total = 50 kVh; GE Healthcare Biosciences, Australia). The second dimension was performed on 11.
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