No bands were observed when the blots were hybridized with a probe corresponding to hynSL, confirming that the genes encoding the hydrogenase were deleted (Fig. 3c). When the
blots were rehybridized with a probe to the kanamycin resistance gene, bands of the expected size were identified, confirming that double recombination had occurred and that the KmR cassette had replaced the hydrogenase genes (Fig. 3d). Mutants confirmed by Southern blot analysis were also found to lack hydrogenase activity in an in vitro hydrogen evolution assay (Fig. 3e). To complement the PW440 hydrogenase mutant, a plasmid, pPW438, containing the wild-type AltDE hydrogenase gene cluster was conjugated into the mutant and a single recombination event in the mutant was selected by resistance to the antibiotics chloramphenicol and
kanamycin. PCR amplification selleck chemicals of hynSL confirmed that the complemented strain contained a copy of the wild-type hydrogenase genes (Fig. 3b). Hydrogenase activity measured by in vitro hydrogen evolution assay with cell extracts indicated that the complemented strain, PW438/PW440, regained hydrogenase activity to almost wild-type levels (Fig. 3e). To learn more about the physiology of A. macleodii, we investigated the ability of AltDE to grow under various Protein Tyrosine Kinase inhibitor conditions. While AltDE grew well in the complete medium (marine broth) under aerobic conditions, growth under anaerobic conditions was inhibited unless nitrate was added to the medium as an electron acceptor (Fig. 4a). No growth was observed when sulfate was provided as an electron acceptor (data not shown). When grown in a complete medium with nitrate under anaerobic conditions containing 3% H2, no differences in the growth rate were observed between the wild type and the hydrogenase mutant strain, PW440 (Fig.
4a). Strains U7, U8, U10, and U12 were isolated by Sass et al. (2001) in the Urania Basin at a depth of 3500 m, where the chloride concentration was measured to be between 2.5 and 3.0 M. Thus, we tested the ability of A. macleodii to grow in the presence of additional salt. When the complete marine medium was supplemented with an additional 2 M NaCl, growth was slowed, but still detectable (Fig. 4b and c). This slower growth in the high-salt medium was detected when grown either aerobically or anaerobically (nitrate was supplied as the electron acceptor) SDHB (Fig. 4b and c). As expected, no growth occurred in minimal seawater media (Fig. 4b and c). No significant differences in the growth rates could be observed between the wild type and the hydrogenase mutant strains of AltDE under all growth conditions tested (Fig. 4b and c). Thus, the presence of the hydrogenase does not appear to affect growth rate in the presence of 3% H2 in a complete medium. The fact that no growth was detected in minimal seawater in the presence of 3% H2 is consistent with the designation of A. macleodii as a chemoheterotroph that requires fixed carbon sources.