, 2005; Ivars-Martinez et al., 2008a, b). When the sequenced genomes

of representative Deep ecotype (AltDE) and surface ecotype (ATCC 27126) strains were compared, many differences were identified, including the presence of a [NiFe] hydrogenase in AltDE, but not in ATCC 27126 (Ivars-Martinez et al., 2008b). The [NiFe] hydrogenase gene locus is present in a 95-kb gene island and includes hynS and hynL encoding the hydrogenase DMXAA in vitro small and large subunits, respectively, and the genes predicted to encode the accessory proteins that are responsible for maturation of the hydrogenase. An environmental Alteromonas hydrogenase showing 99% identity to the AltDE hydrogenase was heterologously expressed in Thiocapsa roseopersicina

and was confirmed to be active (Maroti et al., 2009). Later, the AltDE hydrogenase was characterized and was found to be active (Vargas et al., 2011). The presence of this hydrogenase in AltDE was suggested to help the organism survive in a nutritionally restricted environment (Ivars-Martinez et al., 2008b), but the physiological role of the hydrogenase in this species is unknown. Genetic tools may supplement metagenomic approaches to study the microbial biochemistry of bathypelagic environments (Martín-Cuadrado et al., 2007; Borin et al., 2009). BIBW2992 Transformation systems for other Alteromonas species

have been described (Kato et al., 1998), but no genetic tools have been described as yet for the A. macleodii Deep ecotype. In this paper, we report a survey of hydrogenases in various A. macleodii Deep ecotype strains, the development of a conjugation system for the A. macleodii Deep ecotype, and the effect of hydrogenase mutations on the growth of A. macleodii Deep ecotype under various conditions. Unless noted otherwise, all Escherichia coli strains were grown at 37 °C in Luria–Bertani (LB) broth or LB agar plates and A. macleodii strains were grown at 28 °C in marine broth (MB, Difco) or MB agar plates. Antibiotic concentrations used for the growth of E. coli cultures were ampicillin (50 μg mL−1), Thalidomide tetracycline (12.5 μg mL−1), kanamycin (50 μg mL−1), spectinomycin (50 μg mL−1), and chloramphenicol (25 μg mL−1). Antibiotic concentrations used for the growth of Alteromonas cultures were kanamycin (100 μg mL−1), spectinomycin (50 μg mL−1), and chloramphenicol (25 μg mL−1). Minimal synthetic seawater, essentially marine broth without peptone or yeast extract, was prepared as described previously (Coolen & Overmann, 2000). The sequenced strain of A. macleodii Deep ecotype (DSMZ 17117) was isolated from the Adriatic Sea at a depth of 1000 m (Lopez-Lopez et al., 2005; Ivars-Martinez et al., 2008a). Other strains of A.