25% w/v NaNO3 for 14 days at 28 °C. Motility was assessed in a hanging-drop preparation at × 1000 magnification from 24-h cultures in MB. The activities of constitutive enzymes and other physiological RGFP966 properties were determined using the API 20E, API 20NE, API 50CH strips
(bioMérieux) and Gram-negative MicroPlates (Biolog), according to the manufacturer’s instructions, except that the inoculum was prepared by suspending cells in sterile (121 °C/15 min) seawater. Susceptibility to antibiotics was investigated by the agar diffusion method using the filter discs containing antibiotics. The cell size and morphology, flagellation pattern and hydroxyalkanoate (PHA) were determined using transmission electron microscopy of negatively stained cells (Tindall et al., 2007) grown on MA at 28 °C for 1 day. Colonies of WH169T used for the examination of the presence of prosthecae and buds were grown on MA at 20 °C for 12 days. Ultrathin sections were prepared as described by Mast et al. (2005). Genomic DNA was extracted from 24-h-old cultures on MA plates using standard methods (Ausubel et al., 1995). The 16S rRNA gene (corresponding MK-1775 cost to positions 8–1510 in the Escherichia coli numbering system) was amplified and sequenced using bacterial universal primers as described previously (Liu & Shao, 2005). The near-complete 16S rRNA gene sequence (1232 nt) of strain WH169T
was submitted to GenBank and EMBL to search for similar sequences using the blast algorithm. The identification of phylogenetic neighbours and the calculation of pairwise 16S rRNA gene sequence similarities were achieved using the EzTaxon server (http://www.eztaxon.org/; Chun et al., 2007). Phylogenetic analysis was performed using the software package molecular evolutionary genetics analysis (mega) version 4.0 (Tamura et al., 2007) after manual edition using bioedit Sequence Alignment Editor version 5.0.9 (Hall, 1999) and multiple alignment of data by clustalx (Thompson et al., 1997). The phylogenetic trees were constructed using the neighbour-joining L-gulonolactone oxidase (NJ) method, the maximum-parsimony (MP) method and the minimum evolution
(ME) method with Kimura 2-parameter model analyses implemented in the program mega version 4 (Tamura et al., 2007). Bootstrap values were calculated based on 1000 replicates. For fatty acid methyl ester, quinones and polar lipid analysis, the cell mass of strain WH169T and its phylogenetically closest species A. salexigens DSM 15300T were harvested after incubation at 28 °C in MB for 48 h. Fatty acid profiles for the two strains were determined as described previously (Xie & Yokota, 2003) using the sherlock system (MIDI). Analyses of respiratory quinones and polar lipid were carried out by the Identification Service of DSMZ and Dr B.J. Tindall, DSMZ. The G+C content of the DNA was determined using the method of Mesbah & Whitman (1989) using reverse-phase HPLC. WH169T was a short rod-shaped (0.6 × 1.1–1.