It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two-to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. Halophilic microorganisms living in high-salt environments may be classified into two main groups: extremely halophilic archaea and moderately halophilic bacteria. Extremely halophilic archaea are adapted to survive in environments with extremely high concentrations of salt, such as the Great Salt Lake, while moderately halophilic bacteria can survive over a wide range of salt concentrations, between 3 and 15% (12, 13).These bacteria may be exposed to naturally occurring xenobiotics. To cope with such potential hazards, many bacteria are equipped with a xenobiotic efflux pump, which exports stereochemically unrelated compounds at the expense of cellular energy. Miyauchi et al. reported the presence of a doxorubicin efflux pump in the archaeon Haloferax volcanii (10,19). This finding was made in an extremely halophilic archaeon, and the study of such efflux pumps in moderately halophilic bacteria has been limited (20). In addition, it has been observed that moderately halophilic bacteria exhibit natural resistance to structurally and functionally diverse compounds (35). This observation and its connection to high-salt environments remained to be examined. We therefore undertook a study by selecting a multiantibiotic-resistant mutant of a moderately halophilic bacterium which showed an enhanced activity of the putative multidrug efflux pump. We report here the cloning of the gene responsible for multidrug resistance and the effects of gene disruption and a high salt concentration on the resistance and expression of the corresponding protein. MATERIALS AND METHODSBacterial strains, growth conditions, and isolation of ofloxacin-resistant mutant. The halophilic bacterium used was strain 160, which was isolated from a seashore specimen (Matsushima, Japan). The physiological properties and nucleotide sequence of the 16S rRNA gene (accession number AB105069) suggested that this strain belongs to the genus Chromohalobacter, and it was found to be identical to strain 560, which was used previously (29). Cells were grown at 37°C in nutrient broth (NB; 1% beef extract and 1% polypeptone, pH 7.0) supplemented with 2 M NaCl. SW medium (32) was used for the conjugation of Chromohalobacter and Escherichia coli. E. coli JM109 and S17-1 were used for DNA manipulations. An ofloxacin-resistant mutant was isolated by plating ca. 5 ϫ 10 8 cells on a nutrient agar containing 2 M NaCl and 0.4 g of ofloxacin/ml. De...

Outer and inner membrane fractions of Chromohalobacter marismortui and Halomonas elongata were isolated by differential detergent solubilization, and profiles of membrane proteins, especially major outer membrane proteins, were analyzed. These type strains possessed one extremely abundant outer membrane protein which showed similarity in amino-terminal amino acid sequence with the outer membrane porin proteins in other Gram-negative bacteria. Three halophilic eubacterial strains isolated from saline environments were also characterized. Strains 160 and 43 were found to be Chromohalobacter spp. and strain 40 to be a Halomonas sp. by sequence analysis of their 16 S ribosomal RNA genes. Extremely abundant porin proteins with an apparent molecular mass of 49 kDa were found in Chromohalobacter sp.160 and Halomonas sp. 40, but no major outer membrane protein was detected in Chromohalobacter sp. 43, suggesting strain 43 was most likely a naturally defective porin mutant. Porin proteins from Chromohalobacter spp. and Halomonas spp. showed the same migration on SDS-polyacrylamide gel electrophoresis with or without heat-treatment, indicating that these porin proteins did not form a SDS-resistant trimeric structure, which was detected in most of the Gram-negative bacterial porin proteins.