291 Barophiles: deep-sea microorganisms adapted to an extreme environment Koki Horikoshi The deep-sea environment is characterized by high pressure adapted to life in the deep-sea environment, particularly and low temperature but in the vicinity of hydrothermal the barophilic bacteria. vents regions of extremely high temperature exist. Deep-sea microorganisms have specially adapted features that Biodiversity of the deep-sea microorganisms enable them to live and grow in this extreme environment. To investigate the biodiversity of the deep-sea envi- Recent research on the physiology and molecular biology of ronment, Yayanos [3] isolated microorganisms from the deep-sea barophilic bacteria has identified pressure-regulated deep sea at a depth of 10,500m. The cultured bacterial operons and shown that microbial growth is influenced by isolates grew at pressures >100MPa at 2°C and >40MPa the relationship between temperature and pressure in the at temperatures >100°C. These cultures comprise the deep-sea environment. foundation for the study of the molecular biology and biotechnology of barophilic bacteria. He discussed how temperature and pressure affect the growth rate of a Addresses The DEEPSTAR group, Japan Marine Science and Technology Center bacterium. (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan. e-mail: [email protected] Kato et aL's [4,5] extensive studies on barophiles also Current Opinion in Microbiology 1998, 1:291-295 helped in the search for relationships among bacteria from habitats differing in temperature and pressure. Under high http://biomednet.com/elecref/1369527400100291 pressure conditions >50 MPa growth of barotolerant strain © Current Biology Ltd ISSN 1369-5274 DSK1 was better at high temperature (15°C) than at low Abbreviation temperature (10°C). A comparison of 16S rRNA sequences ORF open reading frame (Figure 1) showed that barophilic and barotolerant strains belong to the Proteobacteria 7 subgroup [6], except for the barotolerant strain DSK25 which is a Gram-positive Introduction spore-forming bacterium [7]. All of the strictly barophilic The bottom of the deep sea is a world exposed to strains (DB5501, DB6101, DB6705, DB6906, DB172F, extremely high pressure and low temperature (1-2°C) but and Shewanella sp. PT99) and some of the moderately in the vicinity of hydrothermal vents the temperature can barophilic strains (DSS12 and Shewanella benthica) are rise to 400°C. Microorganisms living in the deep sea have grouped together in the same sub-branch of the genus several unique features specifically adapted for such an She~,ane/la E [8°']. Other moderately barophilic strains extreme environment. Although the deep sea is under (Shewanella sp. SC2A, Photobacterium sp. SS9 and DSJ4) extremely high hydrostatic pressure, many organisms are and barotolerant strains (S. hanedai and DSK1) are widely able to live and grow in this environment. ZoBell and distributed throughout the Proteobacteria 7-subgroup. It Morita [1] were among the first to attempt to isolate is of interest that all of the strictly barophilic microbes microorganisms which were specifically adapted to grow analyzed are grouped within a particular branch of a single under high pressures, and they called these bacteria genus. barophities. The first barophilic bacteria to be isolated were reported in 1979 [2]. Barophilic bacteria are defined Microbial diversity in the Mariana Trench as those displaying optimal growth at pressures >40 MPa, Kato et al. [9 °°] have obtained sediment samples from the whereas barotolerant bacteria display optimal growth at world's deepest sea floor, the Mariana Trench, Challenger pressure <40MPa and can grow well at atmospheric point at a depth of 10,898m, using the new unmanned pressure. submersible KAIKO of the Japan Marine Science and Technology Center. DNAs directly extracted from the Most of the deep-sea bottom is stable, cold and dark, sediments were amplified by PCR. The sequencing results therefore, it is possible that very ancient life-forms may identified archaeal 16S rRNAs related to the 16S RNA be present in a state of suspended animation in the of a planktonic marine archaeon and at least two kinds world's largest refrigerator. The study of microorganisms of bacterial 16S rRNAs that are closely related to those isolated from the deep sea promises to provide new of the genus Pseudomonas and deep sea adapted marine information about the origin of life and its evolution. The bacteria. The sequences of the amplified pressure-reg- study of these extremophiles also gives an opportunity ulated clusters were more similar to those of deep sea to investigate how life processes work at some of the barophilic bacteria than those of barotolerant bacteria. highest temperatures and pressures of the biosphere. In These results suggest that deep sea adapted barophilic this review I describe the characteristics of microorganisms bacteria, planktonic marine archaea, and some of the 292 Ecology and industrial microbiology Figure 1 bacteria and various extremophiles such as alkaliphiles, thermophiles, three barophiles and psychrophiles [10°°]. Furthermore, phylogenetic analysis of them based on 16S Aeromonas hydroph#a 0.020 rDNA sequences revealed that a wide range of taxa was Plesiomonas shigelloides represented [10°']. Escherichia coli Proteus vulgaris Serratia rnarcescens Table 1 ~ DB5501DB6101 Recovery of colony-forming extremophilic bacteria isolated DBI?2F from a depth of 10,897m from the Mariana Trench. D86906 Category Conditions Recovery* Shewanella PT9 c, pH "C MPa NaCI % DSS12 Shewanella Alkaliphile 9.5-10.0 25 0.1 4.1-12 x 102 benthica Barophile 7.6 25 1 O0 - DB6705 Thermophile 7.6 55-?5 0.1 5.8-3,5× 102 Shewanella hanedai Psychrophile 7.6 4 0.1 2.0 x 102 --- BSK1 Halophile 7.6 25 1 O0 15 - Moritella mannus 7.6 25 100 0.2 - -- Shewanella SC2A Acidophile 3 25 0.1 - _~ Shewanella putrefaciens 3 25 100 Shewanella alga Non-extremophile 7.2 + 0.4 25 0.1 2.2-23 x 104 -- Shewanella ACAM122 *Colonies g-ldry sea mud. -, no growth obtained. "~~ Photobactedum angustum Photobacterium phosphoreum Bacteria adapted to high pressure and cold Photobacterium SS9 temperature DSJ4 Kato et al. [4,5,7] have reported several high pressure Vibrio anguillarum adapted bacteria isolated from samples of deep-sea ~1 Vibrio vulnificus Proteobacteria y sub-group sediment obtained at a depth of 2500-6500 m. A list of .......................... Gram-positive these isolates is shown in Table 2. Most of the deep Bacillus subtilis .~ DSK25 sea adapted bacteria isolated were not only barophilic or Bacillus cohnii barotolerant but also psychrophilic (i.e. unable to grow at Bacillus stearothermophilos temperatures >20°C). At atmospheric pressure (0.1 MPa) the barophilic strain DB6705 was unable to grow at Current Opinion in Microbiology temperatures above 10°C but was able to grow at 4°C. Under high pressure conditions (>50 MPa) this strain was Phylogenetic tree showing the relationships between bacteria able to grow better at the high temperature than at the adapted to deep-sea conditions and genus Bacillus as determined by comparing 16S ribosomal DNA. Bar indicates inferred substitution lower temperature. Recently, Kato et al. [11 °] isolated per 100 nucleotides. several extremely barophilic bacteria from the Mariana Trench that can not grow at pressures below 50 MPa. As shown in Figure 2, strain DB21MT-2 exhibits optimum world's most widespread bacteria (the genus Pseudomonas) growth at 80MPa and can still grow at 100MPa. The coexist in the world's deepest sea trench. effects of different pressures and temperatures on growth of the deep sea adapted bacteria, including other deep To characterize further the microbial flora on the deepest sea barophilic strains isolated by Yayanos [12], have been sea floor, Takami et al. [10 °°] isolated thousands of studied. All strains tested so far became more barophilic microbes from the mud samples collected from the at higher temperatures [4,12]. Mariana Trench. The mud samples were diluted twofold with sterile marine broth and 100-200ml (5-10mg as Molecular biology of barophily dry weight) of the suspension were spread on the The pressure-regulated promoter of ompH from Photo- marine agar or half-strength nutrient agar plates used bacterium sp. SS9 was first reported by Bartlett et al. as a basal medium. In addition, modified marine agar [13,14]. A promoter activated at high pressure from the plates supplemented with 1% (w/v) starch or 1% (w/v) barophilic bacterium strain DB6705 has been cloned in skim milk with different pH (3, 7 or 10) and NaCI Escherichia coli [15]. Gene expression initiated from this concentrations (0.2 or 15% w/v) were used for isolation. promoter, which has sequence similarity to the promoter The agar plates were incubated at 4-75°C at atmospheric of ompH, was induced at the level of transcription by pressure (0.1 MPa) or at 100 MPa for 1--4 weeks. Table 1 high pressure in both the barophilic strain DB6705 shows recovery of colony-forming extremophilic bacteria. and in E. coli transformants harboring this promoter. The microbial flora found at a depth of 10,897m was Downstream from this promoter, two open reading frames composed of actinomycetes, fungi, non-extremophilic (ORF 1 and 2) were identified which together function Barophiles Horikoshi 293 Table 2 High pressure adapted bacterial strains that have been isolated in our laboratory. Bacterial strain Optimal growth properties Source Depth Reference MPa "C Barophilic bacteria* DB5501 50 10 Suruga Bay 2485 m [4] DB6101 50 10 Ryukyu Trench 5110 m [4] DB6705 50§ 10 Japan Trench 6356 m [4] DB6906 50§ 10 Japan Trench 6269 m [4] DB172F 70§ 10 Izu-Bonin Trench 6499 m [5] DB172R 60§ 10 Izu-Bonin Trench 6499 m [5] Moderately barophilic bacateriat DSS12 30 8 Ryukyu Trench 5110 m [4] DSJ4 10 10 Ryukyu Trench 5110 m [8 °°] Barotolerant bacteria DSK1 0.1 10 Japan Trench 6356 m [4] DSK25~: 0.1 35 Japan Trench 6500 m [5] *Barophilic bacteria are defined as those displaying optimal growth at a pressure of more than 40 MPa.