Barophiles: Deep-Sea Microorganisms Adapted to an Extreme Environment Koki Horikoshi

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. tModerately barophilic bacteria are defined as displaying optimal growth at a pressure of less than 40 MPa, and are able to grow well at atmosphere pressure. *Strain DSK25 is a Gram-positive spore-forming bacterium, whereas, the other bacterial strains belong to the Gram-negative proteobacteria y subgroup. {}No growth at atmospheric pressure (0.1 MPa).

Figure 2 Another pressure-regulated operon, from the barotolerant bacterium strain DSS12, has also been characterized. On the basis of the deduced amino acid sequence of the ORF3 0.3 product [15], and the results of heterologous complemen- tation studies in E. coil, ORF3 appears to encode the 0.25 cytochrome d dehyrogenase (CydD) protein [16,17].

0.2 Bacteria adapted to high pressure and high temperature

o) 0,15 Many books and reviews on thermophiles have been published [18-20]. The majority of extremely thermophilic 0.1 microorganisms, those growing optimally at temperatures (5 above 80°C, have been isolated from terrestrial and shallow 0.05 marine solfataras.

0 __J Several new species of hyperthermophilic archaea were isolated from samples collected from deep-sea hydrother- t I I I I mal vents in the western Pacific Ocean. Thermococcus 0.1 20 40 60 80 1 O0 120 profundus [21] and Pyrococcus horikoshii [22] were isolated Pressure (MPa) from bacterial mat samples and hot fluid samples, Current Opinion in Microbiology respectively, from hydrothermal vents in the Mid-Okinawa Trough at a depth of 1395m. T. peptonophilus strains Growth profiles of barophilic bacteria strains DB21MT-2 (-O-) and OG1 and SM-2 were isolated from hot fluid samples DB21MT-5 (-(3-) at 10"C. from hydrothcrmal vents in the Izu-Bonin forearc at a depth of 1380m and in the South Mariana Trench at a depth of 1484m, respectively [23]. To survive in these as a pressure-regulated operon [6,16]. According to an hydrothermal vents the microorganisms must presumably analysis of transcription the pressure-regulated operon was have highly thermostable proteins; for example, it has expressed under elevated pressure and the largest amount been shown that T.peptonophilus produces an SDS-resistant of transcript was observed at 70MPa. The sequence of protease which is stable in boiling water. the pressure-regulated promoter from strain DB6705 was similar to that of the promoter of ompH [15]. These highly Canganella et al. [24] studied the effects of high tempera- conserved pressure-regulated operons have been found tures and elevated hydrostatic pressures on the physiologi- in many deep sea adapted bacteria [5,7,8"]. The operon cal behavior and viability of the extremely thermophilic may have an important function in these bacteria, allowing deep-sea archaeon T. peptonophilus. Maximal growth rates them to survive in the deep-sea environment. were observed at 30 and 45 MPa whereas growth at 60 MPa 294 Ecology and industrial microbiology

was slower. The optimal growth temperature shifted from many new discoveries. Microorganisms living in the deep 85°C at 30 MPa to 90-950C at 45 MPa. Cell viability during sea have special features that allow them to live in this the stationary phase of growth was also enhanced under extreme environment, and it seems likely that further high pressure. These results show that the extremely studies of these organisms will provide important insights thermophilic archaeon T. peptonophilus is a barophile [24]. into the origin of life and its evolution. It has been reported that a high-pressure-regulated systcm for gene In the case of P. hoHkoshii, the maximum growth rate was cxprcssion is found not only in deep-sea-adaptcd bacteria, observed at 95°C under pressure conditions of 0.1-15 MPa, but also in bactcria adapted to grow at atmospheric and at 100°C at a pressure of 30 MPa, with a doubling time pressure, such as E. coli [27-29,30••]. These results of =35 rain (C Kato, personal communication). At 90°C suggest that the systcms developed in the high-pressure and 950C, the growth profiles of P horikoshii resembled environment may be conserved in organisms adapted to those of barotolerant strains, and at 100°C and 103°C, life at atmospheric pressure, possibly indicating that life they resembled those of barophiles (C Kato, personal emerged from the deep-sea environmcnt a long time communication). Thus, barophilic characteristics were ago. Some correlation between barophily and thcrmophily evident at temperatures near the maximum temperature among deep-sea bacteria has been found. These micro- for growth of both Thermococcus spp. and Pyrococcus organisms may be very useful in new applications of spp. Microbial growth in the deep-sea environment is biotcchnology. For example, the genes and proteins from influenced by the relationship between temperature and deep-sea barophilic bacteria are adapted to high-pressurc pressure, and this characteristic could be a common conditions, so they could be used for the development property of deep-sea microorganisms. of high-pressure bioreactors. Barophilic enzymes having characteristic substrate specificity that would bc very Enzyme activity and thermal stability under useful for industrial applications such as proteases and high pressure glucanases for detergents and DNA polymerases for PCR amplification. Michels and Clark [25"'] purified and characterized a protease from Methanococcusjannaschii. This enzyme is the first protease to be isolated from an organism adapted References and recommended reading to a high-pressure and high-temperature environment. Papers of particular interest, published within the annual period of review, The partially purified enzyme has a molecular mass of have been highlighted as: 29 kDa and a narrow substrate specificity. Enzyme activity • of special interest increased as the temperature increased up to 116°C and • • of outstanding interest enzyme activity was measurable up to 130°C, one of 1. ZoBell CE, Morita RY: Barophilic bacteria in some deep-sea the highest temperatures reported for the function of sediments. J Bacterio/1957, 73:563-568. any enzyme. In addition, raising the pressure to 500atm 2. Yayanos AA, Dietz AS, Van Boxtel R: Isolation of a deep-sea increased the reaction rate at 125°C by 3.4-fold and the barophilic bacterium and some of its growth characteristics. Science 1979, 205:808-810. thermostability by 2.7-fold. Spin labeling of the active-site 3. Yayanos AA, Dietz AS, Boxtel RV: Obligately barophilic serine revealed that the active-site geometry of the M. bacterium from the Mariana Trench. Proc Natl Acad Sci USA jannaschii protease is not grossly different from that of 1981, 78:5212-5215. several mesophilic proteases; however, the active-site 4. Kato C, Sato T, Horikoshi K: Isolation and properties of structure may be relatively rigid at moderate temperatures. barophilic and barotolerant bacteria from deep-sea mud samples. Biodiv Conserv 1995, 4:1-9. The barophilic and thermophilic behavior of the enzyme 5. Kato C, Masui N, Horikoshi K: Properties of obligatory barophilic is consistent with the barophilic growth of ill. jannaschii bacteria isolated from a sample of deep-sea sediment from observed previously [26]. the Izu-Bonin trench..I Mar Biotechnol 1996, 4:96-99. 6. Kato C, Inoue A, Horikoshi K: Isolating and characterizing deep- sea marine microorganisms. Trends Biotechnol 1996, 14:6-12. These results suggest that enzymes produced by these high pressure adapted bacteria should be more functional 7. Kato C, Suzuki S, Hata S, Ito T, Horikoshi K: The properties of a protease activated by high pressure from Sporosarcina sp. under high pressure conditions than at atmospheric strain DSK25 isolated from deep-sea sediment. JAMSTEC R pressure. Thus, such enzymes may be very useful in 1995, 32:7-13. high pressure bioreactor systems. This is one of the 8. Li L, Kato C, Horikoshi K: Distribution of the pressure regulated • - operons in deep-sea bacteria. FEMS Microbiol Lett 1998, possible biotechnological applications of such deep-sea 159:159-166. microorganisms. DNA regions corresponding to portions of two different pressure-regulated operons previously identified in two deep-sea barophilic bacteria were sep- arately PCR amplified from a variety of deep-sea microorganisms and se- quenced. With the two sets of primers employed, amplification was par- Conclusions ticularly successful from the more barophilic bacteria examined. 16S rRNA The deep-sea environment is a source of unique mi- sequence analysis revealed that these bacteria are all phylogenetically related and belong in a sub-branch of the genus Shewane/la containing only croorganisms with great potential for biotechnological the deep-sea Shewanella barophilic bacteria. This sub-branch as the 'She- exploitation. Very few studies concerning the isolation and wanella barophile branch' contained at least two different species. The results suggest that the DNA sequences of the pressure-regulated operons characterization of deep sea microorganisms have been can be regarded as marker sequences to identify the Shewanella barophilic carried out, and investigations in this field may lead to strains. Barophiles Horikoshi 295

9. Kato C, Li L, Tamaoka J, Horikoshi K: Molecular analyses of the 19. Brock TD lEd): Thermophilic Microorganisms and Life at High • o sediment of the 11000-m deep Mariana Trench. Extremophiles Temperatures. New York: Springer-Verlag; 1978. 199"7, 1:117-123. 20. Brock TD lEd): Thermophiles. General, Molecular and Appfied DNA fragments were extracted from the sediment from the world's deepest Microbiology. New York: John Wiley & Sons; 1986. sea trench, the Mariana Trench challenger point at a depth of 10,898 m. DNAs encoding several prokaryotic ribosomal RNA small-subunit sequences 21. Kobayashi T, Kwak YS, Akiba T, Kudo T, Horikoshi K: and pressure-regulated gone clusters, typically identified in deep-sea- Thermococcus profundus sp. nov., a new hyperthermophilic adapted bacteria, were amplified by the polymerase chain reaction. From archaeon isolated from a deep-sea hydrothermal vent. System the sequencing results, at least two kinds of bacterial 16S rRNAs closely Appl Microbiol 1994, 17:232-236. related to the genus Pseudomonas and deep-sea-adapted marine bacteria, and archaeal 16S rRNAs related to the 16S rRNA of a planktonic marine 22. Gonzalez JM, Masuchi Y, Robb FT, Ammerman JW, Yanagibayashi archaeon, were identified. The sequences of the amplified pressure-regu- M, Tamaoka J, Kate C: Pyrococcus horikoshii sp. nov., a lated clusters were more similar to those of deep-sea barophilic bacteria than hyperthermophilic archaeon isolated from a hydrothermal vent those of barotolerant bacteria. These results suggest that deep-sea-adapted at Okinawa Trough. Extremophi/es 1998, 2:in press. barophilic bacteria, planktonic marine archaea, and some of the world's most 23. Gonzalez JM, Kato C, Horikoshi K: Thermococcus widespread bacteria (the genus Pseudomonas) coexist in the world's deep- peptonophilus sp. nov., a fast-growing, extremely thermophilic est sea trench. archaebacterium isolated from deep-sea hydrothermal vents. 10. Takami H, Inoue A, Fujii F, Horikoshi K: Microorganisms isolated Arch Microbiol 1995, 164:159-164. co from the deepest sea mud of Mariana Trench. FEMS Microbiol 24. Canganella F, Gonzalez JM, Yanagibayashi M, Kato C, Horikoshi Lett 1997, 152:279-285. K: Pressure and temperature effects on growth and viability of Thousands of microbes were isolated from a mud sample collected from the hyperthermophilic archaeon Thermococcus peptonophilus. the Mariana Trench. The microbial flora found at a depth of 10,897 m was Arch Microbiol 199?, 168:1-7. composed of actinomycetes, fungi, no-extremophilic bacteria and various extremophiles such as alkaliphiles, thermophiles, barophiles and psychrophiles. 25. Michels PC, Clark DS: Pressure-enhanced activity and stability Phylogenetic analysis of the isolates based on 16S rDNA sequences re- • . of a hyperthermophilic protease from a deep-sea methanogen. vealed that a wide range of taxa were represented. Appl Environ Microbio1199"7, 63:3985-3991. Properties of a hyperthermophilic, barophilic protease from Methanococcus 11. Kato C, Li L, Nogi Y, Nakamura Y, Tamaoka J, Horikoshi K: jannasehii, an extremely thermophilic deep-sea methanogen, were studied. • Extremely barophilic bacteria isolated from the Mariana Trench This enzyme is the first protease to be isolated from an organism adapted Challenger Deep at a depth of 11,000. Extremophi/es 1998, 2:in to a high-pressure and high temperature environment. The partially purified press. enzyme has a molecular mass of 29 kDa and a narrow substrate specificity Two strains of obligately barophilic bacteria were isolated from sample of the with strong preference for leucine at the pi site of polypeptide substrates. world's deepest sediment, obtained by the unmanned deep-sea submersible Enzyme activity increased as the temperature increased up to 116"C and KAIKO in the Mariana Trench, Challenger Deep at a depth of 10,898 m. was measured up to 130°C, one of the highest temperatures reported for Phylogenetic analysis based on 16S ribosomal RNA gone sequences, a the function of any enzyme. In addition, enzyme activity and thermostability DNA-DNA relatedness study and an analysis of fatty acids composition increased with pressure: raising the pressure to 500atm increased the re- showed that the first strain (DB21MT-2) appears to be highly similar to Shewanefla benthica and close relatives, and the second strain (DB21MT-5) action rate at 125°C by 3.4-fold and the thermostability by 2.?-fold. Spin labeling of the active-site serine revealed that the active-site geometry of the appears to be closely related to the genus Moritella. The optimal pressure M. jannaschfi protease is not grossly different from that of several mesophilic conditions for growth of these isolates were ?0 MPa for strain DB21MT-2 proteases; however, the active-site structure may be relatively rigid at moder- and 80 MPa for strain DB21 MT-5, and no growth was detected at pressures ate temperatures. The barophilic and thermophilic behavior of the enzyme is below 50 MPa with either strain. This is the first evidence of the existence consistent with the barophilic growth of M. jannaschfi observed previously. of an extremely barophilic bacterium of the genus Moritella isolated from the deep-sea environment. 26. Miller JF, Nilesh NN, Nelson CM, Ludlow JM, Clark DS: Pressure 12. Yayanos AA: Evolutional and ecological implications of the and temperature effects on growth and methane production properties of deep-sea barophilic bacteria. Proc Nat/Acad Sci of the extreme thermophile Methanococcus jannaschii. App/ USA 1986, 83:9542-9546. Environ Microbic~ 1988, 54:3039-3042. 13. Bartlett DH, Wright A, Yayanos A, Silverman M: Isolation of 27. Kato C, Sato T, Smorawinska M, Horikoshi K: High pressure a gone regulated by hydrostatic pressure in a deep-sea conditions stimulate expression of chloramphenicol bacterium. Nature 1989, 342:572-574. acetyltransferase regulated by the lac promoter in Escherichia coil FEMS Microbic~ Lett 1994, 122:91-96. 14. Bartlett DH, Welch TJ: ompH gone expression is regulated by multiple environmental cues in addition to high pressure in 28. Sato T, Kato C, Horikoshi K: Effect of high pressure on gone the deep-sea bacterium Phofobacterium species strain SS9. expression by lac and tac promoters in Escherichia coil J Mar J Baeterio/1995, 177:1008-1016. Biotechno/1995, 3:89-92. 15. Kato C, Smorawinska M, Sato T, Horikoshi K: Cloning and 29. Welch TJ, Farewell A, Neidhardt FC, Bartlett DH: Stress response expression in Escherichia coil of a pressure-regulated of Escherichia coil to elevated hydrostatic pressure. J Bacteriol promoter region from a barophilic bacterium, strain DB6705. 1993, 175:7170-7177. J Mar Biotechno/1995, 2:125-129. 30. Welch TJ, Bartlett DH: Isolation and characterization of 16. Kate C, Tamegai H, Ikegami A, Usami R, Horikoshi K: Open • . structural gone for OmpL, a pressure-regulated porin-like reading frame 3 of the barotolerant bacterium strain DSS12 protein from the deep-sea bacterium Photobacterium species is complementary with cydD in Escherichia co/i: cydD functions strain SS9. J Bacterio/1996, 178:5027-5031. are required for cell stability at high pressure. J Biochem 1996, This paper shows inverse pressure regulation of ompH and ompL gone 120:301-305. expression in the genetically manipulatable moderate barophile Photobao- terium sp. SS9. These genes encode outer membrane proteins. The ompH 1 7. Kato C, Ikegami A, Smorawinska M, Usami R, Horikoshi K: protein is maximally abundant when SS9 is grown at its pressure optimum, Structure of genes in a pressure-regulated operon and 28 MPa, whereas the ompL protein is produced in greatest quantity when adjacent regions from a barotolerant bacterium strain DSS12. SS9 is grown at 0.1 MPa. Although ompH mutants are not high-pressure- J Mar Biotechnol 1996, 6:210-218. sensitive and ompL mutants are not low-pressure-sensitive, physiological 18. Stetter KO: Hyperthermophiles: Isolation, classification and experiments with various mutants suggest that ompH may enable the uptake properties. In Extremophiles. Edited by Horikoshi K, Grant WD. of a greater range of nutrients than ompL, a trait which could be important New York: Wiley-Liss, 1998:1-24. in the deep sea where nutrients are frequently limiting.