JOURNAL OF BACTERIOLOGY, Feb. 1995, p. 1008–1016 Vol. 177, No. 4 0021-9193/95/$04.0010 Copyright q 1995, American Society for Microbiology ompH Gene Expression Is Regulated by Multiple Environmental Cues in Addition to High Pressure in the Deep-Sea Bacterium Photobacterium Species Strain SS9 DOUGLAS H. BARTLETT* AND TIMOTHY J. WELCH Center for Marine Biomedicine and Biotechnology, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093-0202 Received 21 July 1994/Accepted 8 December 1994 Photobacterium species strain SS9 is a moderately barophilic (pressure-loving) deep-sea bacterial species which induces the expression of the ompH gene in response to elevated pressure. Here we demonstrate that at 1 atm (1 atm 5 1.01325 3 105 Pa), ompH expression increases with cell density in 2216 marine medium batch culture and is subject to catabolite repression and that OmpH synthesis is inducible by energy (carbon) starvation. Regulatory mutants which are impaired in ompH gene expression at high pressure are also impaired in cell density regulation of ompH gene expression, indicating that the two inducing conditions overlap in their signal transduction pathways. The same promoter was activated by high cell density at 1 atm of pressure as well as during low-cell-density growth at 272 atm. Catabolite repression of ompH gene expression was induced by a variety of carbon sources, and this repression could be partially reversed in most cases by the addition of cyclic AMP (cAMP). Surprisingly, glucose repression of ompH transcription occurred only at 1 atm, not at 272 atm, despite the fact that catabolite repression was operational in SS9 under both conditions. It is suggested that ompH expression is cAMP and catabolite repressor protein dependent at 1 atm but becomes cAMP and perhaps catabolite repressor protein independent at 272 atm. Possible mechanisms of ompH gene activation are discussed. The deep sea accounts for the largest portion of the bio- The moderate barophile Photobacterium species strain SS9 sphere by volume (47). In contrast to surface environments, has proven to be a useful microorganism for studying the which tend to display relatively large fluctuations in tempera- genetic and molecular bases of pressure sensing and adapta- ture but little change in pressure, the majority of deep-sea tion (4, 5). SS9 modulates the production of several outer environments are characterized by a relatively constant low membrane proteins in response to pressure (3, 15). Elevated temperature, 28C, but pressures ranging from 100 atm (1 atm pressure induces the expression of the ompH gene encoding 5 1.01325 3 105 Pa 5 1.01325 bar) at 1-km depth to greater the outer membrane protein OmpH. Both the ompH gene than 1,000 atm in certain deep-sea basins. By promotion of sequence and uptake experiments with ompH insertion mu- reactions resulting in decreased system volumes, high pressure tants indicate that OmpH functions as a porin (6, 7). Although imposes unique constraints on the energetics of biochemical ompH mutants do not possess any discernible differences in processes and, through these selective effects, on the charac- growth phenotype from wild-type SS9, regulatory mutants im- teristics of life found in abyssal and hadal regions (46–48). paired in ompH gene expression which display high-pressure- Barophilic, or more recently termed piezophilic, bacteria are sensitive growth have been isolated (15). These results suggest microbes possessing increased growth rates at pressure above 1 that the ompH gene comprises one member of a high-pressure atm (58, 62). To date, all such extremophiles have been recov- regulon, or collection of genes coordinately regulated by pres- ered from the deep sea (18, 28, 57). Unlike other environmen- sure, which includes genes whose products facilitate baroad- tal extremes such as high osmolarity or high temperature, aptation. which may select for dramatically different organisms, i.e., ar- To better understand the process of pressure-regulated gene chaeal over bacterial domains (56), most characterized bar- expression, we decided to look for additional sensory cues ophiles obtained from cold deep-sea regions are closely related controlling ompH. Here we present data indicating that ompH to culturable shallow-water marine bacteria and include mem- is subject to multiple activation pathways, including respon- bers of the genera Shewanella, Vibrio, Photobacterium, and siveness to cell density and energy (carbon) availability, in Colwellia (reviewed in reference 5). This finding suggests that addition to high pressure. the biochemical adaptations required for deep-sea environ- ments are not as pervasive as those demanded by other ex- MATERIALS AND METHODS treme habitats, and therefore the cellular and molecular mech- anisms of barophily may be more amenable to dissection than Strains and growth conditions. The SS9 strains used were the original SS9 adaptation to other physical or chemical extremes. isolate (17), the b-galactosidase-deficient mutant DB110, and the ompH::lacZ fusion strain EC10. Strains DB110 and EC10 have been described in greater detail by Chi and Bartlett (15). SS9 was routinely cultured at 108C in 2216 marine medium (28 g/liter; Difco Laboratories, Detroit, Mich.). For experiments requir- * Corresponding author. Mailing address: Center for Marine Bio- ing growth in defined media, SS9 was grown in morpholinepropanesulfonic acid (MOPS) minimal marine medium (M4), which is identical in composition to the medicine and Biotechnology, Scripps Institution of Oceanography, MOPS minimal medium described by Neidhardt et al. (36) except for the addi- University of California, San Diego, La Jolla, CA 92093-0202. Phone: tion of 32 g of Sigma sea salts (Sigma Chemical Co., St. Louis, Mo.) per liter and (619) 534-5233. Fax: (619) 534-7313. Electronic mail address: dbartlett the use of one of various carbon sources (typically used at 25 mM). Carbon @ucsd.edu. starvation experiments were performed after growth of SS9 in M4 containing 1008 VOL. 177, 1995 DIFFERENTIAL ompH REGULATION IN PHOTOBACTERIUM SS9 1009 maltose at 11.1 mM (designated M5). For aerobic growth experiments in test throughout the starvation protocol. Cells were pelleted in a microcentrifuge at tubes, stationary-phase cultures were diluted 1/1,000 into fresh 2216 marine 16,000 3 g for 5 min, resuspended in 200 ml of 2% SDS–50 mM Tris (pH 7.5), medium, and A595 (1-cm path length) over time was monitored in a Spectronic and boiled for 10 min. Radioactivity in samples was measured by the method of 20 spectrophotometer (Milton Roy Corp., Rochester, N.Y.). For growth in Smith and Azam (45), and counts were diluted to 5 3 104 cpm/ml. OmpH was pressurizable bulbs, stationary-phase cultures were diluted 1/1,000 into 2216 then quantitatively immunoprecipitated from 20 ml of cell extract as described in marine medium buffered with HEPES (N-2-hydroxyethylpiperazine-N9-2-eth- the PANSORBIN immunological applications handbook radioimmunoprecipi- anesulfonic acid; 100 mM, pH 7.5; Sigma), and the diluted culture was used to fill tation protocol (Calbiochem, San Diego, Calif.). 15-ml polyethylene transfer pipettes (Samco, San Fernanado, Calif.). Pipettes Outer membrane protein preparation. Outer membrane proteins were iso- were filled until there were no visible air spaces, since at increased pressures lated by a Triton X-114 detergent extraction method (10) modified as described gases can be toxic to microorganisms (50). The transfer pipettes were then heat by Chi and Bartlett (15). The amount of outer membrane protein examined in sealed with a hand-held heat sealing clamp (Nalgene, Rochester, N.Y.). Cells each lane of Fig. 4 was adjusted to cell mass by assaying that amount of outer were incubated at 1 or at 272 atm of hydrostatic pressure in stainless steel membrane protein obtained from an equivalent amount (14 mg) of total cellular pressure vessels which could be pressurized by using distilled water and a hy- protein. draulic pump and which were equipped with quick-connect fittings for rapid Primer extension analysis. Total RNA was isolated by the method of von decompression and recompression as described by Yayanos and Van Boxtel (59). Gabain et al. (54), and primer extension was performed essentially as described In the microaerobic environment of the bulb cultures, stationary phase was by Golden et al. (23). RNA (10 mg) was boiled for 3 min and then annealed to reached when the cell density reached an A of 0.20 (1 atm) or 0.21 (272 atm) 125 fmol of 59-end-labeled primer, a reverse complement to sequence positions 595 497 to 513 in Fig. 5, for 90 min at 658C. The primer was 59 end labeled with or if 20 mM glucose was added when cell density reached an A595 of 1.5 (1 atm) 32 or 2.1 (272 atm). Anaerobic culturing of SS9 was accomplished by subculturing [g- P]ATP (ICN) and T4 polynucleotide kinase (Boehringer Mannheim Corp., cells inside a LabLine Programmed Anaerobic Controlled Environment anaer- Indianapolis, Ind.). Reaction mixtures containing 25 U of avian myeloblastosis obic chamber containing 5% carbon dioxide, 10% hydrogen, and 85% nitrogen virus reverse transcriptase (Boehringer Mannheim) were incubated for 30 min at into butyl rubber-stoppered test tubes containing 2216 marine medium buffered 428C, and the product was analyzed on a 6% polyacrylamide–urea sequencing gel with 100 mM HEPES. Resazurin (0.1 mg/100 ml) was added as an oxygen alongside a dideoxy sequencing reaction using the same primer and ompH indicator. single-stranded template DNA. Soluble inducer assay. Induction of ompH gene expression by a soluble ex- tracellular factor was tested by growing SS9 in 2216 marine medium to an A595 RESULTS of 1.0. Cells were pelleted at 3,000 3 g for 5 min. Then, either the culture medium supernatant or fresh 2216 marine medium was added to an equal OmpH abundance is regulated by cell density in addition to volume of log-phase EC10 cells (A595 of 0.15), and the cells were incubated at pressure.