JOURNAL OF BACTERIOLOGY, Feb. 1995, p. 1008–1016 Vol. 177, No. 4 0021-9193/95/$04.00ϩ0 Copyright ᭧ 1995, American Society for

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 ؋ 105 Pa), ompH expression increases with cell density in 2216 marine medium batch 1.01325 ؍ atm (1 atm 1 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, 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 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, 2ЊC, but pressures ranging from 100 atm (1 atm pressure induces the expression of the ompH gene encoding ϭ 1.01325 ϫ 105 Pa ϭ 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, 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 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 ␤-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 10ЊC 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 ϫ g for 5 min, resuspended in 200 ␮l 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 ϫ 104 cpm/␮l. OmpH was pressurizable bulbs, stationary-phase cultures were diluted 1/1,000 into 2216 then quantitatively immunoprecipitated from 20 ␮l of cell extract as described in marine medium buffered with HEPES (N-2-hydroxyethylpiperazine-NЈ-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 ␮g) 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 ␮g) 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 5Ј-end-labeled primer, a reverse complement to sequence positions 595 497 to 513 in Fig. 5, for 90 min at 65ЊC. The primer was 5Ј 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 [␥- 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 42ЊC, 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 ϫ 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. Differential regulation of OmpH synthesis and ompH 10ЊC for 3 h. At 15-min intervals, portions of the culture were withdrawn and ompH::lacZ gene expression was quantitated by ␤-galactosidase activity deter- gene expression were monitored by using previously described mination. antisera and strains. To facilitate quantitating OmpH levels, we Glucose determination. Glucose concentrations in the culture medium were previously prepared polyclonal antisera for use in Western determined after samples were filtered through a Nuclepore polycarbonate filter blotting (3). ompH gene expression has been measured by with a 0.2-␮m pore size. Glucose present in the filtrates was quantitated by the glucose oxidase-peroxidase coupled assay, using o-dianisidine dihydrochloride constructing a fusion between the ompH promoter and the (Sigma Diagnostics) as the chromogen. reporter gene lacZ (15). Briefly, a lacZ-containing restriction Protein quantitation. Total protein was measured to calibrate samples for fragment was cloned into the third codon of ompH, conjugated experiments involving OmpH quantitation, ␤-galactosidase specific activity de- into SS9, and crossed into the SS9 genome by homologous termination, and outer membrane protein analysis. Protein concentration in cell suspensions was determined by a Coomassie brilliant blue–Triton X-100 dye recombination to generate strain EC10. This fusion strain al- binding assay (22). lows the regulation of ompH promoter activity to be monitored OmpH quantitation. To quantitate OmpH abundance, 10 ml of cells was taken in single copy with all possible upstream control sequences from a single 1-liter culture of DB110 or EC10 at various time intervals and present. optical densities throughout growth. Then 1.5 ␮g of protein from each sample was loaded onto a sodium dodecyl sulfate (SDS)-polyacrylamide gel (this small The amount of OmpH produced as a fraction of total cel- amount of protein gave greater discrimination between high and low OmpH lular protein increased with cell density during growth of the concentrations than larger quantities of protein), and the proteins were sepa- OmpHϩ strain DB110 in 2216 marine medium (Fig. 1A). To rated by SDS-polyacrylamide gel electrophoresis (PAGE) (2). The resolving gel ensure sufficient dilution of products from preceding SS9 sta- was 12.5% acrylamide. Acrylamide and SDS were from BDH (Poole, England). Western blot (immunoblot) electrophoretic transfer of proteins (52) after SDS- tionary-phase cultures, cells were diluted 1,000-fold for all PAGE was for 2.5 h at 75 V in 0.096 M glycine–0.125 M Tris–20% methanol onto growth experiments. Over a 24-h period as the culture pro- Nytran membrane (Schleicher & Schuell). Western blots were blocked with gressed from early logarithmic growth to stationary phase, saturant (5% nonfat dry milk and 0.2% NaN3 in phosphate-buffered saline [PBS; OmpH levels increased by greater than ninefold. A similar 20 mM sodium phosphate, pH 7.3, 100 mM NaCl]), reacted with OmpH-specific antiserum (3) diluted in saturant, washed with PBS, incubated with 125I-labeled result was observed when ompH transcriptional activity was protein A (2 ϫ 105 cpm/ml of saturant; Dupont, NEN Research Products), monitored as a function of culture phase of growth (Fig. 1B). washed with PBS, dried, and exposed to X-ray film (Kodak XAR-5). The amount 125 ␤-Galactosidase activity per milligram of total cell protein in of I associated with OmpH was quantitated by excising the radioactive bands the ompH::lacZ transcriptional fusion strain EC10 increased 9- and counting in a LKB 1282 Compugamma CS gamma counter. The amount of OmpH present per unit of cell mass was calculated by converting to 125I counts to 10-fold as the cells progressed into stationary phase. There- per milligram of total cell protein. fore, as with changes in pressure, OmpH regulation by cell ␤-Galactosidase assays. Measurements of ␤-galactosidase specific activity in density occurs at the transcriptional level. SS9 (endogenous ␤-galactosidase activity) and in EC10 (the ompH::lacZ tran- Although many genes which exhibit maximal levels of ex- scriptional fusion strain constructed in a ␤-galactosidase-deficient strain of SS9) was performed from whole-cell extracts as described by Miller (34) except that pression in stationary phase have been described (1, 26, 30, 53), SS9 ␤-galactosidase activity was monitored in Z buffer containing 200 mM KCl ompH expression appeared to be unusual because it correlated at 10ЊC. more to cell density than to a distinct phase of growth. We OmpH induction by energy (carbon) starvation. To prepare carbon-starved considered several regulatory possibilities which are consistent cultures, SS9 cells were grown aerobically in M5 and maintained in exponential with these results. These were that ompH expression is regu- growth (A595 of 0.05 to 0.6) during the course of more than 30 doublings. Then, at an optical density (A595) of 0.5, 10 ml of the culture was rapidly harvested (less lated by (i) growth rate, (ii) cell density through the excretion than 2 min) by filtration through a 0.2-␮m-pore-size polycarbonate filter (Nucle- of a diffusible inducing substance, or (iii) cell density through pore, Pleasanton, Calif.). Harvested cells were quickly washed twice in 10 ml of a process not involving a diffusible inducing substance. ompH M4 lacking a carbon source and then either starved by resuspension in 10 ml of M4 lacking a carbon source or returned to 10 ml of complete M5. One-milliliter regulation by growth rate in batch cultures could correlate with aliquots of cells were then transfered to Dilu-Vials (Fisher Scientific, Tustin, cell density if the cells are in unbalanced growth and possess Calif.) and pulse-labeled by the addition of TRAN-35S-label (1,089 Ci/mmol, 50 growth rates that are increasing with cell density. However, ␮Ci/ml; ICN Biomedicals, Inc., Costa Mesa, Calif.) for 10-min periods postresus- even when EC10 was maintained in unperturbed exponential pension. At the end of the labeling period, labeling was stopped by the addition of nonradioactive methionine (final concentration, 50 mM) and cysteine (final growth for 6 days by preparing daily 20-fold dilutions of the cell concentration, 8 mM). Care was taken to maintain a constant temperature culture, ompH expression, as quantitated by ␤-galactosidase 1010 BARTLETT AND WELCH J. BACTERIOL.

FIG. 1. OmpH abundance and ompH gene expression in SS9 as a function of cell density in 2216 marine medium. (A) OmpH abundance per milligram of cell protein was quantitated by Western blotting of cells harvested at the indicated optical densities from a single culturing experiment. (B) ompH gene expression was monitored by measuring ␤-galactosidase activity per milligram of cell protein from cells of the ompH::lacZ gene fusion strain EC10 harvested at the indicated optical densities from a single culturing experiment.

specific activity, still increased with cell density in a manner which is not the result of the accumulation of some bacterial comparable to that shown in Fig. 1B. These results, therefore, product in the medium. support the conclusion that ompH is under cell density rather Overlap between cell density and high-pressure regulation than growth rate control. Many members of the genus Photo- of ompH gene expression. We have previously reported the bacterium are bioluminescent and exhibit cell density control of isolation of ompH regulatory mutants derived from the bioluminescence through the excretion of diffusible autoin- ompH::lacZ strain EC10 (15). To investigate whether cell den- ducer molecules (35). Possible excretion of an inducer sub- sity and pressure regulation of ompH gene expression overlap stance for ompH was tested by adding cell-free supernatants in signal transduction pathways, these regulatory mutants were from high-density SS9 cultures to low-density EC10 cells. How- examined for defects in ompH gene expression at high cell ever, no induction of ␤-galactosidase activity was observed density and at high pressures. Mutants EC1002, EC1011, under these conditions. In summary, ompH gene expression EC1017, and EC1020 all exhibited negligible ␤-galactosidase induction at 1 atm appears to be the result of a physical or activity at high cell densities (A595 of 1.0) and following trans- chemical signal arising in response to increased cell density but fer to 272 atm of pressure for 36 h (strains EC1002 and VOL. 177, 1995 DIFFERENTIAL ompH REGULATION IN PHOTOBACTERIUM SS9 1011

FIG. 2. OmpH abundance and ompH gene expression as a function of stage of growth in SS9 cultures grown in 2216 marine medium supplemented with glucose. Conditions were identical to those described in the legend to Fig. 1 with the exception that cells were cultured in the presence of 20 mM glucose.

EC1020 exhibit poor growth at high pressure and are not energy source. When 2216 marine medium supplemented with readily grown at 272 atm). In contrast, strain EC10 ␤-galacto- 20 mM glucose as an additional carbon and energy source was sidase activity progressed from 2,680 to 21,190 ␮mol of o- used for culture experiments with the OmpHϩ strain DB110 nitrophenylphosphate (ONP) per min per mg of cell protein as and the ompH::lacZ strain EC10, OmpH abundance and the culture density went from 0.02 to 1.0 and went up to 26,500 ompH gene expression were both dramatically altered. Little ␮mol of ONP per min per mg of cell protein for a 272-atm OmpH per milligram of cell protein was present in the DB110 culture grown to an A595 of 0.145. These results indicate that a culture regardless of cell density, with less than a twofold common regulatory factor must be required for the induction increase in OmpH evident between cells harvested at A595sof of ompH gene expression by increased cell density and by 0.1 and 1.2 (Fig. 2A). ompH gene expression, as reflected by elevated hydrostatic pressure. ␤-galactosidase activity in EC10, also showed a negligible in- Catabolite repression of ompH gene expression. ompH gene crease with growth of cells to high cell densities (Fig. 2B). expression is also influenced by catabolite repression in addi- Because growth of DB110 in the presence of glucose 2216 tion to cell density at 1 atm. 2216 marine medium contains marine medium to an A595 of 1.0 resulted in a change of peptone (approximately 4 g/liter) as the principal carbon and medium glucose concentration only from 20 to 15.8 mM, the 1012 BARTLETT AND WELCH J. BACTERIOL.

TABLE 1. ompH gene expression in stationary-phase EC10 cells supplemented with various carbon and energy sources with and without added cAMP

␤-Galactosidase activity Supplementa cAMPb (␮mol of ONP/min/mg of cell protein) FIG. 3. OmpH induction by carbon starvation. The amount of [35S]methi- None Ϫ 21,200 onine-labeled OmpH synthesized during the indicated time periods after shifting to carbon-replete (lane 1) or carbon-free (lanes 2 to 5) medium was visualized ϩ 15,100 following immunoprecipitation, SDS-PAGE, and autoradiography. Lane 1, 10 to Glucose Ϫ 4,200 20 min; lane 2, 10 to 20 min; lane 3, 60 to 70 min; lane 4, 110 to 120 min; lane ϩ 7,200 5, 160 to 170 min. Mannose Ϫ 2,400 ϩ 6,200 Galactose Ϫ 7,100 ϩ 13,900 diate the catabolite repression control of ompH. ompH expres- Fructose Ϫ 4,200 sion was also repressed by the addition of glycerol, the organic ϩ 6,600 acids succinate and fumarate, and the disaccharide , but Ϫ 4,400 ϩ 5,500 the repression caused by these carbon sources was less than Maltose Ϫ 2,900 that produced by equimolar quantities of all of the previous ϩ 10,200 sugars except for trehalose, and furthermore, the repression Trehalose Ϫ 9,300 caused by these substrates was not partially reversed by the ϩ 17,600 addition of cAMP to the culture medium. The doubling times Sucrose Ϫ 5,200 of SS9 in minimal medium containing 20 mM concentrations ϩ 5,700 of the above carbon and energy sources were as follows: glu- Lactose Ϫ 10,900 cose, 7 h; mannose, 7 h; galactose, 8.5 h; fructose, 7 h; arabi- ϩ 9,100 nose, no growth; maltose, 8.5 h; trehalose, 6.5 h; sucrose, 11 h; Succinate Ϫ 17,000 ϩ 12,500 lactose, no growth; succinate, 12.5 h; fumarate, 11.5 h; and Fumarate Ϫ 11,200 glycerol, 11.3 h. These results reflect a general, but not abso- ϩ 8,800 lute, correlation between the effectiveness of the compounds as Glycerol Ϫ 8,400 growth substrates and as catabolite of ompH gene ϩ 8,500 expression. Because a general feature of catabolite repression documented in other gram-negative bacteria is that its magni- a All cells were grown in 2216 marine medium unsupplemented or supple- mented with 20 mM the indicated substrates. tude depends on the rate of catabolism of the carbon source b Ϫ, no cAMP; ϩ, 10 mM cAMP added at the beginning of the culture period. (reviewed in reference 41), the differential effects of the vari- ous sugars, organic acids, and glycerol on ompH gene expres- sion are qualitatively similar to those of many genes known to be under cAMP-CRP control. The sugars lactose and arabi- cells were never energy (carbon) limited, thus explaining why nose are interesting exceptions to this generalization as they do derepression of ompH expression was not observed, even when not support the growth of SS9 in minimal medium and yet still the cells reached stationary phase. are capable of inhibiting ompH gene expression in EC10. It The repression of ompH expression by glucose appears to may be noteworthy in this regard that it is possible for sugars result from catabolite repression and not from pH changes to cause the catabolite repression of inducible systems associated with glucose-stimulated production of metabolic ac- in the absence of their utilization (39). Curiously, despite the ids. Although growth of strain DB110 did not change the pH of fact that SS9 cannot grow on lactose as the sole carbon and 2216 marine medium, growth in the presence of 20 mM glu- energy source, it possesses discernible ␤-galactosidase activity cose resulted in a pH decrease from 7.8 to 5.1 as cell density (see below). went from an A595 of 0.05 to 1.0. However, repression of ompH Energy (carbon) starvation induction of OmpH. The expres- gene expression by glucose also occurs in the absence of pH sion of many genes under catabolite repression control can be changes. When EC10 was grown in 2216 marine medium con- induced by carbon starvation. To test whether OmpH synthesis taining 100 mM MOPS (pH 6.7) with and without 20 mM could be induced by energy (carbon) starvation, SS9 was cul- glucose to stationary phase, the pH of both cultures remained tured in M5, and the rate of OmpH synthesis after washing 6.7. However, ␤-galactosidase activity decreased more than cells and resuspending them in carbon-free medium was com- fivefold, from 20,000 to 3,500 ␮mol of ONP per min per mg of pared with that of cells resuspended in complete M5. This was cell protein, in the glucose-supplemented culture. These re- performed by pulse-labeling proteins with [35S]methionine fol- sults suggest that the glucose inhibition of ompH gene expres- lowed by OmpH immunoprecipitation, SDS-PAGE, and auto- sion arises from effects on energy (carbon) availability rather radiography (Fig. 3). Maltose removal led to a rapid and sub- than from changes in pH. stantial increase in the rate of OmpH synthesis, indicating that Repression of ompH gene expression was also observed in energy (carbon) starvation leads to OmpH induction. A similar response to the addition of a variety of other sugars to the result was observed upon glucose removal from glucose mini- culture medium (Table 1). Glucose, mannose, galactose, fruc- mal marine medium; however, little if any induction was ob- tose, arabinose, maltose, trehalose, and sucrose at 20 mM all served by nitrogen or phosphate depletion (data not shown). inhibited ompH gene expression even at high cell densities in High pressure abolishes catabolite repression control of cultures of EC10. Addition of cyclic AMP (cAMP) to 10 mM OmpH. Since the cell density-dependent induction of OmpH partially reversed the inhibition of ompH gene expression in 2216 marine medium is prevented by the presence of glu- caused by all of these sugars. These latter results suggest that cose, the high-pressure induction of OmpH in this medium was ompH expression requires cAMP and the cAMP receptor pro- also examined as a function of glucose availability. As ex- tein (CRP) and that these regulatory factors are likely to me- pected, outer membrane proteins prepared from cells har- VOL. 177, 1995 DIFFERENTIAL ompH REGULATION IN PHOTOBACTERIUM SS9 1013

FIG. 4. Levels of SS9 outer membrane proteins OmpH and OmpL as a function of cell density, glucose, and pressure. Lane 1, low-cell-density culture (A595 ϭ 0.045); lane 2, high-cell-density culture (A595 ϭ 0.75); lane 3, high-cell- density culture grown in the presence of 20 mM glucose (A595 ϭ 1.0); lane 4, low-cell-density bulb culture grown at 1 atm of pressure (A595 ϭ 0.215); lane 5, low-cell-density bulb culture grown at 272 atm of pressure (A595 ϭ 0.205); lane 6, medium-cell-density bulb culture grown at 1 atm of pressure with 20 mM glucose (A595 ϭ 0.44); lane 7, medium-cell-density bulb culture grown at 272 atm of pressure with 20 mM glucose (A595 ϭ 0.48). Each lane represents the amount of outer membrane protein isolated from 14 ␮g of total cell protein.

vested at low cell density, high cell density, and high cell den- sity in the presence of 20 mM glucose showed that OmpH induction as a function of cell density could be prevented by the addition of glucose (Fig. 4). A second outer membrane protein, OmpL (15), showed little change under these different growth conditions. Outer membrane proteins were also exam- ined from cells grown in pressurizable bulbs at 1 and 272 atm of pressure in the presence and absence of glucose. Again as expected, in glucose-free medium even at low cell densities, high pressure stimulated OmpH abundance, and this stimula- FIG. 5. Quantitative ompH transcript analysis as a function of culture den- tion was accompanied by a corresponding decrease in the high- sity, addition of glucose, and pressure. An autoradiogram of primer extension pressure-repressible protein OmpL (15). Finally, the effect of experiments and products of the sequencing reaction is shown. Lanes 1 to 6 correspond to transcript analyses from the cultures represented in lanes 1 to 4 concomitant glucose addition and high-pressure treatment of and lanes 6 and 7, respectively, in Fig. 4. Lane 1, low-cell-density culture; lane 2, SS9 cells was examined. While even high-cell-density SS9 bulb high-cell-density culture; lane 3, high-cell-density culture with 20 mM glucose; cultures produced little OmpH at 1 atm in the presence of lane 4, low-cell-density bulb culture grown at 1 atm of pressure; lane 5, low-cell- glucose (Fig. 4, lane 6), surprisingly, glucose addition did not density bulb culture grown at 272 atm of pressure; lane 6, medium-cell-density bulb culture grown at 272 atm of pressure with 20 mM glucose. Arrows indicate inhibit OmpH induction by high pressure, nor did it affect the positions of the transcript start sites. Below the autoradiogram is shown the OmpL repression by high pressure. Indeed, the amounts of operator sequence of the ompH gene. The entire sequence of the ompH gene and OmpL produced at 1 atm and OmpH produced at high pres- upstream DNA has previously been derived (7). The identified transcription start sure were greater in the presence than in the absence of glu- sites are indicated in boldface type with asterisks above and below. The putative ribosome binding site (RBS), Ϫ35 and Ϫ10 promoter sequences, and previously cose, apparently owing to the greater cell densities achieved in identified five sets of hexanucleotide repeats (7) are also shown, and a possible the glucose-supplemented bulb cultures (Fig. 4, lanes 6 and 7). CRP-binding site is delineated. The predicted start codon begins at position 501. When glucose-supplemented cultures were harvested at cell densities similar to those used for the cultures grown in the absence of glucose, identical levels of OmpL and OmpH were observed compared with their counterparts grown in the ab- positively regulated through the use of alternative RNA poly- sence of glucose (data not shown). These data suggest that the merase ␴ subunits (27). These same start sites were observed effects of cell density and high pressure are additive on ompH under all growth conditions, indicating that the same promoter gene expression and that unlike the situation at 1 atm for cell was being used during high-cell-density and high-pressure density-dependent OmpH induction, high-pressure induction ompH gene activation. Moreover, as with the amount of of OmpH synthesis is not subject to catabolite repression. The OmpH, ompH message was elevated during growth to high cell differences in OmpH levels at low and high pressure do not density, repressed at 1 atm by the addition of glucose, elevated result from the differences in oxygen utilization at low and high at high pressure, and not subject to glucose repression at high pressure in the microaerobic environment of the bulbs. Even pressure. anaerobic SS9 cultures are capable of catabolite repression of High pressure does not prevent catabolite repression in OmpH synthesis in the presence of glucose (data not shown). SS9. One possible explanation for the continued expression of The same ompH transcription start sites are used during the ompH gene at high pressure in cultures supplemented with different culture conditions. Primer extension analysis of glucose was that high pressure leads to a general derepression RNAs prepared from cells grown under different conditions of catabolite repression. To test this possibility, we monitored was used to ascertain the in vivo transcription start sites of the the effect of pressure on catabolite repression of a second SS9 ompH gene and to study levels of expression. The cell cultures gene product subject to catabolite repression. SS9 possesses a used for the outer membrane preparations described above weak but measurable ␤-galactosidase activity. This activity was were used for the RNA preparations. Two strong signals were observed to be strongly repressed by the addition of glucose to found from the ompH transcript analyses, and they corre- cultures, regardless of whether they were grown in test tubes or sponded to nucleotides A-373 and T-379 (Fig. 5). Upstream in pressurizable bulbs at low or high pressure (Fig. 6). These from A-373 is the sequence TTGACAN18CAGGAT, which results are consistent with those of Marquis and Keller, who bears similarity, particularly at the Ϫ35 position, to a canonical observed that catabolite repression was operational in E. coli E␴70 promoter sequence (25). E. coli promot- cells incubated at elevated pressure (32). High pressure, there- ers which contain poor homology to the Ϫ10 region are often fore, does not lead to a general attenuation of catabolite re- 1014 BARTLETT AND WELCH J. BACTERIOL.

shares identity with 11 of 14 of the most conserved residues in the E. coli consensus sequence. Finding such a sequence is not surprising, particularly in view of the fact that a cAMP-CRP- binding sequence with homology to the E. coli consensus se- quence has been reported in another marine bacterial member of the family Vibrioniaceae, Vibrio fischeri (20). However, the position of the SS9 sequence, 329 bp upstream of the tran- scription start site, is unique. The position of the center of other CRP-binding sites with respect to the transcript start site ranges from Ϫ40 to Ϫ200 at different promoters (29). In those instances where CRP binds further upstream, activation re- quires interaction with additional ancillary proteins required for promoter activation. The hexanucleotide repeats present between the ompH transcript start site and possible cAMP- CRP-binding site could represent contact points of such regu- latory factors (Fig. 5). Further inspection of the ompH up- stream sequence does not reveal any striking identities with FIG. 6. The endogenous ␤-galactosidase of SS9 is subject to catabolite re- other consensus sequences which have been described for pression at low and high pressure. Columns: 1, 1 atm of pressure (A595 ϭ 0.71); other growth phase- and/or cAMP-CRP-regulated genes, such 2, 1 atm of pressure, 20 mM glucose (A595 ϭ 1.0); 3, 1-atm bulb culture (A595 ϭ as a gearbox promoter, RpoS promoter, or FNR, IHF, or LRP 0.15); 4, 1-atm bulb culture, 20 mM glucose (A595 ϭ 0.50); 5, 272-atm bulb protein-binding sites (1, 24, 31, 55, 61). culture (A ϭ 0.17); 6, 272-atm bulb culture, 20 mM glucose (A ϭ 0.6). 595 595 On the basis of the apparent function of OmpH as a porin protein providing a relatively large, nonspecific diffusion chan- nel in the outer membrane, we have previously suggested that pression within SS9. Rather, it is concluded that ompH expres- OmpH could enhance the growth and survival of cells under sion is regulated by catabolite repression at low pressure but circumstances of nutritional limitation (6). Its induction by not at high pressure. energy starvation conditions is consistent with this possibility. In this regard, OmpH could function in much the same manner DISCUSSION as the E. coli porin OmpF has been suggested to function in dilute nutrient environments. Because of the larger pore di- In this study, we have shown that in addition to hydrostatic ameter of the OmpF porin compared with OmpC, the other pressure, the ompH gene is regulated by culture density and major porin of E. coli, the OmpF channel can provide for catabolite repression. Furthermore, it has been documented substrate diffusion rates at least 10-fold higher in the case of that OmpH synthesis is also induced by energy (carbon) star- larger compounds, such as disaccharides, than that provided by vation. Other genes encoding outer membrane proteins show an equivalent amount of OmpC porin (37). similarities in their modes of regulation. The E. coli genes There are reasons to believe that a connection could exist ompF, encoding a general porin, tsx, whose product is involved between the adaptations of marine bacteria to decreased nu- in nucleoside uptake, and lamB (maltoporin gene), whose trient supply and to increased pressures. Novitsky and Morita product is required for maltose transport, are also subject to (38) observed that starved cells of the marine bacterium Vibrio catabolite repression control and are positively regulated by species strain ANT-300 were more resistant to killing by high cAMP-CRP (12, 43, 44, 51). The enhancement of ompH ex- pressure than cells in logarithmic growth. Such cross-protec- pression at later stages of growth (higher cell densities) may be tion could represent the overlapping effects of two different similar to that of both E. coli ompC and ompF as well as the stressors, as well the evolutionary selection of a bacterial reg- Vibrio cholerae maltoporin gene (31, 51). LamB is the only ulatory process which correlates starvation with high pressure. protein other than OmpH whose synthesis is documented to be Dissolved organic matter in general tends to decrease with regulated by energy limitation as well as cAMP-CRP (16). increasing depth (and thus increasing pressure) in the marine The basis of the high-cell-density regulation of ompH,as environment (8, 19), and it has been suggested that piezophiles with the control of ompH by high pressure, will require addi- are likely to experience wide fluctuations in nutrient availabil- tional investigation. It is distinct from the stationary-phase ity (41). induction of other cAMP-CRP-controlled genes (1, 26, 30, 53) cAMP levels inversely correlate with those of carbon and since ompH expression increases with cell density even during energy within the gram-negative bacterial cell (21). cAMP logarithmic growth. It may be that density-dependent ompH complexed with CRP acts to modulate the levels of alternative induction requires the depletion of some substance from the catabolic pathways and energy metabolism, through effects on medium or chemical or physical interactions among SS9 cells. gene expression (11). Many E. coli and Salmonella typhi- The requirement for physical interactions among SS9 cells is murium starvation-induced genes are positively regulated by an attractive hypothesis in that it could join together culture cAMP-CRP (42, 49). Although these genes are not important density ompH regulation and pressure ompH regulation by a for the formation of the starvation-resistant state, they have common mechanosensory pathway. Indeed, ompH regulatory been suggested to be important for enhancing the cell’s met- mutants exist which possess mutations mapping outside of abolic potential (33). As with mutants possessing mutations in ompH and which are impaired in both high-cell-density and genes regulated by cAMP-CRP, ompH mutants are not re- high-pressure ompH induction (15), indicating that the two duced in survival in carbon-free media (our unpublished re- regulatory processes share at least one factor. sults). The connection between the cAMP-CRP regulon and As shown in Fig. 5, a possible cAMP-CRP-binding sequence ompH gene expression and possibly also high-pressure adap- is present 5Ј to the ompH transcriptional start site. The con- tation will be interesting to examine. A variant of the meso- sensus E. coli cAMP-CRP recognition sequence is AAATGT philic bacterial species Enterococcus hirae which was dere- GATCTAGATCACATTT (9). The identified SS9 sequence pressed for catabolite repression and was capable of growth at VOL. 177, 1995 DIFFERENTIAL ompH REGULATION IN PHOTOBACTERIUM SS9 1015 pressures 250 atm higher than that of the starting strain was 2. Ames, G. F.-L. 1974. Resolution of bacterial proteins by polyacrylamide isolated (13). Experiments are in progress to isolate and char- electrophoresis on slabs. J. Biol. Chem. 249:634–644. 3. Bartlett, D., M. Wright, A. Yayanos, and M. Silverman. 1989. Isolation of a acterize mutants lacking cAMP and CRP in SS9. gene regulated by hydrostatic pressure. Nature (London) 342:572–574. One of the most surprising observations from our studies is 4. Bartlett, D. H. 1991. Pressure sensing in deep-sea bacteria. Res. Microbiol. that the regulation of ompH gene expression by cell density 142:923–925. and by hydrostatic pressure can be uncoupled. High-cell-den- 5. Bartlett, D. H. 1994. Microbial life at high pressures. Sci. Prog. 76:1–13. 6. Bartlett, D. H., and E. Chi. Genetic characterization of ompH mutants in the sity cultures are subject to catabolite repression of ompH ex- deep-sea bacterium Photobacterium species strain SS9. Arch. Microbial, in pression at 1 atm but not at 272 atm. Two points relating to the press. differential regulation of ompH are noteworthy. First, we have 7. Bartlett, D. H., E. Chi, and M. E. Wright. 1993. Sequence of the ompH gene found that the same ompH promoter is induced by high cell from the deep-sea bacterium Photobacterium SS9. Gene 131:125–128. 8. Benner, R., J. D. Pakulski, M. McCarthy, J. I. Hedges, and P. G. Hatcher. density and by high pressure. Second, since SS9’s catabolite 1992. Bulk chemical characteristics of dissolved organic matter in the ocean. repression system is operational at both low and high pressure, Science 255:1561–1564. cAMP levels must certainly be low in glucose-supplemented 9. Berg, O. G., and P. H. von Hippel. 1988. Selection of DNA binding sites by cultures, even at high pressure. Therefore, high-pressure in- regulatory proteins. II. The binding specificity of cyclic AMP receptor pro- tein to recognition sites. J. Mol. Biol. 200:709–723. duction of ompH would appear to be distinct from high-cell- 10. Bordier, C. 1981. Phase separation of integral membrane proteins in triton density induction in at least one of two ways: (i) requiring CRP X-114 solution. J. Biol. Chem. 256:1604–1607. but little if any cAMP or (ii) becoming both CRP and cAMP 11. Botsford, J. L., and J. G. Harman. 1992. Cyclic AMP in . Mi- independent. crobiol. Rev. 56:100–122. 12. Bremer, E., P. Gerlach, and A. Middendorf. 1988. Double negative and In the first case, high pressure could directly or indirectly positive control of tsx expression in Escherichia coli. J. Bacteriol. 170:108– lead to structural changes in CRP which enable it to promote 116. ompH transcription in the absence of its effector molecule. It is 13. Campbell, J., III, G. Bender, and R. E. Marquis. 1985. Barotolerant variant well-known that small changes in CRP primary structure pro- of Streptococcus faecalis with reduced sensitivity to glucose catabolite repres- sion. Can. J. Microbiol. 31:644–650. duce such changes. Specific mutations in CRP (designated 14. Chi, E., and D. H. Bartlett. Unpublished results. CRP*) which allow it to actively affect gene expression in the 15. Chi, E., and D. H. Bartlett. 1993. Use of a reporter gene to follow high absence of cAMP have been isolated by many groups (re- pressure signal transduction in the deep-sea bacterium Photobacterium SS9. viewed in references 11 and 29). CRP* forms appear to possess J. Bacteriol. 175:7533–7540. 16. Death, A., L. Notley, and T. Ferenci. 1993. Derepression of LamB protein a protein conformation similar to that of the cAMP-CRP com- facilitates outer membrane permeation of carbohydrates into Escherichia coli plex which leads to the extrusion of the F␣ helix, allowing it to under conditions of nutrient stress. J. Bacteriol. 175:1475–1483. make sequence-specific DNA contacts. Furthermore, in at 17. DeLong, E. F. 1986. Adaptations of deep-sea bacteria to the abyssal envi- least one instance, CRP has been found to act in the absence ronment. Ph.D. thesis. University of California, San Diego, La Jolla. 18. Deming, J. W., P. S. Tabor, and R. R. Colwell. 1981. Barophilic growth of of cAMP to regulate gene expression (49). If high-pressure bacteria from intestinal tracts of deep-sea invertebrates. Microb. Ecol. 7:85– activation of ompH transcription does require CRP, then those 94. CRP structural changes brought about by high pressure must 19. Druffel, E. R. M., P. M. Williams, J. E. Bauer, and J. R. Ertel. 1992. Cycling not precisely duplicate the structural changes elicited by of dissolved and particulate organic matter in the open ocean. J. Geophys. Res. 97:15639–15659. cAMP. Otherwise, derepression of all genes controlled by ca- 20. Engebrecht, J., and M. Silverman. 1987. Nucleotide sequence of the regu- tabolite repression would occur at high pressure. latory locus controlling expression of bacterial genes for bioluminescence. The second possibility requires that ompH gene activation at Nucleic Acids Res. 15:10455–10467. high pressure occurs by a cAMP- and CRP-independent mech- 21. Epstein, W., L. B. Rothman-Denes, and J. Hesse. 1975. Adenosine 3Ј:5Љ- cyclic AMP monophosphate as a mediator of catabolite repression in Esch- anism. This could be accomplished by a variety of mechanisms, erichia coli. Proc. Natl. Acad. Sci. USA 72:2300–2304. including, for example, the synthesis of an alternative ␴ factor, 22. Gogstad, G. O., and M.-B. Krutnes. 1982. Measurement of protein in cell the modification of an existing ␴ factor, or the induction or suspensions using the coomassie brilliant blue dye-binding assay. Anal. Bio- activation of an activator protein specific to high pressure. It chem. 126:355–359. has been possible to isolate transposon mutants in SS9 which 23. Golden, S. S., J. Brusslan, and R. Haselkorn. 1986. Expression of a family of psbA genes encoding a photosystem II polypeptide in the cyanobacterium possess greatly reduced OmpH levels at 1 atm but not at high Anacystis nidulans R2. EMBO J. 5:2789–2798. pressure (14). These mutants possess insertions in genes which 24. Goodrich, J. A., M. L. Schwartz, and W. R. McClure. 1990. Searching for and are not involved in catabolite repression, indicating that there predicting the activity of sites for DNA binding proteins: compilation and are at least two separable differences in the regulation of ompH analysis of the binding sites for Escherichia coli integration host factor (IHF). Nucleic Acids Res. 18:4493–5000. expression at low and high pressure. Complex dual regulation 25. Harley, C. B., and R. P. Reynolds. 1987. Analysis of E. coli promoter se- of another starvation-inducible secreted protein, the E. coli quences. Nucleic Acids Res. 15:2343–2361. periplasmic OsmY protein, has been reported. osmY promoter 26. Hengge-Aronis, R., W. Klein, R. Lange, M. Rimmele, and W. Boos. 1991. activity appears to be differentially controlled by osmotic pres- Trehalose synthesis genes are controlled by the putative sigma factor en- coded by rpoS and are involved in stationary-phase thermotolerance in sure and stationary phase, and unlike the case for the ompH Escherichia coli. J. Bacteriol. 173:7918–7924. promoter, stationary-phase induction is repressed by cAMP- 27. Hoopes, B. C., and W. R. McClure. 1987. Strategies in the regulation of CRP (30, 60). transcription initiation, p. 1231–1240. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Esch- erichia coli and Salmonella typhimurium: cellular and molecular biology. ACKNOWLEDGMENTS American Society for Microbiology, Washington, D.C. 28. Jannasch, H. W., and C. O. Wirson. 1984. Variability of pressure adaptation We thank Bianca Brahamsha for assistance with the primer exten- in deep-sea bacteria. Arch. Microbiol. 139:281–288. sion assays. 29. Kolb, A., S. 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