Vol. 140, No. 3
JoURNAL OF BACTFRIOLOGY, Dec. 1979, p. 764-762 0021-9193/79/12-0754/09$02.00/0
Regulation of Membrane Peptides by the Pseudomonas Plasmid alk Regulon SPENCER BENSON,t MICHELE OPPICI, JAMES SHAPIRO,* AND MICHAEL FENNEWALDt Department of Microbiology, University of Chicago, Chicago, Illinois 60637 Received for publication 31 July 1979
Pseudomonas putida strains carrying the plasmid alk genes will grow on n-alkanes. Induced alk+ strains contain membrane activities for alkane hydroxylation and dehydrogenation of aliphatic primary alcohols. P. putida cytoplasmic and outer membranes can be separated by sucrose gradient centrifugation after disruption of cells by either mild detergent lysis or passage through a French press. Both the membrane component of alkane hydroxylase and the membrane alcohol dehydrogenase fractionated with the cytoplasmic membrane. Induction of the alk regulon resulted in the appearance of at least three new plasmiddetermined cytoplasmic membrane peptides of about 59,000 (59K), 47,000 (47K), and 40,000 (40K) daltons as well as the disappearance of a pair of chromosomally encoded outer membrane peptides of about 43,000 daltons. The 40K peptide is the membrane component of alkane hydroxylase and the product of the plasmid alkB gene because the alkB1029 mutation altered the properties of alkane hydroxylase in whole cells, reduced its thermal stability in cell extracts, and led to increased electrophoretic mobility of the inducible 40K peptide. These results are consistent with a model for vectorial oxidation of n-alkanes in the cytoplasmic membrane of P. putida. Pseudomonasputida and most P. aeruginosa strains can grow at the expense of 6- to 10-carbon n-alkanes only when they harbor certain IncP-2 plasmids carrying the alk regulon (10, 15). An important feature of Pseudomonas alkane oxidation is the presence of inducible oxidizing activities in the cell envelope (2, 19, 21). The plasmid alk regulon determines two such activities: the membrane component of alkane hydroxylase, which we have called the AlkB+ activity because it is determined by the plasmid alkB locus (2); and an NAD-independent membrane alcohol dehydrogenase, the synthesis of which specifically requires the expression of at least two plasmid loci, alkC and alkE (2, 4, 7). In addition, alkane hydroxylase activity requires the presence of soluble proteins (17), at least one of which is inducible and determined by the plasmid alkA locus (2, 3). The facts outlined above have led us to postulate a specific model for the assimilation of alkane carbon by induced alk+ Pseudomonas strains (Fig. 1). In our model, alkane molecules spontaneously partition into the interior of the bilayer cytoplasmic membrane without the intervention of a specific transport system (14). t Present address: Frederick Cancer Research Center, Frederick, MD 21701. t Present address: Department of Biochemistry, University of Chicago, Chicago, IL 60637.
(Because the relationship between the outer membrane and cytoplasmic membrane of Pseudomonas cells is not known, we cannot hypothesize a particular mechanism for penetration of the outermost layer.) Once dissolved in the cytoplasmic membrane, the alkane molecule diffiuses laterally until it is bound by the membrane component (oxidase) of alkane hydroxylase, which is in contact with the cytoplasmic rubredoxin protein (17). At the expense of 02 and NADH, alkane hydroxylation occurs, and the alcohol product remains dissolved in the bilayer. The alcohol diffuses until it encounters the membrane alcohol dehydrogenase, dehydrogenation occurs, and the aldehyde product either enters the cytoplasm or is also dehydrogenated in the bilayer to form a fatty acid. The fatty acid carbon is then assimilated into the cytoplasm by the soluble,-oxidation enzymes (20). To summarize the model, we can say that alkane oxidation is a vectorial transformation system with (i) no specific proteins to permit membrane penetration (because of the hydrophobic nature of the substrate) and (ii) at least two membrane enzymes that metabolize substrates dissoved in the hydrophobic interior of the bilayer. In this paper, we present evidence in support of this model. Our results show that both the membrane alkane hydroxylase component and alcohol dehydrogenase activity are located
754
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FIG. 1. Membrane model for alkane oxidation. The details are explained in the text. The figure schematizes the layers of the P. putida cell envelope and steps in alkane oxidation: LPS, lipopolysaccharide; OM, outer membrane; PG, peptidoglycan; CM, cytoplasmic membrane; Cyt, cytoplasm; RCH3, alkane molecule; RCH20H, primary alcohol oxidation product; RCHO, aldehyde oxidation product; RCOOH, fatty acid oxidation product; OX, oxidase or membrane component of alkane hydroxylase, product of the al*B gene; Rub, rubredoxin component of alkane hydroxylase (16), possibly the product of the alkA gene (2); Red, reductase component of alkane hydroxylase (16); Akc Deh., alcohol dehydrogenase (4), possibly the product of the alkE gene (7); T.CA., tricarboxylic acid.
chiefly in the cytoplasmic membrane. We also identify at least three inducible cytoplasmic membrane peptides controlled by the alk regulon and show that one of these is the product of the alkB gene. In addition, we find that the incorporation of at least one major chromosomally encoded peptide into the outer membrane is controlled by the alk regulon in plasmid-bearing strainrs. MATERIALS AND METHODS Bacterial strains. The bacterial strains have all descended from the PpGl P. putida line from the laboratory of . C. Gunsalus, University of Illinois, and are described in Table 1. Microbiological procedures, chemicals, and media. The microbiological procedures, chemicals, and media have been described in earlier publications (1, 2, 4, 15). Cultures were induced for alk expression on solid medium, as described in reference 2, when small amounts were needed for enzyme assays. When larger amounts of induced cells were needed, they were grown in liquid salts-0.2% pyruvate medium containing 0.5 mM dicyclopropylketone (10) and 0.1% (vol/vol) heptane. When we labeled cellular phospholipids, cultures were grown in tryptone (1%)-yeast extract (0.5%)-NaCl (0.5%) medium containing 0.4% glucose and 0.02 M [3H]glycerol (specific activity, 50 mCi/mmol). Isolation of membrane fractions. (i) Total P. putida envelopes were collected as the P200 fraction of a cell-free lysate after centrifugation as described in reference 2.
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(ii) Cytoplasmic and outer membranes were separated by the procedure of Collins and Niederman (6). Overnight induced liquid cultures (250 ml) and [3H]glycerol-labeled cultures (50 ml) were mixed, centrifuged, and frozen as a pellet at -20°C. (Freezing was essential to the success of this separation method.) Frozen cells were suspended in PA buffer (pH 7) (15) containing 25% sucrose, and the optical density (600 nm) was adjusted between 15 and 20. At 10-min intervals during incubation at room temperature, the following additions were made: lysozyme to 700 ,ug/ml, EDTA to 2 mg/ml, Brij 58 to 0.65%, and MgCl2 to 12.5 mM, together with a few crystals of DNase. After a final 10-min incubation, whole cells were removed by centrifugation at 1,000 x g for 15 min. The cell-free supernatant was centrifuged at 250,000 x g for 90 min, and the pellet was suspended in 2 to 3 ml of 0.01 M HEPES buffer (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid, pH 7.5) for overnight dialysis against the same buffer. The dialyzed membranes were then layered on top of a preformed 27-ml linear 20 to 60% or 30 to 60% sucrose gradient in 0.01 M HEPES buffer (pH 7.5) and centrifuged for 12 h in a Sorvall TV850 vertical rotor at 35,000 rpm. After centrifugation, gradients were either fractionated by dripping or visualized under a dissecting microscope lamp that individual bands could be collected in a syringe. (iii) Cytoplasmic and outer membranes were also separated by an adaptation of the procedure of Hancock and Nikaido (11). Frozen cell pellets were thawed and suspended in 0.038 M Tris buffer (pH 7.4) at an optical density of 15 to 20, and the cells were broken by two passages through a French pressure cell at 8,000 lb/in2. A few crystals of DNase and MgCl2 (to a 12 mM final concentration) were added, and the mixture was incubated at room temperature for 10 min. The P200 fraction of the extract was prepared and centrifuged in a 30 to 60% sucrose gradient in 0;08 M Tris buffer (pH 7.4) as outlined above (12 hr at 35,000 rpm in a Sorvall TV850 rotor). Separated fractions were visualized by scattered light and removed with a syringe. For electrophoresis, the proteins in each fraction were concentrated as a pellet by centrifugation at 250,000 x g for 2 h and suspended in electrophoresis buffer. Biochemical assays. (i) Alkane hydroxylase assays were performed as previously described (2, 3). The ALkB membrne component of hydroxylase was measured in a complementation assay with cell extract of the alkB strain PpS181 (3). Activity is expressed as nanomoles of ['4C]nonane or ['4C]octane oxidized per so
TABLE 1. P. putida strains Strain
PpS124 PpS145 PpS181 PpS338 PpS380 PpS1029
Genotype
Wild type (CAM-OCT) alcA81 met-145(CAM-OCT) alcA81(CAM-OCT alkB181) alcA81 trp-338 alcA81 tip-338(CAM-OCT) alcA437 met-598(CAM-OCT::Tn40l alkB1029) PpS1339 alcA81 tip-338(CAM-OCT::Tn40l alkBl029)
Source or reference 10 10 10 10 This paper 7
This paper
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BENSON ET AL.
minute per milligram of protein (or sometimes per milliliter of separated membrane fraction). (ii) Membrane alcohol dehydrogenase was determined as previously described (4). Activity is expressed as micromoles of 2,6-dichlorophenol-indophenol reduced per minute per milligram of protein (or per milliliter of separated membrane fraction) in response to substrate. (iii) Succinic dehydrogenase was assayed by the procedure of King (12). Activity is expressed as nanomoles of 2,6-dichlorophenol-indophenol reduced per minute per milligram of protein in response to substrate. (iv) NADH oxidase activity was determined by the procedure of Osborn et al. (16), except that 0.01 M HEPES buffer (pH 7.5) was sometimes substituted for Tris buffer. Activity is expressed as nanomoles of NADH oxidized per minute per milligram of protein. (v) 2-Keto-3-deoxyoctonate (KDO) was determined by the method of Weissbach and Horwitz (23) as described by Osborn et al. (16). It is expressed as absorbance at 540 nm per milliliter of separated membrane fraction. (vi) Proteins were determined by the 225- to 215nm absorbance method of Waddel (22). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membrane proteins were solubilized by incubation at 30°C in 2% sodium dodecyl sulfate-0.1 M Tris (pH 6.8)-0.01 M MgCl2 for 70 min. Solubilized proteins were collected as the supernatant after centrifugation at 47,000 x g for 1 h and were stored at -20°C before use. Gels were prepared by the method of Laemmli (13), except that sometimes the pH of the running bath was lowered from 8.8 to 7.8. Linear 15 to 20% gradient gels were cross-linked at a bis/acrylamide ratio of 0.0058. Gels were allowed to polymerize at room temperature after addition of 0.03% ammonium persulfate and 0.033% N,N,N',N'tetramethylethylenediamine as catalysts. Proteins were mixed either 1:1 or 1:3 with a disruption mixture of 8% sodium dodecyl sulfate, 20% ,B-mercaptoethanol, 10% glycerol, 0.025% bromophenol blue, and 0.2 M Tris (pH 6.5) and heated for 2 min in a boiling-water bath. Standards of bovine serum albumin, ovalbumin, pancreatic DNase, and lysozyme were run on each gel. Gels were stained in 50% trichloroacetic acid-5% isopropanol-0.1% Coomassie brilliant blue and destained in 7% acetic acid-5% isopropanol containing a few beads of anionic-exchange resin. Gels were photographed without drying through an orange filter. Staphylococcal protease redigestion. Coomassie brillaint blue-stained bands were extracted from gels and subjected to limited proteolysis with staphylococcal protease by the method of Cleveland et al.
J. BACTrERIOL.
before lysis. Figure 2 shows -an equilibrium sucrose gradient of P. putida membranes prepared in this way and photographed under visible and UV illumination. In such preparations, the fluorescent material bands at the top of the denser fraction, and the lighter fraction has a reddishorange color indicative of a high cytochrome content. Figure 3 shows the results of fractionating three separate membrane preparations from a mixture of two cultures: one grown in nutrient broth plus [3H]glycerol to label phospholipids and the other induced for alkane hydroxylase activity. The cytoplasmic membrane markers succinic dehydrogenase and NADH oxidase banded with the lighter phospholipid peak (panels A and B). The outer membrane lipopolysaccharide marker 2-keto-3-deoxyoctonate banded with the denser phospholipid peak. The alkane hydroxylase membrane component (AlkB+ activity) banded principally with the lighter (cytoplasmic membrane) phospholipid peak (panels B and C). The displacement of the AlkB+ activity to the light side of the cytoplasmic membrane peak apparently reflects stimulation of activity by excess lipids (S. Benson, Ph.D. thesis University of Chicago, Chicago, Ill., 1978), because gel electrophoresis of pooled fractions showed that most of the AlkB+ protein (identified below) was located under the main cytoplasmic membrane peak (data not shown). After we had devised the above separation procedure, Hancock and Nikaido worked out
(5). RESULTS Separation of P. putida inner (cytoplasmic) and outer membranes. After unsuccessful attempts to fractionate P. putida membranes by methods based on the Osborn technique (16), we found that the mild detergent lysis method of Collins and Niederman (6) would work provided that the cells were frozen and thawed
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FIG. 2. Sucrose gradient of P. putida membrane fractions after Brij lysis. Separated membranes of strain PpS124 wereprepared by the Collins and Niederman procedure (6) and centrifuged for 12 h as described in the text. The tube was photographed through an orange filter under UV illumination from the side (B) or under visible light from the top (A). The photos are aligned, although the tubes were supported differently for photography under different
lighting.
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methods to separate P. aeruginosa membranes after lysis in a French press (11). Their procedure is more rapid than the Collins-Niederman method and involves no detergent treatment (which inactivates the inducible membrane alcohol dehydrogenase). Freezing, thawing, and two successive passages through a French pressure cell yielded P. putida membranes which separated into two visible bands in equilibrium sucrose gradients. Assays of the NADH oxidase and succinic dehydrogenase and 2-keto-3-deoxyoctonate markers in the two bands showed that the lighter band contained predominantly cytoplasmic membrane and that the denser band contained predominantly outer membrane (Table 2). Assays of the alkane hydroxylase AlkB component and membrane alcohol dehydrogenase showed that the majority of these activities fractionated with the cytoplasmic membrane (Table 2). Fractionation of a gradient of French press membranes showed that alcohol dehydrogenase activity also banded at the leading edge of the cytoplasmic membrane peak (data not shown). Membrane peptides controlled by the alk regulon. Figure 4 shows the pattern of total membrane peptides from induced and uninduced alk+ cells after electrophoresis through a sodium dodecyl sulfate-polyacrylamide gradient
TABLE 2. P. putida membrane separationa Enzyme activity per Expt
Activity
membrane fraction:
determined OM
I
NADHoxidase
CM 55 (17)
5 (1.6)
II
Succinic dehydrogenase Ketodeoxyoctonate Alcohol dehydrogenase
23.4 (43.3) 0.13 19.9 (36.9)
3.9 (7.05) 1.43 3.4 (6.18)
37.1 (26.9) 4.8 (7.6) NADH oxidase 171 (49.3) 19.3 (12.2) Succinic dehydrogenase 16.7 (10.6) 122 (35.2) Alcohol dehydrogenase 16.8 (0.49) 0.18 (0.12) Alkane hydroxylase (AlkB) a Cytoplasmic membranes (CM) and outer membranes (OM) of PpS124-induced cultures were separated by the French press method, visualized in centrifuge tubes by scattered light, and collected in syringes for assay. Enzyme activities are given as total activity per milliliter of separated membrane fraction, and the specific activity per milligram protein is given in parentheses. Units are defined in the text. (Activity per unit volume is a better reflection of how a given enzyme fractionates than specific activity, because the latter value depends both on the amount of enzyme present and the amount of all other proteins present.) Ketodeoxyoctonate is expressed in optical density units as defined in the text. The membrane component of alkane hydroxylase (AlkB activity) was measured by a complementation assay (2, 3).
III
gel. At least three inducible peptides are clearly distinguishable in this and other gels at the
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following positions: 59,000 daltons (59K peptide); 47,000 daltons (47K peptide); and 40,000 daltons (40K peptide). In addition, there was a
pair of peptides at about the 43,000-dalton position (43K) which disappeared after induction. These two peptides (which are seen as a single band in other gels) are present in strains that contain no plasmid (data not shown) and so must be encoded by the P. putida chromosome. The 59K, 47K, and 40K peptides are not found in plasmid-free strains or strains which carry either the CAM plasmid or polar insertion alkB: :Tn7 mutations of CAM-OCT (Benson, Oppici, and Shapiro, unpublished data). Hence, the appearance of these peptides is encoded by the alk regulon. We have not yet analyzed the inducible 19K band in any detail. Electrophoresis of separated cytoplasmic and outer membrane fractions from induced and uninduced alk+ cells showed that the 59K, 47K, and 40K peptides were located in the cytoplasmic membrane and that the 43K peptide(s) was located in the outer membrane (Fig. 5). Identification of the 40K peptide as the alkB product. Our collection of plasmid alk mutations isolated from CAM-OCT plasmids contains a single example, alkB1029, which determines the phenotype expected of a transportdefective mutant: low activity in whole cells but high activity in cell extracts (Table 3). Despite this phenotype, however, we could show that this mutation is really an allele of alkB that modifies its gene product. First, genetic mapping showed that alkB1029 is located between other alkB mutations (7). Second, gel electrophoresis showed that an alkB1029 strain synthesized an altered 40K peptide with slightly increased electrophoretic mobility which we called 40*K (Fig. 4 and 6). Digestion of the alk + 40K and alkB1029 40*K bands with staphylococcal protease showed that the peptides were virtually identical (Fig. 7). Third, the alkBl029 mutation reduced the thermal stability of AlkB+ activity (Fig. 8). One possible explanation for the cryptic phenotype of alkB1029 strains is that the membrane localization of the 40*K peptide is altered so that it is inserted in the outer membrane, where it cannot interact with the soluble hydroxylase proteins. However, analysis of separated alkB1029 membranes showed that the 40*K peptide was located in the cytoplasmic membrane (Fig. 9).
DISCUSSION Our results support the model presented in Fig. 1 by establishing the following characteristics of P. putida alkane oxidation. (i) Membrane alkane hydroxylase and alcohol dehydrogenase activities are located in the cytoplasmic membrane (Fig. 3 and Table 2). (ii) Induction of the
alk MEMBRANE PROTEINS
VOL. 140, 1979
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FIG. 5. Gel electrophoresis of separated P. putida membranes. PpS124 was grown under inducing (Ind) or noninducing conditions (Non), and membranes were separated into cytoplasmic membrane (CM) and outer membrane (OM) fractions by an adaptation of the Hancock and Nikaido procedure (11) as described in the text. The separated fractions were then electrophoresed in 15 to 20% gradient gels (pH 8.8), stained, and photographed as described in the text. Panels A and B represent different preparations. The labeled arrows and stars indicate the peptides affected by induction.
TABLE 3. Crypticity of alkB1029 mutants for alkane hydroxylase activitya Genotype alk+ (expt 1)
alkBl029 (expt 1)
Prepn Whole cells Sonic extract
Alkane hydroxylase activity 0.84 1.82
Whole cells
0.02
Sonic extract
0.47
QltWT
59K
47K
I,
I
40K
*.
:
alkB1029 (expt 2)
Whole cells 0.01 Sonic extract 0.33 French press extract 0.34 Strains PpS145 (alk*) and PpS1029 (aIkBl029) were
grown under inducing conditions and assayed for alkane hydroxylase activity before and after cellular disruption.
alk regulon results in the appearance of at least three new cytoplasmic membrane peptides (Fig. 4, 5, and 9). (iii) Accessibility of alkane hydroxylase activity to substrate in whole cells is governed by the properties of the membrane hydroxylase protein (Table 3 and Fig. 4). In addition, our results permitted us to identify the 40K inducible peptide as the product of the alkB gene because an alkB mutation altered the electrophoretic mobility of the 40K peptide and the
plk4O29
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FIG. 6. Densitometer tracings of lanes 1 and 3 of Fig. 4. The superposition clearly shows that only the migration of the 40Kpeptide was altered by the alk1029 mutation. WT, Wild type.
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(alkB1029, O) were incubated at 48°C for the times indicated, cooled on ice, and then assayed for alkane hydroxylase activity in the presence of an induced PpS181 (AlkB-) extract to measure only the AlkB membrane component. The ordinate shows the percentage of original activity remaining.
14K - o a
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FIG. 7. Staphylococcal protease redigestion of the alk wild type (alkWT) 40K and alk-1029 40*K bands from a gel similar to the one shown in Fig. 4. The labeled arrows indicate the positions of marker proteins on this 15 to 20% gradient gel (pH 8.8).
thermal stability of AlkB+ activity (Fig. 4, 6, and 8). This result is in excellent agreement with the biochemical results of Ruettinger et al., who identified a 40,800-dalton phospholipid-requiring component of alkane hydroxylase (18). It also agrees with analysis of insertion and deletion mutants of the alkBAE operon because there is a strict correlation between the presence of AlkB+ activity and the 40K peptide (unpublished data). The work presented here raises at least three interesting questions. First, what are the functions of the inducible 59K and 47K peptides? We suspect they are involved in the membrane alcohol dehydrogenase activity because polar alkB::Tn7 and alkA::Tn7 insertions (7) elimi-
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VOL. 140, 1979
nate both this activity and the appearance of these two peptides (unpublished data). However, we do not yet have conclusive evidence to show a strict correlation between the appearance of one or both peptides and membrane alcohol dehydrogenase activity. At least the 47K peptide is clearly not the product of the alkC gene
required for alcohol dehydrogenase activity because alkC::Tn7 strains synthesize this peptide (unpublished data). Second, why do the 43K outer membrane peptides disappear from induced cells? This could be due to a specific regulatory effect of some alk regulon product on synthesis of these proteins, altered processing of 43K precursors due to the presence of new peptides in the envelope, or degradation of the 43K peptides by specific or nonspecific proteases. We incline to the first explanation for two reasons. Polar mutations of the alkBAE operon prevent the disappearance of the 43K peptides (unpublished datum), so that specific alk gene products appear to be involved. Second, the 43K peptides are major outer membrane peptides (Fig. 4 and 5). By analogy with Escherichia coli and P. aeruginosa outer membrane proteins, they are likely to influence outer membrane permeability (11). Therefore, it is conceivable that a cell adapting to growth on a hydrophobic substrate which must enter the cytoplasmic membrane will change the permeability of the outer membrane. Gilleland and Lyle (9) report the identification of a major membrane peptide of about 44,000 daltons in P. aeruginosa PAO, and this peptide fractionates with the outer membrane. Third, why is an alkB1029 strain cryptic for alkane hydroxylase activity (Table 3)? We eliminated one explanation by showing that the mutant 40*K peptide is located in the cytoplasmic membrane (Fig. 9). There are trivial explanations, such as altered pH or osmotic optima for activity so that in vitro conditions are much more favorable than in vivo conditions. However, there is also an interesting possible explanation, namely, that the 40*K protein is not properly oriented in the cytoplasmic membrane and cannot interact correctly with rubredoxin (Fig. 1) unless the membranes have been scrambled during extraction. One additional feature of our results merits emphasis. In highly induced cultures, the 59K, 47K, and 40K peptides are the major membrane peptides together with a 42,000-dalton outer membrane protein (Fig. 4 through 6). As Fig. 6 shows, they are not all made in equimolar amounts, and our gels consistently showed the 40K peptide to be the major P. putida membrane protein after induction. We do not yet
alk MEMBRANE PROTEINS
761
understand the consequence of such major alterations in the composition of the Pseudomonas envelope after induction of the alk regulon. Nonetheless, we can safely predict that a thorough understanding of alk regulation will lead to deeper insights into the synthesis of the P. putida envelope. ACKNOWLEDGMENTS This research was supported by grants from the donors of the Petroleum Research Fund administered by the American Chemical Society, the National Science Foundation (PCM 7708591), and the Louis Block Fund of the University of Chicago. S. Benson and M. Fennewald were the recipients of Public Health Service predoctoral traineeships (GM-00090 and GM07197, respectively), and J. Shapiro was the recipient of a Public Health Service Research Career Development Award (1 K04 AI-00118). LITERATURE CITED 1. Benedik, M., M. Fennewald, and J. Shapiro. 1977. Transposition of a ,B-lactamase locus from RP1 into Pseudomonas putida degradative plasmids. J. Bacteriol. 129:809-814. 2. Benson, S., M. Fennewald, J. Shapiro, and C. Huettner. 1977. Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase-negative plasmid mutations. J. Bacteriol. 132:614-621. 3. Benson, S., and J. Shapiro. 1975. Induction of alkane hydroxylase proteins by unoxidized alkane in Pseudomonasputida. J. Bacteriol. 123:759-760. 4. Benson, S., and J. Shapiro. 1976. Plasmid-determined alcohol dehydrogenase in alkane-utilizing strain of Pseudomonas putida. J. Bacteriol. 126:794-798. 5. Cleveland, C. W., S. G. Fisher, M. W. Kirschner, and U. K. Laemmli. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 262:1102-1106. 6. Collins, M. L, and R. Niederman. 1976. Membranes of Rhodospirillum rubrum: isolation and physicochemical properties of membranes from aerobically grown cells. J. Bacteriol. 126:1316-1326. 7. Fennewald, M., S. Benson, M. Oppici, and J. Shapiro. 1979. Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon. J. Bacteriol. 139: 940-952. 8. Fennewald, M., and J. Shapiro. 1977. Regulatory mutations of the Pseudomonas plasmid alk regulon. J. Bacteriol. 132:622-627. 9. Gilleland, H. E., and R. D. Lyle. 1979. Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates. J. Bacteriol. 138:839845. 10. Grund, A., J. Shapiro, M. Fennewald, P. Bacha, J. Leahy, K. Markbreiter, M. Nieder, and M. Toepfer. 1975. Regulation of alkane oxidation in Pseudomonas putida. J. Bacteriol. 123:546-556. 11. Hancock, R. E. W., and H. Nikaido. 1978. Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier. J. Bacteriol. 136:381-390. 12. King, T. E. 1963. Reconstitution of respiratory chain enzymes systems. XI. Use of artificial electron acceptors in the assay of succinate dehydrogenase. J. Biol. Chem.
238:4032-4036. 13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685.
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14. Mira, Y., M. Okazaki, S. I. Hamada, S. I. Marakawa, and R. Yugen. 1977. Assimilation of liquid hydrocarbon by microorganisms. I. Mechanism of hydrocarbon
uptake. Biotechnol. Bioeng. 13:701-714. 15. Nieder, M., and J. Shapiro. 1975. Physiological function of the Pseudomonas putida PpG6 (Pseudomonas oleovorans) alkane hydroxylase: monoterminal oxidation of alkanes and fatty acids. J. Bacteriol. 122:93-98. 16. Osborn, M. J., J. E. Cander, E. Parisi, and J. Carson. 1972. Mechanisms of assembly of the outer membrane of Salmonella typhimurium: isolation and characterization of cytoplasmic and outer membranes. J. Biol. Chem. 247:3967-3972. 17. Peterson, J. A., D. Basu, and M. Coon. 1966. Enzymatic u-oxidation. I. Electron carriers in fatty acid and hydrocarbon hydroxylation. J. Biol. Chem. 241:5162-5164. 18. Ruettinger, R. T., G. R. Griffith, and M. J. Coon. 1977. Characterization of the w-hydroxylase of Pseudomonas oleovorans as a nonheme iron protein. Arch. Biochem.
Biophys. 183:528-537. 19. Tassin, J. P., C. Celier, and J. P. Vandercasteele. 1973. Purification and properties of a membrane bound alcohol dehydrogenase involved in oxidation of long chain hydrocarbons by Pseudomonas aeruginosa.
Biochim. Biophys. Acta 315:220-232. 20. Trust, T. J., and N. F. Millis. 1971. Activation of the C2 to Cig monocarboxylic acids by Pseudomonas. J. Bacteriol. 105:1216-1218. 21. van Eyk, J., and T. J. Bartells. 1970. Paraffin oxidation in Pseudomonas aeruginosa. II. Gross fractionation of the enzyme system into soluble and particulate components. J. Bacteriol. 104:1065-1073. 22. Waddel, W. J. 1956. A simple ultraviolet spectrophotometric method for determination of proteins. J. Lab. Clin. Med. 48:311-314. 23. Weissbach, A., and J. Hurwitz. 1959. The formation of 2-keto-3-deoxyheptanoic acid in extracts of Escherichia coli B. J. Biol. Chem. 234:705-709.
JoURNAL OF BACTFRIOLOGY, Dec. 1979, p. 764-762 0021-9193/79/12-0754/09$02.00/0
Regulation of Membrane Peptides by the Pseudomonas Plasmid alk Regulon SPENCER BENSON,t MICHELE OPPICI, JAMES SHAPIRO,* AND MICHAEL FENNEWALDt Department of Microbiology, University of Chicago, Chicago, Illinois 60637 Received for publication 31 July 1979
Pseudomonas putida strains carrying the plasmid alk genes will grow on n-alkanes. Induced alk+ strains contain membrane activities for alkane hydroxylation and dehydrogenation of aliphatic primary alcohols. P. putida cytoplasmic and outer membranes can be separated by sucrose gradient centrifugation after disruption of cells by either mild detergent lysis or passage through a French press. Both the membrane component of alkane hydroxylase and the membrane alcohol dehydrogenase fractionated with the cytoplasmic membrane. Induction of the alk regulon resulted in the appearance of at least three new plasmiddetermined cytoplasmic membrane peptides of about 59,000 (59K), 47,000 (47K), and 40,000 (40K) daltons as well as the disappearance of a pair of chromosomally encoded outer membrane peptides of about 43,000 daltons. The 40K peptide is the membrane component of alkane hydroxylase and the product of the plasmid alkB gene because the alkB1029 mutation altered the properties of alkane hydroxylase in whole cells, reduced its thermal stability in cell extracts, and led to increased electrophoretic mobility of the inducible 40K peptide. These results are consistent with a model for vectorial oxidation of n-alkanes in the cytoplasmic membrane of P. putida. Pseudomonasputida and most P. aeruginosa strains can grow at the expense of 6- to 10-carbon n-alkanes only when they harbor certain IncP-2 plasmids carrying the alk regulon (10, 15). An important feature of Pseudomonas alkane oxidation is the presence of inducible oxidizing activities in the cell envelope (2, 19, 21). The plasmid alk regulon determines two such activities: the membrane component of alkane hydroxylase, which we have called the AlkB+ activity because it is determined by the plasmid alkB locus (2); and an NAD-independent membrane alcohol dehydrogenase, the synthesis of which specifically requires the expression of at least two plasmid loci, alkC and alkE (2, 4, 7). In addition, alkane hydroxylase activity requires the presence of soluble proteins (17), at least one of which is inducible and determined by the plasmid alkA locus (2, 3). The facts outlined above have led us to postulate a specific model for the assimilation of alkane carbon by induced alk+ Pseudomonas strains (Fig. 1). In our model, alkane molecules spontaneously partition into the interior of the bilayer cytoplasmic membrane without the intervention of a specific transport system (14). t Present address: Frederick Cancer Research Center, Frederick, MD 21701. t Present address: Department of Biochemistry, University of Chicago, Chicago, IL 60637.
(Because the relationship between the outer membrane and cytoplasmic membrane of Pseudomonas cells is not known, we cannot hypothesize a particular mechanism for penetration of the outermost layer.) Once dissolved in the cytoplasmic membrane, the alkane molecule diffiuses laterally until it is bound by the membrane component (oxidase) of alkane hydroxylase, which is in contact with the cytoplasmic rubredoxin protein (17). At the expense of 02 and NADH, alkane hydroxylation occurs, and the alcohol product remains dissolved in the bilayer. The alcohol diffuses until it encounters the membrane alcohol dehydrogenase, dehydrogenation occurs, and the aldehyde product either enters the cytoplasm or is also dehydrogenated in the bilayer to form a fatty acid. The fatty acid carbon is then assimilated into the cytoplasm by the soluble,-oxidation enzymes (20). To summarize the model, we can say that alkane oxidation is a vectorial transformation system with (i) no specific proteins to permit membrane penetration (because of the hydrophobic nature of the substrate) and (ii) at least two membrane enzymes that metabolize substrates dissoved in the hydrophobic interior of the bilayer. In this paper, we present evidence in support of this model. Our results show that both the membrane alkane hydroxylase component and alcohol dehydrogenase activity are located
754
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FIG. 1. Membrane model for alkane oxidation. The details are explained in the text. The figure schematizes the layers of the P. putida cell envelope and steps in alkane oxidation: LPS, lipopolysaccharide; OM, outer membrane; PG, peptidoglycan; CM, cytoplasmic membrane; Cyt, cytoplasm; RCH3, alkane molecule; RCH20H, primary alcohol oxidation product; RCHO, aldehyde oxidation product; RCOOH, fatty acid oxidation product; OX, oxidase or membrane component of alkane hydroxylase, product of the al*B gene; Rub, rubredoxin component of alkane hydroxylase (16), possibly the product of the alkA gene (2); Red, reductase component of alkane hydroxylase (16); Akc Deh., alcohol dehydrogenase (4), possibly the product of the alkE gene (7); T.CA., tricarboxylic acid.
chiefly in the cytoplasmic membrane. We also identify at least three inducible cytoplasmic membrane peptides controlled by the alk regulon and show that one of these is the product of the alkB gene. In addition, we find that the incorporation of at least one major chromosomally encoded peptide into the outer membrane is controlled by the alk regulon in plasmid-bearing strainrs. MATERIALS AND METHODS Bacterial strains. The bacterial strains have all descended from the PpGl P. putida line from the laboratory of . C. Gunsalus, University of Illinois, and are described in Table 1. Microbiological procedures, chemicals, and media. The microbiological procedures, chemicals, and media have been described in earlier publications (1, 2, 4, 15). Cultures were induced for alk expression on solid medium, as described in reference 2, when small amounts were needed for enzyme assays. When larger amounts of induced cells were needed, they were grown in liquid salts-0.2% pyruvate medium containing 0.5 mM dicyclopropylketone (10) and 0.1% (vol/vol) heptane. When we labeled cellular phospholipids, cultures were grown in tryptone (1%)-yeast extract (0.5%)-NaCl (0.5%) medium containing 0.4% glucose and 0.02 M [3H]glycerol (specific activity, 50 mCi/mmol). Isolation of membrane fractions. (i) Total P. putida envelopes were collected as the P200 fraction of a cell-free lysate after centrifugation as described in reference 2.
755
(ii) Cytoplasmic and outer membranes were separated by the procedure of Collins and Niederman (6). Overnight induced liquid cultures (250 ml) and [3H]glycerol-labeled cultures (50 ml) were mixed, centrifuged, and frozen as a pellet at -20°C. (Freezing was essential to the success of this separation method.) Frozen cells were suspended in PA buffer (pH 7) (15) containing 25% sucrose, and the optical density (600 nm) was adjusted between 15 and 20. At 10-min intervals during incubation at room temperature, the following additions were made: lysozyme to 700 ,ug/ml, EDTA to 2 mg/ml, Brij 58 to 0.65%, and MgCl2 to 12.5 mM, together with a few crystals of DNase. After a final 10-min incubation, whole cells were removed by centrifugation at 1,000 x g for 15 min. The cell-free supernatant was centrifuged at 250,000 x g for 90 min, and the pellet was suspended in 2 to 3 ml of 0.01 M HEPES buffer (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid, pH 7.5) for overnight dialysis against the same buffer. The dialyzed membranes were then layered on top of a preformed 27-ml linear 20 to 60% or 30 to 60% sucrose gradient in 0.01 M HEPES buffer (pH 7.5) and centrifuged for 12 h in a Sorvall TV850 vertical rotor at 35,000 rpm. After centrifugation, gradients were either fractionated by dripping or visualized under a dissecting microscope lamp that individual bands could be collected in a syringe. (iii) Cytoplasmic and outer membranes were also separated by an adaptation of the procedure of Hancock and Nikaido (11). Frozen cell pellets were thawed and suspended in 0.038 M Tris buffer (pH 7.4) at an optical density of 15 to 20, and the cells were broken by two passages through a French pressure cell at 8,000 lb/in2. A few crystals of DNase and MgCl2 (to a 12 mM final concentration) were added, and the mixture was incubated at room temperature for 10 min. The P200 fraction of the extract was prepared and centrifuged in a 30 to 60% sucrose gradient in 0;08 M Tris buffer (pH 7.4) as outlined above (12 hr at 35,000 rpm in a Sorvall TV850 rotor). Separated fractions were visualized by scattered light and removed with a syringe. For electrophoresis, the proteins in each fraction were concentrated as a pellet by centrifugation at 250,000 x g for 2 h and suspended in electrophoresis buffer. Biochemical assays. (i) Alkane hydroxylase assays were performed as previously described (2, 3). The ALkB membrne component of hydroxylase was measured in a complementation assay with cell extract of the alkB strain PpS181 (3). Activity is expressed as nanomoles of ['4C]nonane or ['4C]octane oxidized per so
TABLE 1. P. putida strains Strain
PpS124 PpS145 PpS181 PpS338 PpS380 PpS1029
Genotype
Wild type (CAM-OCT) alcA81 met-145(CAM-OCT) alcA81(CAM-OCT alkB181) alcA81 trp-338 alcA81 tip-338(CAM-OCT) alcA437 met-598(CAM-OCT::Tn40l alkB1029) PpS1339 alcA81 tip-338(CAM-OCT::Tn40l alkBl029)
Source or reference 10 10 10 10 This paper 7
This paper
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BENSON ET AL.
minute per milligram of protein (or sometimes per milliliter of separated membrane fraction). (ii) Membrane alcohol dehydrogenase was determined as previously described (4). Activity is expressed as micromoles of 2,6-dichlorophenol-indophenol reduced per minute per milligram of protein (or per milliliter of separated membrane fraction) in response to substrate. (iii) Succinic dehydrogenase was assayed by the procedure of King (12). Activity is expressed as nanomoles of 2,6-dichlorophenol-indophenol reduced per minute per milligram of protein in response to substrate. (iv) NADH oxidase activity was determined by the procedure of Osborn et al. (16), except that 0.01 M HEPES buffer (pH 7.5) was sometimes substituted for Tris buffer. Activity is expressed as nanomoles of NADH oxidized per minute per milligram of protein. (v) 2-Keto-3-deoxyoctonate (KDO) was determined by the method of Weissbach and Horwitz (23) as described by Osborn et al. (16). It is expressed as absorbance at 540 nm per milliliter of separated membrane fraction. (vi) Proteins were determined by the 225- to 215nm absorbance method of Waddel (22). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Membrane proteins were solubilized by incubation at 30°C in 2% sodium dodecyl sulfate-0.1 M Tris (pH 6.8)-0.01 M MgCl2 for 70 min. Solubilized proteins were collected as the supernatant after centrifugation at 47,000 x g for 1 h and were stored at -20°C before use. Gels were prepared by the method of Laemmli (13), except that sometimes the pH of the running bath was lowered from 8.8 to 7.8. Linear 15 to 20% gradient gels were cross-linked at a bis/acrylamide ratio of 0.0058. Gels were allowed to polymerize at room temperature after addition of 0.03% ammonium persulfate and 0.033% N,N,N',N'tetramethylethylenediamine as catalysts. Proteins were mixed either 1:1 or 1:3 with a disruption mixture of 8% sodium dodecyl sulfate, 20% ,B-mercaptoethanol, 10% glycerol, 0.025% bromophenol blue, and 0.2 M Tris (pH 6.5) and heated for 2 min in a boiling-water bath. Standards of bovine serum albumin, ovalbumin, pancreatic DNase, and lysozyme were run on each gel. Gels were stained in 50% trichloroacetic acid-5% isopropanol-0.1% Coomassie brilliant blue and destained in 7% acetic acid-5% isopropanol containing a few beads of anionic-exchange resin. Gels were photographed without drying through an orange filter. Staphylococcal protease redigestion. Coomassie brillaint blue-stained bands were extracted from gels and subjected to limited proteolysis with staphylococcal protease by the method of Cleveland et al.
J. BACTrERIOL.
before lysis. Figure 2 shows -an equilibrium sucrose gradient of P. putida membranes prepared in this way and photographed under visible and UV illumination. In such preparations, the fluorescent material bands at the top of the denser fraction, and the lighter fraction has a reddishorange color indicative of a high cytochrome content. Figure 3 shows the results of fractionating three separate membrane preparations from a mixture of two cultures: one grown in nutrient broth plus [3H]glycerol to label phospholipids and the other induced for alkane hydroxylase activity. The cytoplasmic membrane markers succinic dehydrogenase and NADH oxidase banded with the lighter phospholipid peak (panels A and B). The outer membrane lipopolysaccharide marker 2-keto-3-deoxyoctonate banded with the denser phospholipid peak. The alkane hydroxylase membrane component (AlkB+ activity) banded principally with the lighter (cytoplasmic membrane) phospholipid peak (panels B and C). The displacement of the AlkB+ activity to the light side of the cytoplasmic membrane peak apparently reflects stimulation of activity by excess lipids (S. Benson, Ph.D. thesis University of Chicago, Chicago, Ill., 1978), because gel electrophoresis of pooled fractions showed that most of the AlkB+ protein (identified below) was located under the main cytoplasmic membrane peak (data not shown). After we had devised the above separation procedure, Hancock and Nikaido worked out
(5). RESULTS Separation of P. putida inner (cytoplasmic) and outer membranes. After unsuccessful attempts to fractionate P. putida membranes by methods based on the Osborn technique (16), we found that the mild detergent lysis method of Collins and Niederman (6) would work provided that the cells were frozen and thawed
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FIG. 2. Sucrose gradient of P. putida membrane fractions after Brij lysis. Separated membranes of strain PpS124 wereprepared by the Collins and Niederman procedure (6) and centrifuged for 12 h as described in the text. The tube was photographed through an orange filter under UV illumination from the side (B) or under visible light from the top (A). The photos are aligned, although the tubes were supported differently for photography under different
lighting.
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methods to separate P. aeruginosa membranes after lysis in a French press (11). Their procedure is more rapid than the Collins-Niederman method and involves no detergent treatment (which inactivates the inducible membrane alcohol dehydrogenase). Freezing, thawing, and two successive passages through a French pressure cell yielded P. putida membranes which separated into two visible bands in equilibrium sucrose gradients. Assays of the NADH oxidase and succinic dehydrogenase and 2-keto-3-deoxyoctonate markers in the two bands showed that the lighter band contained predominantly cytoplasmic membrane and that the denser band contained predominantly outer membrane (Table 2). Assays of the alkane hydroxylase AlkB component and membrane alcohol dehydrogenase showed that the majority of these activities fractionated with the cytoplasmic membrane (Table 2). Fractionation of a gradient of French press membranes showed that alcohol dehydrogenase activity also banded at the leading edge of the cytoplasmic membrane peak (data not shown). Membrane peptides controlled by the alk regulon. Figure 4 shows the pattern of total membrane peptides from induced and uninduced alk+ cells after electrophoresis through a sodium dodecyl sulfate-polyacrylamide gradient
TABLE 2. P. putida membrane separationa Enzyme activity per Expt
Activity
membrane fraction:
determined OM
I
NADHoxidase
CM 55 (17)
5 (1.6)
II
Succinic dehydrogenase Ketodeoxyoctonate Alcohol dehydrogenase
23.4 (43.3) 0.13 19.9 (36.9)
3.9 (7.05) 1.43 3.4 (6.18)
37.1 (26.9) 4.8 (7.6) NADH oxidase 171 (49.3) 19.3 (12.2) Succinic dehydrogenase 16.7 (10.6) 122 (35.2) Alcohol dehydrogenase 16.8 (0.49) 0.18 (0.12) Alkane hydroxylase (AlkB) a Cytoplasmic membranes (CM) and outer membranes (OM) of PpS124-induced cultures were separated by the French press method, visualized in centrifuge tubes by scattered light, and collected in syringes for assay. Enzyme activities are given as total activity per milliliter of separated membrane fraction, and the specific activity per milligram protein is given in parentheses. Units are defined in the text. (Activity per unit volume is a better reflection of how a given enzyme fractionates than specific activity, because the latter value depends both on the amount of enzyme present and the amount of all other proteins present.) Ketodeoxyoctonate is expressed in optical density units as defined in the text. The membrane component of alkane hydroxylase (AlkB activity) was measured by a complementation assay (2, 3).
III
gel. At least three inducible peptides are clearly distinguishable in this and other gels at the
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following positions: 59,000 daltons (59K peptide); 47,000 daltons (47K peptide); and 40,000 daltons (40K peptide). In addition, there was a
pair of peptides at about the 43,000-dalton position (43K) which disappeared after induction. These two peptides (which are seen as a single band in other gels) are present in strains that contain no plasmid (data not shown) and so must be encoded by the P. putida chromosome. The 59K, 47K, and 40K peptides are not found in plasmid-free strains or strains which carry either the CAM plasmid or polar insertion alkB: :Tn7 mutations of CAM-OCT (Benson, Oppici, and Shapiro, unpublished data). Hence, the appearance of these peptides is encoded by the alk regulon. We have not yet analyzed the inducible 19K band in any detail. Electrophoresis of separated cytoplasmic and outer membrane fractions from induced and uninduced alk+ cells showed that the 59K, 47K, and 40K peptides were located in the cytoplasmic membrane and that the 43K peptide(s) was located in the outer membrane (Fig. 5). Identification of the 40K peptide as the alkB product. Our collection of plasmid alk mutations isolated from CAM-OCT plasmids contains a single example, alkB1029, which determines the phenotype expected of a transportdefective mutant: low activity in whole cells but high activity in cell extracts (Table 3). Despite this phenotype, however, we could show that this mutation is really an allele of alkB that modifies its gene product. First, genetic mapping showed that alkB1029 is located between other alkB mutations (7). Second, gel electrophoresis showed that an alkB1029 strain synthesized an altered 40K peptide with slightly increased electrophoretic mobility which we called 40*K (Fig. 4 and 6). Digestion of the alk + 40K and alkB1029 40*K bands with staphylococcal protease showed that the peptides were virtually identical (Fig. 7). Third, the alkBl029 mutation reduced the thermal stability of AlkB+ activity (Fig. 8). One possible explanation for the cryptic phenotype of alkB1029 strains is that the membrane localization of the 40*K peptide is altered so that it is inserted in the outer membrane, where it cannot interact with the soluble hydroxylase proteins. However, analysis of separated alkB1029 membranes showed that the 40*K peptide was located in the cytoplasmic membrane (Fig. 9).
DISCUSSION Our results support the model presented in Fig. 1 by establishing the following characteristics of P. putida alkane oxidation. (i) Membrane alkane hydroxylase and alcohol dehydrogenase activities are located in the cytoplasmic membrane (Fig. 3 and Table 2). (ii) Induction of the
alk MEMBRANE PROTEINS
VOL. 140, 1979
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TABLE 3. Crypticity of alkB1029 mutants for alkane hydroxylase activitya Genotype alk+ (expt 1)
alkBl029 (expt 1)
Prepn Whole cells Sonic extract
Alkane hydroxylase activity 0.84 1.82
Whole cells
0.02
Sonic extract
0.47
QltWT
59K
47K
I,
I
40K
*.
:
alkB1029 (expt 2)
Whole cells 0.01 Sonic extract 0.33 French press extract 0.34 Strains PpS145 (alk*) and PpS1029 (aIkBl029) were
grown under inducing conditions and assayed for alkane hydroxylase activity before and after cellular disruption.
alk regulon results in the appearance of at least three new cytoplasmic membrane peptides (Fig. 4, 5, and 9). (iii) Accessibility of alkane hydroxylase activity to substrate in whole cells is governed by the properties of the membrane hydroxylase protein (Table 3 and Fig. 4). In addition, our results permitted us to identify the 40K inducible peptide as the product of the alkB gene because an alkB mutation altered the electrophoretic mobility of the 40K peptide and the
plk4O29
A
FIG. 6. Densitometer tracings of lanes 1 and 3 of Fig. 4. The superposition clearly shows that only the migration of the 40Kpeptide was altered by the alk1029 mutation. WT, Wild type.
760
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alkB1029 alkane hydroxylase. Crude extracts of induced cultures of PpS380 (alk+, 0) and PpS1339 .
18K - -i
(alkB1029, O) were incubated at 48°C for the times indicated, cooled on ice, and then assayed for alkane hydroxylase activity in the presence of an induced PpS181 (AlkB-) extract to measure only the AlkB membrane component. The ordinate shows the percentage of original activity remaining.
14K - o a
O
FIG. 7. Staphylococcal protease redigestion of the alk wild type (alkWT) 40K and alk-1029 40*K bands from a gel similar to the one shown in Fig. 4. The labeled arrows indicate the positions of marker proteins on this 15 to 20% gradient gel (pH 8.8).
thermal stability of AlkB+ activity (Fig. 4, 6, and 8). This result is in excellent agreement with the biochemical results of Ruettinger et al., who identified a 40,800-dalton phospholipid-requiring component of alkane hydroxylase (18). It also agrees with analysis of insertion and deletion mutants of the alkBAE operon because there is a strict correlation between the presence of AlkB+ activity and the 40K peptide (unpublished data). The work presented here raises at least three interesting questions. First, what are the functions of the inducible 59K and 47K peptides? We suspect they are involved in the membrane alcohol dehydrogenase activity because polar alkB::Tn7 and alkA::Tn7 insertions (7) elimi-
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VOL. 140, 1979
nate both this activity and the appearance of these two peptides (unpublished data). However, we do not yet have conclusive evidence to show a strict correlation between the appearance of one or both peptides and membrane alcohol dehydrogenase activity. At least the 47K peptide is clearly not the product of the alkC gene
required for alcohol dehydrogenase activity because alkC::Tn7 strains synthesize this peptide (unpublished data). Second, why do the 43K outer membrane peptides disappear from induced cells? This could be due to a specific regulatory effect of some alk regulon product on synthesis of these proteins, altered processing of 43K precursors due to the presence of new peptides in the envelope, or degradation of the 43K peptides by specific or nonspecific proteases. We incline to the first explanation for two reasons. Polar mutations of the alkBAE operon prevent the disappearance of the 43K peptides (unpublished datum), so that specific alk gene products appear to be involved. Second, the 43K peptides are major outer membrane peptides (Fig. 4 and 5). By analogy with Escherichia coli and P. aeruginosa outer membrane proteins, they are likely to influence outer membrane permeability (11). Therefore, it is conceivable that a cell adapting to growth on a hydrophobic substrate which must enter the cytoplasmic membrane will change the permeability of the outer membrane. Gilleland and Lyle (9) report the identification of a major membrane peptide of about 44,000 daltons in P. aeruginosa PAO, and this peptide fractionates with the outer membrane. Third, why is an alkB1029 strain cryptic for alkane hydroxylase activity (Table 3)? We eliminated one explanation by showing that the mutant 40*K peptide is located in the cytoplasmic membrane (Fig. 9). There are trivial explanations, such as altered pH or osmotic optima for activity so that in vitro conditions are much more favorable than in vivo conditions. However, there is also an interesting possible explanation, namely, that the 40*K protein is not properly oriented in the cytoplasmic membrane and cannot interact correctly with rubredoxin (Fig. 1) unless the membranes have been scrambled during extraction. One additional feature of our results merits emphasis. In highly induced cultures, the 59K, 47K, and 40K peptides are the major membrane peptides together with a 42,000-dalton outer membrane protein (Fig. 4 through 6). As Fig. 6 shows, they are not all made in equimolar amounts, and our gels consistently showed the 40K peptide to be the major P. putida membrane protein after induction. We do not yet
alk MEMBRANE PROTEINS
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understand the consequence of such major alterations in the composition of the Pseudomonas envelope after induction of the alk regulon. Nonetheless, we can safely predict that a thorough understanding of alk regulation will lead to deeper insights into the synthesis of the P. putida envelope. ACKNOWLEDGMENTS This research was supported by grants from the donors of the Petroleum Research Fund administered by the American Chemical Society, the National Science Foundation (PCM 7708591), and the Louis Block Fund of the University of Chicago. S. Benson and M. Fennewald were the recipients of Public Health Service predoctoral traineeships (GM-00090 and GM07197, respectively), and J. Shapiro was the recipient of a Public Health Service Research Career Development Award (1 K04 AI-00118). LITERATURE CITED 1. Benedik, M., M. Fennewald, and J. Shapiro. 1977. Transposition of a ,B-lactamase locus from RP1 into Pseudomonas putida degradative plasmids. J. Bacteriol. 129:809-814. 2. Benson, S., M. Fennewald, J. Shapiro, and C. Huettner. 1977. Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase-negative plasmid mutations. J. Bacteriol. 132:614-621. 3. Benson, S., and J. Shapiro. 1975. Induction of alkane hydroxylase proteins by unoxidized alkane in Pseudomonasputida. J. Bacteriol. 123:759-760. 4. Benson, S., and J. Shapiro. 1976. Plasmid-determined alcohol dehydrogenase in alkane-utilizing strain of Pseudomonas putida. J. Bacteriol. 126:794-798. 5. Cleveland, C. W., S. G. Fisher, M. W. Kirschner, and U. K. Laemmli. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 262:1102-1106. 6. Collins, M. L, and R. Niederman. 1976. Membranes of Rhodospirillum rubrum: isolation and physicochemical properties of membranes from aerobically grown cells. J. Bacteriol. 126:1316-1326. 7. Fennewald, M., S. Benson, M. Oppici, and J. Shapiro. 1979. Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon. J. Bacteriol. 139: 940-952. 8. Fennewald, M., and J. Shapiro. 1977. Regulatory mutations of the Pseudomonas plasmid alk regulon. J. Bacteriol. 132:622-627. 9. Gilleland, H. E., and R. D. Lyle. 1979. Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates. J. Bacteriol. 138:839845. 10. Grund, A., J. Shapiro, M. Fennewald, P. Bacha, J. Leahy, K. Markbreiter, M. Nieder, and M. Toepfer. 1975. Regulation of alkane oxidation in Pseudomonas putida. J. Bacteriol. 123:546-556. 11. Hancock, R. E. W., and H. Nikaido. 1978. Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier. J. Bacteriol. 136:381-390. 12. King, T. E. 1963. Reconstitution of respiratory chain enzymes systems. XI. Use of artificial electron acceptors in the assay of succinate dehydrogenase. J. Biol. Chem.
238:4032-4036. 13. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685.
762
J. BACTERIOL.
BENSON ET AL.
14. Mira, Y., M. Okazaki, S. I. Hamada, S. I. Marakawa, and R. Yugen. 1977. Assimilation of liquid hydrocarbon by microorganisms. I. Mechanism of hydrocarbon
uptake. Biotechnol. Bioeng. 13:701-714. 15. Nieder, M., and J. Shapiro. 1975. Physiological function of the Pseudomonas putida PpG6 (Pseudomonas oleovorans) alkane hydroxylase: monoterminal oxidation of alkanes and fatty acids. J. Bacteriol. 122:93-98. 16. Osborn, M. J., J. E. Cander, E. Parisi, and J. Carson. 1972. Mechanisms of assembly of the outer membrane of Salmonella typhimurium: isolation and characterization of cytoplasmic and outer membranes. J. Biol. Chem. 247:3967-3972. 17. Peterson, J. A., D. Basu, and M. Coon. 1966. Enzymatic u-oxidation. I. Electron carriers in fatty acid and hydrocarbon hydroxylation. J. Biol. Chem. 241:5162-5164. 18. Ruettinger, R. T., G. R. Griffith, and M. J. Coon. 1977. Characterization of the w-hydroxylase of Pseudomonas oleovorans as a nonheme iron protein. Arch. Biochem.
Biophys. 183:528-537. 19. Tassin, J. P., C. Celier, and J. P. Vandercasteele. 1973. Purification and properties of a membrane bound alcohol dehydrogenase involved in oxidation of long chain hydrocarbons by Pseudomonas aeruginosa.
Biochim. Biophys. Acta 315:220-232. 20. Trust, T. J., and N. F. Millis. 1971. Activation of the C2 to Cig monocarboxylic acids by Pseudomonas. J. Bacteriol. 105:1216-1218. 21. van Eyk, J., and T. J. Bartells. 1970. Paraffin oxidation in Pseudomonas aeruginosa. II. Gross fractionation of the enzyme system into soluble and particulate components. J. Bacteriol. 104:1065-1073. 22. Waddel, W. J. 1956. A simple ultraviolet spectrophotometric method for determination of proteins. J. Lab. Clin. Med. 48:311-314. 23. Weissbach, A., and J. Hurwitz. 1959. The formation of 2-keto-3-deoxyheptanoic acid in extracts of Escherichia coli B. J. Biol. Chem. 234:705-709.
