6
JOURNAL OF BACTERIOLOGY, Apr. 1983, p. 413-418 0021-9193/83/040413-06$02.00/0 Copyright 0 1983, American Society for Microbiology Vol. 154, No. 1 Intracellular Accumulation of Extracellular Proteins by Pleiotropic Export Mutants of Aeromonas hydrophila S. P. HOWARD AND J. T. BUCKLEY* Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada V8W 2 Y2 Received 7 October 1982/Accepted 30 December 1982 Pleiotropic export mutants of Aeromonas hydrophila were obtained which are unable to release protease, hemolysin, and glycerophospholipid:cholesterol acyltransferase. The synthesis of the proteins was not impaired; they were accumulated in active forms inside the mutant cells. The hemolysin could be isolated from cell contents by immunoprecipitation in a form with the same apparent molecular weight as the wild-type extracellular product, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Because both the protease and the hemolysin could be released from the mutant cells by osmotic shock, it was concluded that they were accumulated in the periplasmic space. Some mutants were missing two major outer membrane proteins, both of which reappeared in revertants with the wild-type excretory phenotype. Another mutant class had a normal outer membrane protein profile. That two different mutant classes could be obtained indicates that at least two gene products may be needed for export after protein translocation through the inner membrane. The accumula- tion of proteins which can be released by osmotic shock suggests that the periplasm may be part of the normal route for protein export. The mechanism of exporting protein into extracellular growth medium by gram-negative bacteria has received little attention (14, 25), most probably because neither of the species which have been most studied, Escherichia coli and Salmonella typhimurium, normally releases soluble protein products. It seems likely that exported proteins cross the inner membrane, in a manner similar to periplasmic and outer mem- brane proteins, by means of a leader sequence which aids in attachment to and penetration of the inner membrane (12). An important question is whether exported proteins cross inner and outer membranes together, perhaps at zones of adhesion (2), and thus bypass the periplasm or whether they must pass through the periplasm and then through the outer membrane. Aeromonas hydrophila, a pathogenic member of the family Vibrionaceae, is a prolific producer of extracellular proteins of degradative capacity, toxic capacity, or both. Thus, hemolysins (5, 30), proteases (20, 29), phospholipases (3, 20), an acyltransferase (17), a leucocidin (24), and an enterotoxin (1, 4, 27) have been reported in cell- free supematants of A. hydrophila cultures. This secretory capacity, along with the availability of wide-host-range plasmids, make the organism potentially useful as a vehicle for the production of pharmaceutically important protein products, the genes of which have already been implanted into plasmid vectors in well-understood but non- exporting members of the family Enterobacteri- aceae (7). For this reason, and because of a more specific interest in the function and mecha- nism of extracellular export of a potent cytolytic toxin (aerolysin) and an unusual phospholipase that have been purified and studied in this labo- ratory (5, 6, 11), we have attempted to obtain mutants which are unable to export proteins. We report here the isolation and partial character- ization of several such mutants. MATERIULS AND METHODS Materals. All chemicals used were analytical rea- gent grade. 1"5I was from Amersham Corp.; [1- 14C]cholesterol, "C-labeled E. coli protein, and [3H]polyadenylate were from New England Nuclear Corp. All prepared media used were from Difco Labo- ratories. Phosphatidylcholine and bovine serum albu- min were from Sigma Chemical Co. Staphylococcus aureus, containing protein A on the surface, was from Calbiochem Behring. 1,3,4,6-Tetrachloro-3a,6a-diphen- ylglycoluril (lodogen) was from Pierce Chemical Co. Bacterial strains and culture condidons. A. hydro- phila Ah65 (18) was the parent of all strains used in this study. The bacteria were grown (30°C) in 200-ml volumes in 2-liter flasks, shaken at 250 rpm in a New Brunswick Gyrotory shaker. The inoculum was 1% (vol/vol) of an overnight culture containing 2 x 109 to 5 x 109 viable cells ml-'. Media used were penicillinase assay broth, tryptic soy broth, tryptic soy agar with 5% human erythrocytes, the defined medium de- 413 on January 6, 2021 by guest http://jb.asm.org/ Downloaded from

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JOURNAL OF BACTERIOLOGY, Apr. 1983, p. 413-4180021-9193/83/040413-06$02.00/0Copyright 0 1983, American Society for Microbiology

Vol. 154, No. 1

Intracellular Accumulation of Extracellular Proteins byPleiotropic Export Mutants of Aeromonas hydrophila

S. P. HOWARD AND J. T. BUCKLEY*Department ofBiochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada

V8W 2 Y2

Received 7 October 1982/Accepted 30 December 1982

Pleiotropic export mutants of Aeromonas hydrophila were obtained which areunable to release protease, hemolysin, and glycerophospholipid:cholesterolacyltransferase. The synthesis of the proteins was not impaired; they wereaccumulated in active forms inside the mutant cells. The hemolysin could beisolated from cell contents by immunoprecipitation in a form with the sameapparent molecular weight as the wild-type extracellular product, as determinedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Because both theprotease and the hemolysin could be released from the mutant cells by osmoticshock, it was concluded that they were accumulated in the periplasmic space.Some mutants were missing two major outer membrane proteins, both of whichreappeared in revertants with the wild-type excretory phenotype. Another mutantclass had a normal outer membrane protein profile. That two different mutantclasses could be obtained indicates that at least two gene products may be neededfor export after protein translocation through the inner membrane. The accumula-tion of proteins which can be released by osmotic shock suggests that theperiplasm may be part of the normal route for protein export.

The mechanism of exporting protein intoextracellular growth medium by gram-negativebacteria has received little attention (14, 25),most probably because neither of the specieswhich have been most studied, Escherichia coliand Salmonella typhimurium, normally releasessoluble protein products. It seems likely thatexported proteins cross the inner membrane, ina manner similar to periplasmic and outer mem-brane proteins, by means of a leader sequencewhich aids in attachment to and penetration ofthe inner membrane (12). An important questionis whether exported proteins cross inner andouter membranes together, perhaps at zones ofadhesion (2), and thus bypass the periplasm orwhether they must pass through the periplasmand then through the outer membrane.Aeromonas hydrophila, a pathogenic member

of the family Vibrionaceae, is a prolific producerof extracellular proteins ofdegradative capacity,toxic capacity, or both. Thus, hemolysins (5,30), proteases (20, 29), phospholipases (3, 20),an acyltransferase (17), a leucocidin (24), and anenterotoxin (1, 4, 27) have been reported in cell-free supematants ofA. hydrophila cultures. Thissecretory capacity, along with the availability ofwide-host-range plasmids, make the organismpotentially useful as a vehicle for the productionof pharmaceutically important protein products,the genes of which have already been implanted

into plasmid vectors in well-understood but non-exporting members of the family Enterobacteri-aceae (7). For this reason, and because of amore specific interest in the function and mecha-nism of extracellular export of a potent cytolytictoxin (aerolysin) and an unusual phospholipasethat have been purified and studied in this labo-ratory (5, 6, 11), we have attempted to obtainmutants which are unable to export proteins. Wereport here the isolation and partial character-ization of several such mutants.

MATERIULS AND METHODSMaterals. All chemicals used were analytical rea-

gent grade. 1"5I was from Amersham Corp.; [1-14C]cholesterol, "C-labeled E. coli protein, and[3H]polyadenylate were from New England NuclearCorp. All prepared media used were from Difco Labo-ratories. Phosphatidylcholine and bovine serum albu-min were from Sigma Chemical Co. Staphylococcusaureus, containing protein A on the surface, was fromCalbiochem Behring. 1,3,4,6-Tetrachloro-3a,6a-diphen-ylglycoluril (lodogen) was from Pierce Chemical Co.

Bacterial strains and culture condidons. A. hydro-phila Ah65 (18) was the parent of all strains used in thisstudy. The bacteria were grown (30°C) in 200-mlvolumes in 2-liter flasks, shaken at 250 rpm in a NewBrunswick Gyrotory shaker. The inoculum was 1%(vol/vol) of an overnight culture containing 2 x 109 to 5x 109 viable cells ml-'. Media used were penicillinaseassay broth, tryptic soy broth, tryptic soy agar with5% human erythrocytes, the defined medium de-

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414 HOWARD AND BUCKLEY

scribed by Riddle et al. (23), and casein agar (0.4%sodium caseinate, 0.1% glucose, 0.02% K2HPO4,0.02% MgSO4, 0.0001% FeSO4, 1.5% agar). One opti-cal density unit (660 nm) in the defined mediumcorresponded to ca. 3 x 108 viable cells ml-'.

Mutagenesis. An overnight culture of strain Ah65 inpenicillinase assay broth was diluted 20-fold into freshmedium and incubated at 37°C with shaking for 2.5 h.The cells were harvested, washed once in isotonicsaline, and suspended in one-half of the original vol-ume of phosphate-buffered saline (10 mM NaH2PO4,0.85% NaCl; pH 7.0) to which 70 p.1 of ethyl methanesulfonate was added (15). This mixture was incubatedfor 1 h at 30°C, and the cells were harvested andwashed twice with 40 ml of saline. The final pellet wasinoculated into 20 ml of penicillinase assay broth andincubated overnight at 30°C to allow segregation, afterwhich the culture was diluted and plated on humanblood agar.

Suboellular fractionation. Shock fluids were ob-tained by the sucrose-EDTA method of Willis et al.(28). Shocked cells were disrupted by sonication (three30-s bursts with a Branson Sonifier at the microtiplimit). Supernatants were assayed directly for enzy-matic activities or concentrated for electrophoresis bythe addition of ammonium sulfate to 85% saturation at0°C followed by centrifugation at 10,000 x g for 30min. The pellets were suspended to 1/100 of theoriginal volume in 20 mM Tris-hydrochloride (pH 7.4)and dialyzed overnight against this buffer.Enzyme assays. RNase activity was measured by the

method of Lopes et al. (16), and lactate dehydrogenasewas measured by the method of Stambaugh and Post(26). Protease activity was determined using "4C-labeled E. coli protein (New England Nuclear) dilutedwith bovine serum albumin to a specific activity of0.005 pLCi/100p.g. Each assay contained 100 ,ug ofprotein in 100 ,ul of 50 mM phosphate buffer (pH 7.0).Samples were incubated for 10 min at 37°C, afterwhich 100 p.1 of ice-cold 10o trichloroacetic acid wasadded. Samples were further incubated for 10 min at4°C and centrifuged at 15,000 x g for 5 min. A 100-lpvolume of the suprnatant was added to scintillationfluid; samples were then counted. Glycerophospholip-id:cholesterol acyltransferase (GCAT) activity wasassayed using [4-14C]cholesterol-phosphatidylcholineliposomes as previously described (6). Hemolytic ac-tivity was measured with 0.8% human erythrocytes inphosphate-buffered saline on microtiter plates. Serialdilutions of the samples to be assayed in phosphate-buffered saline containing 1 mg of bovine serum albu-min per ml were made on the plates, and to each wellan equal volume (100 pul) of the erythrocyte solutionwas added. The plates were incubated for 1 h at 37°C,and the number of wells in which the blood had beencompletely lysed were counted. The titer of the sam-ples was equal to the inverse of the highest dilution ofthe original sample at which 100%6 lysis was observed.

Isolation of outer membranes. Cell envelopes wereisolated by passing late-log-phase bacterial cellsthrough a French pressure cell and then centrifuging at40,000 x g for 40 min. Outer membranes were pre-pared, as previously described (18), by solubilizationof the bacterial envelopes by using the sodium laurylsarcosinate method of Filip et al. (8). The outermembranes were centrifuged from the solubilizationmixture at 40,000 x g for 40 min and washed once in 20

mM Tris-hydrochloride (pH 7.4). We have found (datanot shown) that wild-type outer membranes preparedin this manner are very similar in protein compositionto those prepared by sucrose gradient centrifugation(31).Sodium dodecyl sulfate-polyacrylamide gel electro-

phoresis (SDS-PAGE). Samples were prepared forelectrophoresis and separated in 12% acrylamide slabswhich had been prepared by the method of Neville(19). Proteins were stained with Coomassie blue.

Radiolabeling and hmmunoprecipitation. Pure aeroly-sin and whole-cell extracts were labeled with "1,using lodogen, by the method of Fraker and Speck (9).From the iodinated samples, aerolysin was immuno-precipitated with anti-aerolysin immunoglobulin G andS. aureus (Cowan) cells containing protein A on theirsurfaces, as described by Ito et al. (13).

RESULTSExport mutants of strain Ah65 were obtained

at a frequency of ca. 10-4 by ethyl methanesulfonate treatment of the bacteria followed bydirect plating on blood agar. Clones with re-duced extracellular aerolysin exhibited either areduction in size or an absence of the normalzone of hemolysis around the colonies. Pre-sumptive mutants were picked and further plat-ed onto both human blood agar and 0.4% caseinagar. Clones which appeared to lack both theneutral protease and aerolysin as secretoryproducts were thus identified. Four clones (Si,S7, S9, and S11) were further studied in brothculture.Growth curves and aerolysin and protease

production by a representative secretory mutant(Ah65-S9) and its wild-type progenitor areshown in Fig. 1. The growth patterns of the twostrains are very similar, whereas the locations ofthe toxins differ markedly. Aerolysin was pro-duced by the mutant, as it was by the wild type,but it remained associated with the cells, ratherthan being exported into the extracellulargrowth medium. Neutral protease productionwas similarily affected in the mutant; the activitymissing from the mutant supernatant was foundto be associated with the cells (Fig. lB and C).Comparable results were obtained with each ofthe other mutants (data not shown). These re-sults indicated that each mutant was defectivenot in the synthesis of the extracellular proteins,but rather in the ability to direct them to theoutside. Growth experiments were then under-taken to determine the time of production andcompartment distribution of the normally ex-ported proteins in the wild type and the secre-tory mutants. To discover where in the mutantcells aerolysin and protease were accumulated,each mutant was subjected to cold osmoticshock, a treatment that has been shown to resultin the release of the periplasmic space contentsof other gram-negative bacteria (21, 28). Al-though a large fraction of the total cellular

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PLEIOTROPIC AEROMONAS EXPORT MUTANTS 415

10 20HOURS

10 20 30HOURS

10 20HOURS

30

30

150I-.

o10 Iz

0

co5 4

0

IL

75'r

0

50

25

0cOL

FIG. 1. Growth and extracellular protein produc-tion by wild-type and mutant strains. (A) Wild type (0)and S9 (0) growth. (B) Aerolysin (U) and protease (V)in supernatants. Solid symbols refer to the wild type,and open symbols refer to S9. (C) Aerolysin andprotease in celis; symbols as in (B).

RNase (a periplasmic marker) was releasedfrom both the mutant and the wild type byosmotic shock, all of the lactate dehydrogenase(normally cytoplasmic) remained associatedwith the cells (Table 1). These results indicate

TABLE 1. Periplasmic and cytoplasmic enzymeactivities in subcellular fractions'

Shock fluid Shocked cellsStrain Enzyme

/ % /

Ah65 RNase 430 49 452 51LDHb 0 0 453 100

Ah65-S9 RNase 693 70 290 30LDH 0 0 342 100

a Cultures of Ah65 and Ah65-S9 were grown for 18 hin minimal medium and fractionated as described inthe text. There was no LDH or RNase activity in thesupernatant fractions of either Ah65 or Ah65-S9.

b LDH, Lactate dehydrogenase.

that a periplasmic fraction was released by os-motic shock and that it was not contaminatedwith cytoplasm. By this fractionation procedure,the distribution of aerolysin, protease, and athird normally exported protein, GCAT, weremeasured (Fig. 2; Table 2). Release of aerolysin

Ec

4c

'u

z4

0CD4

HOURS

Ec

I-4

z.40

o0

FIG. 2. Distribution of aerolysin during growth.(A) Wild-type growth (0); aerolysin in the supernatant(U), in the shock fluid (0), and in the cytoplasmicfraction (A). (3) Mutant S9 growth; symbols as in (A).

EC26

A

/1o@0

t'0/

A0'

4c

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41

21

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VOL. 154, 1983

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416 HOWARD AND BUCKLEY

TABLE 2. Distribution of degradative enzymeactivities in subcellular fractionsa

Protease (U/ml) GCAT (U/ml)Fraction

Ah65 Ah65-S9 Ah65 Ah65-S9

Supernatant 800 120 17 0Shock fluid 0 766 0 0Shocked cells 48 553 0 14

a See Table 1, footnote a.

by the wild type began in the early log phase andpeaked in the late log phase; the levels had fallenmarkedly in culture supernatants by the latestationary phase. Very little, if any, of the threeactivities were detected either in the periplasmor in the cytoplasm of the wild type (Fig. 2A). Asbefore, the mutant did not release either aeroly-sin or neutral protease, and in addition it did notsecrete the acyltransferase, indicating that it wasa pleiotropic export mutant (Fig. 2B). Bothaerolysin and neutral protease were releasedfrom each mutant by cold osmotic shock withapproximately the same efficiency as was RNaseactivity (Table 2), suggesting that they are accu-mulated in the periplasm and not in either theinner or the outer membrane, nor within thecytoplasm. The third normally exported protein,the acyltranferase, could not be released fromthe cells by osmotic shock (Table 2). Neutralprotease activity began to appear in the supema-tant as the mutant cultures approached the deathphase (data not shown). This phenomenon waspresumably due to leakage from the periplasm,perhaps caused by the very high concentrationsof the potentially toxic proteins which had accu-mulated. However, because aerolysin is as un-stable in the periplasm as it is in the supernatant,its activity had dropped to a low level by thisstage of the growth cycle (Fig. 2B).Because the periplasmic RNase activities of

the mutant and wild type were comparable (Ta-ble 1), it would appear that although the mutantis impaired in its export of extracellular proteins,it is not also impaired in the secretion of peri-plasmic proteins. In fact, SDS-PAGE analysis ofthe periplasmic fractions of mutant S9 and thewild type (Fig. 3A) indicated that although themutant periplasm shared all the prominent pro-tein bands with that of the wild type, it alsocontained extra proteins. One of these proteins,which corresponded to aerolysin in molecularweight, is identified by the arrow in Fig. 3A. Inaddition, the results in Fig. 3B indicated that alarge number of proteins were missing from theextracellular fluid of the mutant. Similar resultswere obtained with each of the other mutants(data not shown).That at least some of the export proteins

missing from the supematant were accumulated

Aa b

94-672-

43-

30- .

20.1-

14.4-

Ba b

94-

67-

43-

30-

FIG. 3. SDS-PAGE of periplasmic and extracellu-lar supernatant proteins. (A) Periplasmic proteins of(a) wild type and (b) S9. The arrow points to positionof aerolysin. (B) Supernatant proteins of (a) wild typeand (b) S9. Periplasmic and supernatant fractions wereisolated from cells in the late log phase. Approximate-ly 25 pLg of protein was applied to each lane except inpart B, lane b, where a volume corresponding to theamount applied in part B, lane a, was used. Thestandards were: phosphorylase B (94k), bovine serumalbumin (63k), ovalbumin (43k), carbonic anhydrase(30k), soybean trypsin inhibitor (20.1k), and a-lactal-bumin (14.4k).

in the periplasm suggested that their exportthrough the outer membrane could be blocked,and the blockage could be the result of a changein the composition of the outer membrane. Itwas of interest, therefore, to compare the outermembranes of the mutant strains with that of thewild type. The protein profiles of outer mem-branes separated by SDS-PAGE are presentedin Fig. 4. It can be seen that the outer mem-

a b c d e f g h

94-_ _67-_N43 -_---">-nt4M a

30-_ _ ___-- f

20.1--

FIG. 4. SDS-PAGE of outer membrane proteins.Lanes: (a), standards; (b), S1; (c), S7; (d), Sli; (e),wild type; (f), R91, revertant of S9; (g), R93, anotherS9 reverant; (h), wild type; (i), S9. Approximately 25,ug of protein was applied in each lane. See the legendto Fig. 3 for standard identification.

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PLEIOTROPIC AEROMONAS EXPORT MUTANTS

branes of three of the mutants, Si, S9, and Si1,are markedly deficient in two major proteins ofmolecular weight 42,000 (42k) and 28k, whichare found in the wild type under these growthconditions, and that each mutant contains amuch more intense band of 23k protein thandoes the wild type. The mutant S11 membranealso contains an additional large band of 22kprotein. In addition, the wild-type outer mem-brane profile was restored in two revertants thatwere independently derived from cultures ofmutant S9. Thus, both the 42k and 28k proteinbands were restored, and the 23k protein bandwas diminished. Both revertants were also iden-tical to the wild type in growth and in theextracellular production of the three proteinsmeasured. In addition, neither accumulated anyexport proteins in the periplasm. In contrast tothe other three export mutants, S7 had the sameouter membrane protein profile as the wild type,suggesting that there are at least two differentlesions which can lead to the accumulation ofexport proteins in the periplasm.

Pleiotropic E. coli mutants that are unable toproperly sequester periplasmic proteins havebeen shown to accumulate higher-molecular-weight periplasmic and outer membrane proteinprecursors on the cytoplasmic side of the innermembrane (22). Although it seemed unlikely thatthis was the case with the A. hydrophila exportmutants because the accumulated aerolysin andprotease could be released from the mutants inactive forms by osmotic shock, the possibilitythat aerolysin was accumulated as a precursorwas investigated more closely. Whole-cell ex-tracts of mutant S9 were radioiodinated andimmuneprecipitated with rabbit anti-aerolysinimmunoglobulin G and S. aureus (Cowan) con-taining protein A on their surface. The precip-itated material was compared to purified aeroly-sin by SDS-PAGE followed by autoradiography(Fig. 5). The apparent molecular weight of theanti-aerolysin-precipitable material from the mu-tant cells was the same as that of purifiedextracellular aerolysin obtained from wild-typeAh65.

DISCUSSIONThe export mutants described are capable of

producing and accumulating active forms ofeach normally exported protein we measured. Inaddition, at least one protein, aerolysin, couldbe isolated from mutant cells in a form indistin-guishable from the normal extracellular product,indicating that the mutants may be able to re-move any leader sequences that are required forinner membrane translocation. At least two ofthe proteins could be released from the mutantcells by osmotic shock, suggesting either thatthey normally pass through the periplasm during

a b

M_-

67-

43-.

24-

18.4-14.3-

FIG. 5. SDS-PAGE of immunoprecipitated aeroly-sin from S9 cells: comparison with aerolysin from thewild-type cell-free supernatant. (a) "MI-labeled pro-teins precipitated from S9 cells with anti-aerolysinantibody. (b) "2I-labeled aerolysin precipitated fromthe wild-type supernatant.

the excretory process or that they are divertedto this compartment when export is blocked.Passage of neutral protease through the peri-plasm has been similarly observed during pro-tein export by Pseudomonas aeruginosa (14),and, in addition, a hemolysin of E. coli (25)appears to pass through the periplasm duringexport. There are several ways to explain theobservation that the third export protein, theacyltransferase GCAT, was not released by os-motic shock. We have previously shown that insome circumstances GCAT has a high affinityfor membranes (6), and it is possible that underthe conditions in the mutant periplasm, theenzyme binds to either the inner or the outermembrane. Alternatively, there may be somedifferences in the secretory pathways followedby this protein, although in our case, both path-ways must have been blocked by the lesion inthe mutants.

If exported proteins do traverse the periplas-mic space on their way to the exterior, then theypresumably must also cross the outer mem-brane. One class of mutants described was miss-ing two major outer membrane proteins, both ofwhich reappeared in revertants with the wild-type excretory phenotype (Fig. 4). These pro-

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418 HOWARD AND BUCKLEY

teins may play some necessary role in outermembrane passage, although, of course, it isalso possible that the step which blocks proteinexport also prevents their proper location in theouter membrane. The other class of excretorymutant contained a normal outer membraneprotein profile, indicating that the above-men-tioned lack of membrane proteins is not the solerequirement for preventing protein export. Inthis mutant class, some other outer membranecomponent, such as phospholipid or lipopoly-saccharide, may be altered, thus affecting thephysical properties of the outer membrane andthereby preventing the translocation process.Another possibility is that mutants in this classmay be missing some other necessary compo-nent of the export process.

Further characterization of these mutants willlead to an improved understanding of the geneproducts which affect protein export by gram-negative bacteria.

ACKNOWLEDGMENTThis research was supported by a grant from the Natural

Sciences and Engineering Research Council of Canada.

LITERATURE CITED1. Annapurna, E., and S. C. Sanyal. 1977. Enterotoxicity of

Aeromonas hydrophila. J. Med. Microbiol. 10:317-323.2. Bayer, M. E. 1979. The fusion sites between the outer

membrane and cytoplasmic membrane of bacteria; theirrole in membrane assembly and virus infection, p. 167-202. In M. Inouye (ed.), Bacterial outer membranes:biogenesis and functions. John Wiley & Sons, Inc., NewYork.

3. Bernheimer, A. W., L. S. Avigad, and G. Avigad. 1975.Interactions between aerolysin, erythrocytes, and eryth-rocyte membranes. Infect. Immun. 11:1312-1319.

4. Boulanger, Y., R. Lallier, and G. Coulneau. 1977. Isola-tion of enterotoxigenic Aeromonas from fish. Can. J.Microbiol. 23:1161-1164.

5. Buckley, J. T., L. N. Halasa, K. D. Lund, and S. MacIn-tyre. 1981. Purification and some properties of the hemo-lytic toxin aerolysin. Can. J. Biochem. 59:430-435.

6. Buckley, J. T., L. N. Halasa, and S. Maclntyre. 1982.Purification and partial characterization of a bacterialphospholipid:cholesterol acyltransferase. J. Biol. Chem.257:3320-3325.

7. Chan, S. J., J. Weiss, M. Konrad, T. White, C. Bahl, S.-D.Yu, D. Marks, and D. F. Steiner. 1981. Biosynthesis andperiplasmic segregation of human proinsulin in Escherich-ia coli. Proc. Natl. Acad. Sci. U.S.A. 78:5401-5405.

8. Filip, C., G. Fletcher, J. L. Wulff, and C. F. Earhart. 1973.Solubilization of the cytoplasmic membrane of Escherich-ia coliby the ionic detergent sodium-lauryl sarcosinate. J.Bacteriol. 115:717-722.

9. Fraker, P. J., and J. C. Speck, Jr. 1978. Protein and cellmembrane iodinations with a sparingly soluble chloram-ide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Bio-chem. Biophys. Res. Commun. 50:849-857.

10. Gray, G. L., R. M. Berka, and M. L. Vasl. 1982. APseudomonas aeruginosa mutant non-derepressible fororthophosphate-regulated proteins. J. Bacteriol. 147:675-678.

11. Howard, S. P., and J. T. Buckley. 1982. Membraneglycoprotein receptor and hole-forming properties of acytolytic protein toxin. Biochemistry 21:1662-1667.

12. Inouye, M., and S. Halegoua. 1980. Secretion and mem-brane localization of proteins in Escherichia coli. Crit.

Rev. Biochem. 7:339-371.13. Ito, K., P. J. Bassford, Jr., and J. Beckwith. 1981. Protein

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