JOURNAL OF BACrERIOLOGY, May 1974, p. 454-464Copyright 0 1974 American Society for Microbiology

Vol. 118, No, 2Printed in U.S.A.

Outer Membrane Proteins of Escherichia coliIV. Differences in Outer Membrane Proteins Due To Strain and Cultural

DifferencesCARL A. SCHNAITMAN

Department of Microbiology, The University of Virginia, School of Medicine, Charlottesville, Virginia 22901

Received for publication 12 December 1973

When the 42,000-dalton major outer membrane protein of Escherichia coli0111 is examined on alkaline polyacrylamide gels containing sodium dodecylsulfate, it is resolved into three distinct bands designated as proteins 1, 2, and 3.Band 3 consists of two distinct polypeptides, proteins 3a and 3b. E. coli K-12 doesnot make any protein 2, but makes proteins similar to 1, 3a, and 3b as indicatedby comparison of cyanogen bromide peptide patterns. Several Shigella speciesand most other strains of E. coli resemble E. coli K-12 in that they lack protein 2,whereas Salmonella typhimurium is more similar to E. coli 0111. In addition.tothese species and strain differences, cultural differences resulted in differences inthe outer membrane protein profiles. Under conditions of catabolite repression,the level of protein 2 in E. coli 0111 decreased while the level of protein 1increased. An enterotoxin-producing strain similar to E. coli 0111 produced noprotein 1 and an elevated level of protein 2 under conditions of low cataboliterepression. The levels of proteins 1 and 3 are also different in different phases ofthe growth curve, with protein 1 being the major species in the exponential-phasecells and protein 3 being the major species in stationary-phase cells. A multiplyphage-resistant mutant of E. coli K-12 with no obvious cell wall defects producedno protein 1 or 2, but made increased amounts of protein 3. Thus, the major outermembrane proteins of E. coli and related species may vary considerably withoutaffecting outer membrane integrity.

Previous studies in this series (14-16) haveshown that the 42,000-dalton major protein ofEscherichia coli 0111 consists of a mixture of atleast four distinct major polypeptides. Thesepolypeptides can be distinguished by their mi-gration on sodium dodecyl sulfate (SDS)-poly-acrylamide gels with an alkaline upper buffer(3) and by their distinctive cyanogen bromidepeptide patterns (15, 16).

All of the previous studies in this series havedealt with strain J-5, a derivative of E. coli0111 B4 that is lacking the enzyme uridine-diphosphate-galactose-4-epimerase. This mu-tant is convenient for cell wall studies since itpermits the specific labeling of the cell walllipopolysaccharide with galactose. Since thislabeling requires the function of the gal operon,which is subject to catabolite repression, cul-tures have been routinely grown with succinateor glycerol as the carbon source. This routineuse of a single strain of E. coli grown under asingle, rather limiting set of culture conditionsfor several years prevented the discovery of tworather interesting phenomena which are de-

scribed in this report, namely, that the outermembrane protein composition of a singlestrain may vary greatly as a consequence ofculture conditions, and that all strains of E. coli(and other enteric bacteria) do not have thesame polypeptides present in their outer mem-branes.

In addition to the profound differences ob-tained with different SDS-polyacrylamide gelmethods (1, 8, 14, 15), the differences notedabove have certainly contributed to the ratherconfusing lack of agreement in the literatureconcerning the outer membrane proteins ofgram-negative bacteria.

MATERIALS AND METHODSStrains and culture conditions. Except where

noted, cultures were grown in minimal medium (23)with 1% Casamino Acids, 1% sodium succinate, or0.5% glucose or glycerol as the carbon source. Cultureswere grown at 37 C in a rotary shaker and harvested atlate exponential phase (60% maximal turbidity).The bacterial strains used in this study are de-

scribed in Table 1.

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VARIATIONS IN OUTER MEMBRANE PROTEINS

TABLE 1. Bacterial strainsa

Strain or species designation Source Relevant characteristics, references

E. coli O111A1 E. Heath Forms extensive intracytoplasmic membranes (17)E. coli 0111B4 E. Heath Parent of strain J-5 (17)E. coli J-5 E. Heath galE mutant from 0111B4 (14, 23)E. coli 0111B4ATCC 12015 ATCCE. coli 041 ATCC 23976 ATCCE. coli 0127 ATCC 12740 ATCCE. coli 055 ATCC 12014 ATCCE. coli B (Hill) ATCC 23225 ATCCE. coliW ATCC 9637 ATCCE. coli ML308 H. Winkler Widely used in transport and membrane vesicle studiesE. coli K-12 R. Kadner F- revertant of strain KL 96; T6'E. coli AB 1859 CGSC K-12 derivative, lacY-, strr (2)E. coli AB 1621 CGSC Derived from AB 1859 by selection for resistance to phages

T4 and T6; lacY-, tfr-5, tsx-57 (2)E. coli 334 R. Guerrant Produces heat-labile enterotoxin; serotype 015:H11; clini-

cal isolate, Calcutta, India (4)E. coli H10407 R. Guerrant Produces heat-labile enterotoxin; serotype 078 (20);

clinical isolate, Dacca, Bangladesh

Salmonella typhimurium U. Va. SC Clinical isolate, U. Va. Hospital

Shigella sonnei ATCC 11060 U. Va. SCS. schmitzii U. Va. SC Clinical isolate, U. Va. HospitalS. flexneri ATCC 9380 U. Va. SCS. dysenteriae ATCC 9665 U. Va. SC

a Source abbreviations: ATCC, American Type Culture Collection; CGSC, E. coli genetic stock center, YaleUniversity; U. Va. SC, Stock culture collection, Microbiology Department, University of Virginia MedicalSchool.

Isolation and analysis of outer membraneprotein. Harvested cells were treated in an Omni-Mixer to remove flagella, and broken in a Frenchpressure cell and extracted with Triton X-100 toremove the cytoplasmic membrane from the crudeenvelope fraction (14). The Triton-insoluble outermembrane protein was dissolved in SDS solution at37 C (first step, method II, reference 14) and appliedto SDS-polyacrylamide gels without further treat-ment, or the sample was then dialyzed against aSDS-urea solution and boiled briefly (completemethod II). Samples of intact protein were analyzedin 7.5% polyacrylamide gels, as described previously(14-16), with either the pH 7.2 Maizel buffer system(9) or the pH 11.4 to 4.1 Bragg-Hou gel system (3). Aand C proteins were isolated by two cycles of SDS-Sephadex G-200 chromatography and cleaved withcyanogen bromide as described previously (16). Cya-nogen bromide peptides were analyzed either with theSwank-Munkres gel system (16, 21) or on conven-tional SDS-polyacrylamide gels containing 12% acryl-amide, 0.35% bis-acrylamide, and 0.5 M urea thatwere prepared and run in 0.1 M sodium phosphatebuffer (pH 7.2) containing 0.1% SDS. Gels werescanned or sliced as described previously (14).

RESULTSComparison of outer membrane protein

from E. coli 0111 and E. coli K-12. Identicalcultures of strain J-5 (derived from E. coli

0111B) and E. coli K-12 were grown on minimalmedium with succinate as the carbon source.The J-5 culture was labeled with [4C ]leucineand the K-12 culture was labeled with [3H]leu-cine. The cultures were mixed prior to the iso-lation of the outer membrane protein.When samples of outer membrane protein

prepared by boiling in SDS (complete methodII) were analyzed on SDS-polyacrylamide gelswith the Bragg-Hou buffer system, the resultsshown in Fig. 1 were obtained. Protein 2 ap-peared to be missing entirely from strain K-12,whereas proteins 1 and 3 appeared to be identi-cal in terms of migration in the two strains. Lesspronounced differences can also be observedwith the Maizel buffer system (Fig. 2). Insteadof the single, broad peak observed with strainJ-5, strain K-12 gives two very closely spacedbands. These two closely spaced bands can alsobe observed in stained gels containing onlystrain K-12 outer membrane protein (notshown). The spacing between these bands alsodepends upon the sample size and the pH of thegel buffer, since overloading causes the bands tomerge and even a slight increase in the pHabove neutrality causes the bands to moveapart.These two strains also give very different gel

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SCHNAITMAN J. BACTERIOL.

0 10 20 30 40 50SLICE NUMBER

60 70 80

FIG. 1. Comparison of [3H]leucine-labeled outermembrane protein from E. coli K-12 (solid line) and[I4C]leucine-labeled outer membrane protein from E.coli J-5 (dashed line) on a Bragg-Hou gel. Thecultures were grown with succinate as the carbonsource (14), and the outer membrane protein was

dialyzed against SDS-urea solution and boiled priorto electrophoresis (14). The numbers identify thethree major protein bands (14).

40-

N

230-xU'

Z20-

0U

0-

-8

N

0

6 x

U'

z

-4 =0

-2-

-0

10 20 30 40 50 60 TOSLICE NUMBER

FIG. 2. Comparison of 3H-labeled outer membraneprotein from E. coli K-12 (solid line) and "4C-labeledouter membrane protein from E. coli J-5 (dashed line)on a gel with the Maizel (pH 7.2) buffer system. Thesample was prepared by boiling as in Fig. 1.

patterns when the outer membrane protein isdissolved without urea treatment or boiling andanalyzed with the Maizel buffer system (Fig. 3).The A protein (16) from strain J-5 gives severalpeaks near the top of the gel, whereas the Aprotein from strain K-12 gives a single peakwith an apparent molecular mass of about60,000 daltons. The C peak is identical in bothstrains, with an apparent molecular mass of25,000 to 30,000 daltons. This indicates that theA protein from strain J-5 is either more aggre-gated or more unfolded than its counterpartfrom strain K-12. It should be noted that in a

previous experiment (15) it was observed that

protein 1 was present in both the leading andtrailing edge of the broad region of A proteinfrom strain J-5.

Since there is an obvious difference in protein2, it was important to determine whether therewere also differences among proteins 1, 3a, and3b of these two strains. To answer this question,I dissolved a sample of outer membrane proteinfrom the mixed culture described above in SDSsolution at 37 C and chromatographed it on

SDS-Sephadex G-200 (16) to separate the A andC proteins. These were then boiled in SDSsolution and rechromatographed on SDS-Sephadex G-200, and the protein from eachfraction which shifted to peak B after boiling(16) was cleaved with cyanogen bromide.

Figure 4 shows a comparison of the cyanogenbromide peptides from the A protein of strainsJ-5 and K-12. This was analyzed on a conven-

tional 12% polyacrylamide gel rather than withthe Swank-Munkres gel system to facilitatecomparison of the larger polypeptides charac-teristic of protein 2 (16). The numbering of thepolypeptides is the same as was used previously(Fig. 8, reference 16). Peptides 1 and 2, whichare characteristic of protein 2 (16), appear to bemissing from the A protein from strain K-12.Peptides 3 and 6, which are characteristic ofprotein 1 (16), are present in both cultures,although they are present in reduced amountsas would be predicted from the relative amountsof protein 1 present in these two strains (Fig. 1).Peptides 4 and 6 are somewhat ambiguous, butthis is as to be expected since there are overlap-

C

36 A 12

30 -IONu

x

z~~ ~ ~ ~ ~ ~0~

U12 4

30 40 50SLICE NUMBER

FIG. 3. Comparison of E. coli K-12 and E. coli J-5outer membrane protein dissolved in SDS solution at37 C and run in the Maizel gel system without urea or

heat treatment. The sample is the same as in Fig. 1

and 2. The solid line represents 3H-labeled proteinfrom strain K-12, and the dashed line represents'4C-labeled protein from strain J-5. The letters A, B,and C denote the various regions on the gel, as

described previously (14).

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VARIATIONS IN OUTER MEMBRANE PROTEINS

z ID ~~~~2o

04

FIG. 4. Cyngnboidpetesfo thA

I 5

protein from a mixture of outer membrane proteinfrom E. coli K-12 (dashed line) and E. coli J-5 (solidline). The envelope sample is the same as shown inFig. 1-3. The sample was run on a Maizel system gelcontaining 12% acrylamide.

ping peptides from proteins 1 and 2 (in the caseof strain J-5) present in these regions of the gel(16). The conclusion which can be drawn fromFig. 4 is that protein 1 is very similar oridentical in both strains, and protein 2 ispresent only in strain J-5.

Figure 5 shows a comparison of the cyanogenbromide peptides from the C proteins of the twostrains. It appears that strain K-12 also con-tains two polypeptides, proteins 3a and 3b, andthese are present in the same ratio as in strainJ-5. In this figure, peptides 1, 2, and 4 areindicative of protein 3a and peptides 3 and 5 ofprotein 3b (16). It may be concluded then thatstrain K-12 contains three of the four majorpolypeptides identified in strainbdn.Outer membrane protein patterys of other

E. coli strains and other enteric bacteria.Since it is clear that a derivative of E. colit1aiBs (strain J-5) contained a a 3lypeptidewhich was absent in E. coli K-12, it was ofinterest to examine other commonly studied E.coli strains and some related enteric speciesboth to see whether this unique protein wasmissing and whether other major differencescould be detected. To avoid nutritional prob-lems, I grew all of the cultures in minimalmedium containing a small amount of thiamineand Casamino Acids as the carbon source. All ofthe cultures grew well in this medium.

Figures 6 and 7 show the appearance ofboiled, outer membrane protein from a numberof E. coli strains, two Shigella species, andSalmonella typhimurium analyzed with theBragg-Hou gel system. All of the E. coli strains

except the two 0111 strains were found to bemissing protein 2. In general, these E. colistrains appeared similar, with the exception of ahigh-molecular-weight protein that was ob-served in strain MI308 and was absent in theother strains. The migration of proteins 1 and 3

0 10 20 30 40 50SLICE NUMBER

60 70

FIG. 5. Cyanogen bromide peptides from the Cprotein from the same sample of envelope as shown inFig. 4. The solid line represents 3H-labeled proteinfrom strain K-12, and the dashed line represents"4C-labeled protein from strain J-5. The sample wasrun with the Swank-Munkres gel system. The insetshows a scan of the stained gel prior to slicing andillustrates the resolution of this gel system.

nigh -. 32 - --AI ^ ___

I B(Hill) 041

3

0127 ML3081 3

13

FIG. 6. Scans of Bragg-Hou gels of outer mem-brane protein from a number of the E. coli strainsdescribed in Table 1. All of these cultures were grownon minimal medium with Casamino Acids as thecarbon source.

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3 E. coli W ShigellaI schmitzii

E. coli 0111B4 Shigella2 3 dysenteriae

3

Salmonellatyphimurium

2

FIG. 7. Scans of Bragg-Hou gels of outer mem-

brane protein from two E. coli strains, two representa-tive Shigella species, and Salmonella typhimurium.The E. coli 0111B4 strain in this case is ATCC 12015.Note the multiple protein 1 bands in S. typhimurium.All of the cultures were grown on minimal mediumwith Casamino Acids as the carbon source.

appeared to be identical in all of the strains.The two 0111 strains had identical patterns tostrain J-5 (and to the 0111 B4 strain, which is theparent of J-5 [not shown]). Since these repre-sent three different isolates of this serotype, thissuggests that protein 2 is a common feature of0111 serotypes.The Shigella species were all generally similar

to E. coli K-12 (including the two species notshown in the figures) in that they exhibited twomajor bands on Bragg-Hou gels. There wereslight differences in the relative migration ofprotein 1, both among the different Shigellaspecies themselves, and between Shigella andE. coli. However, the migration of protein 3 wasidentical in all of the species of Shigella, Sal-monella, and E. coli tested. The pattern givenby S. typhimurium was quite different from theShigella species, and resembled E. coli 0111 inhaving protein 2 or a protein which migratedsimilar to protein 2. lin addition, there were twoother bands, both of which coincided partiallywith protein 1.

All of the strains and species tested in thisseries gave both A and C bands when unboiledprotein was analyzed with the Maizel buffersystem (not shown). Again, the C band wasidentical from all of the cultures, whereas the A

band was broad and variable in migration (asshown for the two E. coli strains in Fig. 3).

Strain and species differences were notedwhen the boiled samples were analyzed with theMaizel buffer system (Fig. 8). As noted previ-ously, E. coli K-12 gives a pair of closely spacedbands when analyzed with this system. Thiswas also true for most of the other E. coli strainsthat were lacking protein 2 (not shown). How-ever, this was not true for E. coli ML308, whichgave a single band on Maizel gels even though itexhibited no protein 2 on Bragg-Hou gels.Similar differences were noted among the vari-ous Shigella species. Although S. schmitzii andS. dysenteriae gave similar patterns on Bragg-Hou gels (Fig. 7), on Maizel gels S. schmitziigave a single, sharp peak and S. dysenteriaegave a broad double band (Fig. 8). S.typhimurium gave a single, broad band with atrailing shoulder in the Maizel gel system. Tofacilitate these comparisons, all gels were pre-pared at the same time and run in the samebath to eliminate slight variations.A summary of the Bragg-Hou gel patterns of

all of these strains, plus some additional strainswhich are described later in this report, is givenin Table 2.

It was of interest to determine whether therewas any relationship between the presence ofprotein 2 and the production of enterotoxinsoccasionally associated with E. coli strains. On

E.coliOll1a4 E.coli ML308

Shigella Shigellaschmitzii dysenterias

Salmonellatyphimurium

FIG. 8. Scans of Maizel gels of some of the outermembrane protein samples shown in Fig. 6 and 7.Note the difference between the two Shigelta speciesthat appear to be identical on Bragg-Hou gels (Fig. 7).

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TABLE 2. Summary of major outer membraneproteins observed in various strains and species by

Bragg-Hou gel electrophoresis:Strain or species Protein 1 Protein 2 Protein 3

Various E. coli strains:0111AI + + +OlliBI (Heath) + + +J-5 + + +0111B4 (ATCC) + + +041 + _ +0127 + _ +055 + _ +B (Hill) + _ +w + _ +ML 308 + _ +K-12 (KL 96) + _ +K-12 (AB 1859) + _ +K-12 (AB 1621) - +334 + _ +H10407 _ + +

S. typhimurium + + +

Shigella (all species in + _ +Table 1)

a All strains and species are shown as they wouldappear when grown to late exponential phase onminimal medium with Casamino Acids as the carbonsource.

the basis of the data collected to date, theredoes not appear to be a correlation between theproduction of heat-labile enterotoxin and theproduction of protein 2. E. coli 334, a knownenterotoxin producer (4), exhibited an outermembrane profile similar to E. coli K-12. Thethree E. coli Olf1 strains described in Table 1were assayed for enterotoxin production, andnone was detected. These assays were con-ducted-by R. Guerrant, Department of InternalMedicine, University of Virginia MedicalSchool, using a new assay which will be de-scribed elsewhere.

Effect of filament formation on outer mem-brane proteins. Henning et al. (5) have pre-sented evidence recently that suggests that theouter membrane proteins play a role in deter-mining or maintaining the characteristic shapeof E. coli cells. If this is true, one might predictthat the major outer membrane proteins wouldbe different or present in different proportionsat the hemispherical ends of the cell as opposedto the cylindrical mid-section of the cell. To testthis, I compared the major proteins synthesizedby cells growing as drug-induced filaments (inwhich no new ends are formed) to the proteinssynthesized by cells which were dividing nor-mally.

This experiment was done in the followingway. An early exponential-phase culture wasdivided into two equal parts. One part receivedno addition (control culture) and the other partreceived either 2 gg of 5-diazouracil per ml (11)or 1 ,ug of mitomycin C per ml. These levels ofdrugs are sufficient to cause virtually completeconversion of the rod-shaped cells to filamentswithin one generation time with only a slightinhibition of growth (as measured by the in-crease in turbidity at 550 nm) over two genera-,tion times. The control and drug-treated cul-tures were allowed to grow for one-half genera-tion time (to allow completion of septation inthe drug-treated culture), and then [14C ]leucinewas added to the control culture and ['H]leu-,cine was added to the drug-treated culture. Thecultures were allowed to grow for one generationtime after addition of the isotope, protein syn-thesis was stopped by the addition of chloram-phenicol (0.5 mg/ml), and the cultures weremixed and harvested. The cells were then frac-tionated, and the outer membrane protein wasexamined with the Bragg-Hou gel system.

Several combinations of strains, drugs, andculture media were tested in this fashion. Theeffect "of both mitomycin C and 5-diazouracilwas examined with strain J-5 grown either onminimal medium.with succinate as the carbonsource (generation time, 90 min) or on minimalmedium with glucose as the carbon source,

3 3

2 GLUCOSE SUCCINATEJI(7) J

GLUCOSEL-SROTH +

I mM cAMP3

23

1

GLUCOSE+ GLYCEROL

5 mM cAMP3

3

2- jl

FIG. 9. Effect of catabolite repression on E. coliJ-5. the scans are of Bragg-Hou gels of cultures grownon minimal medium with succinate, glycerol, orglucose plus various levels of cAMP. L broth is acomplex, tryptone-based medium containing glucose.For simplicity, only the major peaks are.shown in thelower scans, although minor differences were observedin other regions of the gels.

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supplemented with the 20 essential amino acidsand the B vitamins (generation time, 30 min).On both media, there was no difference betweenthe relative amounts of proteins 1, 2, and 3 inthe control or in either of the drug-treatedcultures. In all of the drug-treated cultures,virtually all of the cells had formed filaments bythe time the cultures were harvested.This experiment was repeated with strain

K-12 grown on minimal medium with glucose asthe carbon source (generation time, 60 min). Inthis case, only mitomycin C was used. Therewas no difference in the relative amounts ofproteins 1 and 3 between the control anddrug-treated cultures. In addition, protein Cwas isolated and cleaved with cyanogen bro-mide as in the experiment described previouslyand shown in Fig. 5. The comparison of thecontrol and drug-treated cultures was identicalto that shown in Fig. 5 (data not shown),indicating that filament formation induced bymitomycin C did not alter the ratio of proteins3a and 3b. Although these experiments do notrule out a role for these proteins in the determi-nation of the cell shape, they do suggest thatnone of the major proteins (1, 2, 3a or 3b) isfound only on the ends of the cells or is formedonly during cell division.

Effect of catabolite repression on the outermembrane protein profile of E. coli 01l1. Theouter membrane protein profile of E. coli 0111strains is very strongly influenced by the condi-tions of growth. This was discovered quite byaccident in the experiment described above, inwhich strain J-5 was grown both in minimalmedium with succinate as the carbon sourceand in an enriched minimal medium with glu-cose as the carbon source.The differences that are observed as a conse-

quence of culture medium are shown in Fig. 9.All of the cultures shown in this figure weregrown to mid-exponential phase. When cultureswere grown on minimal medium with succinateor glycerol as the carbon source, there is consid-erably more protein 2 than protein 1 in the outermembrane. When cultures are grown to mini-mal medium with glucose as the carbon source,or on L broth (a rich tryptone-based mediumcontaining glucose), the situation is reversedand there is much more protein 1 than protein 2.To demonstrate that this was due to catabo-

lite repression and not to differences in growthrate, cyclic 3'-5'-adenosine monophosphate(cAMP) was added to cultures growing onminimal medium with glucose as the carbonsource. A low level of cAMP (1 mM) partiallyreversed the effect of glucose, and 5 mM cAMP

resulted in an outer membrane profile essen-tially identical to that obtained with culturesgrown on non-fermentable carbon sources (Fig.9). Neither concentration of cAMP affected thegrowth rate on glucose minimal medium.Hence, it can be concluaed that the reduction inprotein 2 is due to catabolite repression.More dramatic effects of catabolite repression

have been observed with certain enterotoxin-producing strains of E. coli that. have outermembrane proteins similar to E. coli 0111. Onesuch strain is E. coli H10407, reported bySkerman et al. (20) to be a serotype 078 and toproduce plasmid-associated enterotoxin. Thisstrain exhibited a very strong rabbit ileal loopreaction (20) and a very strong, positive reactionfor heat-labile enterotoxin in the tissue cultureassay which we have employed.When outer membrane protein from strain

H10407 grown on minimal medium with Casa-mino Acids as the carbon source was examinedin the Bragg-Hou gel system (Fig. 10), noprotein 1 was detected, and the amount ofprotein 2 was increased relative to that seen

2

10407

CAA

2A

J-5 I

CAA

3'

AGLUCOSE

NH4

GLUCOSENH4

3

GLUCOSEGLUTAMATE

12^

GLUCOSEGLUTAMATE

FIG. 10. Comparison of the effect of various levelsof catabolite repression on E. coli H 10407 and E. coliJ-5. Cultures were grown on minimal medium withCasamino Acids as the carbon source (minimal catab-olite repression), glucose as the carbon source andNH4+ as the nitrogen source (intermediate cataboliterepression), and glucose as the carbon source with 500Ag of L-glutamate per ml as the nitrogen source(maximal catabolite repression). The scans show onlythe major peak region of the gel. Gels are the same asin Fig. 9.

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with E. coli 0111 (for comparison, the outermembrane protein profile of strain J-5 grownunder the same conditions is shown in the lowerpart of Fig. 10). However, strain H10407 doeshave the capability to produce protein 1 whencatabolite repression is increased. When thisstrain is grown with glucose as the carbon sourceand NH,+ as the nitrogen source, the amount ofprotein 1 is comparable to that which strain J-5makes when grown on Casamino Acids. StrainH10407 will produce a large amount of protein 1when grown under conditions of severe catabo-lite repression (glucose as the carbon sourceplus 500 ug of L-glutamate per ml as thenitrogen source). However, under these condi-tions, it grows very poorly in comparison withstrain J-5, having a generation time of about 5 has opposed to 75 min for strain J-5. The reasonfor this phenomenon is not known, but onepossibility is that it reflects an abnormally highlevel of intracellular cAMP in this strain. Thisphenomenon is not unique to this strain, sincewe have now observed this in some enterotoxin-producing strains of porcine origin. StrainH10407 has no obvious cell wall defect, whichillustrates the point that protein 2 can com-pletely replace protein 1 in this strain withoutaffecting the integrity of the cell.

Effect of growth phase on the outer mem-brane protein profile. The stage of growth atwhich cultures are harvested also affects theouter membrane protein profile that is observedin the Bragg-Hou gel system. This phenomenonhas been observed with all of the enteric strainsand species, and is particularly pronounced inthose strains which lack protein 2. An exampleis the experiment shown in Fig. 11. This wasdone with E. coli B grown on minimal mediumwith glycerol as the carbon source. The culturewas sampled in the late exponential phase ofgrowth and again after the stationary phase wasbegun. The total amount of the major proteins(sum of proteins 1 and 3) remained constant,but the exponential-phase cells had much moreprotein 1 than 3, whereas the stationary-phasecells had more protein 3 than 1. This phenome-non is even more pronounced in cultures grow-ing on complex media such as Trypticase soybroth, where the termination of growth is lessabrupt and growth may continue for severalgenerations after the end of the exponentialphase. We do not know whether this representstrue turnover of these proteins (i.e., catabolismof protein 1 and replacement by protein 3) orwhether it is simply due to the fact that thesynthesis of protein 1 is terminated much earlierin the growth cycle than the synthesis of protein

2.0-

0 1.0-100.8-/

0.6-

0.4-

0.2-

0 I 2 3 4 5 6 7HOURS

FIG. 11. Effect of the growth phase on the outermembrane protein profile. The graph shows theturbidity at 550 nm of a culture of E. coli B growing onminimal medium with glycerol as the carbon source.The culture samples were taken at the times shown bythe arrows, and the scans show the Bragg-Hou gelprofiles of outer membrane protein from these sam-ples. Note the relative change in amounts ofproteins 1and 3.

3. We do know that the "signal" for this changeis probably not cell septation, the terminationof rounds of deoxyribonucleic acid replication,or the actual rate of deoxyribonucleic acidreplication, since these changes were not ob-served in cultures treated with mitomycin C or5-diazouracil.Outer membrane protein changes associ-

ated with multiple phage resistance. Duringthe course of several experiments with strainsconstructed from E. coli AB 1621, it was ob-served that the outer membrane from thesestrains lacked A protein when chromatographedon SDS-Sephadex G-200 and protein 1 whenexamined on Bragg-Hou gels. Strain AB 1621was derived from strain AB 1859 by selection forresistance to phages T4 and T6 (2) and containstwo mutations designated by Taylor and Trot-ter (22) as tfr (pleiotropic resistance to phagesT3, T4, T7, and X) and tsx (resistance to phage

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T6). This strain has normal sensitivity tophages P1 and T5.

Strain AB 1621 and its parent AB1859 werecompared (Fig. 12) and, to rule out differencesin extractability of outer membrane proteins byTriton X-100, the outer membrane was isolatedboth by Triton extraction (Fig. 12B and D) andcentrifugation of the crude envelope fraction ona continuous sucrose gradient (Fig. 12A and C).Both methods of preparation yielded identicalresults-a complete absence of protein 1 instrain AB 1621. This pair of cultures wasgrown to late exponential phase on Trypticasesoy broth, and the growth rates of the two cul-tures were identical. Similar outer membrane

3

B

C

D_kAS

FIG. 12. Outer membrane protein profiles of a

multiply phage-resistant strain of E. coli (AB 1621)and its phage-sensitive parent (AB 1859). The upperpair of scans shows protein from strain AB 1859 (A, B)and the lower pair of scans shows protein from strainAB 1621 (C, D). The outer membrane protein shownin scans A and C was isolated from the crude envelopeby sucrose gradient centrifugation (23), and theprotein shown in scans B and D was isolated by TritonX-100 extraction (13). Both preparations gave identi-cal results. The cultures were grown to late exponen-tial phase on Trypticase soy broth. The scans are ofBragg-Hou gels.

protein profiles have been obtained with AB1621 grown on minimal medium.The density of the outer membrane fraction

was identical for both cultures, as determinedby the position of the bands in the sucrosegradients, and the phospholipid/protein ratiowas the same in these fractions from the gradi-ents, indicating that there was no net decreasein outer membrane protein. The amount ofprotein 3 in strain AB 1621 is similar to the sumof proteins 1 and 3 in strain AB 1859. The Cprotein from strain AB 1621 was isolated andcleaved with cyanogen bromide, and the pep-tide profile was identical to that shown in Fig. 5,indicating that the ratio of proteins 3a and 3bwas normal.

Revertants of the tsx mutation accumulateduring storage of strain AB 1621 on slants, butother than this there is no obvious defect in theouter membrane of this strain. Cultures grownormally, with no evident lysis during growth.Since the strain is lacY-, it was possible to testfor detergent-induced leakiness by growing theculture on plates of lactose-MacConkey me-dium and on lactose-eosine methylene blue agarcontaining 0.5% sodium deoxycholate. The cul-ture grew and exhibited normal lac- colonies onboth media.To determine whether both tfr and tsx muta-

tions were required for this phenotype, I exam-ined the outer membrane protein profile of aT6-sensitive revertant from strain AB 1621.This revertant contained a normal amount ofprotein 1. Seven independent, T6-resistantclones derived from this revertant were exam-ined, and all were missing protein 1. However,five independent T6-resistant clones isolatedfrom the parent strain AB 1859 were examinedand all contained normal or near-normalamounts of protein 1. Thus, both mutationsappear to be required for the phenotype.These data indicate that protein 3 can substi-

tute for protein 1 without any drastic effectsnoted in laboratory culture. Although a strainmissing protein 1 might not survive in nature,the loss of protein 1 is clearly not lethal.

DISCUSSIONTo simplify discussion of the rather complex

changes observed in the outer membrane pro-teins of enteric bacteria, I should like to beginwith an overview of the properties of the majorouter membrane proteins. I shall use the term"major protein complex" to denote the entiregroup of major proteins (and perhaps someminor proteins as well), with molecular massesof about 40,000 daltons, which occur in the

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VARIATIONS IN OUTER MEMBRANE PROTEINS

outer membrane of E. coli and other relatedspecies. The general properties of the majorprotein complex may be summarized as follows.

(i) These proteins represent integral proteinsof the bacterial outer membrane and are diffi-cult to solubilize without dissolution of themembrane. They remain insoluble at neutralpH in aqueous solution except in the presence ofdetergents or chaotropic agents.

(ii) These proteins as a group may representas much as 70% of the total outer mem-braneprotein of E. coli, and comparative studies onother species (13) indicate that they are widelydistributed and may be a universal componentof the outer membranes of gram-negative bacte-ria.

(iii) All of these proteins exhibit an anoma-lous behavior on SDS-polyacrylamide gels orupon gel filtration in the presence of SDS (14),depending upon the conditions that have beenused to solubilize the proteins in SDS solution.

(iv) When these proteins are exposed to SDSunder maximally denaturing conditions (i.e.,exposure to urea, boiling in SDS solution, ororganic solvent treatment), they appear more orless homogeneous on neutral pH SDS-polya-crylamide gels of the Maizel (9) type, but theyexhibit anomalous behavior on SDS gels withan alkaline upper buffer system, as described byBragg and Hou (3), or on SDS gels run with astacking or electrofocusing buffer system (1, 8).Although this appears to be related to chargedensity rather than size (14, 15, 24), it has led touncertainty about the true molecular mass ofthese proteins.

It is evident that these proteins can beseparated quite clearly into two classes byexamining their filtration properties on SDS-polyacrylamide gels, or on Sephadex columnswhen the proteins have been dissolved in SDSunder mild conditions (disaggregation in theabsence of urea at a temperature of 50 C or less).I have designated these classes as A and Cproteins, these letters referring to the proteinbands which are observed on gels (14). Whenthe A proteins are dissolved in SDS solutionunder mild conditions. they exhibit apparentmolecular masses of 60,000 to 100,000 daltons(depending upon the strain or species), andthese appear to be aggregates or polymericforms (14). When the C proteins are dissolved inSDS solution under mild conditions, they give arather sharp peak and an apparent molecularmass of about 30,000 daltons; this appears toconsist of monomeric protein which is not fullyunfolded and reacted with SDS (14).As a group, the A proteins are heterogeneous

with respect to properties and distribution in

various gram-negative species. In E. coli 0111two major A proteins have been described (15,16), and these vary in amount depending oncultural conditions. In E. coli K-12 and mostother E. coli strains we have examined, thereappears to be only a single major A protein,protein 1. There may also be minor species of Aproteins with specific functions for the cell andwhich may function as phage or colicin recep-tors. These cannot be detected by our rathercoarse methods.The A proteins do not play any obvious role in

maintaining the structural integrity of the outermembrane under normal laboratory growthconditions, although they may be important tothese organisms in their normal ecologicalniches. These proteins are greatly reduced inamount when cultures enter the stationaryphase of growth (Fig. 11) and are missing in anotherwise normal multiply phage-resistantstrain (Fig. 12).

In contrast, the C proteins appear homogene-ous as a group. In all of the strains and species Ihave examined, these proteins exhibit a similarmigration on gels when they are dissolved inSDS under mild conditions. When dissolvedunder harsh conditions, they give an identical,single sharp band on Bragg-Hou gels (protein3). In E. coli 0111 and E. coli K-12 there appearto be two distinct species of C protein, proteins3a and 3b, and under the limited sets ofconditions where we have determined theseproteins they are always present in the sameratio. Analysis of the relative amounts of theseproteins is quite difficult and tedious, since itinvolves determination of the cyanogen bromidepeptide profile of the isolated C protein.

In contrast to the A proteins, the C proteinsmay be essential for normal outer membranefunction. In exponential-phase cultures, protein3 is reduced to about one-third of the totalprotein in the major protein complex, but it isnever less than that minimal value. Althoughour sampling has been limited, no strain hasbeen found to be missing protein 3 entirely. Therecent studies of Ames et al. (1) and Koplowand Goldfine (8) also pertain to this point. Theseauthors have studied heptose-deficient mutantsof Salmonella and E. coli. These mutants havedefective cell walls, as indicated by a sensitivityto bile salts and by an alteration in the lipid/protein ratio of the outer membrane, and theentire major protein complex (including protein3) appears to be missing or greatly reduced.The normal physiological change in the outer

membrane proteins which occurs when mac-romolecular synthesis is being shut down at theend of exponential-phase growth is interesting

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SCHNAITMAN

in terms of cellular regulation (Fig. 11), andexplains some of the observations made ontemperature-sensitive dnaA and dnaB mutantsby Shapiro et al. (19) and Siccardi et al. (18).These authors examined the changes in enve-lope proteins that occurred when these mutantswere shifted to nonpermissive temperatures.When they dissolved the envelope samples inSDS solution under mild conditions, they ob-served a decrease in a 60,000-dalton component(A protein, Fig. 3) and an increase in a 30,000-dalton component (C protein, Fig. 3). Thus,these mutations must affect early steps in theregulation of macromolecular synthesis in sucha way that they mimic the normal shutdownthat occurs at the end of exponential-phasegrowth. It was noted that some of the differ-ences in the envelope from the dnaA mutantand all of the differences in the envelope fromthe dnaB mutant (18) disappeared when thesamples were boiled in SDS solution prior toelectrophoresis. It is more difficult to reconcileour data with the X and Y proteins described ina similar mutant by Inouye et al. (6, 7), sincethese authors dissolved their envelope prepara-tions in SDS solution at a temperature (70 C)that leads to only partial dissociation andunfolding of the A and C proteins (14). Thesestudies point out the necessity for understand-ing the way in which the various proteins of themajor protein complex behave under differentconditions of solubilization and electrophoresis,and also the way normal physiological condi-tions affect the relative amounts of these pro-teins.

ACKNOWLEDGMENTSI acknowledge the skilled assistance of Robert McIver,

Anne Summers, and Montserrat Salsas, and the generoushelp in obtaining E. coli strains provided by Richard Guer-rant and John Cronan.

This research was supported by research grant GB-25273from the National Science Foundation, and by Public HealthService Research grant GM18006 and Career DevelopmentAward GM22053 from the National Institute of GeneralMedical Sciences.

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2. Bachmann, B. J. 1972. Pedigrees of some mutant strainsof Escherichia coli K-12. Bacteriol. Rev. 36:525-557.

3. Bragg, P. D., and C. Hou. 1972. Organization of proteinsin the native and reformed outer membrane of Esche-richia coli. Biochim. Biophys. Acta 274:478-488.

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15. Schnaitman, C. 1973. Outer membrane proteins of Esche-richia coli. II. Heterogeneity of major outer membranepolypeptides. Arch. Biochem. Biophys. 157:553-560.

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17. Schnaitman, C., and J. W. Greenawalt. 1966. Intracyto-plasmic membranes in Escherichia coli. J. Bacteriol:92:780-783.

18. Siccardi, A. G., B. M. Shapiro, Y. Hirota, and F. Jacob.1970. On the process of cellular division in Escherichiacoli. IV. Altered protein composition and turnover ofthe membranes of thermosensitive mutants defectivein chromosomal replication. J. Mol. Biol. 56:475-490.

19. Shapiro, B. M., A. G. Siccardi, Y. Hirota, and F. Jacob.1970. On the process of cellular division in Escherichiacoli. II. Membrane protein alterations associated withthe mutations affecting the initiation of DNA synthe-sis. J. Mol. Biol. 52:75-89.

20. Skerman, F. J., S. B. Formal, and S. Falkow. 1972.Plasmid-associated enterotoxin production in a strainof Escherichia coli isolated from humans. Infect. Im-munity 5:622-624.

21. Swank, R. T., and K. D. Munkres. 1971. Molecularweight of oligopeptides by electrophoresis in polyacryl-amide gel with sodium dodecyl sulfate. Anal. Biochem.39:462.

22. Taylor, A. L., and C. D. Trotter. 1972. Linkage map ofEscherichia coli strain K-12. Bacteriol. Rev.36:504-524.

23. White, D. A., W. J. Lennarz, and C. Schnaitman. 1972.Distribution of lipids in the wall and cytoplasmicmembrane subfractions of the cell envelope of Esche-richia coli. J. Bacteriol. 109:686-690.

24. Wu, H. C. 1972. Isolation and characterization of anEscherichia coli mutant with alteration in the outermembrane proteins of the cell envelope. Biochim.Biophys. Acta 290:274-289.

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