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

Transcription

1 JOURNAL OF BACrERIOLOGY, May 1974, p Copyright American Society for Microbiology Vol. 118, No, 2 Printed in U.S.A. Outer Membrane Proteins of Escherichia coli IV. Differences in Outer Membrane Proteins Due To Strain and Cultural Differences CARL A. SCHNAITMAN Department of Microbiology, The University of Virginia, School of Medicine, Charlottesville, Virginia Received for publication 12 December 197 When the 42,000-dalton major outer membrane protein of Escherichia coli 0111 is examined on alkaline polyacrylamide gels containing sodium dodecyl sulfate, it is resolved into three distinct bands designated as proteins 1, 2, and. Band consists of two distinct polypeptides, proteins a and b. E. coli K-12 does not make any protein 2, but makes proteins similar to 1, a, and b as indicated by comparison of cyanogen bromide peptide patterns. Several Shigella species and 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 In addition.to these species and strain differences, cultural differences resulted in differences in the outer membrane protein profiles. Under conditions of catabolite repression, the level of protein 2 in E. coli 0111 decreased while the level of protein 1 increased. An enterotoxin-producing strain similar to E. coli 0111 produced no protein 1 and an elevated level of protein 2 under conditions of low catabolite repression. The levels of proteins 1 and are also different in different phases of the growth curve, with protein 1 being the major species in the exponential-phase cells and protein being the major species in stationary-phase cells. A multiply phage-resistant mutant of E. coli K-12 with no obvious cell wall defects produced no protein 1 or 2, but made increased amounts of protein. Thus, the major outer membrane proteins of E. coli and related species may vary considerably without affecting outer membrane integrity. Previous studies in this series (14-16) have shown that the 42,000-dalton major protein of Escherichia coli 0111 consists of a mixture of at least four distinct major polypeptides. These polypeptides can be distinguished by their migration on sodium dodecyl sulfate (SDS)-polyacrylamide gels with an alkaline upper buffer () and by their distinctive cyanogen bromide peptide patterns (15, 16). All of the previous studies in this series have dealt with strain J-5, a derivative of E. coli 0111 B4 that is lacking the enzyme uridinediphosphate-galactose-4-epimerase. This mu- 454 tant is convenient for cell wall studies since it permits the specific labeling of the cell wall lipopolysaccharide with galactose. Since this labeling requires the function of the gal operon, which is subject to catabolite repression, cultures have been routinely grown with succinate or glycerol as the carbon source. This routine use of a single strain of E. coli grown under a single, rather limiting set of culture conditions for several years prevented the discovery of two rather interesting phenomena which are described in this report, namely, that the outer membrane protein composition of a single strain may vary greatly as a consequence of culture conditions, and that all strains of E. coli (and other enteric bacteria) do not have the same polypeptides present in their outer membranes. In addition to the profound differences obtained with different SDS-polyacrylamide gel methods (1, 8, 14, 15), the differences noted above have certainly contributed to the rather confusing lack of agreement in the literature concerning the outer membrane proteins of gram-negative bacteria. MATERIALS AND METHODS Strains and culture conditions. Except where noted, cultures were grown in minimal medium (2) with 1% Casamino Acids, 1% sodium succinate, or 0.5% glucose or glycerol as the carbon source. Cultures were grown at 7 C in a rotary shaker and harvested at late exponential phase (60% maximal turbidity). The bacterial strains used in this study are described in Table 1.

2 VOL. 118, 1974 VARIATIONS IN OUTER MEMBRANE PROTEINS 455 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, 2) E. coli 0111B4ATCC ATCC E. coli 041 ATCC 2976 ATCC E. coli 0127 ATCC ATCC E. coli 055 ATCC ATCC E. coli B (Hill) ATCC 2225 ATCC E. coli W ATCC 967 ATCC E. coli ML08 H. Winkler Widely used in transport and membrane vesicle studies E. 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 4 R. Guerrant Produces heat-labile enterotoxin; serotype 015:H11; clinical 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 U. Va. SC S. schmitzii U. Va. SC Clinical isolate, U. Va. Hospital S. flexneri ATCC 980 U. Va. SC S. dysenteriae ATCC 9665 U. Va. SC a Source abbreviations: ATCC, American Type Culture Collection; CGSC, E. coli genetic stock center, Yale University; U. Va. SC, Stock culture collection, Microbiology Department, University of Virginia Medical School. 0111B) and E. coli K-12 were grown on minimal Isolation and analysis of outer membrane protein. Harvested cells were treated in an Omni- Mixer to remove flagella, and broken in a French pressure cell and extracted with Triton X-100 to remove the cytoplasmic membrane from the crude envelope fraction (14). The Triton-insoluble outer membrane protein was dissolved in SDS solution at 7 C (first step, method II, reference 14) and applied to SDS-polyacrylamide gels without further treatment, or the sample was then dialyzed against a SDS-urea solution and boiled briefly (complete method II). Samples of intact protein were analyzed in 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 (). A and C proteins were isolated by two cycles of SDS- Sephadex G-200 chromatography and cleaved with cyanogen bromide as described previously (16). Cyanogen bromide peptides were analyzed either with the Swank-Munkres gel system (16, 21) or on conventional SDS-polyacrylamide gels containing 12% acrylamide, 0.5% bis-acrylamide, and 0.5 M urea that were prepared and run in 0.1 M sodium phosphate buffer (ph 7.2) containing 0.1% SDS. Gels were scanned or sliced as described previously (14). RESULTS Comparison of outer membrane protein from E. coli 0111 and E. coli K-12. Identical cultures of strain J-5 (derived from E. coli medium with succinate as the carbon source. The J-5 culture was labeled with [4C ]leucine and the K-12 culture was labeled with [H]leucine. The cultures were mixed prior to the isolation of the outer membrane protein. When samples of outer membrane protein prepared by boiling in SDS (complete method II) were analyzed on SDS-polyacrylamide gels with the Bragg-Hou buffer system, the results shown in Fig. 1 were obtained. Protein 2 appeared to be missing entirely from strain K-12, whereas proteins 1 and appeared to be identical in terms of migration in the two strains. Less pronounced differences can also be observed with the Maizel buffer system (Fig. 2). Instead of the single, broad peak observed with strain J-5, strain K-12 gives two very closely spaced bands. These two closely spaced bands can also be observed in stained gels containing only strain K-12 outer membrane protein (not shown). The spacing between these bands also depends upon the sample size and the ph of the gel buffer, since overloading causes the bands to merge and even a slight increase in the ph above neutrality causes the bands to move apart. These two strains also give very different gel

3 SLICE NUMBER FIG. 1. Comparison of [H]leucine-labeled outer membrane 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. The cultures were grown with succinate as the carbon source (14), and the outer membrane protein was dialyzed against SDS-urea solution and boiled prior to electrophoresis (14). The numbers identify the three major protein bands (14). 40- N 20- x U' Z20-0 U SCHNAITMAN -8 N 0 6 x U' z -4 = J. BACTERIOL. protein 1 was present in both the leading and trailing edge of the broad region of A protein from strain J-5. Since there is an obvious difference in protein 2, it was important to determine whether there were also differences among proteins 1, a, and b of these two strains. To answer this question, I dissolved a sample of outer membrane protein from the mixed culture described above in SDS solution at 7 C and chromatographed it on SDS-Sephadex G-200 (16) to separate the A and C proteins. These were then boiled in SDS solution and rechromatographed on SDS- Sephadex G-200, and the protein from each fraction which shifted to peak B after boiling (16) was cleaved with cyanogen bromide. Figure 4 shows a comparison of the cyanogen bromide peptides from the A protein of strains J-5 and K-12. This was analyzed on a conventional 12% polyacrylamide gel rather than with the Swank-Munkres gel system to facilitate comparison of the larger polypeptides characteristic of protein 2 (16). The numbering of the polypeptides is the same as was used previously (Fig. 8, reference 16). Peptides 1 and 2, which are characteristic of protein 2 (16), appear to be missing from the A protein from strain K-12. Peptides and 6, which are characteristic of protein 1 (16), are present in both cultures, although they are present in reduced amounts as would be predicted from the relative amounts of protein 1 present in these two strains (Fig. 1). Peptides 4 and 6 are somewhat ambiguous, but this is as to be expected since there are overlap TO SLICE NUMBER FIG. 2. Comparison of H-labeled outer membrane protein from E. coli K-12 (solid line) and "4C-labeled outer membrane protein from E. coli J-5 (dashed line) on a gel with the Maizel (ph 7.2) buffer system. The sample was prepared by boiling as in Fig. 1. patterns when the outer membrane protein is dissolved without urea treatment or boiling and analyzed with the Maizel buffer system (Fig. ). The A protein (16) from strain J-5 gives several peaks near the top of the gel, whereas the A protein from strain K-12 gives a single peak with an apparent molecular mass of about 60,000 daltons. The C peak is identical in both strains, with an apparent molecular mass of 25,000 to 0,000 daltons. This indicates that the A protein from strain J-5 is either more aggregated or more unfolded than its counterpart from strain K-12. It should be noted that in a previous experiment (15) it was observed that C 6 A IO Nu z~~ ~ ~ ~ ~ ~ 0~ U SLICE NUMBER FIG.. Comparison of E. coli K-12 and E. coli J-5 outer membrane protein dissolved in SDS solution at 7 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 H-labeled protein from 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). 4 x

4 VOL. 118, 1974 VARIATIONS IN OUTER MEMBRANE PROTEINS 457 z o 0 4 D ~~~~2 I except the two 0111 strains were found to be missing protein 2. In general, these E. coli strains appeared similar, with the exception of a high-molecular-weight protein that was observed in strain MI08 and was absent in the other strains. The migration of proteins 1 and I 5 FIG. 4. Cyngnboidpetesfo tha protein from a mixture of outer membrane protein from E. coli K-12 (dashed line) and E. coli J-5 (solid line). The envelope sample is the same as shown in Fig. 1-. The sample was run on a Maizel system gel containing 12% acrylamide. ping peptides from proteins 1 and 2 (in the case of strain J-5) present in these regions of the gel (16). The conclusion which can be drawn from Fig. 4 is that protein 1 is very similar or identical in both strains, and protein 2 is present only in strain J-5. Figure 5 shows a comparison of the cyanogen bromide peptides from the C proteins of the two strains. It appears that strain K-12 also contains two polypeptides, proteins a and b, and these are present in the same ratio as in strain J-5. In this figure, peptides 1, 2, and 4 are indicative of protein a and peptides and 5 of protein b (16). It may be concluded then that strain K-12 contains three of the four major polypeptides 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. coli t1aibs (strain J-5) contained a a lypeptide which was absent in E. coli K-12, it was of interest to examine other commonly studied E. coli strains and some related enteric species both to see whether this unique protein was missing and whether other major differences could be detected. To avoid nutritional problems, I grew all of the cultures in minimal medium containing a small amount of thiamine and Casamino Acids as the carbon source. All of the cultures grew well in this medium. Figures 6 and 7 show the appearance of boiled, outer membrane protein from a number of E. coli strains, two Shigella species, and Salmonella typhimurium analyzed with the Bragg-Hou gel system. All of the E. coli strains SLICE NUMBER FIG. 5. Cyanogen bromide peptides from the C protein from the same sample of envelope as shown in Fig. 4. The solid line represents H-labeled protein from strain K-12, and the dashed line represents "4C-labeled protein from strain J-5. The sample was run with the Swank-Munkres gel system. The inset shows a scan of the stained gel prior to slicing and illustrates the resolution of this gel system. nigh AI ^ I B(Hill) ML08 FIG. 6. Scans of Bragg-Hou gels of outer membrane protein from a number of the E. coli strains described in Table 1. All of these cultures were grown on minimal medium with Casamino Acids as the carbon source. 1 05s

5 458 SCHNAITMAN J. BACTERIOL. E. coli W Shigella I schmitzii E. coli 0111B4 Shigella 2 dysenteriae 2 Salmonella typhimurium FIG. 7. Scans of Bragg-Hou gels of outer membrane protein from two E. coli strains, two representative Shigella species, and Salmonella typhimurium. The E. coli 0111B4 strain in this case is ATCC Note the multiple protein 1 bands in S. typhimurium. All of the cultures were grown on minimal medium with Casamino Acids as the carbon source. appeared to be identical in all of the strains. The two 0111 strains had identical patterns to strain J-5 (and to the 0111 B4 strain, which is the parent of J-5 [not shown]). Since these represent three different isolates of this serotype, this suggests that protein 2 is a common feature of 0111 serotypes. The Shigella species were all generally similar to E. coli K-12 (including the two species not shown in the figures) in that they exhibited two major bands on Bragg-Hou gels. There were slight differences in the relative migration of protein 1, both among the different Shigella species themselves, and between Shigella and E. coli. However, the migration of protein was identical in all of the species of Shigella, Salmonella, and E. coli tested. The pattern given by S. typhimurium was quite different from the Shigella species, and resembled E. coli 0111 in having protein 2 or a protein which migrated similar to protein 2. lin addition, there were two other bands, both of which coincided partially with protein 1. All of the strains and species tested in this series gave both A and C bands when unboiled protein was analyzed with the Maizel buffer system (not shown). Again, the C band was identical from all of the cultures, whereas the A band was broad and variable in migration (as shown for the two E. coli strains in Fig. ). Strain and species differences were noted when the boiled samples were analyzed with the Maizel buffer system (Fig. 8). As noted previously, E. coli K-12 gives a pair of closely spaced bands when analyzed with this system. This was also true for most of the other E. coli strains that were lacking protein 2 (not shown). However, this was not true for E. coli ML08, which gave a single band on Maizel gels even though it exhibited no protein 2 on Bragg-Hou gels. Similar differences were noted among the various Shigella species. Although S. schmitzii and S. dysenteriae gave similar patterns on Bragg- Hou gels (Fig. 7), on Maizel gels S. schmitzii gave a single, sharp peak and S. dysenteriae gave a broad double band (Fig. 8). S. typhimurium gave a single, broad band with a trailing shoulder in the Maizel gel system. To facilitate these comparisons, all gels were prepared at the same time and run in the same bath to eliminate slight variations. A summary of the Bragg-Hou gel patterns of all of these strains, plus some additional strains which are described later in this report, is given in Table 2. It was of interest to determine whether there was any relationship between the presence of protein 2 and the production of enterotoxins occasionally associated with E. coli strains. On E.coliOll1a4 Shigella schmitzii Salmonella typhimurium E.coli ML08 Shigella dysenterias FIG. 8. Scans of Maizel gels of some of the outer membrane protein samples shown in Fig. 6 and 7. Note the difference between the two Shigelta species that appear to be identical on Bragg-Hou gels (Fig. 7).

6 VOL. 118, 1974 VARIATIONS IN OUTER MEMBRANE PROTEINS 459, TABLE 2. Summary of major outer membrane proteins observed in various strains and species by Bragg-Hou gel electrophoresis: Strain or species Protein 1 Protein 2 Protein Various E. coli strains: 0111AI OlliBI (Heath) J B4 (ATCC) _ _ _ + B (Hill) + _ + w + _ + ML 08 + _ + K-12 (KL 96) + _ + K-12 (AB 1859) + _ + K-12 (AB 1621) _ + H10407 _ + + S. typhimurium Shigella (all species in + _ + Table 1) a All strains and species are shown as they would appear when grown to late exponential phase on minimal medium with Casamino Acids as the carbon source. the basis of the data collected to date, there does not appear to be a correlation between the production of heat-labile enterotoxin and the production of protein 2. E. coli 4, a known enterotoxin producer (4), exhibited an outer membrane profile similar to E. coli K-12. The three E. coli Olf1 strains described in Table 1 were assayed for enterotoxin production, and none was detected. These assays were conducted-by R. Guerrant, Department of Internal Medicine, University of Virginia Medical School, using a new assay which will be described elsewhere. Effect of filament formation on outer membrane proteins. Henning et al. (5) have presented evidence recently that suggests that the outer membrane proteins play a role in determining or maintaining the characteristic shape of E. coli cells. If this is true, one might predict that the major outer membrane proteins would be different or present in different proportions at the hemispherical ends of the cell as opposed to the cylindrical mid-section of the cell. To test this, I compared the major proteins synthesized by cells growing as drug-induced filaments (in which no new ends are formed) to the proteins synthesized by cells which were dividing normally. This experiment was done in the following way. An early exponential-phase culture was divided into two equal parts. One part received no addition (control culture) and the other part received either 2 gg of 5-diazouracil per ml (11) or 1,ug of mitomycin C per ml. These levels of drugs are sufficient to cause virtually complete conversion of the rod-shaped cells to filaments within one generation time with only a slight inhibition of growth (as measured by the increase in turbidity at 550 nm) over two genera-, tion times. The control and drug-treated cultures were allowed to grow for one-half generation time (to allow completion of septation in the drug-treated culture), and then [14C ]leucine was added to the control culture and ['H]leu-, cine was added to the drug-treated culture. The cultures were allowed to grow for one generation time after addition of the isotope, protein synthesis was stopped by the addition of chloramphenicol (0.5 mg/ml), and the cultures were mixed and harvested. The cells were then fractionated, and the outer membrane protein was examined with the Bragg-Hou gel system. Several combinations of strains, drugs, and culture media were tested in this fashion. The effect "of both mitomycin C and 5-diazouracil was examined with strain J-5 grown either on minimal medium.with succinate as the carbon source (generation time, 90 min) or on minimal medium with glucose as the carbon source, JI(7) J 2 SUCCINATE L-SROTH + I mm camp GLYCEROL 5 mm camp 2- jl FIG. 9. Effect of catabolite repression on E. coli J-5. the scans are of Bragg-Hou gels of cultures grown on minimal medium with succinate, glycerol, or glucose plus various levels of camp. L broth is a complex, tryptone-based medium containing glucose. For simplicity, only the major peaks are.shown in the lower scans, although minor differences were observed in other regions of the gels.

7 460 SCHNAITMAN J. BACTERIOL. supplemented with the 20 essential amino acids and the B vitamins (generation time, 0 min). On both media, there was no difference between the relative amounts of proteins 1, 2, and in the control or in either of the drug-treated cultures. In all of the drug-treated cultures, virtually all of the cells had formed filaments by the time the cultures were harvested. This experiment was repeated with strain K-12 grown on minimal medium with glucose as the carbon source (generation time, 60 min). In this case, only mitomycin C was used. There was no difference in the relative amounts of proteins 1 and between the control and drug-treated cultures. In addition, protein C was isolated and cleaved with cyanogen bromide as in the experiment described previously and shown in Fig. 5. The comparison of the control and drug-treated cultures was identical to that shown in Fig. 5 (data not shown), indicating that filament formation induced by mitomycin C did not alter the ratio of proteins a and b. Although these experiments do not rule out a role for these proteins in the determination of the cell shape, they do suggest that none of the major proteins (1, 2, a or b) is found only on the ends of the cells or is formed only during cell division. Effect of catabolite repression on the outer membrane protein profile of E. coli 01l1. The outer membrane protein profile of E. coli 0111 strains is very strongly influenced by the conditions of growth. This was discovered quite by accident in the experiment described above, in which strain J-5 was grown both in minimal medium with succinate as the carbon source and in an enriched minimal medium with glucose as the carbon source. The differences that are observed as a consequence of culture medium are shown in Fig. 9. All of the cultures shown in this figure were grown to mid-exponential phase. When cultures were grown on minimal medium with succinate or glycerol as the carbon source, there is considerably more protein 2 than protein 1 in the outer membrane. When cultures are grown to minimal medium with glucose as the carbon source, or on L broth (a rich tryptone-based medium containing glucose), the situation is reversed and there is much more protein 1 than protein 2. To demonstrate that this was due to catabolite repression and not to differences in growth rate, cyclic '-5'-adenosine monophosphate (camp) was added to cultures growing on minimal medium with glucose as the carbon source. A low level of camp (1 mm) partially reversed the effect of glucose, and 5 mm camp resulted in an outer membrane profile essentially identical to that obtained with cultures grown on non-fermentable carbon sources (Fig. 9). Neither concentration of camp affected the growth rate on glucose minimal medium. Hence, it can be concluaed that the reduction in protein 2 is due to catabolite repression. More dramatic effects of catabolite repression have been observed with certain enterotoxinproducing strains of E. coli that. have outer membrane proteins similar to E. coli One such strain is E. coli H10407, reported by Skerman et al. (20) to be a serotype 078 and to produce plasmid-associated enterotoxin. This strain exhibited a very strong rabbit ileal loop reaction (20) and a very strong, positive reaction for heat-labile enterotoxin in the tissue culture assay which we have employed. When outer membrane protein from strain H10407 grown on minimal medium with Casamino Acids as the carbon source was examined in the Bragg-Hou gel system (Fig. 10), no protein 1 was detected, and the amount of protein 2 was increased relative to that seen A 2 ' J-5 CAA I 2A CAA NH4 NH4 GLUTAMATE 12^ GLUTAMATE FIG. 10. Comparison of the effect of various levels of catabolite repression on E. coli H and E. coli J-5. Cultures were grown on minimal medium with Casamino Acids as the carbon source (minimal catabolite repression), glucose as the carbon source and NH4+ as the nitrogen source (intermediate catabolite repression), and glucose as the carbon source with 500 Ag of L-glutamate per ml as the nitrogen source (maximal catabolite repression). The scans show only the major peak region of the gel. Gels are the same as in Fig. 9.

8 VOL. 118, 1974 VARIATIONS IN OUTER MEMBRANE PROTEINS 461 with E. coli 0111 (for comparison, the outer membrane protein profile of strain J-5 grown under the same conditions is shown in the lower part of Fig. 10). However, strain H10407 does have the capability to produce protein 1 when catabolite repression is increased. When this strain is grown with glucose as the carbon source and NH,+ as the nitrogen source, the amount of protein 1 is comparable to that which strain J-5 makes when grown on Casamino Acids. Strain H10407 will produce a large amount of protein 1 when grown under conditions of severe catabolite repression (glucose as the carbon source plus 500 ug of L-glutamate per ml as the nitrogen source). However, under these conditions, it grows very poorly in comparison with strain J-5, having a generation time of about 5 h as opposed to 75 min for strain J-5. The reason for this phenomenon is not known, but one possibility is that it reflects an abnormally high level of intracellular camp in this strain. This phenomenon is not unique to this strain, since we have now observed this in some enterotoxinproducing strains of porcine origin. Strain H10407 has no obvious cell wall defect, which illustrates the point that protein 2 can completely replace protein 1 in this strain without affecting the integrity of the cell. Effect of growth phase on the outer membrane protein profile. The stage of growth at which cultures are harvested also affects the outer membrane protein profile that is observed in the Bragg-Hou gel system. This phenomenon has been observed with all of the enteric strains and species, and is particularly pronounced in those strains which lack protein 2. An example is the experiment shown in Fig. 11. This was done with E. coli B grown on minimal medium with glycerol as the carbon source. The culture was sampled in the late exponential phase of growth and again after the stationary phase was begun. The total amount of the major proteins (sum of proteins 1 and ) remained constant, but the exponential-phase cells had much more protein 1 than, whereas the stationary-phase cells had more protein than 1. This phenomenon is even more pronounced in cultures growing on complex media such as Trypticase soy broth, where the termination of growth is less abrupt and growth may continue for several generations after the end of the exponential phase. We do not know whether this represents true turnover of these proteins (i.e., catabolism of protein 1 and replacement by protein ) or whether it is simply due to the fact that the synthesis of protein 1 is terminated much earlier in the growth cycle than the synthesis of protein / I HOURS FIG. 11. Effect of the growth phase on the outer membrane protein profile. The graph shows the turbidity at 550 nm of a culture of E. coli B growing on minimal medium with glycerol as the carbon source. The culture samples were taken at the times shown by the arrows, and the scans show the Bragg-Hou gel profiles of outer membrane protein from these samples. Note the relative change in amounts ofproteins 1 and.. We do know that the "signal" for this change is probably not cell septation, the termination of rounds of deoxyribonucleic acid replication, or the actual rate of deoxyribonucleic acid replication, since these changes were not observed in cultures treated with mitomycin C or 5-diazouracil. Outer membrane protein changes associated with multiple phage resistance. During the course of several experiments with strains constructed from E. coli AB 1621, it was observed that the outer membrane from these strains lacked A protein when chromatographed on SDS-Sephadex G-200 and protein 1 when examined on Bragg-Hou gels. Strain AB 1621 was derived from strain AB 1859 by selection for resistance to phages T4 and T6 (2) and contains two mutations designated by Taylor and Trotter (22) as tfr (pleiotropic resistance to phages T, T4, T7, and X) and tsx (resistance to phage

9 462 SCHNAITMAN J. BACTERIOL. T6). This strain has normal sensitivity to phages P1 and T5. Strain AB 1621 and its parent AB1859 were compared (Fig. 12) and, to rule out differences in extractability of outer membrane proteins by Triton X-100, the outer membrane was isolated both by Triton extraction (Fig. 12B and D) and centrifugation of the crude envelope fraction on a continuous sucrose gradient (Fig. 12A and C). Both methods of preparation yielded identical results-a complete absence of protein 1 in strain AB This pair of cultures was grown to late exponential phase on Trypticase soy broth, and the growth rates of the two cultures were identical. Similar outer membrane 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 upper pair of scans shows protein from strain AB 1859 (A, B) and the lower pair of scans shows protein from strain AB 1621 (C, D). The outer membrane protein shown in scans A and C was isolated from the crude envelope by sucrose gradient centrifugation (2), and the protein shown in scans B and D was isolated by Triton X-100 extraction (1). Both preparations gave identical results. The cultures were grown to late exponential phase on Trypticase soy broth. The scans are of Bragg-Hou gels. protein profiles have been obtained with AB 1621 grown on minimal medium. The density of the outer membrane fraction was identical for both cultures, as determined by the position of the bands in the sucrose gradients, and the phospholipid/protein ratio was the same in these fractions from the gradients, indicating that there was no net decrease in outer membrane protein. The amount of protein in strain AB 1621 is similar to the sum of proteins 1 and in strain AB The C protein from strain AB 1621 was isolated and cleaved with cyanogen bromide, and the peptide profile was identical to that shown in Fig. 5, indicating that the ratio of proteins a and b was normal. Revertants of the tsx mutation accumulate during storage of strain AB 1621 on slants, but other than this there is no obvious defect in the outer membrane of this strain. Cultures grow normally, with no evident lysis during growth. Since the strain is lacy-, it was possible to test for detergent-induced leakiness by growing the culture on plates of lactose-macconkey medium and on lactose-eosine methylene blue agar containing 0.5% sodium deoxycholate. The culture grew and exhibited normal lac- colonies on both media. To determine whether both tfr and tsx mutations were required for this phenotype, I examined the outer membrane protein profile of a T6-sensitive revertant from strain AB This revertant contained a normal amount of protein 1. Seven independent, T6-resistant clones derived from this revertant were examined, and all were missing protein 1. However, five independent T6-resistant clones isolated from the parent strain AB 1859 were examined and all contained normal or near-normal amounts of protein 1. Thus, both mutations appear to be required for the phenotype. These data indicate that protein can substitute for protein 1 without any drastic effects noted in laboratory culture. Although a strain missing protein 1 might not survive in nature, the loss of protein 1 is clearly not lethal. DISCUSSION To simplify discussion of the rather complex changes observed in the outer membrane proteins of enteric bacteria, I should like to begin with an overview of the properties of the major outer membrane proteins. I shall use the term "major protein complex" to denote the entire group of major proteins (and perhaps some minor proteins as well), with molecular masses of about 40,000 daltons, which occur in the

10 VOL. 118, 1974 VARIATIONS IN OUTER MEMBRANE PROTEINS outer membrane of E. coli and other related species. The general properties of the major protein complex may be summarized as follows. (i) These proteins represent integral proteins of the bacterial outer membrane and are difficult to solubilize without dissolution of the membrane. They remain insoluble at neutral ph in aqueous solution except in the presence of detergents or chaotropic agents. (ii) These proteins as a group may represent as much as 70% of the total outer mem-brane protein of E. coli, and comparative studies on other species (1) indicate that they are widely distributed and may be a universal component of the outer membranes of gram-negative bacteria. (iii) All of these proteins exhibit an anomalous behavior on SDS-polyacrylamide gels or upon gel filtration in the presence of SDS (14), depending upon the conditions that have been used to solubilize the proteins in SDS solution. (iv) When these proteins are exposed to SDS under maximally denaturing conditions (i.e., exposure to urea, boiling in SDS solution, or organic solvent treatment), they appear more or less homogeneous on neutral ph SDS-polyacrylamide gels of the Maizel (9) type, but they exhibit anomalous behavior on SDS gels with an alkaline upper buffer system, as described by Bragg and Hou (), or on SDS gels run with a stacking or electrofocusing buffer system (1, 8). Although this appears to be related to charge density rather than size (14, 15, 24), it has led to uncertainty about the true molecular mass of these proteins. It is evident that these proteins can be separated quite clearly into two classes by examining their filtration properties on SDSpolyacrylamide gels, or on Sephadex columns when the proteins have been dissolved in SDS under mild conditions (disaggregation in the absence of urea at a temperature of 50 C or less). I have designated these classes as A and C proteins, these letters referring to the protein bands which are observed on gels (14). When the A proteins are dissolved in SDS solution under mild conditions. they exhibit apparent molecular masses of 60,000 to 100,000 daltons (depending upon the strain or species), and these appear to be aggregates or polymeric forms (14). When the C proteins are dissolved in SDS solution under mild conditions, they give a rather sharp peak and an apparent molecular mass of about 0,000 daltons; this appears to consist of monomeric protein which is not fully unfolded and reacted with SDS (14). As a group, the A proteins are heterogeneous with respect to properties and distribution in 46 various gram-negative species. In E. coli 0111 two major A proteins have been described (15, 16), and these vary in amount depending on cultural conditions. In E. coli K-12 and most other E. coli strains we have examined, there appears to be only a single major A protein, protein 1. There may also be minor species of A proteins with specific functions for the cell and which may function as phage or colicin receptors. These cannot be detected by our rather coarse methods. The A proteins do not play any obvious role in maintaining the structural integrity of the outer membrane under normal laboratory growth conditions, although they may be important to these organisms in their normal ecological niches. These proteins are greatly reduced in amount when cultures enter the stationary phase of growth (Fig. 11) and are missing in an otherwise normal multiply phage-resistant strain (Fig. 12). In contrast, the C proteins appear homogeneous as a group. In all of the strains and species I have examined, these proteins exhibit a similar migration on gels when they are dissolved in SDS under mild conditions. When dissolved under harsh conditions, they give an identical, single sharp band on Bragg-Hou gels (protein ). In E. coli 0111 and E. coli K-12 there appear to be two distinct species of C protein, proteins a and b, and under the limited sets of conditions where we have determined these proteins they are always present in the same ratio. Analysis of the relative amounts of these proteins is quite difficult and tedious, since it involves determination of the cyanogen bromide peptide profile of the isolated C protein. In contrast to the A proteins, the C proteins may be essential for normal outer membrane function. In exponential-phase cultures, protein is reduced to about one-third of the total protein in the major protein complex, but it is never less than that minimal value. Although our sampling has been limited, no strain has been found to be missing protein entirely. The recent studies of Ames et al. (1) and Koplow and Goldfine (8) also pertain to this point. These authors have studied heptose-deficient mutants of Salmonella and E. coli. These mutants have defective cell walls, as indicated by a sensitivity to bile salts and by an alteration in the lipid/ protein ratio of the outer membrane, and the entire major protein complex (including protein ) appears to be missing or greatly reduced. The normal physiological change in the outer membrane proteins which occurs when macromolecular synthesis is being shut down at the end of exponential-phase growth is interesting

11 464 SCHNAITMAN J. BACTERIOL. in terms of cellular regulation (Fig. 11), and explains some of the observations made on temperature-sensitive dnaa and dnab mutants by Shapiro et al. (19) and Siccardi et al. (18). These authors examined the changes in envelope proteins that occurred when these mutants were shifted to nonpermissive temperatures. When they dissolved the envelope samples in SDS solution under mild conditions, they observed a decrease in a 60,000-dalton component (A protein, Fig. ) and an increase in a 0,000- dalton component (C protein, Fig. ). Thus, these mutations must affect early steps in the regulation of macromolecular synthesis in such a way that they mimic the normal shutdown that occurs at the end of exponential-phase growth. It was noted that some of the differences in the envelope from the dnaa mutant and all of the differences in the envelope from the dnab mutant (18) disappeared when the samples were boiled in SDS solution prior to electrophoresis. It is more difficult to reconcile our data with the X and Y proteins described in a similar mutant by Inouye et al. (6, 7), since these authors dissolved their envelope preparations in SDS solution at a temperature (70 C) that leads to only partial dissociation and unfolding of the A and C proteins (14). These studies point out the necessity for understanding the way in which the various proteins of the major protein complex behave under different conditions of solubilization and electrophoresis, and also the way normal physiological conditions affect the relative amounts of these proteins. ACKNOWLEDGMENTS I acknowledge the skilled assistance of Robert McIver, Anne Summers, and Montserrat Salsas, and the generous help in obtaining E. coli strains provided by Richard Guerrant and John Cronan. 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