JOURNAL OF BACTERIOLOGY, Aug. 1979, p. 646-651 0021-9193/79/08-0646/06$02.00/0
Vol. 139, No. 2
Ultrastructure of the Outer Membrane of Salmonella typhimurium Bacteriocin-Resistant Mutants Deficient in the 33K Protein K. LOUNATMAA
Department of Electron Microscopy, University of Helsinki, Malminkatu 20, SF-O0O00 Helsinki 10, Finland
Received for publication 12 June 1979
Outer membrane mutants of Salmonella typhimurium deficient in one, two, or three of the 33,000-dalton (33K), 34K, and 36K outer membrane proteins (7) were studied by using thin sectioning and freeze-fracturing electron microscopy techniques. The outer concave fracture face of all mutants deficient in the 33K protein had numerous particleless patches. In contrast to all previously examined 34K to 36K-deficient mutants, the 33K-deficient mutants showed marked heterogeneity in the size and distribution of such "empty" patches between cells of a culture. One mutant was deficient in both the 33K and the 34K to 36K "porin" protein complex; its outer membrane had very large particleless smooth areas. It is concluded that the 33K protein on one hand and the porin on the other are both able to form intramembraneous particles. fresh broth, and grown under aeration to early logarithmic phase. The pellet collected by centrifugation was used for electron microscopy as described earlier
We have previously studied the ultrastructure of bacteriophage-resistant Salmonella typhimurium mutants that are deficient in the 34,000dalton (34K), 35K, and 36K outer membrane (OM) proteins, the so-called porins (3, 5). Mutants deficient in all of these were found to have particleless patches in the outer concave fracture face of the OM (OM), whose intramembraneous particles (IMP) appeared reduced in number and probably also in size. Mutants deficient in only two of the porin proteins showed no alterations in their IMP. It was concluded that the porins are important constituents of the particles of the OM. Recently, mutants deficient in the fourth major OM protein (33K) were isolated by selecting for resistance to the bacteriocin 4-59 (7), whose receptor the 33K protein appears to be. This made it possible to examine whether this protein also is a constituent of the particles of OM. For this purpose I have studied 33K-deficient derivatives of S. typhimurium strains varying in their complement of the porin (34K to 36K) proteins.
(2, 5).
Two kinds of controls were performed to check for
possible reversion or new mutations in the omp genes. Envelopes were prepared from 20 ml of the culture and analyzed for OM proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (5). A subculture was made on nutrient agar plates from which 50 single colonies were tested for sensitivity to OM protein-specific bacteriophages (5) and bacteriocin 459 (7).
MATERLALS AND METHODS Bacterial strains (Table 1). All S. typhimurium strains were derivatives of the subline SD14, which has normally large amounts of the 33K, 34K, and 36K proteins, but very little of the 35K protein (4). The bacteriocin- and bacteriophage-resistant mutants have been described before (5, 7). SL1995 to 1997 are pyrD+ ompA transductants of SL1941 (7). Samples for electron microscopy. All cultures were started from a freshly isolated single colony, grown overnight in 5 ml of Luria broth, diluted 1:20 in 646
RESULTS Thin sections. The OM of the parent strain S. typhimurium SH5014 (Fig. 1) has a typical wavy appearance (1). In contrast, the OM of its 33K-deficient mutant SL1909 is more irregular (Fig. 2). In many cells of the culture the OM appeared loose from the underlying parts of the cell wall (arrow, Fig. 2). Large OM vesicles were commonly seen in the preparations. The 33Kdeficient strain SH9097 (Fig. 3) shows large numbers of vesicles of varying size but with less irregularity of the OM. The 33K+ parent of this strain is the porin-deficient strain SH6261 (Fig. 4), which has less-pronounced changes (numerous but small vesicles) in its OM. All the above strains had the same kind of mutant lipopolysaccharide (LPS) based on an rfaJ mutation. Another series of strains, with a somewhat more defective LPS because of a galE mutation, is seen in Fig. 5 to 7. These strains are all pyrD+ transductants of the same galE parent and differ in only in respect of the 33K protein.
VOL. 139, 2
STRUCTURE OF OUTER MEMBRANE MUTANTS
Strain SL1995 (Fig. 5) is 33K+, whereas strains SL1996 and SL1997 (Fig. 6-7) are 33K deficient. In thin section the OM of all these strains looks similar to that of the rfaJ parent strain SH5014 in Fig. 1. Freeze-fractured cells. Extensive changes are seen in the OM of all 33K-deficient strains as compared to their parents. Figure 8 shows the typical wild-type appearance of the OM of strain SH5014 with densely packed IMP of homogeneous size. In contrast, the (M of its 33K-deficient mutant SL1909 has numerous particleless patches, although the particles appear as if clustered together and standing out from the background (Fig. 9). The OM of the 33K-, 34K-, (35K-), and 36K- mutant SH9097 is distorted and has very large, smooth particleless patches (Fig. 10). This mutant shows, however, structural heterogeneity so that cells with (M identical to that of SL1909 are also found in the same sample (Fig. 11). The same amount of heterogeneity was seen in several independently prepared samples. In all cells the changes are larger than in the porin-deficient parent SH6261 (Fig. 12). In the galE series of strains, small particleless patches are already seen in the (MM of strain SL1995 (Fig. 13), although it has both the 33K protein and the porins like those of strain SH5014, where no particleless patches appear (Fig. 8). These smooth patches are larger and more conspicuous in the OM of the 33K-deficient strains SL1996 and SL1997 (Fig. 14 and 15). Also in these three strains the GM showed heterogeneity so that the smooth patches were larger in some cells than in others. No alterations could be seen in the TM structure of all these mutants. Neither did these studies give information of the structure of the cytoplasmic membrane because the fracturing took place exclusively through the outer fracture plane (OM); this seems to be a feature common to OM protein-deficient mutants (5, 8). All cultures used for electron microscopy were tested for possible reversion of their omp mutations or other changes in their OM protein content. Gel electrophoresis of these samples always gave the expected pattern (7) with no indication of new peptides in the 20K to 40K size range. A total of 50 colonies grown from each sample and tested for sensitivity to a set of bacteriophages and bacteriocin 4-59 as indicators of OM proteins always behaved alike, indicating a revertant frequency of less than 1:50. DISCUSSION To study whether the loss of the 33K major OM protein has any effect on the structure of the OM, three congenic lines of S. typhimurium
647
were used in which the 33K protein only was
manipulated. One of these lines was an rfaJ line (SH5014), whose LPS deficiency does not affect the ultrastructure of the OM (5), whereas deeper defects in the LPS are known to affect the OM structure and decrease the amount of OM proteins (6). This was probably the case also with the galE line in this study (SL1995) in which there was some loss of IMP as compared with SH5014. The third line (SH6261) was defective in the porins (34K, [35K], and 36K) and its GM showed correspondingly particleless patches, as described earlier (5). A comparison of the 33K-deficient mutants in each case with their immediate parent showed fairly extensive changes in the structure of the OM. This could in some mutants be seen in thin sections as instability and vesicle formation, but could more consistently be seen as changes in the IMP of the GM. All 33K-deficient mutants indicated a loss of IMP resulting in particleless patches. These were largest in the mutant SH9097, which was also porin-deficient, and the least conspicuous in the galE line. Strain SL1909, a 33K-deficient derivative of SH5014, showed extensive changes both in thin section and in the appearance of the GM (Fig. 2 and 9). It is not clear what is the cause of the varying severity of the OM alterations in the 33K-deficient mutants. It could in each case be a characteristic of the mutation causing the 33K deficiency, since the four strains originated from separate mutational events (see description of strains, Table 1). However, the OM protein pattern as examined in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7) was very similar in all these strains, with a fairly complete lack of material in the region of the 33K protein. It could be due to the slightly different structure of LPS-the least alterations were seen in the galE line with more defective LPS. It could also be due to unspecified effects of the genetic background of the bacteria-the galE line differing extensively from the others
(7).
All the 33K-deficient mutants exhibited heterogeneity between individual bacteria of a logarithmic phase culture: an extreme example is shown in Fig. 10 and 11 taken from the same culture. I have not seen this heterogeneity in the many porin-deficient mutants that I have studied. The possibility that the mutants' heterogeneity was due to a high frequency of revertants or other mutants that would have restored a more normal amount of the major OM proteins was considered. The controls performed did not, however, support this hypothesis. No revertants
648
LOUNATMAA
J. BACTERIOL.
2
3
5
FIG. 1 to 7. Thin-section electron micrographs of S. typhimurium strains. Magnification in these and subsequent electron micrographs is X50,000. FIG. 1. Structure of the OM of the parent strain SH5014. FIG. 2. Altered OM of a 33K-deficient mutant SL1909. FIG. 3. Vesiculation of the OM of a 33K-, 34K-, and 36K-deficient mutant SH9097. FIG. 4. OM of a 34K, 36K-deficient mutant SH6261. FIG. 5. OM of the 33K' galE strain SL1995. FIG. 6. OM of the 33K-deficient galE strain SL1996. FIG. 7. OM of the 33K-deficient galE strain SL1997.
VOL. 139, 2
STRUCTURE OF OUTER MEMBRANE MUTANTS
649
FIG. 8 to 15. Freeze-fracturing electron micrographs of S. typhimurium strains. The arrowhead in these micrographs indicates the direction of the platinum shadowing. FIG. 8. .M of the parent strain SH5014 with closely packed intramembraneous particles. FIG. 9. Oi' of a 33K-deficient mutant SL1909 with numerous particleless patches. FIG. 10. OK of a 33K-, 34K-, and 36K-deficient mutant SH9097 with very large smooth, particleless patches. FIG. 11. 0M of SH9097 varies, and the particleless areas can be even as small as in this micrograph. FIG. 12. OIk of SH6261 deficient in the 34K and 36Kporin proteins.
650
LOUNATMAA
J. BACTERIOL.
FIG. 13. 014 of the 33K' galE strain SL1995 with small particleless patches. FIG. 14. 0Mr of the 33K-deficient galE strain SL1996 with numerous large particleless patches. FIG. 15. O& of the 33K-deficient galE strain SL1997 with numerous large particleless patches.
observed among several hundred control colonies tested; furthermore, stock cultures of the strains have remained stable in the laboratory for over a year. Neither did the gel electro-
were
phoresis show the appearance of extra bands in these experiments or at other times (M. Nurminen, personal communication). The definite reduction in the number of the particles in all
STRUCTURE OF OUTER MEMBRANE MUTANTS
VOL. 139, 2
651
TABLE 1. Mutant strains of S. typhimurium LT2 Strain
Protein deficiency
Mutations affecting OM structure
SH5014 SL1909 SH6261
(35K-) 33K-, (35K-) 34K-, 36K-, (35K-)
SH9097
33K-, 34K-, 36K-, (35K-)
SL1995 SL1996
(35K-) 33K-, (35K-)
rfaJ4041 rfaJ4041 ompA201 rfaJ4041 ompC336 ompD115 rfaJ4041 ompA204 ompC336 ompD115 galE706 galE706 ompA202
SL1997
33K-, (35K-)
galE706 ompA203
33K-deficient mutants as compared with their parents indicates that the 33K protein is normally a constituent of the IMP. Since previously porin-deficient mutants have also been shown to have lost part of their IMP, it is apparent that both the 33K protein and the porins are each able to form intramembraneous particles. These findings are consistent with corresponding studies in Escherichia coli, in which Verkleij et al. (8) also found that the loss of protein d (which in E. coli corresponds to the 33K protein in Salmonella) causes loss of IMP, whereas all the porins (proteins b and c in E. coli) have to be lost before a structural change is seen in the OM. They also described the heterogeneity of the OM structure in the protein d-deficient population. ACKNOWLEDGMENTS I thank Sirkku Waarala and Tuire Koro for skilled technical assistance, M. Nurminen for gel electrophoresis, and P. Helena Makela for discussion and critical reading of the manuscript.
LITERATURE CITED 1. Lounatmaa, K. 1977. Effect of acrolein-glutaraldehyde on the outer membrane of Salmonella typhimurium
Derivative of:
Reference
SH5014 SH6017
4 7 4
SH6261
7
SL1941 SL1941 with ompA202 from SL1917 SL1941 with ompA203 from SL1918
7 7 7
smooth and rough strains. FEMS Lett. 2:167-121. 2. Lounatmaa, K., and N. Nanninga, 1976. Effect of polymyxin on the outer membrane of Salmonella typhimurium: freeze-fracture studies. J. Bacteriol. 128: 665-667. 3. Lounatmaa, K., and M. Nurminen. 1977. Electron microscopy of bacteriophage resistant mutants of Salmonella typhimurium deficient in major outer membrane proteins. FEMS Lett. 2:317-322. 4. Nikaido, H., Sun An Sing, L. Shaltiel, and M. Nurminen. 1977. Outer membrane of Salmonella. XIV. Reduced transmembrane diffusion rates in porin-deficient mutants. Biochem. Biophys. Res. Commun. 76: 324-330. 5. Nurminen, M., K. Lounatmaa, M. Sarvas, P. H. Makela, and T. Nakae. 1976. Bacteriophage-resistant mutants of Salmonella typhimurium deficient in two major outer membrane proteins. J. Bacteriol. 127:941955. 6. Smit, J., Y. Kamio, and H. Nikaido. 1975. Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J. Bacteriol. 124:942-958. 7. Stocker, B. A. D., M. Nurminen, and P. H. Makela. 1979. Mutants defective in the 33K outer membrane protein of Salmonella typhimurium. J. Bacteriol. 139: 376-383. 8. Verkleij, A., L. van Alphen, J. Bijvelt, and B. Lugtenberg. 1977. Architecture of the outer membrane of Escherichia coli K-12. II. Freeze-fracture morphology of wild type and mutant strains. Biochim. Biophys. Acta 466:269-282.
Vol. 139, No. 2
Ultrastructure of the Outer Membrane of Salmonella typhimurium Bacteriocin-Resistant Mutants Deficient in the 33K Protein K. LOUNATMAA
Department of Electron Microscopy, University of Helsinki, Malminkatu 20, SF-O0O00 Helsinki 10, Finland
Received for publication 12 June 1979
Outer membrane mutants of Salmonella typhimurium deficient in one, two, or three of the 33,000-dalton (33K), 34K, and 36K outer membrane proteins (7) were studied by using thin sectioning and freeze-fracturing electron microscopy techniques. The outer concave fracture face of all mutants deficient in the 33K protein had numerous particleless patches. In contrast to all previously examined 34K to 36K-deficient mutants, the 33K-deficient mutants showed marked heterogeneity in the size and distribution of such "empty" patches between cells of a culture. One mutant was deficient in both the 33K and the 34K to 36K "porin" protein complex; its outer membrane had very large particleless smooth areas. It is concluded that the 33K protein on one hand and the porin on the other are both able to form intramembraneous particles. fresh broth, and grown under aeration to early logarithmic phase. The pellet collected by centrifugation was used for electron microscopy as described earlier
We have previously studied the ultrastructure of bacteriophage-resistant Salmonella typhimurium mutants that are deficient in the 34,000dalton (34K), 35K, and 36K outer membrane (OM) proteins, the so-called porins (3, 5). Mutants deficient in all of these were found to have particleless patches in the outer concave fracture face of the OM (OM), whose intramembraneous particles (IMP) appeared reduced in number and probably also in size. Mutants deficient in only two of the porin proteins showed no alterations in their IMP. It was concluded that the porins are important constituents of the particles of the OM. Recently, mutants deficient in the fourth major OM protein (33K) were isolated by selecting for resistance to the bacteriocin 4-59 (7), whose receptor the 33K protein appears to be. This made it possible to examine whether this protein also is a constituent of the particles of OM. For this purpose I have studied 33K-deficient derivatives of S. typhimurium strains varying in their complement of the porin (34K to 36K) proteins.
(2, 5).
Two kinds of controls were performed to check for
possible reversion or new mutations in the omp genes. Envelopes were prepared from 20 ml of the culture and analyzed for OM proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (5). A subculture was made on nutrient agar plates from which 50 single colonies were tested for sensitivity to OM protein-specific bacteriophages (5) and bacteriocin 459 (7).
MATERLALS AND METHODS Bacterial strains (Table 1). All S. typhimurium strains were derivatives of the subline SD14, which has normally large amounts of the 33K, 34K, and 36K proteins, but very little of the 35K protein (4). The bacteriocin- and bacteriophage-resistant mutants have been described before (5, 7). SL1995 to 1997 are pyrD+ ompA transductants of SL1941 (7). Samples for electron microscopy. All cultures were started from a freshly isolated single colony, grown overnight in 5 ml of Luria broth, diluted 1:20 in 646
RESULTS Thin sections. The OM of the parent strain S. typhimurium SH5014 (Fig. 1) has a typical wavy appearance (1). In contrast, the OM of its 33K-deficient mutant SL1909 is more irregular (Fig. 2). In many cells of the culture the OM appeared loose from the underlying parts of the cell wall (arrow, Fig. 2). Large OM vesicles were commonly seen in the preparations. The 33Kdeficient strain SH9097 (Fig. 3) shows large numbers of vesicles of varying size but with less irregularity of the OM. The 33K+ parent of this strain is the porin-deficient strain SH6261 (Fig. 4), which has less-pronounced changes (numerous but small vesicles) in its OM. All the above strains had the same kind of mutant lipopolysaccharide (LPS) based on an rfaJ mutation. Another series of strains, with a somewhat more defective LPS because of a galE mutation, is seen in Fig. 5 to 7. These strains are all pyrD+ transductants of the same galE parent and differ in only in respect of the 33K protein.
VOL. 139, 2
STRUCTURE OF OUTER MEMBRANE MUTANTS
Strain SL1995 (Fig. 5) is 33K+, whereas strains SL1996 and SL1997 (Fig. 6-7) are 33K deficient. In thin section the OM of all these strains looks similar to that of the rfaJ parent strain SH5014 in Fig. 1. Freeze-fractured cells. Extensive changes are seen in the OM of all 33K-deficient strains as compared to their parents. Figure 8 shows the typical wild-type appearance of the OM of strain SH5014 with densely packed IMP of homogeneous size. In contrast, the (M of its 33K-deficient mutant SL1909 has numerous particleless patches, although the particles appear as if clustered together and standing out from the background (Fig. 9). The OM of the 33K-, 34K-, (35K-), and 36K- mutant SH9097 is distorted and has very large, smooth particleless patches (Fig. 10). This mutant shows, however, structural heterogeneity so that cells with (M identical to that of SL1909 are also found in the same sample (Fig. 11). The same amount of heterogeneity was seen in several independently prepared samples. In all cells the changes are larger than in the porin-deficient parent SH6261 (Fig. 12). In the galE series of strains, small particleless patches are already seen in the (MM of strain SL1995 (Fig. 13), although it has both the 33K protein and the porins like those of strain SH5014, where no particleless patches appear (Fig. 8). These smooth patches are larger and more conspicuous in the OM of the 33K-deficient strains SL1996 and SL1997 (Fig. 14 and 15). Also in these three strains the GM showed heterogeneity so that the smooth patches were larger in some cells than in others. No alterations could be seen in the TM structure of all these mutants. Neither did these studies give information of the structure of the cytoplasmic membrane because the fracturing took place exclusively through the outer fracture plane (OM); this seems to be a feature common to OM protein-deficient mutants (5, 8). All cultures used for electron microscopy were tested for possible reversion of their omp mutations or other changes in their OM protein content. Gel electrophoresis of these samples always gave the expected pattern (7) with no indication of new peptides in the 20K to 40K size range. A total of 50 colonies grown from each sample and tested for sensitivity to a set of bacteriophages and bacteriocin 4-59 as indicators of OM proteins always behaved alike, indicating a revertant frequency of less than 1:50. DISCUSSION To study whether the loss of the 33K major OM protein has any effect on the structure of the OM, three congenic lines of S. typhimurium
647
were used in which the 33K protein only was
manipulated. One of these lines was an rfaJ line (SH5014), whose LPS deficiency does not affect the ultrastructure of the OM (5), whereas deeper defects in the LPS are known to affect the OM structure and decrease the amount of OM proteins (6). This was probably the case also with the galE line in this study (SL1995) in which there was some loss of IMP as compared with SH5014. The third line (SH6261) was defective in the porins (34K, [35K], and 36K) and its GM showed correspondingly particleless patches, as described earlier (5). A comparison of the 33K-deficient mutants in each case with their immediate parent showed fairly extensive changes in the structure of the OM. This could in some mutants be seen in thin sections as instability and vesicle formation, but could more consistently be seen as changes in the IMP of the GM. All 33K-deficient mutants indicated a loss of IMP resulting in particleless patches. These were largest in the mutant SH9097, which was also porin-deficient, and the least conspicuous in the galE line. Strain SL1909, a 33K-deficient derivative of SH5014, showed extensive changes both in thin section and in the appearance of the GM (Fig. 2 and 9). It is not clear what is the cause of the varying severity of the OM alterations in the 33K-deficient mutants. It could in each case be a characteristic of the mutation causing the 33K deficiency, since the four strains originated from separate mutational events (see description of strains, Table 1). However, the OM protein pattern as examined in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7) was very similar in all these strains, with a fairly complete lack of material in the region of the 33K protein. It could be due to the slightly different structure of LPS-the least alterations were seen in the galE line with more defective LPS. It could also be due to unspecified effects of the genetic background of the bacteria-the galE line differing extensively from the others
(7).
All the 33K-deficient mutants exhibited heterogeneity between individual bacteria of a logarithmic phase culture: an extreme example is shown in Fig. 10 and 11 taken from the same culture. I have not seen this heterogeneity in the many porin-deficient mutants that I have studied. The possibility that the mutants' heterogeneity was due to a high frequency of revertants or other mutants that would have restored a more normal amount of the major OM proteins was considered. The controls performed did not, however, support this hypothesis. No revertants
648
LOUNATMAA
J. BACTERIOL.
2
3
5
FIG. 1 to 7. Thin-section electron micrographs of S. typhimurium strains. Magnification in these and subsequent electron micrographs is X50,000. FIG. 1. Structure of the OM of the parent strain SH5014. FIG. 2. Altered OM of a 33K-deficient mutant SL1909. FIG. 3. Vesiculation of the OM of a 33K-, 34K-, and 36K-deficient mutant SH9097. FIG. 4. OM of a 34K, 36K-deficient mutant SH6261. FIG. 5. OM of the 33K' galE strain SL1995. FIG. 6. OM of the 33K-deficient galE strain SL1996. FIG. 7. OM of the 33K-deficient galE strain SL1997.
VOL. 139, 2
STRUCTURE OF OUTER MEMBRANE MUTANTS
649
FIG. 8 to 15. Freeze-fracturing electron micrographs of S. typhimurium strains. The arrowhead in these micrographs indicates the direction of the platinum shadowing. FIG. 8. .M of the parent strain SH5014 with closely packed intramembraneous particles. FIG. 9. Oi' of a 33K-deficient mutant SL1909 with numerous particleless patches. FIG. 10. OK of a 33K-, 34K-, and 36K-deficient mutant SH9097 with very large smooth, particleless patches. FIG. 11. 0M of SH9097 varies, and the particleless areas can be even as small as in this micrograph. FIG. 12. OIk of SH6261 deficient in the 34K and 36Kporin proteins.
650
LOUNATMAA
J. BACTERIOL.
FIG. 13. 014 of the 33K' galE strain SL1995 with small particleless patches. FIG. 14. 0Mr of the 33K-deficient galE strain SL1996 with numerous large particleless patches. FIG. 15. O& of the 33K-deficient galE strain SL1997 with numerous large particleless patches.
observed among several hundred control colonies tested; furthermore, stock cultures of the strains have remained stable in the laboratory for over a year. Neither did the gel electro-
were
phoresis show the appearance of extra bands in these experiments or at other times (M. Nurminen, personal communication). The definite reduction in the number of the particles in all
STRUCTURE OF OUTER MEMBRANE MUTANTS
VOL. 139, 2
651
TABLE 1. Mutant strains of S. typhimurium LT2 Strain
Protein deficiency
Mutations affecting OM structure
SH5014 SL1909 SH6261
(35K-) 33K-, (35K-) 34K-, 36K-, (35K-)
SH9097
33K-, 34K-, 36K-, (35K-)
SL1995 SL1996
(35K-) 33K-, (35K-)
rfaJ4041 rfaJ4041 ompA201 rfaJ4041 ompC336 ompD115 rfaJ4041 ompA204 ompC336 ompD115 galE706 galE706 ompA202
SL1997
33K-, (35K-)
galE706 ompA203
33K-deficient mutants as compared with their parents indicates that the 33K protein is normally a constituent of the IMP. Since previously porin-deficient mutants have also been shown to have lost part of their IMP, it is apparent that both the 33K protein and the porins are each able to form intramembraneous particles. These findings are consistent with corresponding studies in Escherichia coli, in which Verkleij et al. (8) also found that the loss of protein d (which in E. coli corresponds to the 33K protein in Salmonella) causes loss of IMP, whereas all the porins (proteins b and c in E. coli) have to be lost before a structural change is seen in the OM. They also described the heterogeneity of the OM structure in the protein d-deficient population. ACKNOWLEDGMENTS I thank Sirkku Waarala and Tuire Koro for skilled technical assistance, M. Nurminen for gel electrophoresis, and P. Helena Makela for discussion and critical reading of the manuscript.
LITERATURE CITED 1. Lounatmaa, K. 1977. Effect of acrolein-glutaraldehyde on the outer membrane of Salmonella typhimurium
Derivative of:
Reference
SH5014 SH6017
4 7 4
SH6261
7
SL1941 SL1941 with ompA202 from SL1917 SL1941 with ompA203 from SL1918
7 7 7
smooth and rough strains. FEMS Lett. 2:167-121. 2. Lounatmaa, K., and N. Nanninga, 1976. Effect of polymyxin on the outer membrane of Salmonella typhimurium: freeze-fracture studies. J. Bacteriol. 128: 665-667. 3. Lounatmaa, K., and M. Nurminen. 1977. Electron microscopy of bacteriophage resistant mutants of Salmonella typhimurium deficient in major outer membrane proteins. FEMS Lett. 2:317-322. 4. Nikaido, H., Sun An Sing, L. Shaltiel, and M. Nurminen. 1977. Outer membrane of Salmonella. XIV. Reduced transmembrane diffusion rates in porin-deficient mutants. Biochem. Biophys. Res. Commun. 76: 324-330. 5. Nurminen, M., K. Lounatmaa, M. Sarvas, P. H. Makela, and T. Nakae. 1976. Bacteriophage-resistant mutants of Salmonella typhimurium deficient in two major outer membrane proteins. J. Bacteriol. 127:941955. 6. Smit, J., Y. Kamio, and H. Nikaido. 1975. Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J. Bacteriol. 124:942-958. 7. Stocker, B. A. D., M. Nurminen, and P. H. Makela. 1979. Mutants defective in the 33K outer membrane protein of Salmonella typhimurium. J. Bacteriol. 139: 376-383. 8. Verkleij, A., L. van Alphen, J. Bijvelt, and B. Lugtenberg. 1977. Architecture of the outer membrane of Escherichia coli K-12. II. Freeze-fracture morphology of wild type and mutant strains. Biochim. Biophys. Acta 466:269-282.
