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Electron microscopy of bacteriophage resistant mutants of Salmonella typhimurium deficient in major outer membrane proteins
317
FEMSMi~obiolosy Letters 2 (1977) 317-322 © Copylisht Federationof EuropeanMicrobiologicalSocieties Publishedby Elsevier/North-HollandBiomedica[Press
E L E C T R O N MICROSCOPY O F B A C T E R I O P H A G E R E S I S T A N T M U T A N T S O F T Y P H I M U R I U M D E F I C I E N T IN M A J O R O U T E R MEMBRANE P R O T E I N S
SALMONELLA
K. LOUNATMAA and MARJATTA NURMINEN Depcartment o f Electron MLcroscopy, University of H,ffslnki, Malminkatu 20, SF-O0100 HelMnki l O, and Central Public Health laboratory. Helsinkl, Finland
Received4 October 1977
~. Introduction Severa~ Salmonella typhimurium mutants d~ficient in "major" outer membrane (OM) proteins (3,~K and 36K) have been isolated as resistant to bacteri,~phages that use these proteins as their receptors [1,2]. The inotpholosy of the OM was found altered in these mutants compared with their parents [ 1]. Thinswas ,~'en in election micrographs both in thin sections and in fre, ze.fractured cellS. We describe here the ultrastructure of the OM of a different class of mutants deficient in one (36K) or ~wo (34K, 36K) major band proteins but containing a relatively larse amwJnt of the third major band pro~ein (35K), Both these and the previously deu;ribed mutants e~o derivatives of& typhlmurium line LT2, "#/hose major OM proteins have been characterized as 33K, 34K, 35K and 36K [3]. The immediate parent .~f all them mutants is 5115014, a rough (rfa) strain whlcJt hat a low amount of the 35K protein []!,4].
2. Methods
The mutants were Isolated as resistant to phages PHI05 and PH51 [2] and characterized in the same way as the previous mutants [I] (Nurminen, to be 'published). Their OM proteins were characterized by SDS.p-=|¥acwlsmide ~e! electrophoresis of envelope preparations ([ 1], Method It). '.['he thin sectioning, freeze./'ractudng and dectron-microscopic operation were performed as described earlier [l,S].
A
B
C
D
E
Fig. I. SDS-goIelectrophore~is(10%gel) of ly~ozyme-EDTA envelopesof the paxen~SH5014(A); the PHIOS-~'esistant mutant SH6015 (B);and three PHI05 an4 PH5l-resistant double mutants SH6377 (C). SH637&(D). and SH6379(E).
318 3. Results and Discussiou A Pl]!05 resist~nt mutant SH6015 was isolated fi'om S. O,,'~himuril,m Stl5014. It was, as expected, deficient in the 36K protein (Fig. I), but differed from the previousl:/described 36K deficient mutants artd also from the parent SH5014 in having more o f the 35K protein. S milar compensatory chang~.~ in the amounts o f OM proteins have been described in Escherichia coil [6].
From $1i6015 we furH,,er isolated PI-I5 l.resistant ntt, tants SIt6377, S1-'¢3378 and SH6379, which had lost (most of) their 34K protein, The relative amount o f the 35K protein in ti~ese double mutants was even higher than in their immediate parent SH6015. The LI'S o f all these mutants was un;dteted as judged from their sensitivity patten,, to LPS.specific phages [7] and fro~-uat) analysis o f the l.l['S monosaeeharide components [8] in SH5014 and the double mutant SH6378 (ur, published data). The sensitiv-
Figs. 2-4. Ultr~strueture of the OM of the PHlOS,rcsl~tant mutant S|t6015 deftclgnt in ~ major prote~ (36K). l:ar ropresents in all micl ographs 0.2 pm and the arrow in the freeze-fract are microgl'aphs indicates the dit¢ctjor) of platinum shadowinlg, i:ig, 2, The strut;tare of the cell wall in thin section, Th0 waved OM and the straight cytopla~nie membrane ~¢ s0on. Fig. 3. The outer concave (O"]~)fracture f~ce with clo~ly packed fl to l0 I]ln p~tttcl0s, ;:ig. 4. The ot,ter convex ( ( ~ ) fracture face with few pits and particles. Note a groove in thi~ face,
3t9
Figs, 5~6. Ultrast~uctute el' the OM of the PHI05 and Ptt51 resistant mutant S!16377 deficient in two major proteins (34K, 36K), Fig. 5. Yhe structure of the cell wa~!in thin section. Fig, 6, Largo areas of the O~ and (:,M with typical particles. The O~Mis slightly undulated. No inner t'tactur¢ faces are ~¢en,
CC, cell content.
ity of all the other strains to a number of detergents and antibiotics [9] was unaltered, whereas one of the_ double mutants, SH6378, showed increased ~nsitivity to benzalconiumchloride (to be published). The morphology of the cell wall of SH6015 was similar to that of the parent strain SH5014 [1 ] both in thin sections (Fig. 2) and in freeze-fractured cells (Figs. 3 and 4), and therefore SH5014 is not shown. In this sense it differed to some extent from the previous 36K-negative, low 35K mutants which showed some, although not extensive, morphological changes in their OM [1]. The three double mutants show different degrees of changes in their OM (Figs. 5 - 1 4 ) . In thin sections one can sea vesicles or even loosening of a large part of the Ol~~. in freeze-fractured cells the fracturing takes place almost exclusively throu~l OM (-- the ot:ter fracture plane) in contrast to cells with nomlal OM proteins. This apparently increased fra~lity of the- OM has been previously described in double mutants of both Salmonella and E. coli[ 1 ,I0]. However, in the concave fracture face ( ~ l ) there are no particleless patches and no decrease in the size of particles, which were changes typical to the previously described double mutants, also low in the 35K protein [1]. In this sense our findings correspond to studies of E. coil mutants (deficient in one major protein) among which the density oF OM particles was reduced in some but not all [6,10]. SH6377 shows only small changes, in thin sections (Fig. 5) the OM is similar to that of the parent straha. The O~M looks slightly undulated (Fig. 6). The cell surface of SH6379 (Figs. 7 - l O ) is covered by numerous small vesicles, which seem to peel off as little clusters in the division area of the cell [Fig. 8). where they are also more numerous. These vesicles are small and homogeneous in size ( 1 0 - 2 0 nm) and differ thus from the large vesicles previously described in a deep rough mutant [11,12] or in other OM mutants I l l . The fracture faces of OM (Figs. 0 and 10) appear unchanged. SH6378 shows the largest changes. In fact the OM looks structurally quite peculiar (Figs. 1 I - 1 4 ) : it is peeling off as vesicles (Figs. 11 and 13) and in many cells appears to (ome loose from the underlying structures over a large area of the cell (Figs. 12 and 14). We have fo~.nd this morphology regul~ly in separate batches of the bacteria and both in thin-see-
320
Fig. 7-10. UlUastructure of ('he OM of PHI05 and I'1t51 resistant stzaJn SH6379 deficient in two major proteins (34K, 36K). Fig. 7. The cell surface is covered by rows of numerous small vesicles (10-20 nm in size). Fig. 8. The vesicles are more numerous and clustered in the division area of the cell. l:ig. 9. Numerous strlall vesicles outside the O"~1. ]:ig. ]0. The O~Mwith typical particles.
tioned cells (which had been fixed with glutaraldehyde) azzd in freeze-fractured cells not treated w~th a chemical fixative. It could therefore not be a fixation artefact. The O"~1of these cells is also in many cases altered so that it looks flat (Figs. 13 and 14) and often broken (Fig. 14). Even the convex fracture face (O, Jl) shows changes: it has fewer particles but
more numerous pits than seen in other strain~ (Fig, 13). These alterations seem to us unexpectedly large in view of the apparently normal growth and only slightly increased sensitivity to a cationic detergent. This mutant may be quite informative for further studies ot the rule of the OM proteins in nsembrane structure,
321
Figs, 11 --14, Ultrastructure of the OM of the Pt1105 and PH51 resistant strain SH6378 deficient in two major proteins t34K. 36K). Fig, I 1, Nuntetous vesicles peeling off from tile OM. Fig. i 2. The outer leallet loose from the OM. Fig, i 3, Numerous vesicles peeling off from the OM corresponding to Fig. 1 I. The O"~1has n~t its t.~pical concave appearance and O"M has many pits and few p~rticles. Fig, 14. The outer leaflet loose from the OM corresponding to Fig. 12. Tile O"~,1 is also brok~n; no inner fracture fa,.es are seen_
322
^ ~ t s We th.nnk Mrs. Tulle Koro for skilled ~echnicid assistance, Dr. P. He.term MakelJ for clis~sslons and a cdtkad reading of fl, e manuscript and Emil Aaltonen Foundation for economical support (to K.L).
Refmences [ 1] Nurminen, M., Lounamum, K., Sarvas,M., MIkel& F.H. and Nakae, T. (1976) J. BacterJoL 127,941-955. [2] Slitonen, A., Johanmon, V., Nurminen, M. and MIkelt', P.H. (1977) FEMS Microbiol. Left. 1,141-144. [3J Ames,G.F., Spudich, E.N. and Niluddo, H. (1974) J. Bacteriol. 117,406-416.
[4] N~clddo,H., Sun Ah Sing,~liudtlel,L. lind Nurmiiwa, M. (1977) Blochem.Biophys. Res.Commun.76, 324-330. [.5] Loumtmu, K. and Ntnninp, N. (1976) J. Baoteriol. 128.6C~5-667. [6] Schwelzar,M., Schw,,'z,H, Sonntq, i. ~ H m ~ , U. (1976) Biochkn. Blophys.Acta 448, 474-491. [7] Wilklmon, R.G.,Gmmkl, P. and ~odc~r, B.A.D. (19'/2) J. GM. ~ 70, .524-554. [81 Nunulmm, M., H~iet,qvist, C,G., V,,ltomm, Y.Y. and Mlkdli, P.H. (19"/1) Eur. J. Binchem. 22° 500-505. [9] Niluddo, H. (1976)Blocldm. Blophy8. Acta 433,118132. [101 V e f l ~ , A., van Alphen, L., BJ~51t,J. and Lujteaborl, B. (1977) Bioddm. Blophy8.Acta 466, 269-282. I 11J irwin, P,.T.,C'hatte~jeo,A.K, Sanderson,K £. and Costerton,LW. (197.5)J. hctetioL 124,930-941. [ 12] Smith,J., Kumio, Y. and Nlkaido, H. (1975) I. BtctetioL 124,942-9.58.
FEMSMi~obiolosy Letters 2 (1977) 317-322 © Copylisht Federationof EuropeanMicrobiologicalSocieties Publishedby Elsevier/North-HollandBiomedica[Press
E L E C T R O N MICROSCOPY O F B A C T E R I O P H A G E R E S I S T A N T M U T A N T S O F T Y P H I M U R I U M D E F I C I E N T IN M A J O R O U T E R MEMBRANE P R O T E I N S
SALMONELLA
K. LOUNATMAA and MARJATTA NURMINEN Depcartment o f Electron MLcroscopy, University of H,ffslnki, Malminkatu 20, SF-O0100 HelMnki l O, and Central Public Health laboratory. Helsinkl, Finland
Received4 October 1977
~. Introduction Severa~ Salmonella typhimurium mutants d~ficient in "major" outer membrane (OM) proteins (3,~K and 36K) have been isolated as resistant to bacteri,~phages that use these proteins as their receptors [1,2]. The inotpholosy of the OM was found altered in these mutants compared with their parents [ 1]. Thinswas ,~'en in election micrographs both in thin sections and in fre, ze.fractured cellS. We describe here the ultrastructure of the OM of a different class of mutants deficient in one (36K) or ~wo (34K, 36K) major band proteins but containing a relatively larse amwJnt of the third major band pro~ein (35K), Both these and the previously deu;ribed mutants e~o derivatives of& typhlmurium line LT2, "#/hose major OM proteins have been characterized as 33K, 34K, 35K and 36K [3]. The immediate parent .~f all them mutants is 5115014, a rough (rfa) strain whlcJt hat a low amount of the 35K protein []!,4].
2. Methods
The mutants were Isolated as resistant to phages PHI05 and PH51 [2] and characterized in the same way as the previous mutants [I] (Nurminen, to be 'published). Their OM proteins were characterized by SDS.p-=|¥acwlsmide ~e! electrophoresis of envelope preparations ([ 1], Method It). '.['he thin sectioning, freeze./'ractudng and dectron-microscopic operation were performed as described earlier [l,S].
A
B
C
D
E
Fig. I. SDS-goIelectrophore~is(10%gel) of ly~ozyme-EDTA envelopesof the paxen~SH5014(A); the PHIOS-~'esistant mutant SH6015 (B);and three PHI05 an4 PH5l-resistant double mutants SH6377 (C). SH637&(D). and SH6379(E).
318 3. Results and Discussiou A Pl]!05 resist~nt mutant SH6015 was isolated fi'om S. O,,'~himuril,m Stl5014. It was, as expected, deficient in the 36K protein (Fig. I), but differed from the previousl:/described 36K deficient mutants artd also from the parent SH5014 in having more o f the 35K protein. S milar compensatory chang~.~ in the amounts o f OM proteins have been described in Escherichia coil [6].
From $1i6015 we furH,,er isolated PI-I5 l.resistant ntt, tants SIt6377, S1-'¢3378 and SH6379, which had lost (most of) their 34K protein, The relative amount o f the 35K protein in ti~ese double mutants was even higher than in their immediate parent SH6015. The LI'S o f all these mutants was un;dteted as judged from their sensitivity patten,, to LPS.specific phages [7] and fro~-uat) analysis o f the l.l['S monosaeeharide components [8] in SH5014 and the double mutant SH6378 (ur, published data). The sensitiv-
Figs. 2-4. Ultr~strueture of the OM of the PHlOS,rcsl~tant mutant S|t6015 deftclgnt in ~ major prote~ (36K). l:ar ropresents in all micl ographs 0.2 pm and the arrow in the freeze-fract are microgl'aphs indicates the dit¢ctjor) of platinum shadowinlg, i:ig, 2, The strut;tare of the cell wall in thin section, Th0 waved OM and the straight cytopla~nie membrane ~¢ s0on. Fig. 3. The outer concave (O"]~)fracture f~ce with clo~ly packed fl to l0 I]ln p~tttcl0s, ;:ig. 4. The ot,ter convex ( ( ~ ) fracture face with few pits and particles. Note a groove in thi~ face,
3t9
Figs, 5~6. Ultrast~uctute el' the OM of the PHI05 and Ptt51 resistant mutant S!16377 deficient in two major proteins (34K, 36K), Fig. 5. Yhe structure of the cell wa~!in thin section. Fig, 6, Largo areas of the O~ and (:,M with typical particles. The O~Mis slightly undulated. No inner t'tactur¢ faces are ~¢en,
CC, cell content.
ity of all the other strains to a number of detergents and antibiotics [9] was unaltered, whereas one of the_ double mutants, SH6378, showed increased ~nsitivity to benzalconiumchloride (to be published). The morphology of the cell wall of SH6015 was similar to that of the parent strain SH5014 [1 ] both in thin sections (Fig. 2) and in freeze-fractured cells (Figs. 3 and 4), and therefore SH5014 is not shown. In this sense it differed to some extent from the previous 36K-negative, low 35K mutants which showed some, although not extensive, morphological changes in their OM [1]. The three double mutants show different degrees of changes in their OM (Figs. 5 - 1 4 ) . In thin sections one can sea vesicles or even loosening of a large part of the Ol~~. in freeze-fractured cells the fracturing takes place almost exclusively throu~l OM (-- the ot:ter fracture plane) in contrast to cells with nomlal OM proteins. This apparently increased fra~lity of the- OM has been previously described in double mutants of both Salmonella and E. coli[ 1 ,I0]. However, in the concave fracture face ( ~ l ) there are no particleless patches and no decrease in the size of particles, which were changes typical to the previously described double mutants, also low in the 35K protein [1]. In this sense our findings correspond to studies of E. coil mutants (deficient in one major protein) among which the density oF OM particles was reduced in some but not all [6,10]. SH6377 shows only small changes, in thin sections (Fig. 5) the OM is similar to that of the parent straha. The O~M looks slightly undulated (Fig. 6). The cell surface of SH6379 (Figs. 7 - l O ) is covered by numerous small vesicles, which seem to peel off as little clusters in the division area of the cell [Fig. 8). where they are also more numerous. These vesicles are small and homogeneous in size ( 1 0 - 2 0 nm) and differ thus from the large vesicles previously described in a deep rough mutant [11,12] or in other OM mutants I l l . The fracture faces of OM (Figs. 0 and 10) appear unchanged. SH6378 shows the largest changes. In fact the OM looks structurally quite peculiar (Figs. 1 I - 1 4 ) : it is peeling off as vesicles (Figs. 11 and 13) and in many cells appears to (ome loose from the underlying structures over a large area of the cell (Figs. 12 and 14). We have fo~.nd this morphology regul~ly in separate batches of the bacteria and both in thin-see-
320
Fig. 7-10. UlUastructure of ('he OM of PHI05 and I'1t51 resistant stzaJn SH6379 deficient in two major proteins (34K, 36K). Fig. 7. The cell surface is covered by rows of numerous small vesicles (10-20 nm in size). Fig. 8. The vesicles are more numerous and clustered in the division area of the cell. l:ig. 9. Numerous strlall vesicles outside the O"~1. ]:ig. ]0. The O~Mwith typical particles.
tioned cells (which had been fixed with glutaraldehyde) azzd in freeze-fractured cells not treated w~th a chemical fixative. It could therefore not be a fixation artefact. The O"~1of these cells is also in many cases altered so that it looks flat (Figs. 13 and 14) and often broken (Fig. 14). Even the convex fracture face (O, Jl) shows changes: it has fewer particles but
more numerous pits than seen in other strain~ (Fig, 13). These alterations seem to us unexpectedly large in view of the apparently normal growth and only slightly increased sensitivity to a cationic detergent. This mutant may be quite informative for further studies ot the rule of the OM proteins in nsembrane structure,
321
Figs, 11 --14, Ultrastructure of the OM of the Pt1105 and PH51 resistant strain SH6378 deficient in two major proteins t34K. 36K). Fig, I 1, Nuntetous vesicles peeling off from tile OM. Fig. i 2. The outer leallet loose from the OM. Fig, i 3, Numerous vesicles peeling off from the OM corresponding to Fig. 1 I. The O"~1has n~t its t.~pical concave appearance and O"M has many pits and few p~rticles. Fig, 14. The outer leaflet loose from the OM corresponding to Fig. 12. Tile O"~,1 is also brok~n; no inner fracture fa,.es are seen_
322
^ ~ t s We th.nnk Mrs. Tulle Koro for skilled ~echnicid assistance, Dr. P. He.term MakelJ for clis~sslons and a cdtkad reading of fl, e manuscript and Emil Aaltonen Foundation for economical support (to K.L).
Refmences [ 1] Nurminen, M., Lounamum, K., Sarvas,M., MIkel& F.H. and Nakae, T. (1976) J. BacterJoL 127,941-955. [2] Slitonen, A., Johanmon, V., Nurminen, M. and MIkelt', P.H. (1977) FEMS Microbiol. Left. 1,141-144. [3J Ames,G.F., Spudich, E.N. and Niluddo, H. (1974) J. Bacteriol. 117,406-416.
[4] N~clddo,H., Sun Ah Sing,~liudtlel,L. lind Nurmiiwa, M. (1977) Blochem.Biophys. Res.Commun.76, 324-330. [.5] Loumtmu, K. and Ntnninp, N. (1976) J. Baoteriol. 128.6C~5-667. [6] Schwelzar,M., Schw,,'z,H, Sonntq, i. ~ H m ~ , U. (1976) Biochkn. Blophys.Acta 448, 474-491. [7] Wilklmon, R.G.,Gmmkl, P. and ~odc~r, B.A.D. (19'/2) J. GM. ~ 70, .524-554. [81 Nunulmm, M., H~iet,qvist, C,G., V,,ltomm, Y.Y. and Mlkdli, P.H. (19"/1) Eur. J. Binchem. 22° 500-505. [9] Niluddo, H. (1976)Blocldm. Blophy8. Acta 433,118132. [101 V e f l ~ , A., van Alphen, L., BJ~51t,J. and Lujteaborl, B. (1977) Bioddm. Blophy8.Acta 466, 269-282. I 11J irwin, P,.T.,C'hatte~jeo,A.K, Sanderson,K £. and Costerton,LW. (197.5)J. hctetioL 124,930-941. [ 12] Smith,J., Kumio, Y. and Nlkaido, H. (1975) I. BtctetioL 124,942-9.58.