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Structure of the proximal ends of bacterial flagella
J. Mol. Biol. (1965) 14,297-299
Structure of the Proximal Ends of Bacterial Flagella Recent investigations with the electron microscope using negatively stained preparations have revealed that, depending on the species or strain, bacterial flagella show at least two types of surface structure (Lowy & Hanson, 1964, 1965). The A form has globular units arranged in a hexagonal pattern (Plate I(a)); in the B form one sees a number of lines running practically parallel to the long axis of the flagellum (Plate I(b)). A comparative survey indicated that, with one exception, the type of surface structure is constant for a given strain of bacteria. The exception was the case of sheathed flagella in Pseudomonas rhodos, where both A and B structures appeared to be present along the length of the same flagellum. Thus it seemed conceivable that under certain conditions one type of structure turns into the other. The question was raised (Lowy & Hanson, 1965) as to whether or not such a structural transformation might have some relevance to the functioning of the flagellum. Using the same techniques as in the previous study, further work has now produced evidence which does not support this idea; and it leads to a different interpretation of the observations made on the sheathed flagella of Ps. rhodos. In preparations from strain B9 of this organism, Lowy & Hanson (1965) found B form flagella showing four or five lines, and flagella with apparently similar structure around which was a sheath in the form of two helically wound ribbons. Such sheathed B form flagella frequently had at one end a short length which lacked the sheath and showed a surface structure that resembled the A form. Examination of another Ps. rhodos strain (B19, obtained from Professor W. Heumann) has confirmed these observations and revealed a number of additional features. Flagella of both sheathed and sheatWess B form were found on the same organism. Their over-all widths were about 180 A and 130 A, respectively. The sheathless flagella occur in coiled bunches of 10 to 20, and such an assembly shows the sinusoidal shape ofthe individual flagellum with a wavelength of about 0·6 to 0·75 tt (Plate II(a)). The sheathed flagella occur singly and (as in the previous work with strain B9) it was found that four or five lines could often be seen through the sheath, their spacing being much the same as in the sheatWess B form flagella. Most of the sheathed flagella had at one end a sheatWess region showing globular units (Plates I(b) and II(b)). The length of this region (about 2000 A) was fairly constant. In the same preparations, sheatWess B form flagella were found which also had at one end a region showing globular units, but here the length of this region was only about 700 A (Plate II(c)). In both sheathed and sheatWess flagella the region showing globular units often had a somewhat bulbous appearance with a terminal taper (Plate II(b) and (c)). Endings about 600 A long and showing globular units were also found in the B form flagella of Pseudomonas fiuorescens (National Collection of Type Cultures strain 10038) (Plate II(d)). Studies with a variety of techniques on Proteus vulgaris (van Iterson, 1953), Spirillum serpens (Houwink, 1953), Salmonella typhimurium (Kerridge, Horne & Glauert, 1962) and Vibrio metchnikovii (Glauert, Kerridge & Horne, 1963) have shown that flagella from these organisms possess at one end a blunt hook-shaped 297
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J. LOWY
structure of about the same width as the flagellum. Furthermore, several of these investigators produced evidence that, after passing through the bacterial cell wall, the flagella attach themselves by their hook-shaped ends to basal circular discs in the cytoplasm. The observation that hook-shaped endings are inserted into the bacterial body was confirmed in the present work for the A form flagella of Salmonella typhimurium (strain SL 735) and Proteus vulgaris (NCTC strain 10020). Examination of detached flagella with hooks showed that there is a recognizable zone which separates the flagellum from the hook-shaped ending (Plate I(a)). As in the case of the endings in the B form flagella, one sees within the hook globular units arranged in a hexagonal pattern which resembles the A form structure. The question now naturally arises whether or not in the B form flagella of Ps. jluororescens, and in the sheathed and sheathless B form flagella of Ps. rhodos, the part of the flagellum which is inserted in the cell body is the only region showing globular units. But because in these flagella that region is relatively straight, and because in negatively stained preparations structural details cannot be seen where the flagellum lies within the bacterial body, the evidence cannot be as direct as in the case of A form flagella, the hook-shaped endings of which are readily located in the cytoplasm. Preparations were made in which most of the flagella remained attached to the cell body. When detached flagella situated very near the organism were examined, it was found in most instances that their proximal ends showed globular unit structure. This was most convincingly seen in the case of coiled bunches of sheathless B form Ps. rhodos flagella lying very near the bacterial body: the apposed individual flagella all end proximally in a region with globular unit structure. It would seem reasonable to conclude therefore that in B form flagella the region showing globular units is analogous with the hook-shaped ending present in A form flagella. As regards the possible function of these terminal regions, the only information available so far comes from work with Spirillum serpens, where the flagella have hooks (Martinez, 1963). From structural and biochemical evidence, Martinez suggested that there is RNA associated with the hooks. In all types of flagella described here the proximal ends show a structure which resembles the A form in that (as judged by their spacing) the globular units have about the same dimensions and appear to be connected along oblique rows, the axial spacing of which is about 50 A. The significance of this resemblance remains unknown, as does the puzzling finding that the proximal region in the sheathed flagella of Ps. rhodos is about three times longer than in the other flagella. It also has yet to be explained why the proximal region in some B form flagella is relatively straight, bulbous and tapering, whereas in A form flagella the analogous region is hookshaped. If it turns out, as appears conceivable for hook-shaped endings from the work of Martinez (1963), that the proximal region in B form flagella represents a specialized structural modification somehow concerned in the synthesis of flagella, then it seems unlikely that the transformation seen in this region could provide clues to what might happen during the functioning of the flagellum. I am grateful to Professor Sir John Randall, F.R.S. for providing facilities, to Dr Jean Hanson for helpful discussion, and to Professor B. A. D. Stocker, Professor W. Heumann and Dr 1\'1. W. McDonough for generously supplying the bacterial cultures. This work
PLATE 1. Electron micrographs of flagella negativey-lstained with uranyl acetate as described by Lowy & Hanson (1965). The line represents 1000 A. (a) Flagella of Salmonella typhimurium. The arrows point to the zone separating the flagellum from the hook-shaped ending. (b) Pseudomonas rhodos. One sheathed flagellum showing the proximal end with globular unit structure, and part of a coiled bunch of sheath less flagella showing B form structure.
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(0 ) PLATE
II. Electron micrographs of flagella negatively-stained with uranyl acetate as described
hy Lowy & Hanson (1965). (a), (b) and (c) Flagella of Pseudomonas rhodos,
(a) Coiled bunch of sheathless B form flagella. The line represents 2000 A. (b) Sheathed flagellum ending in a region showing globular unit structure. 'I'he line represents 1000 A. (c) Arrow points to the proximal end of a sheathless flagellum. The line represents 1000 A. (d) Proximal end of a flagellum of Pseudomonas fluorescens, The line represents 1000 A.
LETTERS TO THE EDITOR
299
was supported in part by a r esearch grant (AN 06166·01) from the Institute of Arthritis and Metabolic Di seases, U.S. Public H ea lth Service. Medic a l R esearch Council Bioph ysics R esearch Unit Univer sity of Lond on King's College 26·29 Drury Lane London, \V.C.2, E ngla nd
J .Lowy
Receiv ed 24 August 1965 REFERENCES Glauert, A . M., K err idge, D. & H orne, R. W. (1963). J. Cell B ioI. 18, 327 . H ouwink, A. L . (1953) . B iochim. biophys. Acta, 10, 360. Itorson, W ., van (1953 ). Bacterial Cytology , Symp. 6th Congr. Int. M icrobial. p. 24. Oxford: B lackwell Scien t ific Publications. K erridge, D ., Horne, R. W. & Glauert, A. M. (1962).J. Mol. tu«, 4, 227. Lowy , J . & Hanson, J. (1964). Nature , 202, 538. Lowy , J. & Hanson, J. (1965). J. Mol . tu«. 11, 293. Mar t inez, R. J. (196 3). Bioch em. Biophys. R es. Com m, 12,1 80.
Structure of the Proximal Ends of Bacterial Flagella Recent investigations with the electron microscope using negatively stained preparations have revealed that, depending on the species or strain, bacterial flagella show at least two types of surface structure (Lowy & Hanson, 1964, 1965). The A form has globular units arranged in a hexagonal pattern (Plate I(a)); in the B form one sees a number of lines running practically parallel to the long axis of the flagellum (Plate I(b)). A comparative survey indicated that, with one exception, the type of surface structure is constant for a given strain of bacteria. The exception was the case of sheathed flagella in Pseudomonas rhodos, where both A and B structures appeared to be present along the length of the same flagellum. Thus it seemed conceivable that under certain conditions one type of structure turns into the other. The question was raised (Lowy & Hanson, 1965) as to whether or not such a structural transformation might have some relevance to the functioning of the flagellum. Using the same techniques as in the previous study, further work has now produced evidence which does not support this idea; and it leads to a different interpretation of the observations made on the sheathed flagella of Ps. rhodos. In preparations from strain B9 of this organism, Lowy & Hanson (1965) found B form flagella showing four or five lines, and flagella with apparently similar structure around which was a sheath in the form of two helically wound ribbons. Such sheathed B form flagella frequently had at one end a short length which lacked the sheath and showed a surface structure that resembled the A form. Examination of another Ps. rhodos strain (B19, obtained from Professor W. Heumann) has confirmed these observations and revealed a number of additional features. Flagella of both sheathed and sheatWess B form were found on the same organism. Their over-all widths were about 180 A and 130 A, respectively. The sheathless flagella occur in coiled bunches of 10 to 20, and such an assembly shows the sinusoidal shape ofthe individual flagellum with a wavelength of about 0·6 to 0·75 tt (Plate II(a)). The sheathed flagella occur singly and (as in the previous work with strain B9) it was found that four or five lines could often be seen through the sheath, their spacing being much the same as in the sheatWess B form flagella. Most of the sheathed flagella had at one end a sheatWess region showing globular units (Plates I(b) and II(b)). The length of this region (about 2000 A) was fairly constant. In the same preparations, sheatWess B form flagella were found which also had at one end a region showing globular units, but here the length of this region was only about 700 A (Plate II(c)). In both sheathed and sheatWess flagella the region showing globular units often had a somewhat bulbous appearance with a terminal taper (Plate II(b) and (c)). Endings about 600 A long and showing globular units were also found in the B form flagella of Pseudomonas fiuorescens (National Collection of Type Cultures strain 10038) (Plate II(d)). Studies with a variety of techniques on Proteus vulgaris (van Iterson, 1953), Spirillum serpens (Houwink, 1953), Salmonella typhimurium (Kerridge, Horne & Glauert, 1962) and Vibrio metchnikovii (Glauert, Kerridge & Horne, 1963) have shown that flagella from these organisms possess at one end a blunt hook-shaped 297
298
J. LOWY
structure of about the same width as the flagellum. Furthermore, several of these investigators produced evidence that, after passing through the bacterial cell wall, the flagella attach themselves by their hook-shaped ends to basal circular discs in the cytoplasm. The observation that hook-shaped endings are inserted into the bacterial body was confirmed in the present work for the A form flagella of Salmonella typhimurium (strain SL 735) and Proteus vulgaris (NCTC strain 10020). Examination of detached flagella with hooks showed that there is a recognizable zone which separates the flagellum from the hook-shaped ending (Plate I(a)). As in the case of the endings in the B form flagella, one sees within the hook globular units arranged in a hexagonal pattern which resembles the A form structure. The question now naturally arises whether or not in the B form flagella of Ps. jluororescens, and in the sheathed and sheathless B form flagella of Ps. rhodos, the part of the flagellum which is inserted in the cell body is the only region showing globular units. But because in these flagella that region is relatively straight, and because in negatively stained preparations structural details cannot be seen where the flagellum lies within the bacterial body, the evidence cannot be as direct as in the case of A form flagella, the hook-shaped endings of which are readily located in the cytoplasm. Preparations were made in which most of the flagella remained attached to the cell body. When detached flagella situated very near the organism were examined, it was found in most instances that their proximal ends showed globular unit structure. This was most convincingly seen in the case of coiled bunches of sheathless B form Ps. rhodos flagella lying very near the bacterial body: the apposed individual flagella all end proximally in a region with globular unit structure. It would seem reasonable to conclude therefore that in B form flagella the region showing globular units is analogous with the hook-shaped ending present in A form flagella. As regards the possible function of these terminal regions, the only information available so far comes from work with Spirillum serpens, where the flagella have hooks (Martinez, 1963). From structural and biochemical evidence, Martinez suggested that there is RNA associated with the hooks. In all types of flagella described here the proximal ends show a structure which resembles the A form in that (as judged by their spacing) the globular units have about the same dimensions and appear to be connected along oblique rows, the axial spacing of which is about 50 A. The significance of this resemblance remains unknown, as does the puzzling finding that the proximal region in the sheathed flagella of Ps. rhodos is about three times longer than in the other flagella. It also has yet to be explained why the proximal region in some B form flagella is relatively straight, bulbous and tapering, whereas in A form flagella the analogous region is hookshaped. If it turns out, as appears conceivable for hook-shaped endings from the work of Martinez (1963), that the proximal region in B form flagella represents a specialized structural modification somehow concerned in the synthesis of flagella, then it seems unlikely that the transformation seen in this region could provide clues to what might happen during the functioning of the flagellum. I am grateful to Professor Sir John Randall, F.R.S. for providing facilities, to Dr Jean Hanson for helpful discussion, and to Professor B. A. D. Stocker, Professor W. Heumann and Dr 1\'1. W. McDonough for generously supplying the bacterial cultures. This work
PLATE 1. Electron micrographs of flagella negativey-lstained with uranyl acetate as described by Lowy & Hanson (1965). The line represents 1000 A. (a) Flagella of Salmonella typhimurium. The arrows point to the zone separating the flagellum from the hook-shaped ending. (b) Pseudomonas rhodos. One sheathed flagellum showing the proximal end with globular unit structure, and part of a coiled bunch of sheath less flagella showing B form structure.
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zv«
(0 ) PLATE
II. Electron micrographs of flagella negatively-stained with uranyl acetate as described
hy Lowy & Hanson (1965). (a), (b) and (c) Flagella of Pseudomonas rhodos,
(a) Coiled bunch of sheathless B form flagella. The line represents 2000 A. (b) Sheathed flagellum ending in a region showing globular unit structure. 'I'he line represents 1000 A. (c) Arrow points to the proximal end of a sheathless flagellum. The line represents 1000 A. (d) Proximal end of a flagellum of Pseudomonas fluorescens, The line represents 1000 A.
LETTERS TO THE EDITOR
299
was supported in part by a r esearch grant (AN 06166·01) from the Institute of Arthritis and Metabolic Di seases, U.S. Public H ea lth Service. Medic a l R esearch Council Bioph ysics R esearch Unit Univer sity of Lond on King's College 26·29 Drury Lane London, \V.C.2, E ngla nd
J .Lowy
Receiv ed 24 August 1965 REFERENCES Glauert, A . M., K err idge, D. & H orne, R. W. (1963). J. Cell B ioI. 18, 327 . H ouwink, A. L . (1953) . B iochim. biophys. Acta, 10, 360. Itorson, W ., van (1953 ). Bacterial Cytology , Symp. 6th Congr. Int. M icrobial. p. 24. Oxford: B lackwell Scien t ific Publications. K erridge, D ., Horne, R. W. & Glauert, A. M. (1962).J. Mol. tu«, 4, 227. Lowy , J . & Hanson, J. (1964). Nature , 202, 538. Lowy , J. & Hanson, J. (1965). J. Mol . tu«. 11, 293. Mar t inez, R. J. (196 3). Bioch em. Biophys. R es. Com m, 12,1 80.