JouRNAL OF BACTEIOLOGY, July 1977, p. 288-292 Copyright 0 1977 American Society for Microbiology
Vol. 131, No. 1 Printed in U.S.A.
Ultrastructural Surface Changes Associated with Dextran Synthesis by Leuconostoc mesenteroides B. E. BROOKER National Institute for Research in Dairying, Shinfield, Reading, England
Received for publication 5 April 1977
When Leuconostoc mesenteroides NCDO 1875 was grown in MRS broth and fixed for electron microscopy in the presence of ruthenium red, the cell wall appeared as a triple-layered structure similar to other, gram-positive bacteria. When such logarithmic-phase cultures were exposed to sucrose, the appearance and growth of a uniform layer of electron-dense material was evident on the surface of the cell wall. After 2 h in the presence of sucrose, the formation of this surface coat (110 to 130 nm thick) was complete. For 85 to 90%o of the cells, continued exposure to sucrose did not produce any further change in their appearance, but the rest of the population began to accumulate insoluble capsular dextran at the surface of their coat material. Within 18 h, these cells had produced a large capsule (maximum diameter, 6 um) composed mainly of an extensive reticulum of fine filaments. Periodate-reactive carbohydrate was localized cytochemically in the capsular dextran and in the surface coat of all cells. It is suggested that the surface coat of sucrose-grown cells represents a cellbound dextran-dextransucrase complex and that the acapsulate cells produce the relatively soluble S dextran reported by previous workers. In a recent ultrastructural study of Leuconostoc mesenteroides NCDO 523, it was found that the formation of extracellular dextran in the presence of sucrose was accompanied by qualitative and quantitative morphological changes in a coat of surface material enveloping the cell wall (2). It was suggested that these changes resulted from the appearance on the cell surface of the dextran-dextransucrase complex reported by earlier workers (5, 9). In addition, it was shown that some cells produce a capsule of insoluble dextran containing at least two morphologically distinct components. L. mesenteroides NCDO 523 produces dextran in which the non-(1--36) glucosidic linkages have been reported to be mainly similar to (1->3) (6). Since the chemical structure and properties of the dextrans produced by L. mesenteroides are strain variable (6, 8), it was considered of interest to examine a strain that produces insoluble dextran whose chemical structure differs from that synthesized by NCDO 523 to see (i) if the surface changes accompanying dextran synthesis, previously reported from NCDO 523, are common to other strains and (ii) if dextrans that differ in their proportions of various glucosidic linkages can be distinguished by electron microscopy. L. mesenteroides NCDO 1875 was chosen for this purpose since, in addition to producing a water-soluble dextran (S), it elabo288
rates a much less soluble dextran (L) recoverable from the gelatinous "insoluble dextran" sediment found at the bottom of the culture vessel. Since microscopic examination of this sediment shows it to consist very largely of capsulated cells (unpublished data), the L dextran almost certainly corresponds to the capsular dextran. Although the L dextran contains a(1-3) as well as a(l- 2) glucosidic linkages, the former represent terminal linkages on nonreducing chain ends and/or occur in unbranched segments of the dextran molecule (1) so that they are unlikely to be of central importance in determining the ultrastructure of the dextran. On the other hand, almost all of the a(1-32) linkages occur at branch points (1) and are therefore more likely to be major determinants of dextran fine structure. The surface changes that occur when this strain is exposed to sucrose, and the appearance of its insoluble dextran, are reported in this paper. MATERIALS AND METHODS Bacteria and growth conditions. L. mesenteroides NCDO 1875 (NRRL B-1299) was grown at 22°C for 18 h in MRS broth (4), harvested in the exponential phase of growth, and compared by light and electron microscopy with cells grown under identical conditions in MRS broth supplemented with 3.6% (wt/vol) sucrose (Analar, British Drug House). The sequence
VOL. 131, 1977
SURFACE CHANGES IN LEUCONOSTOC MESENTEROIDES
of morphological changes that occurred when cells exposed to sucrose was studied using a 9- or 18-h logarithmic-phase culture of L. mesenteroides grown in MRS broth. The cells were sedimented by centrifugation at 1,000 x g for 15 min, the supernatant was discarded, and part of the pellet was fixed for electron microscopy as described below. The remainder of the pelleted organisms was suspended in MRS broth containing 3.6% (wt/vol) sucrose to give an initial optical density of 0.5 (X, 580 nm; path length, 15 mm). Samples were withdrawn and fixed for electron microscopy after 1, 2, and 5 h. In an attempt to obtain separate isolates of capsule-forming and noncapsule-forming bacteria, 30 individual colonies were picked from MRS agar plates, grown for 18 h at 22°C in MRS broth supplemented with 3.6% (wt/vol) sucrose, and examined by phase-contrast light microscopy. Light microscopy. Bacteria were examined by phase-contrast light microscopy. For this purpose, a small drop of culture medium was placed on a microscope slide coated with a thin layer of MRS agar, a cover slip was carefully applied, and the preparation was examined with a Zeiss photomicroscope. This procedure ensured optically flat preparations and provided optimal conditions for the examination of bacteria. Electron microscopy. As in a previous study of L. mesenteroides (2), all cells were fixed in the presence of ruthenium red (EMscope) to achieve maximum contrast of the dextran (7). A preliminary study showed that conventional glutaraldehyde-osmium tetroxide fixation in the absence of ruthenium red failed to produce sufficient contrast in either the surface coat or dextran, described below, for their meaningful examination. The fixation and embedding schedule used was that reported in an earlier study (2). Sections were cut on a Reichert Om U2 ultramicrotome, mounted on naked or carbon-coated copper grids, and stained with lead citrate and uranyl acetate before examination in an Hitachi HUliE electron microscope. Periodate-reactive carbohydrates were localized cytochemically by the periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-Ag) technique described by Thi6ry (10). Control sections were treated similarly, but aldehyde groups were blocked either before or after periodic acid oxidation by immersion in an aqueous 0.1% (wt/vol) solution of sodium borohydride for 0.5 h at room temperature as recommended by Craig (3). Experimental and control sections were examined by electron microscopy without additional staining in lead or uranium salts.
were
RESULTS
Light microscopy. When grown in MRS broth at 22°C for 18 h, L. mesenteroides NCDO 1875 appeared as pairs and short chains of ovoid cells. For most bacteria, there was little change in appearance when grown under the same conditions in the presence of sucrose. However, 10 to 15% of the population produced a prominent, phase-dense capsule 4 to 6 ,um in diameter.
289
Single colonies picked from MRS agar plates and then grown in MRS broth supplemented with sucrose always produced cultures containing both capsulate and acapsulate cells. In all cases, only 10 to 15% of the bacteria produced a capsule. Electron microscopy. The appearance of cells grown in MRS broth is shown in Fig. 1. The cell wall was composed of three distinct layers and closely resembled that of other, gram-positive bacteria. Additional surfacebound material was never observed. The cytoplasm of most cells contained a single, electrondense granule of circular profile (Fig. 1). Exposure of 9- and 18-h logarithmic-phase cultures to sucrose resulted in the appearance and growth of a uniform layer of electron-dense material on the surface of the cell wall. After 1 h, this surface coat was visible on most cells, but there was considerable cell to cell variation in its thickness. When bacteria had been exposed to sucrose for 2 h, it was apparent that surface coat formation was already complete, since in all cells it was a conspicuous layer 110 to 130 nm thick, and there was no further visible increase in its thickness with time (Fig. 2). For 85 to 90% of the cells, there was little or no further change in their appearance, even when exposed to sucrose for 18 h; however, for the remainder, the completion of the surface coat marked the beginning of capsule formation and the first appearance of insoluble dextran. Occasional evidence of extracellular dextran was seen after 1 h of exposure to sucrose on the surface of bacteria that had apparently completed their surface coat in advance of most other cells. The dextran appeared as patches of short filaments radiating from the surface coat. By 2 h, larger amounts of dextran had accumulated on the surface of many more bacteria and, in some cases, had already formed a capsule that was visible by light microscopy. The dextran at this stage consisted of a compact reticulum of filaments but was not usually of uniform thickness over the entire surface of the cell. Further exposure to sucrose resulted in the progressive accumulation of dextran and the gradual increase in thickness of the capsule. As the capsule increased in size, there appeared within its uniform reticular structure small circular areas of dextran with increased electron density (Fig. 3). Within 18 h, the capsules had reached their final size, with a maximum diameter of 6 ,um (Fig. 3). The surface of each capsule was clearly defined, and there was little evidence to suggest the loss of dextran from the surface into the surrounding medium. After they were stained with PA-TSC-Ag,
290
J. BACTERIOL.
BROOKER
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FIG. 1. MRS-grown cell. The cell wall is composed of three distinct layers, but there is no additional surface material. In all figures, the bar represents 0.2 ,um. FIG. 2. Completed surface coat after 2 h of exposure to sucrose. FIG. 3. Completed capsule from a culture grown in sucrose-supplemented MRS broth for 18 h. This capsule contains at least two bacteria (bl and b2). Numerous dense areas occur throughout the reticular matrix of the capsule.
the surface coats of all bacteria contained large amounts of reaction product (Fig. 4). In the case of capsulate bacteria, the fibrillar network and denser areas of the capsule were intensely
stained (Fig. 5). An identical staining pattern was observed when sections were exposed to sodium borohydride before periodic acid oxidation. However, sections of bacteria treated with
VOL. 131, 1977
SURFACE CHANGES IN LEUCONOSTOC MESENTEROIDES
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FIG. 4. Acapsulate cell grown for 18 h in the presence of sucrose. Stained with PA-TSC-Ag. Reaction product is confined to the surface coat. Not stained with lead citrate and uranyl acetate. FIG. 5. Capsulate cell from the same culture as that in Fig. 4. Stained with PA-TSC-Ag. The surface coat of the bacterium and the capsular dextran contains abundant reaction product.
sodium borohydride after oxidation were free of reaction product. DISCUSSION These results show that L. mesenteroides NCDO 1875 produces a prominent coat of surface material when exposed to sucrose and that its completion, in those cells destined to form a capsule, precedes the appearance of insoluble dextran. However, there is no visible precursor of this thick surface coat on MRS-grown cells. This situation is quite different from that observed in NCDO 523 (2) where MRS-grown cells possess a thin surface coat that increases in thickness and undergoes internal differentiation in the presence of sucrose. Although the significance of these strain differences in terms of dextran production is unclear, it may be supposed that in both cases the formation of a thick surface coat corresponds to the appearance of the cell-bound dextran-dextransucrase complex reported from earlier biochemical studies (5, 9). It would therefore be attractive to interpret the presence of periodate-reactive carbohydrate in the surface coat of sucrose-grown NCDO 523 (2) as the dextran moiety of a cellbound enzyme-polymer complex. However, since MRS-grown cells of the same strain do not
produce dextransucrase, but nevertheless possess a thin surface coat that is also periodate reactive, the additional possibility that the surface carbohydrate of sucrose-grown cells is derived directly from that of MRS-grown bacteria precludes any firm conclusion. No such confusion exists in the case of NCDO 1875 where the surface coat arises de novo in the presence of sucrose. It is suggested, therefore, that the periodate-reactive carbohydrate of the surface coat demonstrated in this study represents the cellbound dextran referred to above. It has been observed here that although all cells undergo morphological surface changes in the presence of sucrose, not all of them produce a capsule of insoluble dextran. Since L. mesenteroides NCDO 1875 is known to produce dextrans that differ in their solubility (6, 9), this observation may be explained by postulating that the acapsulate cells produce the relatively soluble S dextran. The biosynthetic relationship between the soluble and insoluble dextrans produced by this strain has been investigated by Smith (9), who found that more than 60% of the insoluble polymer consisted of a complex between cell-bound dextransucrase and a water-soluble dextran in various stages of synthesis. When synthesis of a dextran chain
292
J. BACTERIOL.
BROOKER
was complete, this complex broke down and liberated the water-soluble dextran into the medium. Smith (9) suggested that a large proportion of the gelatinous (i.e., capsular) "insoluble dextran" produced by this strain represented the cell-bound enzyme-soluble polymer complex. However, subsequent examination of the purified dextrans showed that about 10% of the native dextran produced by structurebound enzyme was inherently water insoluble (1). In view of this work and the results of the present study, it now seems more likely that the gelatinous or capsular dextran corresponds to the inherently insoluble polymer reported by Bourne et al. (1) and that the surface coats of the acapsulate cells represent the complex between dextransucrase and the potentially soluble dextran. The coexistence of two variants in cultures of NCDO 1875, one apparently producing soluble dextran and the other producing relatively insoluble dextran, is unexpected in view of previous reports (11), which pointed to population uniformity with regard to dextran synthesis. These reports suggested that the well-established structural heterogeneity of the dextrans produced by some strains of L. mesenteroides (including NCDO 1875) is not due to variation within the population since no essential difference can be detected in the chemical properties of dextrans isolated from a number of single colonies picked from agar plates. This conclusion was based on the erroneous assumption that populations of bacteria derived from individual colony-forming units would be uniform with respect to dextran formation. Thus, in the present study, colonies picked from MRS agar plates and subsequently grown in the presence of sucrose consistently produced capsulate and acapsulate bacteria. The internal structure of the capsule produced by NCDO 1875 differs markedly from that of NCDO 523 (2). In NCDO 1875, unlike NCDO 523, the structure of the capsule is uniform throughout and is never differentiated
into distinct layers, and there is little evidence to suggest the dispersion of peripheral dextran
into the surrounding medium. The dense areas scattered throughout the capsule of NCDO 1875 resemble the more prolific globular dextran of NCDO 523 and may represent coiled chains of predominantly (1->6)-linked dextran. The fine reticulum of filaments, which forms the bulk of
the capsule in NCDO 1875, is in contrast to the mass of much thicker and less branched fibrils of NCDO 523. Since the L dextran of NCDO 1875 has been shown by methylation analysis (1) to contain a relatively large percentage (32%) of (1-*2) linkages, all of which occur at branch points, the reticular structure of the dextran observed by electron microscopy is, perhaps, not unexpected. Although such detailed information of the chemistry of NCDO 523 dextran is not available, it is likely that the clear ultrastructural differences between the capsular dextrans of NCDO 1875 and NCDO 523 reflect corresponding differences in their chemical structure. ACKNOWLEDGMENT Thanks are due to E. I. Garvie for providing the strain used in this study and for much constructive discussion.
LITERATURE CITED 1. Bourne, E. J., R. L. Sidebotham, and H. Weigel. 1972. Studies on dextrans and dextranases. Part X. Types and percentages of secondary linkages in the dextrans elaborated by Leuconostoc mesenteroides NRRL B-1299. Carbohydr. Res. 22:13-22. 2. Brooker, B. E. 1976. Surface coat transformation and capsule formation by Leuconostoc mesenteroides NCDO 523 in the presence of sucrose. Arch. Microbiol. 111:99-104. 3. Craig, A. S. 1974. Sodium borohydride as an aldehyde blocking reagent for electron microscope histochemistry. Histochemistry 42:141-144. 4. deMan, J. C., M. Rogosa, and M. E. Sharpe. 1960. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23:130-135. 5. Ebert, K. H., and G. Schenk. 1968. Mechanisms of biopolymer growth: the formation of dextran and levan. Adv. Enzymol. Relat. Areas Mol. Biol. 30:179221. 6. Jeanes, A., W. C. Haynes, C. A. Wilham, J. C. Rankin, E. H. Melvin, M. J. Austin, J. E. Cluskey, B. E. Fisher, H. M. Tsuchiya, and C. E. Rist. 1954. Characterization and classification of dextrans from ninety-six strains of bacteria. J. Am. Chem. Soc. 76:5041-5052. 7. Luft, J. H. 1971. Ruthenium red and violet. II. Fine structural localization in animal tissues. Anat. Rec. 171:369-415. 8. Sidebotham, R. L. 1974. Dextrans. Adv. Carbohydr. Chem. Biochem. 30:371-444. 9. Smith, E. E. 1970. Biosynthetic relation between the soluble and insoluble dextrans produced by Leuconostoc mesenteroides NRRL B-1299. FEBS Lett. 12:3337. 10. Thiery, J. P. 1967. Mise en evidence des polysaccharides sur coupes fines en microscopie electronique. J. Microsc. (Paris) 6:987-1018. 11. Wilham, C. A., B. H. Alexander, and A. Jeanes. 1955. Heterogeneity in dextran preparations. Arch. Biochem. Biophys. 59:61-75.
Vol. 131, No. 1 Printed in U.S.A.
Ultrastructural Surface Changes Associated with Dextran Synthesis by Leuconostoc mesenteroides B. E. BROOKER National Institute for Research in Dairying, Shinfield, Reading, England
Received for publication 5 April 1977
When Leuconostoc mesenteroides NCDO 1875 was grown in MRS broth and fixed for electron microscopy in the presence of ruthenium red, the cell wall appeared as a triple-layered structure similar to other, gram-positive bacteria. When such logarithmic-phase cultures were exposed to sucrose, the appearance and growth of a uniform layer of electron-dense material was evident on the surface of the cell wall. After 2 h in the presence of sucrose, the formation of this surface coat (110 to 130 nm thick) was complete. For 85 to 90%o of the cells, continued exposure to sucrose did not produce any further change in their appearance, but the rest of the population began to accumulate insoluble capsular dextran at the surface of their coat material. Within 18 h, these cells had produced a large capsule (maximum diameter, 6 um) composed mainly of an extensive reticulum of fine filaments. Periodate-reactive carbohydrate was localized cytochemically in the capsular dextran and in the surface coat of all cells. It is suggested that the surface coat of sucrose-grown cells represents a cellbound dextran-dextransucrase complex and that the acapsulate cells produce the relatively soluble S dextran reported by previous workers. In a recent ultrastructural study of Leuconostoc mesenteroides NCDO 523, it was found that the formation of extracellular dextran in the presence of sucrose was accompanied by qualitative and quantitative morphological changes in a coat of surface material enveloping the cell wall (2). It was suggested that these changes resulted from the appearance on the cell surface of the dextran-dextransucrase complex reported by earlier workers (5, 9). In addition, it was shown that some cells produce a capsule of insoluble dextran containing at least two morphologically distinct components. L. mesenteroides NCDO 523 produces dextran in which the non-(1--36) glucosidic linkages have been reported to be mainly similar to (1->3) (6). Since the chemical structure and properties of the dextrans produced by L. mesenteroides are strain variable (6, 8), it was considered of interest to examine a strain that produces insoluble dextran whose chemical structure differs from that synthesized by NCDO 523 to see (i) if the surface changes accompanying dextran synthesis, previously reported from NCDO 523, are common to other strains and (ii) if dextrans that differ in their proportions of various glucosidic linkages can be distinguished by electron microscopy. L. mesenteroides NCDO 1875 was chosen for this purpose since, in addition to producing a water-soluble dextran (S), it elabo288
rates a much less soluble dextran (L) recoverable from the gelatinous "insoluble dextran" sediment found at the bottom of the culture vessel. Since microscopic examination of this sediment shows it to consist very largely of capsulated cells (unpublished data), the L dextran almost certainly corresponds to the capsular dextran. Although the L dextran contains a(1-3) as well as a(l- 2) glucosidic linkages, the former represent terminal linkages on nonreducing chain ends and/or occur in unbranched segments of the dextran molecule (1) so that they are unlikely to be of central importance in determining the ultrastructure of the dextran. On the other hand, almost all of the a(1-32) linkages occur at branch points (1) and are therefore more likely to be major determinants of dextran fine structure. The surface changes that occur when this strain is exposed to sucrose, and the appearance of its insoluble dextran, are reported in this paper. MATERIALS AND METHODS Bacteria and growth conditions. L. mesenteroides NCDO 1875 (NRRL B-1299) was grown at 22°C for 18 h in MRS broth (4), harvested in the exponential phase of growth, and compared by light and electron microscopy with cells grown under identical conditions in MRS broth supplemented with 3.6% (wt/vol) sucrose (Analar, British Drug House). The sequence
VOL. 131, 1977
SURFACE CHANGES IN LEUCONOSTOC MESENTEROIDES
of morphological changes that occurred when cells exposed to sucrose was studied using a 9- or 18-h logarithmic-phase culture of L. mesenteroides grown in MRS broth. The cells were sedimented by centrifugation at 1,000 x g for 15 min, the supernatant was discarded, and part of the pellet was fixed for electron microscopy as described below. The remainder of the pelleted organisms was suspended in MRS broth containing 3.6% (wt/vol) sucrose to give an initial optical density of 0.5 (X, 580 nm; path length, 15 mm). Samples were withdrawn and fixed for electron microscopy after 1, 2, and 5 h. In an attempt to obtain separate isolates of capsule-forming and noncapsule-forming bacteria, 30 individual colonies were picked from MRS agar plates, grown for 18 h at 22°C in MRS broth supplemented with 3.6% (wt/vol) sucrose, and examined by phase-contrast light microscopy. Light microscopy. Bacteria were examined by phase-contrast light microscopy. For this purpose, a small drop of culture medium was placed on a microscope slide coated with a thin layer of MRS agar, a cover slip was carefully applied, and the preparation was examined with a Zeiss photomicroscope. This procedure ensured optically flat preparations and provided optimal conditions for the examination of bacteria. Electron microscopy. As in a previous study of L. mesenteroides (2), all cells were fixed in the presence of ruthenium red (EMscope) to achieve maximum contrast of the dextran (7). A preliminary study showed that conventional glutaraldehyde-osmium tetroxide fixation in the absence of ruthenium red failed to produce sufficient contrast in either the surface coat or dextran, described below, for their meaningful examination. The fixation and embedding schedule used was that reported in an earlier study (2). Sections were cut on a Reichert Om U2 ultramicrotome, mounted on naked or carbon-coated copper grids, and stained with lead citrate and uranyl acetate before examination in an Hitachi HUliE electron microscope. Periodate-reactive carbohydrates were localized cytochemically by the periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-Ag) technique described by Thi6ry (10). Control sections were treated similarly, but aldehyde groups were blocked either before or after periodic acid oxidation by immersion in an aqueous 0.1% (wt/vol) solution of sodium borohydride for 0.5 h at room temperature as recommended by Craig (3). Experimental and control sections were examined by electron microscopy without additional staining in lead or uranium salts.
were
RESULTS
Light microscopy. When grown in MRS broth at 22°C for 18 h, L. mesenteroides NCDO 1875 appeared as pairs and short chains of ovoid cells. For most bacteria, there was little change in appearance when grown under the same conditions in the presence of sucrose. However, 10 to 15% of the population produced a prominent, phase-dense capsule 4 to 6 ,um in diameter.
289
Single colonies picked from MRS agar plates and then grown in MRS broth supplemented with sucrose always produced cultures containing both capsulate and acapsulate cells. In all cases, only 10 to 15% of the bacteria produced a capsule. Electron microscopy. The appearance of cells grown in MRS broth is shown in Fig. 1. The cell wall was composed of three distinct layers and closely resembled that of other, gram-positive bacteria. Additional surfacebound material was never observed. The cytoplasm of most cells contained a single, electrondense granule of circular profile (Fig. 1). Exposure of 9- and 18-h logarithmic-phase cultures to sucrose resulted in the appearance and growth of a uniform layer of electron-dense material on the surface of the cell wall. After 1 h, this surface coat was visible on most cells, but there was considerable cell to cell variation in its thickness. When bacteria had been exposed to sucrose for 2 h, it was apparent that surface coat formation was already complete, since in all cells it was a conspicuous layer 110 to 130 nm thick, and there was no further visible increase in its thickness with time (Fig. 2). For 85 to 90% of the cells, there was little or no further change in their appearance, even when exposed to sucrose for 18 h; however, for the remainder, the completion of the surface coat marked the beginning of capsule formation and the first appearance of insoluble dextran. Occasional evidence of extracellular dextran was seen after 1 h of exposure to sucrose on the surface of bacteria that had apparently completed their surface coat in advance of most other cells. The dextran appeared as patches of short filaments radiating from the surface coat. By 2 h, larger amounts of dextran had accumulated on the surface of many more bacteria and, in some cases, had already formed a capsule that was visible by light microscopy. The dextran at this stage consisted of a compact reticulum of filaments but was not usually of uniform thickness over the entire surface of the cell. Further exposure to sucrose resulted in the progressive accumulation of dextran and the gradual increase in thickness of the capsule. As the capsule increased in size, there appeared within its uniform reticular structure small circular areas of dextran with increased electron density (Fig. 3). Within 18 h, the capsules had reached their final size, with a maximum diameter of 6 ,um (Fig. 3). The surface of each capsule was clearly defined, and there was little evidence to suggest the loss of dextran from the surface into the surrounding medium. After they were stained with PA-TSC-Ag,
290
J. BACTERIOL.
BROOKER
1
2
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FIG. 1. MRS-grown cell. The cell wall is composed of three distinct layers, but there is no additional surface material. In all figures, the bar represents 0.2 ,um. FIG. 2. Completed surface coat after 2 h of exposure to sucrose. FIG. 3. Completed capsule from a culture grown in sucrose-supplemented MRS broth for 18 h. This capsule contains at least two bacteria (bl and b2). Numerous dense areas occur throughout the reticular matrix of the capsule.
the surface coats of all bacteria contained large amounts of reaction product (Fig. 4). In the case of capsulate bacteria, the fibrillar network and denser areas of the capsule were intensely
stained (Fig. 5). An identical staining pattern was observed when sections were exposed to sodium borohydride before periodic acid oxidation. However, sections of bacteria treated with
VOL. 131, 1977
SURFACE CHANGES IN LEUCONOSTOC MESENTEROIDES
'Y 'i i..
291
I%$.i,
FIG. 4. Acapsulate cell grown for 18 h in the presence of sucrose. Stained with PA-TSC-Ag. Reaction product is confined to the surface coat. Not stained with lead citrate and uranyl acetate. FIG. 5. Capsulate cell from the same culture as that in Fig. 4. Stained with PA-TSC-Ag. The surface coat of the bacterium and the capsular dextran contains abundant reaction product.
sodium borohydride after oxidation were free of reaction product. DISCUSSION These results show that L. mesenteroides NCDO 1875 produces a prominent coat of surface material when exposed to sucrose and that its completion, in those cells destined to form a capsule, precedes the appearance of insoluble dextran. However, there is no visible precursor of this thick surface coat on MRS-grown cells. This situation is quite different from that observed in NCDO 523 (2) where MRS-grown cells possess a thin surface coat that increases in thickness and undergoes internal differentiation in the presence of sucrose. Although the significance of these strain differences in terms of dextran production is unclear, it may be supposed that in both cases the formation of a thick surface coat corresponds to the appearance of the cell-bound dextran-dextransucrase complex reported from earlier biochemical studies (5, 9). It would therefore be attractive to interpret the presence of periodate-reactive carbohydrate in the surface coat of sucrose-grown NCDO 523 (2) as the dextran moiety of a cellbound enzyme-polymer complex. However, since MRS-grown cells of the same strain do not
produce dextransucrase, but nevertheless possess a thin surface coat that is also periodate reactive, the additional possibility that the surface carbohydrate of sucrose-grown cells is derived directly from that of MRS-grown bacteria precludes any firm conclusion. No such confusion exists in the case of NCDO 1875 where the surface coat arises de novo in the presence of sucrose. It is suggested, therefore, that the periodate-reactive carbohydrate of the surface coat demonstrated in this study represents the cellbound dextran referred to above. It has been observed here that although all cells undergo morphological surface changes in the presence of sucrose, not all of them produce a capsule of insoluble dextran. Since L. mesenteroides NCDO 1875 is known to produce dextrans that differ in their solubility (6, 9), this observation may be explained by postulating that the acapsulate cells produce the relatively soluble S dextran. The biosynthetic relationship between the soluble and insoluble dextrans produced by this strain has been investigated by Smith (9), who found that more than 60% of the insoluble polymer consisted of a complex between cell-bound dextransucrase and a water-soluble dextran in various stages of synthesis. When synthesis of a dextran chain
292
J. BACTERIOL.
BROOKER
was complete, this complex broke down and liberated the water-soluble dextran into the medium. Smith (9) suggested that a large proportion of the gelatinous (i.e., capsular) "insoluble dextran" produced by this strain represented the cell-bound enzyme-soluble polymer complex. However, subsequent examination of the purified dextrans showed that about 10% of the native dextran produced by structurebound enzyme was inherently water insoluble (1). In view of this work and the results of the present study, it now seems more likely that the gelatinous or capsular dextran corresponds to the inherently insoluble polymer reported by Bourne et al. (1) and that the surface coats of the acapsulate cells represent the complex between dextransucrase and the potentially soluble dextran. The coexistence of two variants in cultures of NCDO 1875, one apparently producing soluble dextran and the other producing relatively insoluble dextran, is unexpected in view of previous reports (11), which pointed to population uniformity with regard to dextran synthesis. These reports suggested that the well-established structural heterogeneity of the dextrans produced by some strains of L. mesenteroides (including NCDO 1875) is not due to variation within the population since no essential difference can be detected in the chemical properties of dextrans isolated from a number of single colonies picked from agar plates. This conclusion was based on the erroneous assumption that populations of bacteria derived from individual colony-forming units would be uniform with respect to dextran formation. Thus, in the present study, colonies picked from MRS agar plates and subsequently grown in the presence of sucrose consistently produced capsulate and acapsulate bacteria. The internal structure of the capsule produced by NCDO 1875 differs markedly from that of NCDO 523 (2). In NCDO 1875, unlike NCDO 523, the structure of the capsule is uniform throughout and is never differentiated
into distinct layers, and there is little evidence to suggest the dispersion of peripheral dextran
into the surrounding medium. The dense areas scattered throughout the capsule of NCDO 1875 resemble the more prolific globular dextran of NCDO 523 and may represent coiled chains of predominantly (1->6)-linked dextran. The fine reticulum of filaments, which forms the bulk of
the capsule in NCDO 1875, is in contrast to the mass of much thicker and less branched fibrils of NCDO 523. Since the L dextran of NCDO 1875 has been shown by methylation analysis (1) to contain a relatively large percentage (32%) of (1-*2) linkages, all of which occur at branch points, the reticular structure of the dextran observed by electron microscopy is, perhaps, not unexpected. Although such detailed information of the chemistry of NCDO 523 dextran is not available, it is likely that the clear ultrastructural differences between the capsular dextrans of NCDO 1875 and NCDO 523 reflect corresponding differences in their chemical structure. ACKNOWLEDGMENT Thanks are due to E. I. Garvie for providing the strain used in this study and for much constructive discussion.
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