Jul 31, 1975 - don (11) at room temperature, using a Unicam. SP1800 spectrophotometer ... for 30s by the method of Devoe et al. (5) prior to replica formation.
Vol. 124, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Nov. 1975, p. 930-941 Copyright © 1975 American Society for Microbiology
Comparison of the Cell Envelope Structure of a Lipopolysaccharide-Defective (Heptose-Deficient) Strain and a Smooth Strain of Salmonella typhimurium R. T. IRVIN,* A. K. CHATTERJEE, K. E. SANDERSON, AND J. W. COSTERTON
Department of Biology, University of Calgary, Calgary, Alberta, Canada Received for publication 31 July 1975
The cell envelope structure of Salmonella typhimurium LT2, which has a heptose-deficient lipopolysaccharide (LPS), is significantly different from that of an isogenic strain with a normal LPS. The rough strain, when examined by freeze-etching, lacks most surface structures that are routinely present in the smooth strain (surface particles and flagella) and has few transmembrane studs in the cytoplasmic membrane (those present are generally found in aggregates), and the outer membrane cleavage is substantially stronger than that of the smooth strain. These envelope differences were independent of both growth temperature and culture age. Examination of ultrathin sections indicated that the rough strain has an outer membrane which forms a much more defined double-track artifact than the smooth strain. The addition of MgCl2 to the growth medium of the rough strain decreased the extent of outer membrane cleavage, and flagella became evident in freeze-etched preparations. The presence of supplemental MgCl2 in the growth medium, which resulted in these morphological changes in the rough strain, also produced growth at a previously restrictive temperature and a decrease in the leakage of periplasmic enzymes. The smooth strain was unaltered morphologically or physiologically by MgCl2 under identical conditions. It is suggested that the outer membrane of the rough strain is more planar. Mutants of gram-negative bacteria with ge- compare the ultrastructure of the cell envelope netically determined lesions in the lipopolysac- of a smooth strain of Salmonella typhimurium charide (LPS) molecule of the outer membrane, with a mutant having heptose-deficient LPS called "rough" mutants, have been extensively (chemotype Re). Recently Koplow and Goldfine studied (21, 37). Rough mutants lack the 0- (10) predicted an alteration in the architecture specific sugars, normally present as a side of the outer membrane of heptose-deficient muchain on the LPS molecule, and may also lack tants, as a result of the loss of proteins from the sugars of the core region (14, 15, 21, 37). These outer membrane. Shands et al. (33) noted that rough mutants may differ from smooth strains, extracted and purified LPS from smooth Salmowhich have an entire LPS molecule, in many nella strains formed thicker trilaminar strucaspects. The colony morphology (37), maximal tures than LPS from heptose-deficient mutants. growth temperature (A. K. Chatterjee et al., We have, therefore, examined the ultrastrucmanuscript in preparation), sensitivity to ture of a smooth strain of S. typhimurium and phage (12, 40), sensitivity to bile salts (28), pene- a strain with heptose-deficient LPS made isotrability of and thus susceptibility to antibiotics genic by transduction (28, 29) in freeze-etch (30), penetrability of lysozyme (28), accessibility preparations and in fixed and sectioned mateof antibodies to surface antigens (3), sensitivity rial. The studies have been done under growthto complement effects (8), degree of pathogenic- permissive conditions and under conditions ity (23, 25, 32), protein composition of the outer that restricted the growth of the rough strain. membrane (1, 10), and the release of periMATERIALS AND METHODS plasmic enzymes to the culture medium (13; A. Both strains of S. typhimurium LT2 have been K. Chatterjee and K. E. Sanderson, manuscript described (28, 29). Strain SA1377 rfaC630 in preparation) may be modified by a lesion in previously (P22)+ is a "rough" strain with heptose-deficient LPS the LPS molecule. In general, these effects be- (chemotype Re) produced by the transduction of the come more striking as the lesion penetrates rfaC630 mutation into a smooth strain; SA1355 is an more deeply into the core of the LPS. isogenic smooth line produced in the same transducWe have undertaken the present study to tion cross. 930
VOL. 124, 1975
S. TYPHIMURIUM LPS-DEFECTIVE AND SMOOTH STRAINS
Cells were cultured in nutrient broth (Difco) with Mg2+ ions supplemented as described (Fig. 1, legend). Portions of log-phase cultures grown at 30 C were shifted to 42 C (Fig. 1, legend). At stated intervals 10-ml samples were removed and centrifuged (12,500 x g for 10 min at 4 C). Electron microscope sample preparation then involved the freezing of a portion of the pellet on 3-mm gold disks in freon 22 at -170 C (16), followed by storage at -170 C until freeze-etching was performed. Samples for embedding were fixed with glutaraldehyde and osmium tetroxide as described below. Supernatants of centrifuged samples were assayed for enzyme activities without further treatment, unless otherwise indicated. Cell-associated enzyme activities were determined by suspending the pellet obtained by centrifugation in tris(hydroxymethyl)aminomethane-hydrochloride (10 mM, pH 7.0), and subsequently toluenized samples (1% at 37 C for 30 min) were assayed. Acid phosphatase was assayed as described by Neu (20) at 37 C, using p-nitrophenyl phosphate as the substrate; cyclic phosphodiesterase (3'-nucleotidase) was assayed as described by Neu (20) at 37 C, using bis-(p-nitrophenyl) phosphate as the substrate; phosphoglucose isomerase was assayed as described by Reithel (22) at 37 C, using glucose-6phosphate as the substrate; and glucose-6-phosphate dehydrogenase was assayed as described by Langdon (11) at room temperature, using a Unicam SP1800 spectrophotometer equipped with a Unicam A25 linear recorder. Specimens for embedding were prefixed for 20 min at room temperature by the addition of 1/10 volume of 5% glutaraldehyde (purchased as a 70% solution under argon from Ladd Industries, Burlington, Vt.) in veronal acetate buffer (0.24 M, pH 6.2) (27) to a sample from the culture medium. The sample was then centrifuged (12,500 x g for 10 min at 4 C), and the pellet was resuspended in 5% glutaraldehyde in veronal acetate buffer (0.24 M, pH 6.2) and fixed for 2 h at room temperature. After fixation, the samples were enrobed in 4% agar (18) and washed five times (15 min each) with veronal acetate buffer (0.24 M, pH 6.2) prior to postfixation in 2% osmium tetroxide (purchased as a 4% aqueous solution under argon from Polysciences Inc., Rydal, Pa.) for 2 h at room temperature. After five washes (15 min each) in the veronal acetate buffer (0.24 M, pH 6.2), the specimens were dehydrated with an acetone series (30 min in each of 30, 50, 70, 90, and 100% acetone, by volume). Freshly distilled acetone was diluted to 30, 50, and 70% by addition of veronal acetate buffer (0.24 M, pH 6.2), but the 90% acetone was made by dilution of 100% acetone with distilled water. The specimens were then washed twice (20 min each) in 100% propylene oxide (Polysciences Inc.) and subsequently embedded in Vestopal W (Polysciences Inc.). Thin sections were cut using an LKB ultratome III 8800 or a Reichert OM U2 ultramicrotome. Sections were stained with uranyl acetate (1% aqueous solution, pH 4.8) and Reynolds lead citrate (26). Freeze-etching was accomplished with a Balzers BA 360 M apparatus with a specimen temperature of -100 C; after cleaving the specimen was sublimed
931
for 30 s by the method of Devoe et al. (5) prior to replica formation. RESULTS The growth rate of the smooth strain (SA1355) in nutrient broth at 30 or 42 C and that of the rough strain (SA1377) at 30 C was similar (Fig. 1). The rough strain (SA1377) grew at the 30 C rate of growth for 45 min after the shift to 42 C but subsequently slowed and ceased after 90 min. With the addition of 20 mM MgCl2, the growth rate of the rough strain at 42 C was increased to the 30 C rate, whereas other growth rates were not detectably altered. The smooth strain (SA1355) at 30 and 42 C in nutrient broth released very little cyclic phosphodiesterase (1 to 2% of the total activity) and some 7 to 8% of the total acid phosphatase activity (Fig. 2). At 30 C, the rough strain leaked 13% of the total cyclic phosphodiesterase activity and 15% of the total acid phosphatase activity into the culture supernatant, whereas at 42 C the corresponding figures were 38 and 48%, respectively. MgCl2 (20 mM) was effective in preventing the leakage of both enzymes from the rough strain (SA1377) (Fig. 2). In the presence of MgC12 (20 mM) at 30 and 42 C, respectively, 2 and 8% of the total cyclic phosphodiesterase and 1 to 2% of the acid phosphatase activity at both temperatures was present in the culture supernatant. Leakage of enzymes to the culture supernatant was not the result of cell lysis, as glucose-6-phosphate dehydrogenase, a cytoplasmic enzyme, was not detectable in the culture supernatants, but good activity was present in cell extracts. The cell envelope of the smooth strain (SA1355) cleaved in a pattern often seen in gram-negative bacterial cells (2, 4, 19, 36) in that the outer membrane constituted only a very minor cleavage plane, generally in the form of a small shelf (0, in Fig. 3-7), before the cleavage descended to the major cleavage plane of the cytoplasmic membrane (C, in Fig. 3-7). The globular protein "studs" routinely seen in the cytoplasmic membrane cleavage plane appear to be numerous, randomly distributed, of uniform size (ca. 8 nm), and similar to those seen in many bacterial membranes (6, 34, 35, 38, 39). The small areas exposed by the weak cleavage plane of the outer membrane also contain globular particles of a similar size (P, in Fig. 4) (also present in the micrographs of references 6 and 25), but these particles appear to be much more sparsely distributed. The formation of a eutectic layer (5) during freezing obscured a substantial amount of cell surface detail, which is exposed by sublimation (etching), but flagella (F, in Fig. 7) and a pattern of globular
932
J. BACTERIOL.
IRVIN ET AL.
SA1355 (smooth)
SA 1377 (rfac 630) 150 r 120 E C
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0 90 180 90 180 INCUBATION TIME (min) FOLLOWING SHIFT FIG. 1. Growth (optical density change) in nutrient broth (Difco) was measured by a Klett-Summerson colorimeter at 660 nm, using shaken cultures. Cultures grown overnight at 30 C were reinoculated and grown to logarithmic phase at 30 C in Klett side arm flasks at 30 C; at Klett readings of 50 to 60, portions of the cultures were shifted to 42 C. Symbols: 0, 30 C, no added MgC12; A, 42 C, no added MgC12; *, 30 C, with 20 mM MgCl2; and A, 42 C, with 20 mM MgC12. 0
CYCLIC PHOSPHO-DIESTERASE 50
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SA 1377 SA 3S5 SA 1377 into the culture supernatant from strains of S. typhimurium. The cells, grown
FIG. 2. Release of enzymes indicated in the legend to Fig. 1, were sampled at the 120-min interval in Fig. 1, and enzymes of supernatants and cells were assayed as in Materials and Methods. Glucose-6-phosphate dehydrogenase was not detected in the supernatant of any of these samples.
as
particles (ca. 16 nm in diameter) (S, in Fig. 3 and 5) were visualized where the eutectic was thin or absent. The cleavage pattern of the cell envelope of the smooth strain (SA1355) was independent of cell age (cf. Fig. 3, 90 min-log phase, with Fig. 4, 180 min-early stationary phase), or the presence of additional Mg2+ (20 mM) (cf. Fig. 4, no
Mg2+, with Fig. 6, 20 mM Mg2+ and Fig. 5, no Mg2+, with Fig. 7, 20 mM Mg2+), or growth temperature (cf. Fig. 4, 30 C, with Fig. 5, 42 C and Fig. 6, 30 C, with Fig. 7, 42 C). Thus, the cleavage pattern of the cell envelope of this smooth strain (SA1355) was not affected by age, temperature, or Mg2` within the limits of this experiment.
FIG. 3. Smooth strain (SA1355) 90-min sample grown at30 C in nutrient broth with no MgCl2 supplement. In this and subsequent figures 0 is outer membrane cleavage, C is cytoplasmic membrane cleavage, S is surface particles, P is particles observed in outer membrane cleavage, ¢ is direction of shadow, and the bar represents 100 nm. 933
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FIG. 4. FIG. 5. FIG. 6. FIG. 7. Flagella.
Smooth strain (SA1355) 180-min sample grown at 30 C Smooth strain (SA1355) 180-min sample grown at 42 C Smooth strain (SA1355) 180-min sample grown at 30 C Smooth strain (SA1355) 180-min sample grown at 42 n- A
with no MgCl2 supplement. with no MgCl2 supplement. with a 20 mM MgCl2 supplement. C with a 20 mM MgCl2 supplement. F,
VOL. 124, 1975
S. TYPHIMURIUM LPS-DEFECTIVE AND SMOOTH STRAINS
The cleavage pattern of the rough strain (SA1377) is substantially different from that exhibited by the smooth strain (SA1355). In the rough strain large areas of the outer membrane cleaved (0, in Fig. 8-13) before the cleavage plane descended to the cytoplasmic membrane (C, in Fig. 8-13). The globular protein studs seen in the cleavage plane of the cytoplasmic membrane were concentrated in certain limited areas (L, in Fig. 9, 11, and 13), and the total number of these particles was so reduced that the greater part of the area exposed by the cleavage within the cytoplasmic membrane was smooth. The cleavage within the outer membrane exposed a small number of globular particles (P, in Fig. 8-13), which were similar in size to those seen in the cytoplasmic membrane. The surface globular particles found in the smooth strain (SA1355) are entirely absent in the rough strain (SA1377), and the number of flagella present on the rough strain (SA1377) was very greatly reduced (visible on
