JOURNAL OF BACTERIOLOGY, OCt. 1976, p. 325-336 Vol. 128, No.

1
Copyright © 1976 American Society for Microbiology Printed in U.S.A.

Outer Membrane of Gram-Negative Bacteria

XII. Molecular-Sieving Function of Cell Wall
GARY M. DECAD AND HIROSHI NIKAIDO*
Department of Bacteriology and Immunology, University of California, Berkeley, California 94720
Received for publication 1 July 1976

The permeability function of the cell wall of gram-negative bacteria such as

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Salmonella was investigated by producing cells with an expanded periplasmic
volume, and incubating them with radioactive non-utilizable oligo- and polysac-
charides or polyethylene glycols. To quantitate the extent of penetration of these
hydrophilic compounds into the periplasm, the radioactivity of the cell pellet
was determined after centrifugation. We found that only di- and trisaccharides
could fully diffuse into the periplasm, whereas higher-molecular-weight saccha-
rides were nonpenetrable. In addition, low-molecular-weight polyethylene glycols
rapidly diffused across the cell wall. Kinetics experiments also showed that both
sucrose and raffinose in the periplasm exchanged rapidly with sugars in the
medium, even at 0°C. These results suggest that the cell wall acts as a molecular
sieve, with an exclusion limit near 550 to 650 daltons for saccharides. We also
suggest that the diffusion of these hydrophilic compounds most likely occurs
through water-filled pores present in the cell wall of gram-negative bacteria.
The cell envelope of gram-negative bacteria nary account of this work has already been
such as Escherichia coli and Salmonella con- published [G. Decad, T. Nakae, and N. Ni-
tains two membranes, the inner cytoplasmic kaido, Fed. Proc. 33:1240, 1974]. Unknown to
membrane and the outer membrane (15). The us, an isotope penetration study had been per-
outer membrane and the underlying peptido- formed on S. typhimurium, which revealed the
glycan layer constitute the cell wall. The space penetration of sucrose and several other sub-
between the cell wall and the cytoplasmic mem- stances through the cell wall [J. Stock and S.
brane is the periplasm, which contains peri- Roseman, Fed. Proc. 32:517, 1973].)
plasmic enzymes (31) and binding proteins (41).
Since all known active-transport systems MATERIALS AND METHODS
were found in the cytoplasmic membrane (23), Bacterial strains. We used S. typhimurium LT2
we assumed that there must be some mecha- and its derivatives HN202 (galE503) (i.e., strain
nism by which essential molecules can pass LT2M1 of reference 17) and TA2168 (galE506 rfa-
through the cell wall to reach the site of active 1009 hisC3076) (1), which produce incomplete lipo-
transport. Several studies have suggested that polysaccharides of the Rc and Re types (33), respec-
the cell wall can also restrict the diffusion of tively. E. coli B and E. coli K-12 (strain G-6 of
certain molecules, such as oligopeptides beyond reference 21), gifts of Gerald Hazelbauer, University
a certain size (36), (-thiogalactosides (40), and of Uppsala, Sweden, were also used. Pseudomonas
certain antibiotics, dyes, and bile salts (19, 20, aeruginosa 45 was from the culture collection of our
28, 30, 39, 4345). department. Proteus morganii ICPB 2873 (ATCC
25830) and Alcaligenes feacalis ICPB 2003 were
Since, however, the precise permeability given to us by Mortimer P. Starr, Department of
properties of the gram-negative cell wall were Bacteriology, University of California, Davis.
not known, we wanted to find out which mole- Growth conditions. All bacterial strains, unless
cules pass through the cell wall and which mol- otherwise noted, were grown in L broth (5) (glucose
ecules are excluded. We did this by examining omitted) at 37°C in 1,500 ml of medium (in a 6-liter
the penetration of radioactively labeled saccha- Erlenmeyer flask) with vigorous aeration, by shak-
rides and polyethylene glycols of various sizes ing at 200 rpm on a New Brunswick gyratory
across the cell wall and into the expanded peri- shaker, model G-52. P. aeruginosa was also grown in
plasmic space of plasmolyzed cells. minimal medium (10) containing 0.4% glycerol, un-
der the same conditions. When the cell density
(This work was taken in part from a thesis reached 1 mg/ml (wet weight), the cells were har-
submitted by G. M. D. to the Univ. of Cali- vested by centrifugation at 6,000 x g for 5 min (this
fornia, Berkeley, in partial fulfillment of the as well as all following procedures were performed
requirements for the Ph.D. degree. A prelimi- at room temperature, unless otherwise noted). The
32g
326 DECAD AND NIKAIDO J. BACTERIOL.
cells were washed once with 0.025 M sodium phos- bascose was isolated from E. lens (lentils) by a modi-
phate buffer (pH 7.0) suspended in 6 to 8 ml of the fication of published procedures (27, 32). A 100-g
same buffer, and used immediately. amount of finely powdered seed was extracted with
Chemicals. The chemicals used were of the best 60% boiling ethanol (6 parts of ethanol by weight per
grade commercially available. [U-'4C]sucrose (420 1 part of powdered seed) for 10 min. After centrifu-
mCi/mmol) was from Schwarz/Mann, Orangeburg, gation at 6,000 x g for 10 min, the supernatant was
N. Y. [4,5-3H]leucine (50 Ci/mmol), [U-_4C]glycerol saved and the pellet was extracted again, as de-
(10 mCi/mmol), and [2-3H]glycerol (6.48 Ci/mmol) scribed above. Pooled supernatants were filtered
were from the New England Nuclear Corp., Boston, through Whatman no. 50 filter paper, concentrated
Mass. Potassium boro[3Hlhydride (200 mCi/mmol) to 40 ml, and passed through both a cation-exchange
was from the Radiochemical Centre, Amersham, column (AG50W X-8, +H form, 200 to 400 mesh, 5 by
England. Methoxy-[3H]dextran (1 mCi/3 mg) and 8.7 cm, and an anion-exchange column (AG1-X-8,
[3H]inulin, both from New England Nuclear Corp., HCO3- form, 200 to 400 mesh, 5 by 3.7 cm) (both from

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were fractionated by gel filtration as described pre- Bio-Rad Laboratories, Richmond, Calif.). The final
viously (30). Galactose oxidase was from Worthing- eluate was concentrated to 25 ml. After centrifuga-
ton Biochemicals Corp., Freehold, N. J. tion at 15,000 x g for 1 h, a portion of the superna-
Preparation of radioactive oligosaccharides. Raf- tant was applied to a column (2.5 by 40 cm) of Bio-
finose (Baker) (20 mg), stachyose (Sigma Chemical Gel P-2 (200 to 400 mesh; Bio-Rad Laboratories),
Co., St. Louis, Mo.) (20 mg), and verbascose, (20 mg, with 0.1 M NaCl as the eluent. The column was
isolated and purified from Ervum lens as described calibrated with blue dextran (Pharmacia Fine
below) were each dissolved in distilled water along Chemicals, Inc.) as the exclusion marker, NaBr as
with 144 Worthington units of galactose oxidase in a the inclusion marker, [14C]sucrose, [3H]raffinose,
final volume of 1.0 ml. They were incubated over- and [3H]stachyose. Oligosaccharides eluting at the
night at 37°C with 0.07 ml of toluene. The pH of each theoretical elution volume for verbascose were col-
sample was adjusted to 10.0 with NaOH. Potassium lected; the theoretical elution volume was calculated
boro[3Hlhydride (2 mg) was then added, and each by plotting logarithms of molecular weights versus
sample was incubated for 6 h at room temperature Kav. The pooled verbascose fractions were concen-
under a well-ventilated hood. Unlabeled potassium trated and rechromatographed on a higher-resolv-
borohydride (20 mg) was then added to each sample, ing column (see Results). A portion of verbascose
and the mixtures were further incubated for 3 h. The from this column was labeled with 3H as described
reaction was terminated by adding 0.01 ml of glacial above, rechromatographed on the same column (see
acetic acid, the mixtures were applied to a water- Fig. 1), and further purified by descending paper
jacketed, calibrated column (1.27 by 93 cm) of Bio- chromatography in three solvent systems (see
Gel P-2 (minus 400 mesh) (Bio-Rad Laboratories, above). The Rf value was as expected for verbascose
Richmond, Calif.), kept at 65°C as described previ- from plots of Rf values of stachyose, raffinose, su-
ously (22), and the column was eluted with distilled crose, and sucrose versus the numbers of hexose
water (see Results and Fig. 1). Fractions containing units (16).
the 3H-labeled oligosaccharides were pooled, and the Possible utilization of oligosaccharides by cells.
material was purified further by descending paper The possible utilization of the sucrose-raffinose se-
chromatography on Whatman no. 3 paper with 1- ries of oligosaccharides was determined as follows.
butanol-pyridine-water (6:4:3, vol/vol/vol). In addi- A crude extract was prepared from 1 g (wet weight)
tion, analytical paper chromatography was carried of HN202 cells, suspended in 0.025 M sodium phos-
out in propanol-ethyl acetate-water (6:1:3, vol/vol/ phate buffer (pH 7.0), by sonic oscillation (Biosonik
vol) and 1-butanol-acetic acid-water (4:1:5, vol/vol/ IV, Bronwill Scientific Inc., Rochester, N.Y.). In-
vol). Each of the 3H-labeled oligosaccharides pro- tact cells were removed by centrifugation at 1,085 x
duced a single band, with an Rf corresponding to g for 10 min. The crude extract containing 6.7 mg of
that of the unlabeled saccharide standard (47). The total protein in 0.2 ml, or intact cells alone (100 mg,
3H-labeled saccharides were then eluted with dis- wet weight), was incubated with 0.12 j,Ci (0.09 to
tilled water and used. 0.40 ,g) of ['4C]sucrose, [3H]raffinose,
Preparation of radioactive polyethylene glycols. [3Hlstachyose, or [3H]verbascose at 37°C for 30 min.
Two micromoles of K2Cr2O7 in 0.01 ml of 5 N H2SO4 After boiling for 5 min and centrifugation at 14,500
was added to 10 ,imol each of polyethylene glycols x g for 10 min, the supernatants were applied to two
(Baker) having average molecular weights of 1,540, strips of Whatman 3MM paper. A sugar standard
1,000, 600, and 400, respectively. After the reac- containing all of the labeled reference oligosaccha-
tion was complete, 0.5 ml of water was added, and rides was applied to separate strips. Paper chroma-
the reaction mixture was neutralized with 0.05 ml of tography was run in 1-butanol-pyridine-water
1 N NaOH. Potassium boro[3Hlhydride was added (2 (6:4:3, vol/vol/vol) either until the solvent front was
to 3 mg). After 2 h at room temperature, 3 mg of near the bottom of the paper strips or for 36 h. Radio-
KBH4 was added and, after an additional hour, the activity was determined by counting segments of
reaction was stopped by adding 0.01 ml of glacial each strip in Bray solution (8) in a liquid scintilla-
acetic acid. Each reaction mixture was then applied tion spectrometer.
to a column (1.27 by 55 cm) of Sephadex G-15 (Phar- Cell wall permeability. The standard procedure
macia Fine Chemicals, Inc., Uppsala, Sweden), for determining wall permeability with intact cells
which was then eluted with 0.1 M NaCl (see Re- was as follows. To a cell suspension (100 or 150 mg,
sults). total wet weight) in 0.025 M sodium phosphate
Extraction and purification of verbascose. Ver- buffer (pH 7.0), ['4C]sucrose (0.15 ,Ci) and 3H-la-
VOL. 128, 1976
beled oligosaccharides (or [3H]inulin or [3H]dextran
[0.40 gCi] or [3H]polyethylene glycol [0.40 gCi])
were added along with a plasmolyzing agent, either
0.3 M NaCl or 0.5 M sucrose (final concentrations).
To minimize possible adsorption of radioactive com-
pounds to cells, the reaction mixtures contained 0.01
M sucrose and 0.02 M raffinose (or 0.002 M polyeth-
ylene glycols of appropriate molecular weight) (final
concentrations). The above-mentioned reaction mix-
ture (final volume, 0.5 ml) in 1.5-ml plastic centri-
fuge tubes (Eppendorf, no. 3810) was mixed immedi-
ately for 5 s with a Vortex-type mixer and incubated
at room temperature for 5 min without further mix-
ing. The tubes were then centrifuged at 1,790 x g as
described previously (30). After the supernatant was
removed, the inside wall of the centrifuge tube was
wiped with tissue and the pellet was suspended to
1.00 g with 0.025 M sodium phosphate buffer (pH
7.0) by mixing vigorously with a Vortex-type mixer.
After 10 min at room temperature, the tubes were
centrifuged again, as described above.
A portion of the first supernatant (100 IlI) plus 400
,ul of water, and 400 ,ul of the second supernatant
plus 100 ,ul of water, were transferred to separate
vials, and the radioactivity was determined by the
double-label counting procedure described below.
The space (in microliters) permeable to a given
compound in the pellet was calculated according to
the following formula:
permeable space
total radioactivity in
the second supernatant
radioactivity/microliter
of first supernatant
radioactivity in 400 1ld
of second supernatant
x 250
radioactivity in 100 ,ul
of first supernatant
The permeable space could be affected by the
amount of residual supernatant; we solved this
problem by using a double-label technique. Thus,
the permeable space for [14C]sucrose is the sum of a
(periplasmic space) and b (interstitial space; see Re-
sults) plus c (volume of residual first supernatant).
The permeable space for any 3H-labeled compound is
pa + b + c, whenp 0, 0 <p < 1, orp 1 if the
= =
compound is nonpenetrable, partially penetrable, or
fully penetrable, respectively. Thus, the 3H-im-
permeable space (i.e., ['4C]sucrose-permeable space
minus the 3H-permeable space) equals (a + b + c)
(pa + b + c) = (1 p)a, and this value is not affected
-
by the residual first supernatant. If a nonpenetrat-
ing 3H-labeled compound such as dextran is used, a
is obtained by this procedure. Thus, p, the degree of
penetration for any 3H-labeled compound, is ex-
pressed as:
a - (1 p)a
-
P= = grown in Vogel-Bonner citrate medium (48) to a
a density of 1 mg/ml (wet weight), harvested at 6,000
3H-impermeable space for that compound
periplasmic space
OUTER MEMBRANE AS A MOLECULAR SIEVE 327
We usually multiply p by 100 to express it as a
percentage.
Sucrose and raffinose efflux. The efflux of labeled
saccharides from plasmolyzed cells was determined
as follows. A 50-mg amount (wet weight) of HN202
cells in 0.025 M sodium phosphate buffer (pH 7.0)
was plasmolyzed with 0.3 M NaCl (final concentra-
tion) in a reaction mixture containing either 0.03 M
raffinose plus [3H]raffinose (1.7 ,uCi) or 0.005 M
sucrose plus [14C]sucrose (0.5 pCi), final concentra-
tions, in a total volume of 0.2 ml. After 5 min of
incubation at 0 or 25°C, 0.02-ml portions of the mix-
ture were added to flasks each containing 50 ml of
buffer (at 0 or 25°C) that had the same composition
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and conductivity as the reaction mixture minus the
labeled compounds. One suspension was filtered on
a membrane filter (DA, 0.65-,um pore size, 47 mm;
Millipore Corp., Bedford Mass.) immediately upon
dilution. Other samples were filtered after being
kept for varying periods subsequent to dilution. The
filtration took 5 s. For the control, 5 mg of cells (wet
weight) and [3H]raffinose (0.17 uCi) or ['4C]sucrose
(0.05 jCi) were added to 50 ml of the above men-
tioned dilution buffer and filtered. Radioactivity
was determined by liquid scintillation counting in
10 ml of PCS solubilizer (Amersham/Searle, Arling-
ton Heights, Ill.) after the filters were dried at 100°C
for 40 min. The counts in the control were routinely
subtracted from experimental counts.
Permeability of spheroplasts. Cells of strain
HN202 (1 g, wet weight) were washed twice with
0.01 M tris(hydroxymethyl)aminomethane (Tris)-
hydrochloride (pH 8) and suspended in 80 ml of 20%
sucrose-0.03 M Tris-hydrochloride (pH 8) at 25°C
(31). To this solution, lysozyme and pancreatic deox-
yribonuclease (both 10 itg/ml) were added, and after
15 min the cells were recovered by centrifugation at
2,070 x g for 10 min. The pellet was suspended by
slowly adding 0.5 to 1 ml of 0.25 M sucrose-0.02 M
Tris-hydrochloride buffer (pH 7.5) containing 1 mg
each of pancreatic ribonuclease and deoxyribonucle-
ase. This suspension of lysozyme-treated cells was
added to Eppendorf centrifuge tubes that contained,
in 0.25 M sucrose-0.02 M Tris-hydrochloride (pH
7.5), [3H]dextran (16,500 daltons), either [14C]su-
crose or ['4C]glycerol, and disodium ethylenedi-
aminetetraacetate (Na2-EDTA) sufficient to make
the final concentration 1 mM. In this manner, more
than 95% cells were converted into lysozyme-EDTA
spheroplasts, in which the cytoplasm is bounded
only by the cytoplasmic membrane (6). By perform-
ing EDTA treatment in the reaction tubes, we could
minimize the damage to spheroplasts. After 5 min at
-
room temperature, the suspensions were centri-
fuged for 10 min, and the spaces penetrated by ra-
dioactive markers were calculated as in the experi-
ments with plasmolyzed cells.
Integrity of cell wall in plasmolyzed cells. To
determine whether plasmolysis resulted in gross cell
wall damage, we tested for the release of leucine-
binding activity in NaCl- or sucrose-plasmolyzed
HN202 cells as follows. The culture (3 liters) was
x g, and washed once with 0.025 M phosphate buffer
(pH 7.0). One gram (wet weight) suspended in 4.0 ml
328 DECAD AND NIKAIDO J. BACTERIOL.
of buffer was plasmolyzed with 0.5 M sucrose or 0.3 With tritiated polyethylene glycols, we col-
M NaCl (final concentrations). After 5 min of incu- lected only the peak fractions eluted from the
bation at room temperature, the suspensions were gel filtration column. Analytical gel filtration
centrifuged at 6,000 x g for 5 min, sucrose and NaCl of these compounds (Fig. 1 and 2) revealed few
supernatants were removed carefully, and each was
filtered through 0.22-,um Millipore filters. After ex- impurities and showed that the tritiated poly-
tensive dialysis against 0.01 M sodium phosphate ethylene glycols were not too polydisperse.
buffer (pH 7.0) in the cold, portions of the superna- As for the second condition, we expected that
tants were assayed for leucine-binding activity by
the filter assay (1). Another gram of cells (wet
weight) was treated by the osmotic shock procedure
(31). After osmotic shock, the shock fluid was fil-
tered through a 0.22-,um Millipore filter and concen-
trated by ultrafiltration through a UM-2 membrane

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6
(Amicon Corp., Lexington, Mass.), and the concen-
trated shock fluid was dialyzed extensively against I 0
0.01 M sodium phosphate buffer (pH 7.0) in the cold. 2 CL
Leucine-binding activity was assayed as described 0 0
E
0
above.
Liquid scintillation counting. Radioactivity was
determined with an Isocap 300 (Nuclear-Chicago 00
Corp., Des Plaines, Ill.) liquid scintillation spectro-
meter, with windows set manually for double-label
counting, so that the spillover of counts into the
opposite channel was always less than 7%. Ten mil-
liters of Aquasol (New England Nuclear Corp.) or Ve (ml)
PCS solubilizer (Amersham/Searle) was added to
each vial. Since the calculation of 3H-impermeable FIG. 1. Gel filtration of the tritiated sucrose-raffi-
space involved subtraction of a large number from a nose series of oligosaccharies. [3H]verbascose,
larger number, the radioactivity had to be deter- [3H]stachyose, [3H]raffinose, ["4C]sucrose, blue dex-
tran 2000 (Pharmacia Fine Chemicals, Inc.), and
mined with the highest possible accuracy. The use of NaBr
radioactive toluene as an internal standard some- Bio-Gelwere applied to a column ( 27 by 93 cm) of
P-2 (minus 400 mesh) kept at 65°C, and
times gave wrong factors for spillover correction, eluted with
presumably because the seemingly clear counting tran and distilled water. The presence of blue dex-
mixture was actually heterogeneous and contained measuring NaBr in the effluents was determined by
microscopic droplets of the samples, especially when and 220 nm. optical density (OD) ofthe effluents at 650
the samples contained much NaCl. Because of this,
blanks containing all ingredients of the reaction
mixture (except for radioactive markers) were al-
ways run in parallel with experimental samples, 4000X

and these blanks, to which 3H-labeled (0.08 ,ICi) f

saccharides or ['4C]sucrose (0.011 jACi) were added, S
were used as standards for spillover and quench I 3000 ^
correction. .5 7E
Other methods. Protein was determined with the 04 01
phenol reagent (25), and carbohydrate was deter- 2000 X
mined by the phenol-sulfuric acid reaction (13). Car-
bohydrate on chromatograms was detected as de- 1000
scribed previously (47). Bacterial growth was moni-
tored with a Klett-Summerson colorimeter, using a
red filter. 40 - 50
Ve (ml)
RESULTS
FIG. 2. Gel filtration of tritiated polyethylene gly-
Saccharides and polyethylene glycols for cols. 3H-labeled polyethylene glycols of 1,540, 1,000,
studying cell wall permeability. To directly 600, and 400 daltons were each applied to a column
study the permeability properties of the cell (1.27 by 55 cm) of Sephadex G-15 (Pharmacia Fine
wall of gram-negative bacteria, we needed la- Chemicals, Inc.), precalibrated with [14C]sucrose,
beled compounds with defined molecular [3H]stachyose, blue dextran 2000 (Pharmacia Fine
weights which could not diffuse into the cyto- Chemicals, Inc.), and NaBr. The latter two com-
plasm. We prepared the labeled compounds as pounds were measured by determining the optical
described above. With tritiated oligosaccha- density (OD) at 650 and 220 nm, respectively. The
eluant was 0.1 M NaCl. Symbols: A, 0, 0, and 0,
rides, we were careful in removing radioactive 3H-labeled polyethylene glycols of molecular weight
impurities by gel filtration, treatment with ion- 1,540, 1,000, 600, and 400, respectively; A,
exchange resins, and paper chromotography. ["'Cisucrose; *, NaBr.
VOL. 128, 1976 OUTER MEMBRANE AS A MOLECULAR SIEVE
oligosaccharides of the sucrose-raffinose series
might not be transported across the inner (cy-
toplasmic) membrane, since these sugars are not
utilized as carbon sources by the bacterial spe-
cies we used (9). The following data show that
sucrose indeed did not diffuse across the cyto-
plasmic membrane. Spheroplasts were pre-
pared from strain HN202, and the penetration
of labeled saccharides was determined by a pro-
cedure similiar to that used for plasmolyzed
cells (see Materials and Methods and below).
[3Hldextran (16,500 daltons) and [14C]glycerol
were used as markers for interspheroplast and
total spaces, respectively. For example, when
the permeable spaces for ['4C]sucrose,
[3H]dextran, and [14C]glycerol were determined
in a spheroplast pellet (35 mg, wet weight), the
results were 15, 16, and 33 ,ul, respectively,
regardless of the concentration of nonradioac-
tive carrier molecules used (ruling out nonspe-
cific adsorption). Since the space penetrated by
[3H]dextran, which was assumed to be nonpe-
netrable across the cytoplasmic membrane,
was essentially the same as the [14C]sucrose-
permeable space, we assume that both of these
compounds do not penetrate the cytoplasmic
membrane within the time course of this exper-
iment (15 min). The space penetrated by
['4C]glycerol was large, since this compound
diffuses across phospholipid bilayers (3) and
biological membranes (11). However, glycerol
was probably not actively transported by our
spheroplasts prepared from cells grown in the
absence of glycerol (38) since: (i) the total pellet
volume (35 ,ul calculated from pellet weight
assuming a density of 1) is approximately equal
to the total ['4C]glycerol space (33 1,u); and (ii) it
was expected that glycerol would accumulate
as glycerol 3-phosphate in spheroplasts if the
enzymes of glycerol utilization had been in-
duced (38). However, it was not detected by
analytical paper chromotography.
Although sucrose (and presumably also
"higher" oligosaccharides) does not diffuse into
the cytoplasm, there is a possibility that some
ofthe oligosaccharides may become altered dur-
ing incubation through the action of peri-
plasmic enzymes. To rule out this possibility,
we incubated labeled saccharides with crude
cell extracts. Analysis of the radioactive sub-
stances in the incubation mixture by paper
chromotography revealed no alteration or hy-
drolysis of the sucrose-raffinose series of oligo-
saccharides. These oligosaccharides were also
recovered in the unaltered form after incuba-
tion with intact cells of HN202.
Double-label assay for cell wall penetra-
tion. When a suspension of cells is incubated
with [14C]sucrose and [3H]dextran and centri-
329
fuged and 3H knd '4C concentrations in the
supernatant and the pellet are determined, the
fractions of the pellet volume that are pene-
trated by ['4C]sucrose and [3H]dextran can be
calculated. ['4C]sucrose does not penetrate
through the cytoplasmic membrane as de-
scribed above, but it obviously passes through
the cell wall, since high concentrations of su-
crose produce plasmolysis (26). [3H]dextran
(16,500 daltons) was assumed to be completely
nonpenetrable through the cell wall, since it
gave 3H-permeable space identical to the
[3Hldextran of very high molecular weight
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(e.g., 100,000). If these assumptions are correct,
we can calculate: (i) the total cell volume as
3H-impermeable space, i.e., pellet volume mi-
nus [3Hldextran-permeable space; (ii) the cyto-
plasmic volume as '4C-impermeable space, i.e.,
pellet volume minus [14C]sucrose-permeable
space; and (iii) the periplasmic space as cell vol-
ume minus cytoplasmic volume, i.e., [14C]su-
crose-permeable space minus [3H]dextran-per-
meable space.
The correctness of these assumptions was
confirned by measuring spaces under nonplas-
molyzing and plasmolyzing conditions. Thus,
when the exponential-phase cells of strain
HN202 were incubated in 0.025 M sodium phos-
phate buffer (pH 7.0) with [14C]sucrose and
[3Hldextran (16,500 daltons), 109 mg of pellet
after centrifugation (approximate volume =
109 ,ul assuming the density of 1.0) contained 49
and 47 ,u1 of 14C- and 3H-permeable space, re-
spectively. When another portion containing an
equal number of the cells of the same batch was
incubated in a similar manner but in 0.3 M
NaCl, which was found to produce plasmolysis
as judged by phase-contrast microscopy, 88 mg
of pellet contained 58 and 40 ,1u of 14C- and 3H-
permeable space, respectively.
Plasmolysis reduces the volume of cyto-
plasm, and the [14C]sucrose-impermeable
space, which should correspond to the cytoplas-
mic volume according to our assumptions, in-
deed decreased drastically from 60 to 30 al. On
the other hand, the [3Hldextran-impermeable
space, which should correspond to the cell vol-
ume, also showed a reduction (from 62 to 48 ,ul)
upon plasmolysis. This decrease probably is
caused by some shrinkage of the cell, which is
expected because the very thin peptidoglycan
layer (15) of these bacteria probably does not
have enough mechanical rigidity. The peri-
plasmic volume increased, again as expected,
on plasmolysis from 2 to 18 ul.
The addition of varying amounts of nonradio-
active sucrose and dextran to the reaction mix-
ture did not alter results; the penetration of
these compounds is therefore not the result of
330 DECAD AND NIKAIDO
adsorption. In addition, we have shown that
sucrose and higher oligosaccharides were not
altered metabolically by the cells (see above).
That the different degrees of penetration of
[14C]sucrose and [3H]dextran indeed reflect
plasmolysis was also confirmed by measuring
plasmolysis by a completely different tech-
nique. Determination of turbidity of HN202
cells in varying concentrations of NaCl, fol-
lowed by an analysis based on the Mie scatter-
ing theory (G. M. Decad and H. Nikaido, man-
uscript in preparation), showed that in 0.3 M
NaCl the cytoplasmic volume shrank to about
50% of that in nonplasmolyzed cells. This result
agrees well with the conclusions of the double-
label experiments, suggesting that the cyto-
plasmic membrane and the cell wall indeed
constitute the permeation barrier for sucrose
and dextran, respectively.
Conditions of plasmolysis. Since our assay
for cell wall permeability required the use of
plasmolyzed cells, we needed to know whether
the extent of plasmolysis varied with time or
temperature, or was affected by the presence of
nutrients or the concentration of the plasmolyz-
ing agent. Cell suspensions of strain HN202 in
0.025 M sodium phosphate buffer (pH 7.0) were
mixed with ['4C]sucrose and [3Hldextran
(16,500 daltons) and the plasmolyzing agent
(0.3 M NaCl, final concentration) and centri-
fuged immediately and after 1, 6, 15, and 30
min at 25°C. (The centrifugation procedure
took about 5 min). The periplasmic volumes
obtained were 21, 21, 22, 24, and 20 ,ul, respec-
tively. Clearly, plasmolysis reached near maxi-
mal levels within a few minutes, and stayed at
the same level for at least 30 min.
We tested the effect of temperature on plas-
molysis by plasmolyzing cells with 0.3 M NaCl
for 5 min in 0.025 M sodium phosphate buffer
(pH 7.0) in the presence of labeled compounds
at 0, 13 to 16, and 25°C. The [I4C]sucrose-perme-
able space was the same in each case (60 Iul)
compared with the [3Hldextran (16,500 dal-
tons)-permeable space of 35 ,ul. Thus, the plas-
molysis occurs fully, even at 0°C.
We also noted that the periplasmic volume
becomes gradually smaller with time ("deplas-
molysis") if unwashed cells are kept in 0.3 M
NaCl at 25°C. This deplasmolysis is probably
caused by the osmotic pressure-induced inward
transport of K+ (14, 35), and the nutrients in
carried-over L broth are probably supplying the
energy for this process. Therefore, all experi-
ments were done after washing cells once with
a large amount of 0.025 M sodium phosphate
buffer (pH 7.0) and resuspending them in the
same buffer.
J. BACTERIOL.
We routinely used 0.3 M NaCl for producing
plasmolysis. At this concentration, the plas-
molysis-produced gross cell wall damage was
minimal, as seen from the lack of the release of
periplasmic proteins from plasmolyzed cells.
For example, when the osmotic shock fluid and
the supernatants from plasmolyzed HN202 cells
were assayed for leucine-binding activity, less
than 5% of total leucine-binding activity was
found in the 0.3 M NaCl or 0.5 M sucrose plas-
molysis supernatants, whereas 80% of the total
leucine-binding activity was released into the
shock fluid, in close agreement with published
Downloaded from http://jb.asm.org/ on January 24, 2019 by guest
data for E. coli (18).
When 0.43 M NaCl (final concentration) was
used as a plasmolyzing agent, we usually ob-
tained a larger periplasmic volume (50% of the
cell volume) than by using 0.3 M NaCl. How-
ever, the cell wall apparently becomes dam-
aged in a large fraction of cells in 0.43 M NaCl,
as seen from the observation that saccharides of
fairly large size (e.g., 2,000 daltons) partially
penetrate into these cells (see below and Fig. 6).
Penetration of saccharides into plasmo-
lyzed cells. The double-label penetration exper-
iments were performed with [14C]sucrose and
3H-labeled oligosaccharides, [3H]inulin, or
[3Hldextran of various molecular weights.
When the 3H-labeled saccharide is completely
nonpenetrable through the cell wall, the differ-
ence between the 14C-permeable space and the
3H-permeable space gives the periplasmic
space. If the 3H-labeled saccharide partially
penetrates into the periplasmic space, this dif-
ference becomes smaller than the periplasmic
space, and the degree of penetration (p) for this
3H-labeled saccharide can be calculated as de-
scribed in Materials and Methods.
A typical experiment (Fig. 3) for the penetra-
tion of saccharides into the periplasmic volume
of S. typhimurium, E. coli, and P. morganii
shows a sharp exclusion limit near molecular
weights of 550 to 650. The cell wall is essentially
imperneable to larger saccharides. Sucrose and
raffinose penetrate completely into the peri-
plasmic space. A very similar result was also
obtained when 0.5 M sucrose was used as the
plasmolyzing agent (data not shown). The par-
tial penetration of saccharides larger than raffi-
nose, e.g., stachyose, may be the result of slow
penetration into a uniform population of plas-
molyzed cells, or complete penetration into a
portion of a heterogeneous population. When
[3Hlstachyose was incubated with plasmolyzed
cells of strain HN202 for 5, 10, and 30 min, the
[3H]stachyose-permeable space was always 10
to 20% of the total periplasmic volume. Thus,
the penetration reached near maximal levels
VOL. 128, 1976 OUTER MEMBRANE AS A MOLECULAR SIEVE 331
100 - , ,-- .,
E E
0
0

X 1.
80
0.2
a)
CL
CL
0.

-° 60 1. 0

0
0

LO 40 .S-
s
-

c
Q-
a)
0
a->e
0ll
20
0- o
S

-0.2 0.4 0.6 1.0 2.0 4.0 '10.0 20.0 100.0

Molecular weight x 10 3

Downloaded from http://jb.asm.org/ on January 24, 2019 by guest

Molecular weight x 10 I
FIG. 4. Penetration of saccharides into periplams
FIG. 3. Penetration of labeled saccharides into ofplasmolyzed S. typhimurium with lipopolysaccha-
periplasm of plasmolyzed cells of S. typhimurium ride mutations. See legend of Fig. 5. Symbols: 0,
HN202 (a), E. coli B (O), E. coli K-12 (U), and P. LT2; *, HN202; A, TA2168.
morganii (A). The cells were plasmolyzed with 0.3 M
NaCl in 0.025 M sodium phosphate buffer (pH 7.0),
and the percent penetration values were calculated .
100
E
after 5 min of incubation, indicating the latter 0

possibility above (see Discussion). 80k

Since some mutants that were defective in a)

lipopolysaccharide biosynthesis were shown to 0

w
be altered in the barrier properties of the cell Q._
C: 60F
wall (34, 39, 43-45), we examined whether 0

these mutations affected the penetration of sac- A

40k
charides. Figure 4 shows that the penetration of C)

saccharides into S. typhimurium (wild type)
LT2, HN202, and TA2168 (which produce Rc-
type and Re-type lipopolysaccharide, respec-
tively) is essentially the same. A possible expla-
nation for the biphasic response of strain
TA2168 will be considered in the Discussion.
Penetration of polyethylene glycols
through cell wall. We also wanted to know
whether other hydrophilic molecules of differ-
ent structures but similar molecular weights
penetrated to the same extent as the oligosac-
charides. Therefore, the penetration of tritiated
polyethyleneglycols was tested in a similar
manner. Figure 5 shows that low-molecular-
weight polyethylene glycols (400 and 600 dal-
tons) penetrate to an extent similar to that of
the di- and trisaccharides. 3H-labeled polyeth-
ylene glycols of 400 daltons, however, slightly
penetrated the cytoplasmic membrane. This
was shown by a 3H-permeable space that was
slightly larger than the ['4C]sucrose-permeable
space (see Materials and Methods). In addition,
tritiated polyethylene glycol of 1,000 daltons
and polyethylene glycol of 1,540 daltons pene-
trated to a significant extent into the peri-
plasm, whereas saccharides of similar molecu-
lar weight did not penetrate at all. (The pene-
tration of polyethylene glycol was not affected
by adding nonradioactive carrier molecules, a
04
20H
I0.2 0.4 0.6 1.0 2.0
Molecular weight x 10 3
FIG. 5. Penetration of labeled polyethylene glycols
into periplasm of plasmolyzed cells. S. typhimurium
LT2, HN202, and TA2168 producing smooth, Rc-
type and Re-type lipopolysaccharide, respectively,
and E. coli B were plasmolyzed in 0.3 M NaCl-0.025
M sodium phosphate (pH 7.0), and the penetration
values were calculated. Symbols: A, LT2; * and 0,
HN202; A, TA2168; 0, E. coli B.
result ruling out nonspecific adsorption.) The
results were the same for E. coli B and three
strains of S. typhimurium.
Saccharide penetration into Pseudomonas
and Alcaligenes. When P: aeruginosa and A.
faecalis were plasmolyzed with 0.3 M NaCl or 0.5
M sucrose, their cell walls allowed a partial
penetration of much larger oligosaccharides
than those of the enteric bacteria (Fig. 3), pro-
ducing a biphasic curve (see Fig. 7). A similar
biphasic curve could sometimes be obtained
with S. typhimurium HN202, if plasmolysis
was done with 0.43 M NaCl (final concentra-
tion). Thus, in the latter case, a partial pene-
332 DECAD AND NIKAIDO
tration of saccharides larger than stachyose
was sometimes observed (Fig. 6). The biphasic
response was always associated with the more
extensively plasmolyzed cells (periplasmic vol-
ume corresponding to 50% compared to 40% of
cell volume) and may be relhted to plasmolysis-
produced cell wall damage in a portion of the
cell population (see Discussion).
We tried to find conditions that produce plas-
molyzed cells with minimal cell wall damage in
Pseudomonas. Plasmolysis with 0.22 M NaCl
plus 0.01 M MgCl2 had a slight but reproducible
effect in reducing the penetration of higher-mo-
lecular-weight compounds. However, a bi-
phasic curve was still produced (Fig. 7). The cell
wall exclusion limit is close to, if not identical
to, that in the enteric bacteria, if one extrapo-
lates the first portion of the curve to the ab-
scissa. A possible explanation for the sensitiv-
ity of these strains to plasmolysis is considered
in the Discussion.
Efflux of sucrose and raffinose from peri-
plasm. We wanted to know how rapidly
[14C]sucrose or [3H]raffinose diffused across the
cell wall. This was done by plasmolyzing cells
in NaCl and preloading the periplasm with
either [14C]sucrose or [3H]raffinose. After dilu-
tion into a buffer having the same composition
(minus the radioactive compounds) as the reac-
tion mixture, filtering was done at various
times on Millipore filters and took 5 s. Results
show that greater than 90% of the labeled com-
I DISCUSSION
100 _
E rect pieces of evidence, that the cell wall of
.X 80_
CL
I gram-negative bacteria may act as a diffusion
0.
0
0~~~~
40 A ties, which we studied by using hydrophilic
a.o1
0 ~~~~0A
420
C
0
0.2 0.4 0.6 1.0 2.0
Molecular weight x 10 3
FIG. 6. Penetration of labeled saccharides into
periplasm of extensively plasmolyzed S. typhimu-
rium. Strain HN202 was plasmolyzed with 0.43 M
NaCI in the presence of 0.025 M sodium phosphate
buffer, (pH 7.0), and the percent penetration of sac-
charides was calculated. Two kinds of results are
shown: 0 and A, associated with less extensive plas-
molysis (periplasm equals 40% cell volume); 0 and
A, associated with more extensive plasmolysis (peri-
plasm equals 50% cell volume).
J. BACTERIOL.
U 80
0
a1)CL 60
0
A
o 40
Q, 20 \\0
20
0.4 0.6 1.0 2.0 4.0 10.0 20.0 100.0
Molecular weight x 10
Downloaded from http://jb.asm.org/ on January 24, 2019 by guest
FIG. 7. Penetration of labeled saccharides into
periplasm of plasmolyzed cells of P. aeruginosa and
A. faecalis. P. aeruginosa was plasmolyzed with 0.22
M NaCI-0.01 M MgC12, and A. faecalis was plasmo-
lyzed with 0.3 M NaCl, both in 0.025 M sodium
phosphate buffer (pH 7.0). The percent penetration
values were then calculated. Symbols: *, P. aerugi-
nosa; A, A. feacalis.
pounds diffuse out from the periplasm almost
immediately, even at 0°C (Fig. 8). We calcu-
lated theoretical zero-time points based on the
amount of radioactivity added initially and the
fact that 1 mg (wet weight) of cells contains a
0.25-,ul periplasmic volume under our condi-
tions. Very similar results were obtained for
the efflux of ['4C]sucrose, in the presence of 0.5
M sucrose, from cells plasmolyzed by 0.5 M
sucrose (data not shown).
Previous workers have suggested, from indi-
barrier for certain molecules (19, 20, 24, 28, 36,
39, 40, 43-45). Almost nothing, however, was
known about its precise permeability proper-
molecules as permeants.
Before we began our study, several experi-
mental conditions had to be fulfilled. The de-
gree of penetration of hydrophilic molecules
into the periplasm had to be quantitated and
the cell wall damage had to be minimized. In
addition, the permeants could not diffuse into
the cytoplasm nor be acted upon by extracellu-
lar or periplasmic enzymes. We solved these
problems by plasmolyzing cells in the presence
of ['4C]sucrose and tritiated dextrans, inulins,
oligosaccharides of the sucrose-raffinose series,
or polyethylene glycols. Since the periplasm
was extremely small in nonplasmolyzed cells,
we used plasmolysis to increase the sensitivity
of our assay. We could then quantitate the
degree of penetration of labeled compounds into
VOL. 128, 1976 OUTER MEMBRANE AS A MOLECULAR SIEVE 333
20.0 were found also with P. aeruginosa, A faecalis
(Fig. 7), and frequently with a "deep rough"
l1o.c (Re-type) mutant of S. typhimurium (Fig. 4),
and are also assumed to be a consequence of cell
wall damage. Perhaps the absence ofthe Braun
x
5.0 lipoprotein in the former two strains (7) and the
E
drastically reduced protein content in the latter
(2) produce rather unstable outer membranes
0'
._-
that are easily damaged.
These considerations suggest that one should
3.
a') be very careful in the interpretation of the re-
3.
0)
sults.the
If aperiplasmic
compound does
1.0 into space,notit penetrate
is clearlyfully
im-

Downloaded from http://jb.asm.org/ on January 24, 2019 by guest

permeable. On the other hand, successful pene-
0.5 tration may be due to the intrinsic permeability
of the cell wall or to plasmolysis-induced dam-
age in the cell wall. With the saccharides of
sucrose-raffinose series, oligosaccharides larger
t
A* than stachyose (666 daltons) are probably non-
penetrable under physiological conditions, since
I they show little penetration in experiments
2 ^ 4
0.
o 6 8 10 with 0.3 M NaCl, which is believed to produce
Time (minutes) minimal amounts of cell wall damage (Fig. 3
and 4). On the other hand, it seems likely that
8Eflux of [3H]raffinose and [4C]sucrose the cell wall is intrinsically permeable to su-
FIG. 8.
from eriplasm ofplasmolyzed cells. Periplasm was
j

preloaded with labeled saccharides, and the efflux crose (342 daltons) and raffinose (504 daltons),
then d etermined.Theoretical zero-timepoints(0and since these sugars always penetrated nearly
A) WIPere also calculated. Symbols: A and A completelyintoperiplasmic space, even incells
[3H]raffinose; 0 and 0, ["'Cjsucrose. that were plasmolyzed under the least damag-
ing conditions (Fig. 3).
the periplasm by our double-label centrifuga- In these experiments, the saccharides had to
tion procedure. When we used tritiated saccha- diffuse across both the outer membrane and the
rides as permeants, we could show that underlying peptidoglycan layer to reach the
[3HIraffinose (504 daltons) penetrated almost periplasmic space. Recent work from this labo-
i the periplasm of S. typhimurium, E. ratory has shown, however, that the outer
fully into
coli, or P. morganii, whereas [3Hlstachyose membrane alone, and not the peptidoglycan
(666 (daltons) only partially penetrated, and layer, is responsible for the exclusion of larger
highe:r oligosaccharides and polysaccharides saccharides (30). By what mechanism then do
did ncDt penetrate at all. the smaller saccharides diffuse through the
Amlong the conditions listed above, the min- outer niembrane? Dissolution of these mole-
imizaition of cell wall damage was most difficult cules into the hydrophobic membrane interior
to acd hieve. The use of 0.43 M NaCl as the is thermodynamically improbable. The only
plasmolyzing agent most likely damaged the two possible mechanisms would be passive dif-
cell wall. This is not surprising, since plasmoly- fusion through water-filled pores or facilitated
sis mEay pull apart some of the adhesion points diffusion. We favor the former for the following
betwehen the cytoplasmic membrane and the reasons. (i) Facilitated diffusion requires that a
cell wrall (4); also, very high concentrations of component of the system recognizes the sub-
plasmolyzing agents, such as 2 M sucrose, are strate and therefore should show a high degree
indee4 d known to produce enough damage in the of substrate specificity. We showed, however,
cytopllasmic membrane (50). When the cell wall that even the synthetic polyethylene glycols
is danaaged, the plots of penetration versus mo- rapidly penetrate the cell wall. We would not
lecular weight often become biphasic (Fig. 6) expect these compounds to be specifically recog-
and ixndicate the partial penetration of larger nized and transported. (ii) Recent work from
sacchi arides (800 to 1,800 daltons). Since these this laboratory has shown that complexes con-
large saccharides penetrated extremely rapidly taining one to three species of outer-membrane
(see iLesults), their partial penetration is best proteins can be reconstituted with phospholip-
explaiined by diffusion only into damaged cells ids and lipopolysaccharides to produce mem-
withixi the population. Similar biphasic curves brane vesicles permeable to a wide variety of
334 DECAD AND NIKAIDO
hydrophilic substances (29; T. Nakae, manu-
script in preparation). Since facilitated diffu-
sion would require one carrier protein or recog-
nition protein for one species of solute to be
transported, it is inconsistent with these re-
sults. (iii) The rate of diffusion of saccharides is
extremely fast. The efflux experiments showed
that more than 90% of sucrose in the peri-
plasmic space became exchanged with external
sucrose within 20 s when 0.5 M sucrose was
used as the plasmolyzing agent. Thus, the rate
of exchange is higher than 500 x 0.3 x 0.9 x 3
= 405 ,.mol/min per ml if we assume that 30%
of cell volume corresponds to periplasmic space.
This value is at least an order of magnitude
higher than the maximal rate of facilitated dif-
fusion of lactose in a fully derepressed cells of
E. coli, namely 37 ,umol/min per g (wet weight)
(51). It would require unrealistic amounts of
carrier proteins in the outer membrane to cata-
lyze facilitated diffusion of many substances at
such rapid rates. (iv) Facilitated diffusion is
expected to become much slower at a low tem-
perature (37). However, the efflux rate of sac-
charides was extremely rapid even at 0°C.
Penetration through water-filled pores is ex-
pected to be a nonspecific process, limited
mostly by the size of the penetrating solutes.
Payne and Gilvarg (36) found that E. coli can
utilize, for growth, peptides of only up to a
certain size, and attributed this phenomenon to
the molecular-sieving effect of the cell wall.
The maximum size of the peptide utilized is 534
daltons for oligolysines and corresponds closely
to the molecular weight of raffinose (504). The
similar exclusion limits for oligopeptides with
multiple positive charges and uncharged oligo-
saccharides lend further support for the
aqueous pore mechanism of diffusion. With pol-
yethylene glycols, a complete penetration of a
400-dalton sample was observed. However,
even 1,540-dalton polyethylene glycols showed
a partial penetration into the periplasm (Fig.
5). One explanation ofthese results may be that
the exclusion limit for polyethylene glycols lies
around 1,000 daltons, and that 600, 1,000, and
1,540-dalton 3H-labeled polyethylene glycols
show partial penetration because ofthe polydis-
persity of the samples (Fig. 2). That the exclu-
sion limits for oligosaccharides and polyethyl-
ene glycols are different in terms of molecular
weight is not totally unexpected, since saccha-
rides would have quite different conformations
in solution from the long, threadlike polyethyl-
ene glycols. It is improbable that polyethylene
glycols should diffuse by dissolving into the
hydrophobic interior of the membrane, since
their partition coefficients in a 1-octanol-water
J. BACTERIOL.
system were less than 0.01 (H. Nikaido, unpub-
lished data).
In addition to the penetration experiments
using the enteric bacteria, we studied the pene-
tration of saccharides into P. aeruginosa and A .
faecalis. Although large oligosaccharides par-
tially penetrated through the cell wall, the bi-
phasic curve suggests that the true exclusion
limit is close to, if not identical to, that in the
enteric bacteria.
We conclude, therefore, that the cell wall of
the gram-negative bacteria tested acts as a mo-
lecular sieve, with an exclusion limit near mo-
Downloaded from http://jb.asm.org/ on January 24, 2019 by guest
lecular weights 550 to 650 for saccharides. This
property forms a striking contrast to that of the
gram-positive cell wall, which allows the pene-
tration of much larger molecules (e.g., about
100,000-dalton dextrans in Bacillus megate-
rium cell wall [421). The molecular sieving in
the outer membrane is apparently achieved
through the presence of water-filled pores that
permit the rapid, nonspecific diffusion of small,
hydrophilic molecules. This mechanism, how-
ever, may not be the only diffusion pathway
present in the outer membrane. For example,
proteins involved in the specific transport of
molecules too large to penetrate through pores
have been found in the outer membrane (12, 30,
49). The protein involved in maltodextrin diffu-
sion in E. coli may be a special case, since it is
also required for the diffusion of very low con-
centrations of maltose (21, 46). In addition, a
pathway for diffusion of hydrophobic molecules
has been described for the outer membrane of
certain lipopolysaccharide biosynthetic mu-
tants (34).
ACKNOWLEDGMENTS
This study was supported by Public Health Service re-
search grant 5 R01-AI09644 from the National Institute of
Allergy and Infectious Diseases and by American Cancer
Society research grant BC-20. Gary M. Decad was a Public
Health Service Predoctoral Trainee under grant AI-120
from the National Institute of Allergy and Infectious dis-
eases.
We thank Taji Nakae for discussions and for his contri-
bution during the early phase of this work.
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