VIROLOGY66, 268-282 (1975)

The Lipopolysaccharide

Receptor

$X174 S. MICHAL JAZWINSKI, Departments

of Biochemistry

and S13

ALF A. LINDBERC,’

and Medical

Microbiology, California

Accepted

for Bacteriophages

Stanford 94305

February

ANo ARTHUR KORNBERC University

School of Medicine,

Stanford,

14, 1975

The &X174 and S13 receptor is found in the lipopolysaccharide (LPS) of the cell outer membrane of sensitive strains of Escherichia coli and Salmonella typhimurium. Binding of the phage to purified LPS is sufficient to cause the alteration of phage structure called eclipse as manifested by susceptibility to DNase. Based on results with different strains of S. typhimurium and intact and chemically degraded LPS derived from them, the phage binding site can be represented by this structure found in the core and backbone of the LPS: (GlcNAc) Gal Hep P-P-Ethanolamine 1 1 1 5Glc -, Gal+ Glc- Hep-+ Hep-KDO. I P [GlcNAc, N-acetylglucosamine; Glc, glucose; Gal, galactose; Hep, heptose; KDO, 2-keto-3deoxyoctulosonic acid]. Although closely related, #X174 and S13 differ slightly in their receptor requirements. S13 does not require GlcNAc at the nonreducing terminus for optimal binding and eclipse. Removal of the ester-linked fatty acids of the Lipid A moiety of LPS destroys the capability for phage eclipse but not for binding. Physical features of LPS imposed by its lipid components appear to be necessary for the succession of events that lead to eclipse. Regions of fusion between the inner and outer cell membranes may be the locus of the-phage-binding site. INTRODUCTION

@X174 and related phages S13, 6SR, Br2, and U3 infect certain strains of Escherichia coli and Salmonella typhimurium (Lindberg, 1973). These viruses contain a single-stranded circular DNA (SS) of 5500 nucleotides in an icosahedral coat with spikes at the 12 vertices (Sinsheimer, 1968). The phage-adsorption process occurs in two steps: binding of the phage to its cell surface receptor followed by eclipse, the conformational change in the phage which exposes the DNA to DNase attack (Newbold and Sinsheimer, 1970a). Subsequently, the phage DNA penetrates the 1Present address: Department of Bacteriology, National Bacteriological Laboratory, S-10521 Stockholm, Sweden.

cell. Penetration is coupled to the formation of the duplex parental-replicative form (RF), the first step in viral DNA replication (Francke and Ray, 1971). The phage, like the T phages, appears to bind to the cell at points of fusion of cytoplasmic and outer membrane (Bayer and Starkey, 1972), and the parental RF is recovered attached to the cell membrane (Knippers and Sinsheimer, 1968; Loos, Tessler and Salivar, 1971). Our interest in the molecular details of the first step of 4x174 DNA replication (Schekman et al., 1972) has led us to investigate the early events of phage adsorption and penetration. In this paper we consider the cellular receptor for the phage. Subsequent papers deal with the phage protein responsible for adsorption to the

266 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

9X174

AND

receptor (Jazwinski, Lindberg, and Kornberg, 1975) and additional properties of this protein (Jazwinski, Marco, and Kornberg, 1975). With regard to the 4x174 receptor, it was found that isolated cell walls inactivated the phage (Fujimura and Kaesberg, 1962) and released the phage DNA (Brown, MacKenzie, and Bayer, 1971). Since lysozyme destroyed the ability of cell walls to inactivate and eclipse the phage, the @X174 receptor or eclipsing agent was judged to be the mucopeptide (Brown, MacKenzie, and Bayer, 1971). However, more compelling evidence for LPS as receptor was later adduced. Binding and

S13 RECEPTOR

269

eclipse of $X174 phage was demonstrated with purified LPS and specifically the LPS derived from sensitive strains (Jazwinski and Marco, 1973; Incardona and Selvidge, 1973). These observations extended those of Lindberg and Holme (1969) showing that the related phages 6SR and Br2 are inactivated by cell walls and LPS purified from them. The structure of the polysaccharide chain in the LPS was identified as crucially important for receptor activity (Lindberg, 1973) (Fig. 1). Yet, it was not known whether specificity for binding phage resided simply in the polysaccharide chain or if the lipid component was also required, nor were the structural require-

FIG. 1. LPS of S. typhimurium strains (Hellerqvist and Lindberg, 1971; Liideritz et al., 1971; Rietschel et al., 1972; Lindberg, 1973; Hiimmerling, Lehmann and Liideritz, 1973). Abbreviations: Abe, abequose; Man, n-mannose; Glc, n-glucose; Gal, n-galactose; GlcNAc, iV-acetyl-o-glucosamine; Hep, L-glycero-n-mannoheptose; KDO, 2-keto&deoxyoctulosonic acid; EtN, ethanolamine; P, phosphate; FA, fatty acid; BHM, not determined. All strains are &hydroxymyristic acid; GlcN, n-glucosamine; n * 10; x, stoichiometry derivatives of S. typhimurium LT2 (smooth). SL1027 (parent) is a genetically marked LT2 (Wilkinson, Gemski, and Stocker, 1972); the markers have no effect on LPS structure. The salient characteristic of this strain is that it is smooth, as indicated. Strains have been characterized genetically, serologically, biochemically, and by phage-typing (Subbaiah and Stocker, 1964; Gemski and Stocker, 1967; Wilkinson and Stocker, 1968; Osborn, 1968, Nikaido, 1968; Hellerqvist and Lindberg, 1971; Lindberg and Hellerqvist, 1971; Wilkinson, Gemski, and Stocker, 1972; Kuo and Stocker, 1972). Strains SL733, TV161 and TV146 are slightly leaky resulting in the formation of some complete cores to which O-side chains are attached (Lindberg and Holme, 1968; Hellerqvist and Lindberg, 1971). However, the leakiness of SL733 did not affect the number of &X174 attachment sites (see Fig. 4) thus demonstrating that the majority of LPS chains were of mutant character. LPS from rough strains was prepared by phenol-chloroform-petroleum ether extraction, a procedure which does not extract LPS possessing O-side chains (Galanos, Ltideritz, and Westphal, 1969; Hellerqvist and Lindberg, 1971; Lindberg and Holme, 1972), yet the behavior of LPS and the cells from which it was isolated was in close agreement (see Tables 1 and 2). Studies with LPS were not complicated by leakiness of the strains, since only mutant LPS was isolated by the extraction procedure.

270

JAZWINSKI,

LINDBERG

ments for phage eclipse understood. We-report here on the structural features of the LPS receptor sites for $X174 and S13 required for binding and phage eclipse. We also show that +X174 DNA becomes associated with the outer cell membrane upon phage infection of the cell in contrast to Ml3 DNA, which is associated with the inner membrane (Jazwinski, Marco, and Komberg, 1973). We consider the hypothesis that $X174 binding is directed to newly synthesized LPS located on the cell surface where the outer and inner membranes are fused (Bayer and Starkey, 1972; Miihlradt et al., 1973).

AND KORNBERG

phate) to A,““” of 0.5; phage at multiplicity of infection (m.0.i.) of 5 and 5 mCi Hs3!P0, per loo-ml culture were added at the same time. Cells were incubated for 2-3 hr at 37” with aeration, then harvested and washed with 1 culture volume of 50 mM borate-5 mM EDTA, and resuspended in the same buffer to 2.5 x 10” cells/ml. Lysozyme (100 &ml) was added and the cells were frozen in Dry Ice-acetone and thawed at 37” three times. Sarkosyl was then added to a concentration of 0.5% and the cells incubated for 10 min at 37”. The lysates were chilled to 0” and applied to 5-20s linear sucrose gradients containing 10 mMTris-HCl (pH 8)-l mMEDTA-1 M MATERIALS AND METHODS NaCl which were centrifuged in a Spinco Chemicals were as follows: NaB3H, (346 SW27 rotor for 3 hr at 27,000 rpm (5”). mCi/mmol) from Amersham, albumin Gradients were fractionated by puncturing from Miles, and micrococcal nuclease from the bottom of the tube with a 22-gauge Worthington. Sources of other chemicals needle and collecting l-ml fractions. Fracwere as described previously (Jazwinski, tions containing the phage were pooled and Marco, and Kornberg, 1973). alp-labeled f2 dialyzed against 10 mM Tris-HCl (pH and Ml3 phage were a kind gift from Dr. R. 7.5)-l mM EDTA at 5” for 12 hr. [3H]ThyMarco (Marco, Jazwinski, and Kornberg, mine-labeled phage were prepared in the same way except that the thymine concen1974). tration in TPA was reduced to 1 pg/ml and Bacterial and Phage Strains 5 mCi of [3H]thymine was added at time of Sources were as follows: Escherichia coli infection. At least 80% of the particles in phage K12 5274, resistant to +X174 (E. coli”) from the J. Lederberg collection, E. coli preparations were infectious, and the conC/K12 HF4704 thy-, sensitive to 4X174 tent of eclipsed phage was less than 10%. (E. coli’), C/K12 HF4714 .sux+, sensitive to $X174, and K12 HfrC71, resistant to Preparation of Intact and Hydrolyzed LPS 4x174, from Dr. H. Hoffmann-Berling, Bacteria were grown in a complex meand HF4704 F+, sensitive to @X174 from dium with aeration as described earlier Dr. D. Ray, Salmonella strains, all derived (Lindberg and Holme, 1972). LPS was from S. typhimurium strain LT2 (Fig. l), extracted by the phenol-chloroform-pefrom Dr. B. A. D. Stocker, 4X174 am3 troleum ether method (Galanos, Liideritz, (gene E) from Dr. R. L. Sinsheimer, the and Westphal, 1969) from rough strains corresponding S13 mutant amEnl5 from and by the phenol-water method (WestDr. I. Tessman. (Mutants in gene E behave phal, Liideritz, and Bister, 1952) from like the wild-type phage except for a defect smooth and semirough strains. The moin cell lysis). lecular weight of the LPS aggregate has Cells were grown in M medium (Marvin been estimated at 1 to 10 x 10’ (Liideritz and Schaller, 1966) or TPA medium et al., 1971). Alkaline hydrolysis of the (Francke and Ray, 1971). LPS results in the removal of esterlinked fatty acids from Lipid A (Rietschel Labeling of Phage et al., 1972). The product (Alk-PS) obPhages were propagated in log-phase tained from a mutant with the complete cells grown in M medium at 37”. 3zP- core had a molecular weight of 9 x 10’ (Liilabeled 4X174 am3 and S13 amEn were deritz et al., 1971). Hydrolysis and labeling prepared by infecting E. coli HF4704 cells of LPS by tritium exchange were carried grown in TPA (containing 1 mM phos- out simultaneously: lo-15 mg of LPS were

4x174

AND

mixed with 5-7.5 mg of NaB3H,( -100 mCi/mmol) in 2.0 ml of 10 mM NaOH. The reaction mixture was kept at room temperature for 36 hr. Excess NaB3H, was destroyed by dropwise addition of 0.1 M HCl to pH 6. The material was dialyzed against 10 mM Tris-HCl (pH 7.5)-l mM EDTA in the cold. The dialyzed material was applied to a column of Biogel A-l.5 m equilibrated with 50 mM sodium phosphate buffer (pH 7.2)-0.5 M NaCl. The column was eluted with this buffer and the peak fractions (estimated molecular weight 9 x lo* as compared to dextran standards) collected and pooled. The pooled fraction was reduced in volume by flash evaporation, dialyzed against the Tris-EDTA buffer, and lyophilized. Treatment of the LPS with aqueous acetic acid at pH 3.2 for 2 hr at 100” cleaves the acid-labile linkage between KDO residues yielding a polysaccharide fraction (Acid-PS) lacking Lipid A and KDO and retaining only an altered KDO residue attached to heptose (Dtige, Liideritz and Westphal, 1968; mmmerling, Lehmann, and Liideritz, 1973). The Acid-PS was labeled by reduction of KDO with NaB3H, (Hammerling, Lehmann, and Liideritz, 1973); approximately 5 mg of polysaccharide was incubated with 1 mg of NaB3H, in 1 ml of 10 mM NaOH at room temperature for 36 hr after which excess borohydride was destroyed by dropwise addition of 0.1 M HCl to pH 6. Acid-PS was purified by gel filtration through Sephadex G-50 to remove lipid-containing polysaccharide residues and Sephadex G15 to remove KDO residues: columns were equilibrated and eluted with a buffer of pyridine: glacial acetic acid: water (10:4:lOOO v/v); the peak fraction, with an estimated molecular weight of 1500 compared to oligosaccharide standards, was pooled, reduced in volume by flash evaporation, and lyophilized.

271

S13 RECEPTOR

Engineering Co., Alexandria, VA), 25-cl1 loops, and 2% erythrocyte suspensions were used.

Enzyme Assays Phospholipase A and DPNH oxidase activities were determined as described previously (Jazwinski, Marco, and Kornberg, 1973).

Radioactivity

Measurements

Aqueous samples were counted in 5 vol of Triton X-lOO-toluene scintillation fluid (1.8 g PPO and 0.04 g dimethyl-POPOP in 1 liter of toluene-Triton X-100 (2:l) in a scintillation counter. Samples dried on filter discs were counted in 5 ml of toluene scintillation fluid (4 g PPO and 0.1 g dimethyl-POPOP in 1 liter of toluene). RESULTS

The @Xl74 Receptor is Located in the LPS of the Outer Cell Membrane The $X174 receptor in E. co@ cells was

located by ultrafiltration measurements of phage binding to subcellular fractions. Whole membrane (unfractionated cell debris) prepared from cell extracts obtained with a French pressure cell retained the binding capacity of intact cells (Fig. 2). Binding activity was not affected by addition of the soluble fraction of the extract. Fractionation of whole membrane into inner and outer membrane components indicated that the receptor was located in the outer membrane (Fig. 2); the inner membrane contained no 4X174 binding activity, and did not stimulate binding by the outer membrane fraction. LPS is a principal constituent of the outer membrane. When purified from E. coli”cells, it bound $X174 as effectively as did the outer membrane (Fig. 2). The binding capacities of disrupted cells and of LPS were several times greater than those of intact cells (Fig. 2). How disruption of the cell exposes Hemagglutination Inhibition additional receptor sites is not understood. Antisera against LPS of the various The apparent loss of binding sites in the classes of S. typhimurium mutants (Fig. 1) outer membrane as compared to whole and E. coli” LPS were prepared, and pas- membrane and LPS may be due to rearsive hemagglutination was as described rangements in these micellar structures previously (Lindberg and Holme, 1968) during purification. except that a Takatsy microtitrator (Cooke . An irreversible stage which follows bind-

272

JAZWINSKI,

LINDBERG AND KORNBERG

1X

-=

/-

y-

0

1 EDUIVALENT 1 1

1X

1 1 x 10’ 1 x 106

1 1 1 x 10’

I

1111

I 1 x 108

I,, 1 x 109

CELLS OR CELL EQUIVALENTS

FIG. 2. Binding of 9x174 to cells and subcellular fractions of E. coli. Extent of binding of [“*PI phage to E. co@ cells (log phase) and subcellular fractions was determined by incubation with 1 x 10”’ plaque-forming units (PFU) in 2 ml-of M medium containing 2 mM CaCl, for 15 min at 37”. (Results are expressed for 1 ml of incubation mixture.) Samples were placed on ice and then filtered through Millipore filters (HAWP; 0.45 am). Filters were washed three times with 2 ml of deionized water (0% dried, and the radioactivity measured by scintillation counting. Binding of phage to a resistant strain (E. coliR) and subcellular fractions (from lOa to 5 x lOa cells or cell equivalents) prepared from it was also measured. Observed values were all at the same level as the background binding of phage to the filter (approximately 5%); this amount was subtracted from the values observed with sensitive cells and cell fractions. Whole membrane was prepared by disrupting cells suspended in 10 mM Tris-HCl (pH 7.5)-l mi%4EDTA in a French pressure cell at 20,000 psi. Membranes were collected by centrifugation in a Spinco type 30 rotor for 30 min at 30,000 rpm (5”) and resuspended in 10 mM Tris-HCI (pH 7.5)-l mMEDTA. This fraction was then separated into inner and outer membrane as described in Fig. 6. LPS was prepared according to Osborn (1966). The relationship of phage binding and receptor concentration is linear in these plots. At higher receptor concentrations aggregation occurs. This is most pronounced with isolated LPS and causes a deviation from direct proportionality. The number of receptors per cell equivalent is calculated from the values on the curves at the lowest concentrations of receptor.

and 4X174-like

(Lindberg, 1973), we may infer that the core component of LPS is the receptor. Analysis of the LPS core function in E. coli was precluded by lack of knowledge of its structure. However, in S. typhimurium availability of a series of isogenic bacterial mutants (Fig. 1) with known defects in structure of the polysaccharide chain of the LPS enabled us to correlate structure with phage binding. Neither $X174 nor S13 was bound to smooth or semirough strains of S. typhimurium (or a 4X174-resistant E. coli strain) or LPS derived from them (Table 1). Binding was maximal to a rough S.

(S13, 6SR, Br2) infect rough strains of E. coli. and S. typhimurium

typhimurium strain (or its LPS) with a complete core structure possessing N-

ing of 9x174 is eclipse of the phage (Newbold and Sinsheimer, 1970a). Conformational changes in the phage particle make the DNA accessible to nuclease action. The capacity to produce phage eclipse was retained by whole membrane and outer membrane fractions and by LPS (Fig. 3). Thus, the 4X174 receptor is located in the LPS of the outer membrane and binding to purified LPS is sufficient to cause phage eclipse. The Core Component of LPS Is the Receptor for @X174 and S13

Inasmuch phages

as 4x174

273

6x174 AND S13 RECEPTOR

The number of 4X174 binding sites per cell in S. typhimurium did not depend on 3 the presence of an N-acetylglucosamine $ and was similar to the number in E. cob cells (Fig. 4), suggesting that the difference g in extent of phage binding at saturation is E a consequence of different phage affinities for the receptor. The number of binding I 10 sites per cell equivalent of isolated LPS 1 x lo8 1 x 107 ! was also not affected by lack of the NCELLS OR CELL EQUIVALENTS acetylglucosamine (Fig. 4). Thus, in conFIG. 3. Eclipse of 4x174 by cells and subcellular trast to S13, 4X174 requires N-acetylfractions of E. coli. Extent of eclipse of [SzP]phage by glucosamine at the nonreducing end of the E. coli ’ cells (log phase) and subcellular fractions was core for optimal binding. This conclusion is determined by incubation with 1 x 10” PFU in 0.1 ml supported by the extent of eclipse of $X174 of M medium containing 2 mM CaCl, for 15 min at and S13 by cells and purified LPS (Table 37”. Samples were treated in 0.3% Sarkosyl for 5 min at 37”; 2 ml of 50 mM borate-5 mM EDTA-10 mA4 d E

loo

CaCl, were added. Samples were then incubated with micrococcal nuclease (10 fig/ml) for 15 min at 37”, chilled, treated with 0.1 vol of 50% trichloroacetic acid, and after 15 min filtered through Whatman glass-fiber discs. The latter were washed five times with 2 ml of 0.1 M sodium pyrophosphate-1 M HCl, and once with 5 ml of ethanol (0’). Filters were dried and radioactivity was measured by scintillation counting. Whole membrane, outer membrane, and LPS were prepared as described in Fig. 2, Fig. 6, and Materials and Methods, respectively. Results are expressed as percentage of the maximal extent of DNase sensitivity, 66% of the input 32P made acid soluble after treatment with micrococcal nuclease. Values for endogenous content of eclipsed particles in the phage preparation (8%) have been subtracted. Phage eclipse was not detectable after incubation with a resistant strain (E. colia) and subcellular fractions (from 10’ to 10’ cells or cell equivalents) prepared from it.

acetylglucosamine at the terminal nonreducing end; extent of binding was comparable to that with E. coli’ or its LPS. Lack of IV-acetylglucosamine at the nonreducing end of the core (compare SH180 and SL733) reduced the binding capacity of the cells for $X174, but had little effect on Sl3 binding (Table 1). Absence of the terminal N-acetylglucosamine-glucose dissacharide of the core (TV161) eliminated 9X174 binding but still permitted a significant and reproducible, though small binding of S13. Prefiltering S13 through Millipore filters did not eliminate this binding. Mutants with more profound defects in the core failed to bind either of the phages.

TABLE

1

EXTENT OF BINDING OF 6X174 AND S13 TO S. typhimurium AND E. coli CELLS AND LPS” BaC-

terium

Phage sensitivityb

Strain no.

I-I .-; I-

Phagebound, % of input

6X174

:&

S. typhimurium

ReS ReS

Sens Sens Ressens ReS

E. coli

Res Res Sens Res

!

SL1027 SL901 SH180 SL733 TV161

0 0 38 16 0

TV148 SL869 SL1032 HF4704 5274

0 0 0 60 0

r-1_I

s13

LPE 0

24 10

0

1 I -

18 0

Cellr ,PS 0 0 47 47 6 0 0 0 62 0

0 36 27

0

25 0

“32P-Labeled $X174 or S13 phage (1.5 x 10’ PFU) were incubated with 1 x 10’ cells (log phase) or 10 pg of LPS in 1 ml of M medium containing 2 mM CaCl, for 15 min at 37”. Binding was determined as described in Fig. 2. Results are expressed as percent of input phage bound. Background binding of phage to the filter was 4%; this amount was subtracted from all the values. The low value of S13 binding to TV161 cells was reproducible; values of 6,7, 10, and 12%were obtained in four separate experiments. Prefiltering the phage did not reduce this binding; values for prefiltered phage and untreated phage, were 13% and 12%, respectively. bRes, resistant to infection (plaque formation) by @X174 and S13; sens, sensitive to infection (plaque formation) by 4x174 and S13; res-sens, resistant to 4x174, sensitive to S13.

274

JAZWINSKI,

LINDBERG

AND KORNBERG LPS, big

0.01 I

0.1 9

‘If1

1.0 ’

100

10

“‘1

’

111,

1

(II

FIG. 4. Number of 4x174 receptor sites in E. coli and S. typhimurium cells and LPS. Extent of binding of 32P-labeled phage to log phase E. colis and S. typhimurium (SH180 and SL733) cells and LPS was determined with 2 x lo9 PFU in 1 ml of M medium containing 2 mM CaCl, as described in Fig. 2. Background binding of phage to the filter (approximately 4%) was subtracted from these values. The scales on the abscissa (LPS and cells) are not superimposable.

2). Lack of the terminal N-acetylglucosamine reduced the extent of eclipse of 4x174, while having little effect on S13 eclipse. In addition, S13 was eclipsed to a limited extent by cells lacking the terminal disaccharide of the core and this was not eliminated by prefiltering the phage. The results measuring phage sensitivity by the extent of binding were confirmed in studies of kinetics of binding (Table 3). Binding was determined at lo”, since at 37” over 80% of the binding was complete within 1 min. The rate constant for $X174 binding to S. typhimurium (or LPS) with a complete core was of the same order as for E. coli’ cells, but lo-fold higher than for cells (or LPS) lacking N-acetylglucosamine. The differences in binding rate constants for S13 with these three strains (and their LPS) were less striking. rate of S13 to S. typhimurium

The binding

lacking the terminal disaccharide of the core was too low to be measured at 10”. The temperature-dependence curve of 4X174 eclipse by cells possessestwo slopes (Newbold and Sinsheimer, 1970b). This raises the additional question of whether the difference in the extent of eclipse of @X174 and S13 by cells and LPS lacking N-acetylglucosamine is. a function of temperature.

Eclipse

of both

phages by S.

TABLE 2 AND S13 BY S. twhimurium E. coli CELLS AND LPS”

ECLIPSE OF 6x174 Bacterium

Phage sensitivityb

Strain no.

Phage eclipse, % of input

T :e11s LPS

S. typhimurium

ReS Rt?S

Sens Sens Ressens Res Res Res Sens Res

SL1027 SL901 SH180 SL733 TV161

AND

0 0 75 32 0

0 77 19

s13

:e11s ,PS 0 0 67 63 9

0 56 52

0 0 0 0 0 0 0 0 71 72 62 50 E. coli 0 0 0 0 -“32P-labeled 4X174 or S13 phage (2 x lo* PFU) were incubated with 1 x 10’ cells (log phase) or 2 pg of LPS in 0.1 ml of M medium, containing 2 mM CaCI, for 15 min at 37”. Eclipse was determined as described in Fig. 3. Results are expressed as percent of input 3*P made acid soluble after treatment with micrococcal nuclease. Values for endogenous content of eclipsed particles in the phage preparation (6%) have been subtracted. b Res, resistant to #X174 and S13; sens, sensitive to @X174 and S13; res-sens, resistant to @X174, sensitive to S13. TV148 SL869 SL1032 HF4704 5274

4X174 TABLE BINDING

KINETICS

Bacterium

S. typhimurium

E. coli"

OF @X174

AND

3 AND S13 TO SENSITIVE

strain number

SH180 SL733 HF4704

S.

Binding rate constant (x 10’“ml.min-l) 4x174

s13

1.24 0.14 0.69

0.05

0.18 0.09

a %lp-Labeled 6X174 or S13 phage (2 x loo PFU) were incubated with 1 x lo9 cells (log phase) in 1 ml of M medium containing 2 mM CaCl, for 1, 5, 10, 15, and 20 min at 10”. Binding was determined as described in Fig. 2. Background binding of phage to the filter was 4%; this amount was subtracted from all the values.

typhimurium possessing or lacking Nacetylglucosamine, by E. colis cells and by LPS from each type was determined at various temperatures (data not shown). With both cells and LPS, the extent of eclipse of 4X174 by the strain with the incomplete LPS core was significantly lower at all temperatures between 10” and 37”. In contrast, S13 was eclipsed to the same extent by all three strains, regardless of temperature. We conclude that the differences in eclipse of 4X174 and S13 on cells and LPS lacking the terminal Nacetylglucosamine do not depend on temperature. Lipid A is Not Essential for Phage Binding but is Essential for Eclipse LPS was altered by (i) alkaline removal of ester-linked fatty acids (but not amidelinked @-hydroxymyristic acid) disaggregating the LPS and reducing molecular weight from 1 to 10 x lo6 down to approximately 1 x 10” (Alk-PS), and (ii) acidic cleavages between the KDO residues yielding a polysaccharide (Acid-PS) of approximately 1.5 x lo3 molecular weight (Fig. 1). Binding of Alk-PS to 4X174 was measured by velocity sedimentation in a sucrose gradient (Fig. 5). Phage sediment near the bottom of the gradient, while PS fractions remain at the top (data not shown). Alk-PS from S. typhimurium cells with the complete core was bound to the

275

S13 RECEPTOR

phage whereas binding of Alk-PS from E. coliR was not detected (less than 0.1 of the value for S. typhimurium). The binding is, therefore, dependent on the polysaccharide receptor and not on the presence of the ester-linked fatty acids. Binding of Alk-PS to phage requires CaCl, (data not shown). Determination of the number of molecules of Alk-PS from S. typhimurium and E. coli” bound per phage particle is complicated by aggregation of this PS fraction. Acid-PS of S. typhimurium or E. coli’ are murium with complete core) was bound to the phage whereas the Acid-PS from resistant cells (E. coliR) was not (Fig. 5). Approximately 100 moleculesz of Acid-PS of S. typhimurium or E. coli” are bound per phage particle. The binding is specific for the dX174 particle, since Alk-PS and Acid-PS from sensitive S. typhimurium or E. coli’ do not bind to F2, an icosahedral RNA phage (data not shown). Although Alk-PS and Acid-PS retain the capacity for binding to phage, they were inert in supporting eclipse of 4X174 and S13 (Table 4). The Receptor Structure in E. coli The receptor for binding &1X174 and S13 in S. typhimurium is: (GlcNAc) 1 Glc+

Gal Gal+

1 Glc+

Hep I Hep+ I P

P - P - EtN I Hep-+KD0.3

Information on the LPS receptor structure is not available for E. coli. Although E. coli’ and the LPS prepared from it exhibit virtually identical binding and eclipse properties to the appropriate S. typhimurium strains, and the Alk- and Acid-PS prepared from E. coli’bind phage as efficiently as comparable fractions from ‘The number of Alk- and Acid-PS molecules bound per phage particle may be underestimated because the amount of polysaccharide in the phagereceptor complex was not determined at equilibrium. 3 Mild acid hydrolysis cleaves between KDO-residues; the ring of the @-1,5-linked KDO residue remaining with the polysaccharide is opened (Hlmmerling, Lehmann, and Liideritz, 1973).

276

JAZWINSKI,

ALK-PS

LINDBERG AND KORNBERG

FROM PHAGESENSITIVE CELLS 6HlSO)

ALK-PS

I

ACIO-PS

FROM PHAGE-RESISTANT CELLS (&I P

FROM PHAGESENSITIVE CELLS WSO)

ACIO-PS

FROM PHAGE-RESISTANT CELLS (E.EOIIR)

10,cm

o I

8,000

i

loo

I”

i

% Go

1

0 40

FRACTIONS

FIG. 5. Binding of &X174 to LPS lacking ester-linked fatty acids and to LPS lacking KDO and Lipid A. *T-labeled phage to log phase E. colis and S. typhimurium (SHl80 and SL73) cells and LPS was determined with 2 x 10s PFU in 1 ml of M medium containing 2 mM CaCl, as described in Fig. 2. Background binding of phage (8 x 10”’ PFU) was incubated with 10 fig of [aH]Acid-PS (LPS hydrolyzed in acid as in Materials and Methods) in 0.1 ml M medium containing 2 mM CaCl, and 0.3% albumin for 1 hr at 37” (in C and D). Samples were layered onto 5% to 20% linear sucrose gradients containing 10 mMTris-HCl (pH 7.5)-l mMEDTA-2 mM CaCl, with a 5 MCsCl shelf at the bottom and centrifuged in a Spinco SW40 (in A and B) or SW 41 (in C and D) rotor for 2 hr at 40,000 rpm (5”). Gradients were fractionated by puncturing the bottom of the tube with a 22-gauge needle and collecting 6-drop fractions directly into vials; then 0.7 ml water and 5 ml Triton-toluene scintillation fluid were added and radioactivity measured. Sedimentation is from right to left. The vertical arrows indicate the position of eclipsed phage in the gradient, which were present in the phage preparation to begin with and did not arise through the action of Alk- and Acid-PS. The PS fractions from sensitive cells appear to bind to eclipsed phage.

S. typhimurium with the complete core, the LPS structures appear to be unrelated by hemagglutination-inhibition tests. LPS from E. coli’ (>250 pg/ml) did not inhibit hemagglutination employing sheep red blood cells coated with LPS and homologous antisera representative of all classes of

S. typhimurium mutants shown in Fig. 1; homologous inhibition required only l-2 pg LPS/ml (data not shown). Yet this immunochemical distinction could be due to a minor difference in structure, as in the configuration of the anomeric residue at the terminal nonreducing end.

277

$X174 AND S13 RECEPTOR TABLE

4

REQUIREMENT FORLIPID A FORECLIPSEOF 4X174 AND S13” LPS

Source

Phage

Strain no.

Phageeclipse, % of input

sensitivity”

Untreated

S. typhimurium E. coli” E. colP

Al%-PS Acid-PS

All strains’ All strains’

Sens Sens Res

$X174

s13

76 84 0

42 39 0

0 0

0 0

SH180 HF4704 5274

a 3ZP-labeled @X174 or S13 phage (3.8 x 10’ PFU) were incubated with 10 c(gof LPS, Alk-PS, or Acid-PS, in 0.1 ml of M medium containing 2 mM CaCl, for 15 min at 37”. Eclipse was determined as described in Fig. 3. Results are expressed as percent of input s*P made acid soluble after treatment with micrococcal nuclease. Values for endogenous content of eclipsed particles in the phage preparation (6%) have been subtracted. Alk-PS and Acid-PS were prepared as described in Materials and Methods. * I&, resistant to phage; sens, sensitive to phage. e SH180, HF4704, 5274.

Association of @X174 RF with Membrane

the Outer

After binding of $X174 to its LPS receptor on the cell surface, the phage undergoes eclipse and the DNA penetrates the cell becoming associated with the cell membrane (Knippers and Sinsheimer, 1968; Loos, Tessler, and Salivar, 1971). It was not determined to what part of the membrane the DNA becomes attached nor how the DNA penetrates from the outside of the cell to an intracellular site. Is the phage DNA associated with the inner or outer cell membrane? Cells were infected with 4X174 and M13, lysed, and the membrane fractions separated by sedimentation to equilibrium in a sucrose gradient (Fig. 6). The position of the inner membrane is usually defined by DPNH oxidase activity, while that of the outer membrane by phospholipase activity (Osborn et al., 1972). 4X174 DNA banded in a position distinct from that of M13, a marker for the inner membrane (Jazwinski, Marco, and Kornberg, 1973), sedimenting with phospholipase activity. 4X174 DNA, therefore, appears to be attached to the outer cell membrane. Since this fraction of outer membrane contained substantial amounts of DPNH oxidase, it may represent regions of fusion with inner membrane. Such a membrane fraction containing both phospholipase and DPNH

oxidase, is not simply a result of technically poor separation, because it was found in several E. coli C strains tested, infected or not, but not in E. coli Kl2 strains (5274, HfrC71). The superposition of phospholipase activity and 4X174 DNA in gradients was variable in several experiments, the DNA peak being either coincident with the enzyme activity or displaced up to three fractions toward the light side of the enzyme peak. We attribute this to heterogeneity of membrane fragments and vesicles in membrane preparations. Bayer and Starkey’s (1972) electronmicroscopic studies showed $X174 adsorbed at points of fusion of outer and inner membranes; the pattern seen in Fig. 6 may be another demonstration of this. When the outer-membrane fraction containing $X174 DNA was lysed with detergent and the DNA analyzed by velocity sedimentation in a sucrose gradient (Fig. 7) it contained RF as well as phage and SS, the latter derived most probably from disruption of eclipsed phage. DISCUSSION

4X174 and S13 have a broad lytic spectrum. They infect and lyse rough strains of S. typhimurium as well as E. coli C and E. coli with complete cores Rl to 4 and Shigella flexneri with a complete core (Lindberg, Jazwinski, and Kornberg, un-

278

JAZWINSKI,

LINDBERG AND KORNBERG

OUTERMEMBRANE

INNERMEMBRANE

--

FRACTIONS

FIG. 6. Association of 6X174 DNA with outer-membrane fraction. [3H] Thymine-@X174 and [32P]M13 phages were used at m.o.i. of 4 and 20, respectively, to coinfect E. coli HF4704 F+ cells (5 x lOe/ml, preincubated for 10 min at 37” with 200 fig/ml chloramphenicol) in M medium containing 2 mM CaCl, for 20 min at 37”. The cells were chilled to 0” and washed three times with 10 mM Tris-HCl (pH 7.5)-l mM EDTA (O’), and resuspended in the same buffer at 5 x 10B/ml. The cells were disrupted in a French pressure cell at 20,000 psi and the membranes collected by centrifugation in a Spinco type 30 rotor for 30 min at 30,000 rpm (5”). The membranes, resuspended in 10 mM Tris-HCI (pH 7.5)-l mM EDTA, were applied to a gradient formed with these layers: 6.5 M CsCl, 70, 65,60, 55,50,45,40, and 30% (w/v) sucrose (1 ml each) containing 10 mkf Tris-HCl (pH 7.5)-5 mM EDTA. The gradient was centrifuged to equilibrium in a Spinco SW40 rotor for 18 hr at 36,000 rpm (5”) and fractionated by puncturing the bottom of the tube with a 20-gauge needle and collecting ‘i-drop fractions. Aliquots of the fractions were spotted on Whatman 3MM filter discs, dried and radioactivity measured by scintillation counting. The vertical arrow indicates the position where 99% of the host DNA bands in the gradient. Sedimentation is from right to left.

published results). The core structures of these E. coli and Sh. flexneri strains are all different from that of S. typhimurium. These phages initiate infection by binding to specific receptors on the bacterial surface. This binding is followed by an irreversible conformational change in the phage particle (eclipse) manifested by susceptibility of the phage DNA to nuclease attack (Newbold and Sinsheimer, 1970a). Phage DNA penetration into the cell depends on concomitant DNA synthesis (Francke and Ray, 1971). The results to be discussed deal with the structure and location of the 4X174 and S13 binding receptors and the elements of receptor structure required for eclipse. The Phage Receptor The 4X174 and S13 receptors are located

in the lipopolysaccharide (LPS) of the cell outer membrane in E. coli and S. typhimurium (Figs. 2, 3; Tables 1, 2). Binding of the phages to LPS extracted from bacteria susceptible to 4X174 and S13 triggers phage eclipse (Table 2). There are 31-55 @X174 binding sites per cell in E. cob, but disruption of the cell or extraction of the LPS exposes several times that number (Figs. 2, 4). Even so, the number of binding sites determined is only 0.01% of the approximately lo6 LPS molecules in the cell (based on a molecular weight of 12,000) (Rothfield and Romeo, 1971). The number of binding sites per cell determined by the ultrafiltration method is in agreement with the value obtained by sedimentation of phage-cell complexes (Jazwinski, Lindberg and Kornberg, unpublished results). A similar assessment

279

4x174 and S13 Receptor

@X

Ml3 PHACE MARKER

FRACTlONS

defects affecting earlier steps in the biosynthesis of core polysaccharide do not bind the phage, an observation consistent with their known resistance to &X174 infection. Addition to the core of even a single tetrasaccharide unit of the 0 side-chain, as in the semirough strain, prevented 4X174 binding and eclipse (Tables 1, 2). The receptor for phage S13 resembles that for @Xl74 except in two ways: (i) absence of the terminal N-acetylglucosamine does not reduce binding and eclipse, and (ii) absence of the terminal disaccharide still permits the LPS to serve, although at a associ- much reduced level (Tables 1, 2).

FIG. 7. Replicative form of +X174 DNA is ated with the outer-membrane fraction. E. coli HF4704 F+ cells were infected with [“‘PI &X174 phage as in Fig. 6. After 20 min, cells were converted to spheroplasts, lysed by osmotic shock according to Osborn et al. (1972), and then applied to a sucrose equilibrium gradient as in Fig. 6. An aliquot of the outer membrane fraction containing 32P-labeled &X174 DNA was treated with 0.5% Sarkosyl for 10 min at 37O and [WIthymine-labeled Ml3 phage was added as a marker. The sample was applied to a 5%20% linear sucrose gradient containing 10 mM Tris-HCl (pH 8)-l mM EDTA-1 A4 NaCl with a 5 M CsCl shelf at the bottom and centrifuged in a Spinco SW40 rotor for 16 hr at 25,000 rpm (5”). The gradient was fractionated and radioactivity measured as in Fig. 5. Sedimentation is from right to left.

with phage-LPS complexes was difficult to make. Since about 35% of the phage, adsorbed to cells, can detach spontaneously in eclipsed form (Newbold and Sinsheimer, 1970a), our binding-site determinations must be considered semiquantitative estimates. Structure

of the Receptor

The structure of the +X174 and S13 receptors in S. typhimurium was analyzed with a series of isogenic mutants defective in the LPS polysaccharide chain (Fig. 1). Optimal binding and eclipse of 4x174 was observed with cells (or purified LPS) possessing an exposed complete core polysaccharide. A mutant lacking the terminal N-acetylglucosamine, bound phage only half as well (Tables 1, 3). Yet the number of binding sites measured with cells or LPS (expressed in cell equivalents) is approximately the same in strains with or without a complete core (Fig. 4). Mutants with

Lipid A Requirement Binding

for Eclipse

but Not

Polysaccharide fractions, obtained by alkaline or acid hydrolysis (Alk- and Acid-PS) of LPS extracted from $X174susceptible strains, bind to the phage, whereas corresponding fractions from resistant bacteria fail to bind (Fig. 5). As many as 1000 molecules of the Alk-PS may bind per phage particle. This large number may be due to aggregation of the polysaccharide fraction in the presence of the calcium ions required for $X174 binding. (Even in the absence of added divalent cations, the molecular weight of the polysaccharide fraction, determined by gel filtration, indicates aggregation of about 10 Alk-PS molecules). Approximately 100 molecules of the Acid-PS bind per phage, compared to only a tenth as many (near background value) of the polysaccharide from a 4X174-resistant bacterium (Fig. 5). Thus, about 10 molecules of Acid-PS are bound per molecule of the gene H spike protein, the phage component which recognizes the receptor (Jazwinski, Lindberg, and Kornberg, 1975). The following component of the LPS core of S. typhimurium contains the structure essential for binding: (GlcNAc) 1 Glc -+ Gal-r

Gal 1 Glc+

Hep 1 Hep-+

P-P-EtN 1 Hep +KDO.

I P

Possibly, a structure further stripped from

280

JAZWINSKI.

LINDBERG

right to left might still permit phage binding. Removal of the ester-linked fatty acids of the Lipid A component of LPS destroys its capacity to eclipse the phages (Table 4). This loss of activity may reside in change in physical state of the LPS. Extracted LPS has a marked tendency to aggregate and exists as a micellar structure in solution. The molecular weight of LPS from the mutant strain with a complete core (SH180) is from 1 to 10 x lo6 compared to a value of 12,000 estimated for the LPS molecular unit. Loss of fatty acids from Lipid A may prevent the formation of micelles and the interactions they may support in the eclipse of a phage particle. This is consistent with the finding that @X1,74and S13 infectivitiy is inhibited by LPS extracted from phage-susceptible strains but not by the polysaccharide fractions obtained by treatment with alkali or acid (Jazwinski, Lindberg, and Kornberg, 1975). Location of the LPS Receptor LPS together with lipoproteins and phospholipids are the chief components of the outer membrane of gram-negative bacteria. The LPS is not known to be covalently linked to the peptidoglycan network but is bound primarily by physical forces, i.e., ionic or hydrophobic bonds. The molecular architecture of the outer membrane and the distribution of LPS molecules, more specifically the receptor sites, is not understood. Results from studies using ferritin-conjugated antipolysaccharide chain antibodies reveal the surface to be densely populated by polysaccharide chains which would indicate that the entire surface is a mosaic of receptor sites (Miihlradt et al., 1973). We have found, however, only 31-55 binding sites per cell (Figs. 2, 4). This figure is in the same range as the number of $X174 membrane sites in the cell (Knippers and Sinsheimer, 1968), but is in contrast to the large number (more than 1 x 106) of LPS molecules on the cell surface. This suggests that there is only a limited number of binding sites per cell which permit phage infection. Bayer and Starkey (1972) concluded from electron-microscope

AND KORNBERG

analysis of +X174 binding that the phage preferentially binds to points of adhesion or fusion between the cytoplasmic membrane and the cell wall. These adhesions represent less than 6% of the total cell surface. When E. coli HF4704 F+ cells are infected with +X174 and the membrane fractions separated, the phage particles and phage DNA band in the outer membrane region, in contrast to the DNA from Ml3 which is associated with the cytoplasmic membrane fraction (Figs. 6, 7). The outer membrane fraction contains, however, significant levels of cytoplasmic membrane material. The membrane fraction to which 9x174 DNA is bound may represent the wall-membrane fusions. Newly synthesized LPS appears preferentially at these fusions (Mtihlradt et al., 1973). We propose that the phage binds to newly synthesized LPS at the fusions. Binding of the phage at the fusions would explain the facility with which the phage DNA penetrates the cell to reach specific intracellular loci. Furthermore, the invading phage DNA may appropriate the replication enzymes located with replicating host DNA at certain loci (Jazwinski, Marco, and Kornberg, 1973; Worccl and Burgi, 1974). The phage component responsible for directing the phage DNA to these loci may be the gene H spike protein which recognizes the LPS receptor (Jazwinski, Lindberg, and Kornberg, 1975). ACKNOWLEDGMENTS This work was supported in part by grants from the National Institutes of Health and the National Science Foundation. A. Lindberg was an International Fellow of the National Institutes of Health. REFERENCES BAYER, M. E., and STARKEY, T. W. (1972). The adsorption of bacteriophage $X174 and its,interaction with Escherichia coli; a kinetic and morphological study. Virology 49, 236-256. BROWN, D. T., MACKENZIE, J. M., and BAYER, M. E. (1971). Mode of host cell penetration by bacteriophage &X174. J. Virol. 7. 836-846. DR~GE, W., LUDERITZ, O., and WESTPHAL, 0. (1966). Biochemical studies on lipopolysaccharides of Salmonella R mutants 3. The linkage of the heptose units. Eur. J. Biochem. 4. 126-133. FRANCKE, B., and RAY, D. S. (19711. Fate of parental &X174-DNA upon infection of starved thymine-

281

+X174 AND S13 RECEPTOR requiring host cells. Virology 44, 168-187. FUJIMW, R., and K-BERG, P. (1962). The adsorption of bacteriophage 9x174 to its host. Biophys. J. 2, 433-449.

GALANOS,C., LUDERITZ, O., and WESTPHAL, 0. (1969). A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 9,245-249. GEMSKI,P., and STOCKER,B. A. D. (1967). Transduction by bacteriophage P22 in nonsmooth mutants of Salmonella

typhimurium.

J.

Bacterial.

93,

1588-1597. HXMMJSRLING, G., LEHMANN, V., and LUDERITZ, 0.

(1973). Structural studies on the heptose region of Salmonella lipopolysaccharides. Eur. J. Biochem. 38, 453-458.

HELLERGVIST,C. G., and LINDBERG,A. A. (1971). Structural studies of the common-core polysaccharide of the cell-wall lipopolysaccharide from Salmonella typhimurium.

Carbohyd.

Res. 16, 39-48.

INCARDONA,N. L., and SELVIDGE,L. (1973). Mechanism of adsorption and eclipse of bacteriophage &X174. II. Attachment and eclipse with isolated Escherichiu coli cell wall lipopolysaccharide. J. Virol. 11, 775-782.

JAZWINSKI,S. M., LINDBERG,A. A., and KORNBERC,A. (1975). The gene H spike protein of bacteriophages @X174 and S13. I. Functions in phage-receptor recognition and in transfection. Virology 66, 000. JAZWINSKI,S. M., and MARCO, R. (1973). In vitro analysis of the steps in the uncoating and replication of @X174 DNA. Fed. Proc. 32, 491. JAZWINSKI,S. M., MARCO, R., and KORNBERG,A. (1973). A coat protein of the bacteriophage Ml3 virion participates in membrane-oriented synthesis of DNA. Proc. Nat. Acad. Sci. USA 70, 205-209. JAZWINSKI,S. M., MARCO, R., and KORNBERG,A. (1975). The gene H spike protein of bacteriophages 4X174 and S13. II. Relation to synthesis of the parental replicative form. Virology 66, 000. KNIPPERS,R., and SINSHEIMER, R. L. (1968). Process of infection with bacteriophage +X174. XX. Attachment of the parental DNA of bacteriophage +X174 to a fast-sedimenting cell component. J. Mol. Biol. 34, 17-29. Kuo, T. T., and STOCKER,B. A. D. (1972). Mapping of rfa genes in Salmonella typhimurium by ES18 and P22 transduction and by conjugation. J. Bacterial. 112, 48-63.

LINDBERG, A. A. (1973). Bacteriophage receptors. Annu Rev. Microbial. 27, 205-241. LINDBERG,A. A., and HELLERGVIST,C. G. (1971). Bacteriophage attachment sites, serological specificity, and chemical composition of lipopolysaccharides of semi-rough and rough mutants of Salmonella typhimurium.

J. Bacterial

105, 57-64.

LINDBERG,A. A., and HOLME, T. (1968). Immunochemical studies on cell-wall polysaccharide of rough mutants of Salmonella typhimurium. J. Gen.

Microbial.

52, 55-65.

LINDBERG,A. A., and HOLME, T. (1969). Studies on Salmonella lipopolysaccharides using phage inactivation technique. Colloq. Znt. CNRS 174, 141-154. LINDBERG,A. A., and HOLME,T. (1972). Evaluation of some extraction methods for the preparation of bacterial lipopolysaccharides for structural analysis. Acta Pathol. Microbial. Stand. Bt30, 751-759. Loos, L. J., TESSLER,P. M., and SALIVAR,W. 0. (1971). Host cell membrane-associated phage @X174 DNA replication. Virology 45. 339-355. LODERITZ, O., WESTPHAL,O., STAIJB, A. M., and NIKAIDO, H. (1971). Isolation and chemical and immunological characterization of bacterial lipopolysaccharides. In “Microbial Toxins” G. Weinbaum, S. Kadis, and S. J. Ajl, eds., Vol. 4, pp. 145-233. Academic Press, New York. MARCO,R., JAZWINSKI,S. M., and KORNBERG, A. (1974). Binding, eclipse and penetration of the filamentous bacteriophage, Ml3 in intact and disrupted cells. Virology

62, 209-223.

MARVIN,D. A., and SCHALLER, H. (1966). The topology of DNA from the small filamentous bacteriophage fd. J. Mol. Biol. 15, l-7. M~HLRADT, P. F., MENZEL, J., GOLECKI,J. R., and SPETH,V. (1973). Outer membrane of Salmonella. Sites of export of newly synthesized lipopolysaccharide on the bacterial surface. Eur. J. Biochem. 35, 471-481. NEWBOLD,J. E., and SINSHEIMER,R. L. (1970a). The process of infection with bacteriophage 4x174. XXXII. Early steps in the infection process: Attachment, eclipse and DNA penetration. J. Mol. Biol. 49, 49-66.

NEWBOLD,J. E., and SINSHEIMER,R. L. (1970b). Process of infection with bacteriophage 4x174. XXXIV. Kinetics of the attachment and eclipse steps of the infection. J. Virol. 5. 427-431. NIKAIDO,H. (1968). Biosynthesis of cell wall lipopolysaccharide in gram-negative enteric bacteria. Advan. Enzymol.

31, 77-124.

OS~ORN,M. J. (1966). Preparation of lipopolysaccharide from mutant strains of Salmonella. Meth. Enzymol. 8, 161-164. OSBORN,M. J. (1968). Biochemical characterization of lacking glumutants of Salmonella typhimurium cosyl or galactosyl lipopolysaccharide transferases. Nature (London) 217.957-960. OSBORN, M. J., GANDEX, J. E., P~~lsr, E., and

CARSON,J. (1972). Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J. Biol. Chem. 247, 3962-3972. RIETSCHEL,E. T., GOT~ERT,H., LUDERITZ,0.. and WESTPHAL,0. (1972). Nature and linkages of the fatty acids present in the Lipid A component of Salmonella lipopolysaccharides. Eur. J. Biochem. 28, 166-173.

282

JAZWINSKI,

LINDBERG

L., and ROMEO, D. (1971). Role of lipids in the biosynthesis of the bacterial cell envelope.

ROTHFIELD,

Bocteriol.

Reu. 35, 14-38.

R., WICKNER, W., WESTERGAARD, O., D., GEIDER, K., BERTSCH, L. L., and A. (1972). Initiation of DNA synthesis: Synthesis of 9x174 replicative form requires RNA synthesis resistant to rifampicin. Proc. Nat. Acad. Sci. USA 69, 2691-2695. SINSHEIMER, R. L. (1968). Bacteriophage 4x174 and related viruses. Progr. Nucl. Acid Res. 8, 115169. SUBBAMH,T. V., and STOCKER, B. A. D. (1964). Rough (I) Genetics. mutants of Salmonella typhimurium. Nature (London) 201, 1296-1299. WESTPHAL, O., L~~DERITZ, O., and BISTJM, F. (1952).

SCHEKMAN, BRUTLAG, KORNBERC,

AND KORNBERG

Uber die extraktion von bakterien mit phenol/wasser. 2. Naturforsch. 7b, 148-155. WILKINSON, R. G., GEMSKI, P., and STOCKER, B. A. D. (19721. Non-smooth mutants of Salmonella typhimurium: Differentiation by phage sensitivity and genetic mapping. J. Gen. Microbial. 70, 527-554. WILKINSON, R. G., and STOCKER, B. A. D. (1966). Genetics and cultural properties of mutants of Salmonella typhimurium lacking glucosyl or galactosyl lipopolysaccharide transferases. Nature (London) 217.955-957. WORCEI, A., and BURGI, E. (1974). Properties of a membrane-attached form of the folded chromosome of Escherichia coli. J. Mol. Biol. 82, 91-105.