Occurrence and biochemistry of lipoteichoic acids in the genus Listeria

System. Appl. Microbiol. 9,40-46 (1987)

Occurrence and Biochemistry of Lipoteichoic Acids in the Genus

Listeria 1 G. J.

RUHLAND and

F. FIEDLER

Institut fiir Genetik und Mikrob iologie der Universitat Miinchen, 8000 Miinchen 19, Federal Republic of Germany Received June 10, 1986

Summary Lipoteichoic acids were isolated from strains of the genus Listeria, representing all valid species and different serotypes. The lipoteichoic acids consisted of hydrophilic poly(glycerophosphate) chains covalently attached to glyco- or phosphatidylglycolipids (galacrosyl ~ glucosyl ~ acyl-glycerol or phosphatid yl-(galacrosyl ~ glucosyl ~ acyljglycerol), the hydrophobic moieties of the molecules. The glycerophosphate units of the hydrophilic chains were substituted with ester-linked D-alanine and u (I-2) linked galactosyl residues. Only in Listeria grayi and Listeria murrayi were galactosyl substituents missing. In Listeria denitrificans lipoteichoic acid was absent. The results are discussed with regard to their chemotaxonomic and serological implications for the genus Listeria.

Key words: Listeria monocytogenes - Listeria ivanovii - Listeria innocua - Listeria seeligeri - Listeria toelshimeri - Listeria murrayi - Listeria grayi - Listeria denitrificans - Lipotei ch o ic ac id - Serology Chemotax onomy

Introduction

In recent years, much interest has been given to lipoteichoic acids since their interactions with bacterial and mammalian cells have been recognized (Shockman and Wicken, 1981). Lipoteichoic acids are amphiphilic polym ers of the cytoplasmic membrane s of many Grampositive bacteria (Wicken, 19 80). Typically, the hydrophilic moi etie s of the lipoteich oic acid molecules con sist of 1-3 phosphodiester-linked polymers of glycero p hosp hate residues w hich are substitu ted at C-2 w ith glycos yl residues or with ester-linked D- alan ine. The hydrophobic moieties are covalently bound to the terminal phosphomonoester of the hydrophilic ch ain s and consist of eith er glycolipids or phosphatidyl glycolipids. The lipid moiety anchors the polymers to the outside of the cytoplasmic membrane by hydrophob ic interactions . The hydrophilic pol y(glycerophosphat e) cha ins may penetrate the cell wall and cause immunological reactions on the cell surface (Lambert et aI., 19 77 ). In the genera Lactobacillus (Knox et al. , 19 70) and Streptococcus (Wicken et aI., 1 This work was partly presented at the "N inth International Symposium on the Problems of Listeriosis" (Nantes, France,

1985).

Abbrevations: HF, aqueous 60 % (w/v) hydrofluoric acid; TLC, thin-layer chromatography; GLC, gas-liquid chromatography

1963 ), lipoteichoic acids were shown to be group antigens. Recently, lipoteichoic acids were described for a serotype 1 strain of Listeria monocytogenes (Hether and Jackson, 1983). However, in a virulen t serotype 4 b strain of this spe cies lipoteichoic acid wa s not found to be a membr ane component (Wexler and Oppenheim, 19 79 ), although material extracted fro m Listeria sero type 4b ad sorbed ant ibo dies directed aga inst lipoteichoic acids of Lactobacillus (Antonissen et aI., 1981 ). It was the aim of thi s study to examine this ob viou s discrepancy and to characterize the occurrence and struc ture o f lipoteichoic acids in th e genus Listeria with regard to their sero logical and chem otaxonomic impact.

Material and Methods Bacterial strains. The Listeria strains used and their sources are listed in Table 1. Most of the strains were kindly provided by Dr. H. P. R. Seeliger, Wiirzburg, Federal Republic of Germany. Growth of cells. All organisms were grown aerobically at 37°C for 16 h in brain heart infusion broth (Merck) under aeration by shaking in Erlenmeyer flasks or by pressing sterilized air through the medium. Purity of cultures was controlled by phase contrast microscopy and plating on brain heart infusion agar.

Lipoteichoic Acids in Listeria

Extraction and purification of lipoteichoic acids. Cultures were heated for 20 min at 100 DC, in order to kill the cells and inactivate autolytic enzymes. Subsequently the cells were harvested by centrifugation (14,000 X g), washed with 0.1 M sodium acetate (pH 5.0) and disrupted by shaking with glass beads in a cell mill (Buhler, Heidelberg, Federal Republic of Germany). Lipoteichoic acids were extracted by adding an equal volume of hot 80% (w/v) aqueous phenol to the suspension of desintegrated cells and stirring the emulsion at 65 DC for 45 min (Fischer et al., 1980). The cooled mixture was centrifuged at 2000 x g for 30 min. The aqueous lipoteichoic acid containing layer was removed and extensively dialyzed against 0.05 M sodium acetate (pH 4.0). Purification of lipoteichoic acids was achieved by a modified shortened procedure as described by Fischer et al. (1983) applying hydrophobic chromatography on a column of ocryl- or phenyl-Sepharose CL-4B (Pharmacia, Uppsala, Sweden). The procedure proved to be effective because high yields of lipoteichoic acids were obtained essentially free of protein, carbohydrate and nucleic acid. It should be noted that a lipoteichoic acid from Bifidobaeterium bifidum ssp. pennsylvanicum interacted only weakly with octyl-Sepharose (Op den Camp et al., 1984). Column chromatography. Columns (2 x 30 em) of octyl- or phenyl-Sepharose CL-4B were equilibrated with 0.05 M sodium acetate (pH 4.0). Elution of the columns was performed with 0.05 M sodium acetate (pH 4.0) followed by a linear gradient of 0-66% (v/v) propanol in 0.05 M sodium acetate (pH 4.0). At a flow rate of 20 ml-h", 5 ml fractions were collected. Fractions were analyzed for phosphate, neutral sugars and optical density at 280nm. Hydrolysis with hydrofluoric acid (HF). Phosphodiester bonds of isolated lipoteichoic acids were cleaved by 60% (w/v) aqueous HF (Riedel-de Haen, Seelze) at O°C for 16 h (Lipkin et aI., 1969). After incubation, HF was removed by evaporation in vacuum and adsorbed over NaOH pellets (Fiedler et al., 1981). Lipid anchors and water-soluble products of lipoteichoic acids obtained by HF-hydrolysis were separated in chloroform/ methanol/water 1:1:0.9 (v/v) as described by Nakano and Fischer (1978). Alkaline hydrolysis of lipids. The glycolipid anchors of lipoteichoic acids were saponificated in 1 M methanolic KOH for 4 h at 100 DC Uames, 1960). Methanol was removed in vacuum at 30 DC using a rotary evaporator. and the residue dissolved in water. After acidification of the aqueous solution with 2.5 M H 2S0 4 , free fatty acids were extracted three times with an equal volume of petrolether (40-60 DC). Detection of fatty acids was achieved by gas-liquid chromatography as described below. The aqueous layers, containing the glycosidic portions of the glycolipid anchors, were subjected to further structural investigation. Thin-layer chromatography (TLC). Separation of lipids was carried out on silica gel plates (Merck 60) with the following solvent systems: solvent A, chloroform/methanoliwater, 65 :25:4, v/v (Nakano and Fischer, 1977) and solvent B, petrolether/diethylether/acetic acid, 90:20:1, v/v (Stahl, 1967). Reference glycolipids prepared from Lactobacillus casei DSM 20021 were kmdly provided by W. Fischer (University of Erlangen, FRG). Lipids were visualized on TLC plates with phenoliH 2S04 or Rhodamine B spray reagent (Stahl, 1967). Glycolipids were prepared by preparative thin-layer chromatography and eluted from silica gel successively with chloroform/methanol (2:1 and 1: 1, v/v) and chloroform/ methanol/water (65:25:4, v/v), each solvent system containing 0.1 % (v/v) acetic acid (Fischer, (977). Gas-liquid chromatography (GLC). HF-degradation products were analyzed without and after acid hydrolysis (2 M HCI, 3 h,

41

100 DC) by GLC as described by Fiedler et al. (1981). Acetylation of HF-degradation products, glycerol, reduced sugars and amino sugars was accomplished according to Albersheim et al. (1967). Quantitative determination of substances was achieved by the use of an internal standard (mannose) and an external standard mixture containing the components of the sample in defined quantities. Fatty acids were analyzed by GLC, after methylation with Methyl-S TM concentrate (Pierce chemical company, Rockford, Illinois, USA). Fractionation was carried out at 200 DC on 3% SE30 silicone on gaschrom Q, 125-150 mesh (Serva, Heidelberg, FRG) in a metal column (200 x 2 mm). Identification of fatty acids was achieved by comparison with authentic fatty acids and an identified fatty acid mixture isolated from Streptomyces griseus, which was kindly provided by R. M.. Kroppenstedt (Darmstadt, Federal Republic of Germany). Analytical procedures. Components of the lipoteichoic acids were quantified after hydrolysis with 2 M HCl (3 h, 100 DC) and treatment with alkaline posphatase (see below). Peptide bonds were cleaved by hydrolysis with 4 M HCI (16 h, 100 DC). Amino acid analyses were carried out using an animo acid analyzer (Biotronic LC 6001). Glucose, galactose and glycerol were enzymatically quantified (Bergmeyer, 1974). Total neutral sugars were measured using the method of Dubois et al. (1956). Phosphate was determined using the method of Ames and Dubin (1960). The configuration of alanine was determined by treatment of the hydrolysates (4M HCI, 16h, 100 DC) of lipoteichoic acids with D-amino acid oxidase and monitored using an amino acid analyzer. Enzymic hydrolyses. Phosphomonoesters were hydrolyzed with alkaline phosphatase (Boehringer, Mannheim, Federal Republic of Germany) in 0.1 M Tris/HCI, pH 8.0 at 25 DC for 14h. For the determination of glycosyl residues and the anomeric configurations, the following specific glycosidases and reaction conditions were used (Bergmeyer, 1974): a-glucosidase, 0.1 M acetate buffer, 1 mM EDTA, pH 6.0; ~-glucosidase, 0.1 M acetate buffer, pH 5.0; a-galactosidase, 0.1 M phosphate buffer, pH 6.5; ~-galactosidase, 0.1 M phosphate buffer, pH 7.0.(The enzymes were obtained from Sigma, Miinchen, Federal Republic of Germany, except a-galactosidase which came from Boehringer, Mannheim). One drop of toluene was added to the samples to prevent bacterial growth during incubation for 48 h at 37 DC. Assay of lipoteichoic acid carrier (LTC) activity. In order to test LTC activity, D-alanine was removed by dialysis of lipoteichoic acids against 0.1 M Tris-HCI buffer (pH 8.0) for 24 h Alanine-free lipoteichoic acids were tested as described at by Fiedler and Glaser (1974) with poly(ribitolphosphate) polymerase, prepared from Staphylococcus xylosus (Fiedler, 1981).

3rc.

Results

Detection and purification of lipoteichoic acids. For the detection and purification of lipoteichoic acids, the dialyzed aqueous layers of phenol extracts obtained from the desintegrated cells were fractionated by hydrophobic chromatography using columns of octyl- or phenylSepharose (Fig. 1). Similar elution profiles were obtained for all Listeria strains investigated, except Listeria denitrificans. The compounds not bound to the gel (peak I), showed high absorbance at 280 nm and were rich in phosphate and carbohydrate. Material which interacted with t~e ocryl-Sepharose and was eluted by the propanol gradient (peak II) contained phosphate and glycerol in

42

G. ]. Ruhland and F. Fiedler B

C

11

I

1

Table 1. Source of Listeria strains used

JO

f--------j

Species

Strain"

Serovar''

L. monocytogenes (L. m.) L. monocytogenes (L. m. ) L. monocyt ogeues (L. m.) L. ivanovii (L. iv.) L. innocua (L. i.) L. seeligeri (L. s. ) L. welshimeri (L. w. ) L. murrayi (L. mu.) L. grayi (L. g.) L. denitrificans (L. d.)

ATCC 15313c SLCC 5782 d NCTC 10527 ATCC 1911 9 ATCC 33090 SLCC 395 4 SLCC 533 4 ATCC 25401 ATCC 19120 ATCC 14870

1/2a 1/2a 4b

(I)

/

/

/

/

,,

,,

,,

,, ,, 10

Fig. 1. Chromatography of ph en ol-extracted and dialyzed mat erial from L. grayi AT CC 19 120 on octyl-Seph arose CLA B. T he ext rac t of 10 9 wet cells was applied . A: Applica tion of the sample; B: Elut ion w ith 0.05 M so diu m acetate, pH 4.0 ; C : Eluti on with a linear gradient of propanol (0-66% ) in 0.05 M sodium ace ta te, pH 4.0.

equimolar rati os. Ester-linked Dvalanine a ~d. variable amounts of carbohydrate were found to be additional constituents as described below. These findings and the fact that this mat erial did not pass the dialysis membrane were consistent with the presence of lipoteichoic acids. The fractio na tion of the dialyzed phen ol-soluble mat erial fro m broken cells of L. denitrificans yielded only a small amount of amphiphilic material (Fig. 2, peak II). Its composition differed fro m the correspo~ding fracti ~n s from the other stra ins investigated. D-alamn e wa s lacking and glycerol and pho sphate wer e pres:nt. i~ a mol ~ r rat io of 0.2. A high proportion of the arnphiphi lic matenal consisted of carbohydrate containing galactose and glucosamine (data not sho wn). These results ex~luded th: presence of a lipoteichoic acid of the usual type In L. dent-

trificans. B

1 I

c

1

1.5

ts

I

(I)

50 : 1

.w'li '0

10

.w

50

(I)

ro

00

~

100

o

Froct ion Number

Fig. 2. Chro ma to graphy of phe nol -extrac ted and dialyze d ma terial from L. denitrificans AT CC 14870 on octyl -Sepharose C L4B. The ext ract of 109 wet cells was applied. Th e elution procedure wa s equ al to Fig. 1.

5 6a

1/2b 6a

" SLCC, Special Listeria Culture Collection, Wiirzburg, FRG; NCTC, National Collection of Type Cultures, London, UK; ATCC, American Type Culture Collection, Rockville, USA. b Serovars are based on the Seeliger-Donker-Voet antigenic scheme (Donker-Voet, 1972; Seeliger and Hahne, 1979). c Type strain. J Vaccine strain from ]. Potel, Hannover, FRG (R-form derived fWIIl

Sv 1I2a, orig. No. 32146J).

Composition of lipoteichoic acids. Degradati on products of the lipotei choic acids obta ined (T able 1) by H Ftreatment and subsequent acid hyd rolysis (2 M H CI, 3 h, 100°C) were analyzed by gas chromatography. In addi tion to glycerol , galactose and glucose were found in all lipoteichoic acids exam ined (Table 2 ). After ~F-treatme.nt without acid hydrol ysis, glycerol appea red In substantial amounts, whereas galactos e was found only in negligible quantities and glucose was absent. As a prominentHFdegradat ion product, gal actopyranosy l - (a l -2 ) -glyc~ro l was detected in the lipot eichoic acids fro m all the stra ins, except Listeriagrayi and Listeria murrayi. Th e presence of a-linked galactose wa s fur ther demonstrated by the release of monomeric galactose from lipoteichoic acids sub sequent to treatment with a-galactosidase. These findings and a mol ar ratio of glycerol: phosphate = 1 (Tabl e 2) indicated the presence of pol ytglycerophosphate) chains partly substitu ted by galactose at C-2 of the glycerophosphate residu es. Th is is also suggested by the data given in Table 2 showing that maximally 40 % of the glycerophosphate residues were substituted with galactose. D-alanine was present in the lipoteichoic acids of all Listeria str ains and alan yl-glycerol was found among the HF-degrad ation products using an amino acid anal yzer. The molar ra tios of D-alanine: phosphate indicated 20 to 40 % substitution of glycerophosphate residues with D-al anine. Glucose was present in low quantities only and did not seem to be a con stituent of the hydrophilic but of the lipophilic portion of lipoteichoic acids.

Structural analysis of the lipophilic moiety of lipoteichoic acids. To obtai n prepa ration s of the lipophilic moiet y, H F-degradation products of lipot eichoic acids were extracted wit h chloroform/meth anol/wat er. Th e chloroform layers conta ining the lipoph ilic moiety of the lipoteichoic acids wer e subjected to TL C. A major frag ment (B) migrating like a dihexosyl-diacylglycerol (Fig. 3, trace j) was found in alllipoteichoic acids prepared. GLC of acid hydrolysates (2 M HCI, 3 h, 100 °C) of this frag-

Lipoteichoic Acids in Listeria Table 2. Composition and structura l features of lipoteichoic acids isolated from different Listeria strains

Species

Strain

L.m. L.m. L.m. L.iv. L.i. L.s. L.w.

ATCC 15313 SLCC 5782 NCTC 10527 ATCC 19119 ATCC 33090 SLCC 3954 SLCC 5334

L.mu. L. g,

ATCC 25401 ATCC 19120

a

b

C

Components identified by GLC Molar rarios of components to phosphate Glycerol Glucose Galactose D-Alanilh' HF HF degradadegradation tion products products hydrolyzed

Glycerol

Glycerol (Galactose)' Galactose Clactosyl-" (a l- 2)-glycerol Glucose Glycerol

C

•

.

•

•

•

•

••

•

1.08 n. d.' n. d.' 1.06 1.11 1.17 1.] 6

0.04 n.d .' n. d." 0.04 0.05 0.03 0.06

0.21 n. d.' n. d.' 0.15 0.39 0.22 0.39

0.31 n.d .' n.d .' 0.36 0.32 0.26 0.24

1.13 1.J 0

0.03 0.05

0.03 0.05

0.21 0.27

Present in negligible quantities. Authentic galactosyl-(a l - 2)-glycerol prepared from the lipoteichoic acid of Streptococcus lactis NCDO 712 was used as reference. Not determined.

ment revealed glycero l, ga lactose an d glucose in equim olar ratios. The less intensely stai ned spots dete cted on the TLC (Fig. 3, A an d C) contained equimolar amounts of galactose, glucose and glycerol (C) and glucose and glycerol (A), respectively. Th ese spo ts resulted fro m unspecific hydrolysis of acyl (C) and glycosidic link ages (A) by H F (da ta not shown). To determine the stru ctur e and the an omeric form of th e glycosidic link ages, their hydrolysis by specific glycoh ydrol ases was follow ed . The results of the sequential cleavage of the glycolipid pr epared fro m th e lipoteichoic acid of Listeria monocytogenes ATCC 15313 ar e summarized in Table 3. The data are consistent with the struc ture galacto se ~ gluc ose ~ diacylglycerol which o bvio usly is pr esent in the lipoteicho ic acids of all stra ins examined. The lipoteichoi c acids of th e strains L. monocytogens ATCC 15313, Listeria seeligeri, Listeria welshimeri, L. grayi an d L. murrayi were chec ked by TLC for the occur renc e of diacy lglycerol as a lipophilic HF-degrad at ion produ ct. A

B -

43

Table 3. Sequential cleavage of the glycolipid moiety in the lipoteichoic acid of L. mon ocytogenes ATCC 15313 by specific glycohydrolases Step I

Step II

Glycohydrolase

Liberated hexose

Glycohydrolase

a-Galactosidase

Galactose

a-Glucosidase Glucose ~ -G luco sidase -

B-Galactosidase a-Glucosidase

Liberated hexose

~ - G lucosida s e

compound exhibiting th e respecti ve RF values was detected in all the strains examined, except L. grayi and L. murrayi (Fig. 4 ). Th e identitiy of thi s com pound with diacylglycerol is a lso indicat ed by the release of glycero l upon sapo nificatio n. Thus, a substitutio n of the lipophilic portion of the lipoteichoic acids of th e Listeria strains with phosph aridylglycero l is most likely.

•

f-- O 0 0 0 0 0 0 ~.-.

~---

• ••••••

ab

Fig. 3. Identification of the glycolipid portions of lipoteichoic acids by TLC in solvent A. Glycolipids were detected by spraying with phenolIH zS0 4 reagent. a, GIc( ~ 1-6)Gal (a1-2 )GIc­ (a 1- 3)diacylglycero l; b, 1. grayi; c, 1. murrayi; d, L. innocua; e, L. monocytogenes ATCC 15313 ; f, L. ivanovii; g, 1. welshimeri; h, L. seeligeri; i, GIc( ~ 1-6 ) Gal(a1-2) acyl~GIc6(a1-3 ) dia­ cylglycerol; j, Gal(a 1- 2)GIc(al- 3)diacylglycerol.

c

d

e

f

9

h

i

Fig. 4. Detection of diacylglycerol as HF-fragment of lipoteichoic acids in Listeria strains using solvent B. For detection of compounds Rhodamine B reagent was applied. a, Blank ; b, Monopalmitoyl-rac-glyccrol; c, Dipalrnitoyl-rac-glycerol; d, 1. urelsbimeri; e, L. seeligeri, f, L. monocytogens ATe C 153 13; g, L. murrayi; h, L. grayi; i, Gal (al -2)Glc(a l - 3)diacylglycerol.

44

G.]. Ruhland and F. Fiedler

The patterns of fatty acids obtained after saponification of the lipophilic moieties were similar in all the lipoteichoic acids prepared from the Listeria strains (Table 4). The major fatty acids were 15:0 anteiso and 17:0 anteiso.

Chain length of lipoteichoic acids and substitution of glycerol residues. The average numbers of glycerophosphate residues forming the poly(glycerophosphate) chains of the lipoteichoic acids examined were calculated from the molar ratios glucose: phosphate under the assumption that only one glucosyl residue was present in the lipophilic moiety of a lipoteichoic acid molecule. The 16 to 33 glycerophosphate units found per mol of glucose (Table 5) correspond to a chain length of approximately 11 to 23 nm. Table 5 also shows the degree of substitution with galactose and D-alanine of glycerophosphate residues in the hydrophilic chains. The degree of substitution was deduced from the molar ratios substituents: phosphate taking into consideration the presence of only one galactosyl residue in the lipophilic moiety. The substitution of glycerophosphate residues with D-alanine was in a range of 21 to 36% for all the Listeria strains. Galactose substituted 11 to 34% of glycerophosphate residues. However, in 1. grayi and 1. murrayi this substituent was absent.

Lipoteichoic acid carrier activity of lipoteichoic acids.

To prove the presence of lipoteichoic acids in Listeria strains by a different method we have examined the ability of the respective fractions to serve as acceptors in the in Table 4. Fatty acid compositon of the lipoteichoic acid prepared from L. welshimeri SLCC 5334, determined by GLC % of total fatty acids Fatty acid 35.5

15:0 anteiso

2.4

Xli!

2.6 5.9 32.9 4.9 4.9 6.7 4.3

16:0 iso 16:0 17:0 anteiso

18:0

a

Unidentified fatty acids

Table 5. Length of poly(glycerophosphate) chains and substitution of glycerophosphate residues with galactose and D-alanine Species

Strain

Mean chain" length

Molar ratios to glycerophosphate residues Galactose D-Alanine

L.m. L.iv, L. i. L. s. L.w. L. g. L.mu.

ATCC 15313 ATCC 19119 ATCC 33090 SLCC 3954 SLCC 5334 ATCC 19120 ATCC 25401

23 23 20 29 16 20 33

0.17 0.11 0.34 0.19 0.33

vitro biosynthesis ofpoly(ribitolphosphate) employing the purified poly(ribitolphosphate) polymerase from Staphylococcus xylosus DSM 20266 and CDp- 3H-ribitol (Fiedler and Glaser, 1974; Fiedler, 1981). Whereas the D-alanine substituted fractions of teichoic acids showed only low acceptor-activities in accordance with Fischer et aI., 1980, the alanine-free partially galactosyl substitued fractions functioned very well as acceptors in the in vitro polymerization of ribitolphosphate. This finding corroborates the chemical evidence that lipoteichoic acids occur in Listeria strains. Discussion With the exception of 1. denitrificans all Listeria strains including strain NCTC 10527 of serovar 4 b were found to contain lipoteichoic acids. The observation of Wexler and Oppenheim (1979) that no lipoteichoic acid is present in a serotype 4 b strain of 1. monocytogenes seems to be a rather unusual case. The small amount of an amphiphilic polymer detected in 1. denitrificans may indicate the presence of another type of an amphiphile but was not studied in detail. The lipoteichoic acids that occur within the genus Listeria belong to the common structural type schematically depicted in Fig. 5. Their hydrophilic moiety consists of a 1-3 phosphodiester-linked chain of glycerophosphate units, which are partly substituted with ester-linked Dalanine (21-36%) or D-galactopyranosyl residues (11-34%). However, in 1. grayi and 1. murrayi the galactosyl substituents are absent. The occurrence of galactosyl monomers as substituents of glyerophosphate residues is in contrast with the findings of Hether and Jackson (1983), who postulated the presence of onegalactose-containing disaccharide as glycosyl side chain per lipoteichoic acid molecule in 1. monocytogenes. The lipophilic moiety attached to the terminal phosphomonoester of the poly(glycerophosphate) chain corresponds to a o-galactosyl-c-glucosyl-diglyceride, which has been found as a free glycolipid in the cell membranes of 1. monocytogenes (Shaw, 1970; Kosaric and Carroll, 1971). In this lipid, galactose and glucose are 1-2 bound. Since the lipid moieties of lipoteichoic acids were found to be related to free membrane glycolipids (Fischer, 1981), we assume that in the lipid portion of Listeria lipoteichoic acids the galactosyl residue, likeweise, is attached to the hydroxyl group at C-2 of glucose, giving rise to a a-D-

--l-:~t ~~/ HO~c;;H, CH,-DH

o o

0.31 0.36 0.32 0.26 0.24 0.27

0.21

a Mean number of glycerophosphate residues per lipoteichoic acid molecule.

CH;Or

°

n

CH;-OH r~/

~O+ORI OH CH;0R, ~ 9H;o-J,-l

+o~

CH;0R,

Fig. 5. Schematic structure of the lipoteichoic acids in Listeria strains. R: Alanyl-, galactopyranosyl-, or H-, R]: Fatty acid residue, mainly 15:0 anteiso or 17:0 anteiso, n: 16-33.

Lipoteichoic Acids in Listeria ga lactopy ra nosyl(1- 2)a-D-g luco py ra nosy l disa ccharide. Anothe r common featur e of th e lipo teichoic acids in Listeria strai ns is th e fatty acy l substi tutio n, which reflects th e fatty acid composition of th e cytoplas mic membranes ide nt ified for several stra ins of L. monocytogenes (Raines et aI., 196 8). Wi th th e exc eptio n of L. grayi and L. mu rrayi, th e glyco lipid anchors o f th e lipoteichoic acids of Listeria strai ns are subs titu ted wi th a ph osphat idyl residu e. H owever , th e exact location of th e su bstituent was not clarified. Th e occur rence of a phosphatidyl substitution was pre viou sly detected in the lipoteichoic acid of Streptococcus faeca lis N ClB 819 1 (Toon et aI., 1972 ). T he lipoteichoic acids of the Listeria species exhibit struc tu ra l analogies with lip ot eichoic acids from other ba cteria. The substitution of glycero phos p hate units of the hyd rophilic chain with o -D- galactopyranosyi residues wa s also sho wn in the lipotei choi c acid fro m Lactobacillus fermenti (Wicken an d Knox, 1970) and found to be common in lipoteicho ic acids of str eptococci st rains rece ntly reclas si fied in th e new genus Lactococcus (Schleifer et aI., 1985). T he c-Dvga lacto pyra nosylt 1-2)-a-D-glucop yranosyl disaccharide as a struc tural unit in th e lipid mo iet y of a lip ot eichoic acid w as also found in Lb. [ermenti (Wicken and Knox, 19 70 ) and in lip oteichoic acids of other lactobacilli (Shaw, 19 70 ). In th e lipoteich oic acids fro m Lactobacillus casei and Lactobacillus helveticus a related trisa ccha ride has been iden tifie d: ~-D -glucosyl ( l-6) -a-D ­ ga lac tosy l(1- 2 )-a -D -gluco syl(1-3 )diacylglyceride (Nakano and Fischer, 19 78 ; Fischer et aI., 1980 ). However, in strains of Staphylococcus aureus, Streptococcus, and Bacillus th e glycosy l portion s of the lipid an cho rs of lipo teic hoi c acids differ fro m th ose found in Listeria. Whereas in Streptococcus th e disaccharid e kojibiose serves as th e glycosyl unit (Ganfield and Pieringer, 1975), gentiobiose has been found in S. aureus and bacilli (Druckworth et aI., 1975; Fischer et al., 1978 ). Alth ou gh lipoteichoic acids show a distinct structural diver sity in their hydrophilic and lipop hilic portions, a given lipoteicho ic acid is kn own to be a fairl y sta ble character istic and may be used as a taxonomic ma rke r. In th e case of th e genus Listeria the pr esence of a particul ar lipo teicho ic acid provides fur ther evidence th at this genus repr esent s a biochem ically co herent taxon. Th e occurrence of a modifie d lipoteichoic acid in L. grayi and L. murrayi may reflect th e more d ist ant rela tio nship of th ese organisms with th e othe r species of Listeria, which is shown by th e low degree of DNA-DNA rela tedness (Rocourt et al., 1982 ). T he lack of lipot eich oic acid in L. denitrificans and th e pr esence of anothe r type of an amphip hile indica tes agai n th at this orga nism needs to be rea llocated (Fiedler et al. , 1984 ; jones, 19 75; Stuart and

We/shimer, 1974).

Lipo teic ho ic acids may not playa role in th e sero logica l diffe rentia tio n of Listeria strains since thev ar e ident ical in different sero types of Listeria. Th is, in turn, supports the sugges tion that the strucut rally dive rse ribitol tei choic ac ids in th e cell walls of Listeria stra ins are important in deter mining the different O- ant igenic properties existing in th e genus Listeria (Fiedler et al., 198 4 ).

45

The fact that lipoteichoic aci ds ar e abse nt in co ryneform bac te ria (Ruhland an d Fiedler, unpublish ed dat a ) but are found in bacilli, sta ph ylococci , streptococci an d lactobacilli, ind icate s th at th e gen us Listeria sho uld be gro uped among the lat ter organisms . They form the " Clostridium " subbra nch of Gram-positive bacteria characterized by a G + C co nte nt of th e D N A lower th an 55 mo l% . T his grouping is in agreement with th e rece nt resu lts of compa rat ive seq uence ana lyses of 16 S rR N A (Ludwig et al., 1984 ).

Acknowledgement. We are indepted to Prof. Dr. H. P. R. Seeliger(University of Wiirzburg, Federal Republic of Germany) for critical reading of the manuscript.

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Professor Dr. Franz Fiedler, Lehrstuhl fiir Mikrobiologie der Universitat, Maria-Ward-Str. 1 a, D-8000 Miinchen 19