FEMS Microbiology Letters 117 (1994) 203-206 © 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00 Published by Elsevier

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FEMSLE 05883

Effects of sub-minimal inhibitory concentrations of EDTA on growth of Escherichia coli and the release of lipopolysaccharide

C h r i s t o p h e P e l l e t i e r ,,a, P i e r r e B o u r l i o u x a a n d J e a n v a n H e i j e n o o r t b

a Ddpartement de Microbiologie et Immunologie, Facultd de Pharmacie, 92296, Chatenay-Malabry, France and b Unitd de Recherche Associde du CNRS 1131, Biochimie Moldculaire et Cellulaire, Universitd Paris-Sud, 91405 Orsay, France

(Received 16 December 1993; revision received 10 January 1994; accepted 19 January 1994)

Abstract: Release of lipopolysaccharide from E. coli was studied in the presence of sub-minimal inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA). In untreated cells no release was detected with 50 mM Mg 2+ in the medium, but a steady release of over 50% of the synthesized lipopolysaccharide was observed with 0.1 mM Mg 2+. EDTA at MIC/8 led to a 2- to 3-fold higher release, presumably by an adjustment of the concentration of unchelated Mg 2+ to a value still sustaining normal growth but giving rise to a highly unstable outer membrane. No structural difference was observed between cell-bound and released lipopolysaccharide.

Key words: EDTA; LPS; Escherichia coli; Magnesium ion

Introduction

In the course of growth of E. coli cells, ou t e r m e m b r a n e vesicles o r f r agmen t s a re r e l ea sed into the cu l tu re f luid [1-3]. I t has b e e n sugges ted tha t this s p o n t a n e o u s re lease is pe rhaps assoc ia ted with cell division, s ince the shedd ing off of ou t e r m e m b r a n e b lebs has b e e n p re fe ren t i a l ly obse rved f rom the septa l r eg ion [4]. I t is genera l ly a s sumed tha t d iva lent ca t ions a re r equ i r ed for the main te - nance of the n o r m a l s t ruc tura l o rgan iza t ion of the o u t e r m e m b r a n e by br idg ing po lyan ion ic

* Corresponding author.

l ipopo lysacchar ide (LPS) molecu les and thus avo id ing e l e c t r o n i c r e p u l s i o n [5,6]. E D T A che la tes d iva len t cat ions, thus offse t t ing the i r sta- bi l izing effects. Va r ious E D T A t r e a tme n t s of iso- l a ted E . coli cells have been desc r ibed [7-12]. Some lead to an inc reased re lease of ou t e r mem- b r a n e ma te r i a l as well as to an inc reased p e r m e - abil i ty of the o u t e r m e m b r a n e with p rese rva t ion of cell viabi l i ty [7,11], whe reas o the r s have m o r e dras t ic effects such as cell lysis [8]. In all cases these E D T A t r e a tme n t s were brief .

In the p r e se n t p a p e r a no the r a p p r o a c h was fol lowed. T h e effects of sub-min imal inhib i tory concen t ra t ions ( M I C ) of E D T A direct ly a d d e d to cul tures at the onse t of g rowth were inves t igated. In pa r t i cu la r the LPS con ten t of cells and cu l ture

SSDI 0378-1097(94)00028-P

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fluids was quantified at various stages of growth. The correlation between the extent of LPS excre- tion and the Mg 2+ concentration of the culture medium is discussed.

Materials and Methods

Bacterial strain and growth conditions E. coil strain AL46 used throughout this work

was initially isolated from an urinary tract infec- tion and characterized as a rough strain (Ra) on the basis of LPS serotyping and on the LPS mass determined by 252Cf-plasma desorption mass spectrometry [13]. Cultures (300 ml in 2000 ml flasks) were grown at 37°C under strong aeration in MT minimal medium containing per liter Tris (6.06 g), NH4CI (1 g), KzHPO 4 (0.5 g), sodium citrate (0.5 g), MgSO 4 . 7 H 2 0 (24 mg), CaCI 2, 2 H 2 0 (2.9 rag), F e S O 4 . 7 H 2 0 (5 mg) and NaCI (0.5 g). After adjustment to pH 7.2 with HC1, the medium was supplemented with 0.5% glucose. The Mg 2+ concentration, normally 0.1 raM, was in certain experiments set at 50 mM. Growth was monitored by optical density at 600 nm with a Gilford 240 spectrophotometer. The MIC of ED T A (Prolabo, Paris, France) against strain AL46 was determined in MT medium as de- scribed previously [14].

Isolation of lipopolysaccharides from culture fluid and whole cells

Cultures (300 ml) were rapidly chilled in ice and cells were harvested by centrifugation in the cold for 15 min at 10 000 × g. Supernatants were lyophilized, suspended in a small volume of water and dialysed against water for 48 h in the cold. Cell pellets were washed once with water and lyophilized. LPS was extracted from this material according to the method of Galanos et al. [15]. Extracted LPS was finally precipitated by addi- tion of water (0.3 ml). After centrifugation (20 min at 4000 × g) LPS pellets were washed once with 0.5 ml 80% phenol and twice with 0.5 ml acetone, suspended in 1.5 ml water, and cen- trifuged for 45 min at 90000 x g to remove nu- cleic acids. Pellets were suspended in 0.5 ml of water.

Quantification of LPS The LPS content of culture fluids or cell ex-

tracts was expressed in terms of their ketodeoxy- octonic acid (KDO) content, which was deter- mined according to Warren [16]. Some E. coli strains possess KDO-containing capsules [17,18]. In order to ascertain that KDO determinations were specific for LPS it was essential to verify that strain AL46 contained no capsular KDO- containing exopolysaccharide. LPS serotyping carried out by I. Orskov (Statens Seruminstitut, Denmark) failed to reveal the presence of such a structure. Its absence was further confirmed by optical microscopy examination of cells after their staining with Indian ink and by SDS-PAGE.

Results

LPS content of untreated cells and their culture fluid

The KDO content of LPS extracts from un- treated exponential and stationary phase cells was similar (Table 1) and estimated at 4 ~mol per g dry weight. This value compared well with previous data [1]. When the KDO content of culture fluids was considered (Table 1), the excre- tion of LPS appeared as an important continuous process involving 52 and 61% of the LPS synthe- sized after 6.5 and 10 h of growth, respectively. During the stationary phase synthesis and excre- tion of LPS continued. After 18 h the total amount of excreted LPS was two-fold higher than the cellular LPS content. These experiments were carried out with low Mg 2+ and Ca 2+ concentra- tions in the growth medium (10 -4 M and 2.10 -s M, respectively). To assess the role of Mg 2+ in LPS excretion, cells were grown with 50 mM Mg 2+. Under these conditions the rate and ex- tent of growth were not modified (Fig. 1), nor the cell morphology. However, praticaIly no LPS ex- cretion was detected by KDO-analysis even after a 100-fold concentration of the extracts (Table 1).

LPS contents of EDTA-treated cells and their cul- ture fluid

The MIC of ED TA against strain AL46 was estimated at 128 ~g m1-1 (3.4 × 10 -4 M) (A MIC

E C

o o ul

i - I - ra f4 z l U

i i

l i m

l - B. 0

0 . 1

a b c

0 . 0 2 i l i l l . . . . . . . , i

5 I 0

H O U R S

Fig. 1. Growth of E. coli AL46 with 0.1 mM or 50 mM Mg 2+ (a), with EDTA at MIC/16 (b) or M I C / 8 (c). EDTA was

added to cultures at the same time as the inoculum.

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cells), but to a considerable increase of the amount of LPS released into the culture fluid (Table 1). After 18 h excreted LPS accounted for approximately four times the cellular LPS con- tent. At M I C / 4 growth was greatly impeded.

Analysis of LPS To determine whether the ED TA treatment

led to modification of the LPS structure, analysis of isolated LPS material was undertaken by SDS-PAGE [19] and thin-layer chromatography [20]. Identical electrophoretic profiles were ob- served for both cellular and culture fluid LPS in controls and after ED TA treatment at M I C / 8 or MIC/16. No modification was observed by thin- layer chromatography or by 252Cf-plasma desorp- tion mass spectrometry of the polysaccharide se- cured by mild acid hydrolysis of the isolated LPS [131.

value of 256 /xg m1-1 was estimated against a K12 strain). At MIC/16 or M I C / 8 EDTA had little effect on the rate of growth, but some one on the extent of growth (Fig. 1). The presence of E D T A led to a small increase of the cellular LPS content (at most 30% for late stationary-phase

Discussion

Clearly, the release of LPS in the culture fluid was dependent on the concentration of divalent cations in the growth medium as shown by the three Mg 2+ concentrations considered. The ab-

Table 1

KDO content of E. coli cells and their culture fluid a,c

Time of harvest Control EDTA b (hours) M I C / 8 MIC/16

Cells 6.5 0.81 + 0.09 1.02 + 0.03 0.96 + 0.07 10 0.77+0.04 1.11 +0.04 0.84_+0.10 18 0.88 + 0.03 1.29 + 0.08 1.20 + 0.19 18 0.86 + 0.03

(50 mM Mg 2+)

Culture 6.5 0.87 + 0.06 1.17 5:0.09 1.25 5:0.02 fluids 10 1.23 5:0.03 3.10 + 0.10 2.44 5:0.23

18 1.68 + 0.04 4.70 + 0.14 4.63 + 0.16 18 0.00 d

(50 mM Mg 2÷)

a The KDO content of cells was expressed as mg per g bacterial dry weight and that of culture fluids was expressed as mg for a volume yielding I g dry weight of bacteria.

b EDTA was added to the culture at the same time as the inoculum (1% from an overnight culture). c The values are means with standard derivations of at least three separate experiments. d No detectable KDO even after a 100-fold concentration of the samples.

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sence of excretion at high concentration (50 mM) was undoubtly a consequence of a complete stabi- lization of the outer membrane [5,6]. The steady release of LPS material from ceils growing in minimum medium with a low Mg 2÷ content (0.05-0.4 mM) has also been observed previously with two other E. coli strains [1,2]. In the pres- ence of EDTA at MIC/8 (4.3 x 10 -5 M) the concentration of unchelated divalent cations was presumably adjusted to a critical value (ca. 2 x 10 -5 M) which could still sustain growth at a normal rate but which led to a highly unstabilized outer membrane. The extensive continuous loss of LPS was compensated by a greatly increased rate of biosynthesis. The possibility of modulating the rate of LPS synthesis at constant growth rate by varying the Mg 2+ concentration or by using sub-MICs of EDTA suggested that the stabiliza- tion of the outer membrane has a regulatory effect on LPS biosynthesis.

Acknowledgements

This work was supported by grants from the Centre National de la Recherche Scientifique (URA 1131) and the Universit6 Paris-Sud (Ac- tions Interdisciplinaires 1990). We are indebted to Dr. A. Ledur and M. Caroff for expert advice concerning the KDO determination and chemical analysis of LPS.

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