1 VTT Biotechnology, PO Box 1500, FIN-02044 VTT, Espoo, Finland
2 Department of Applied Chemistry and Microbiology, Division of Microbiology, University of Helsinki, PO Box 56, FIN-00014 University of Helsinki, Finland
Correspondence
H.-L. Alakomi
Hanna-Leena.Alakomi{at}vtt.fi
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The permeabilizing action of EDTA is usually considered to result from LPS release and from consequent perturbations of OM structure and function (Vaara, 1992), but there are still unexplained features of the mechanism. It is not known, for instance, why only a certain proportion of LPS is released. It was shown by Hukari et al. (1986)
that the macromolecular quality (LPS chain length distribution) of EDTA-released LPS vs cell-bound LPS was identical. It can, however, be postulated that the releasable fraction differs from the non-releasable one in some structural aspect that is related to the stabilizing effect of divalent cations, perhaps at the level of anionic groups in the core oligosaccaharide and lipid A.
In our studies concerning permeabilization of Gram-negative bacteria we have utilized several methods to unravel the mechanisms underlying these phenomena. In addition to measuring LPS release, the sensitization of bacteria to lysozyme and detergents is used to measure alterations in OM function (Helander et al., 1997a, 1998
; Alakomi et al., 2000
). Yet another method is the application of a nonpolar hydrophobic probe, 1-N-phenylnaphthylamine (NPN). NPN fluoresces strongly in glycerophospholipid environments, but only weakly in aqueous environments (Träuble & Overath, 1973
). This is utilized in fluorometric studies of the permeability of OMs, as increased fluorescence in suspensions of Gram-negative bacteria can be used as a measure of functional changes of the OM (Loh et al., 1984
; Helander & Mattila-Sandholm, 2000
).
By applying several independent methods to the study of EDTA-induced permeabilization in S. enterica we have noticed that mere LPS release does not account for OM alterations. The results leading to this conclusion are presented in this paper. These results have been in part presented in a poster by H.-L. Alakomi & I. M. Helander at the 6th Conference of the International Endotoxin Society, Paris, 2427 August 2000.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Test strain and growth conditions.
Salmonella enterica serovar Typhimurium VTT E-95582T (ATCC 13311T) cells were grown in LuriaBertani broth (LB) at 37 °C as described previously (Helander et al., 1997a). Experiments in LB were performed with either 2 mM CaCl2 or 2 mM MgCl2 addition or in LB without added salts. The amounts of calcium and magnesium in the LB without added salts were 0·26 mM and 0·23 mM, respectively (determined by atomic absorption spectrometry: AAS). The effect of the growth phase on EDTA sensitivity was studied by using cells harvested in different growth phases: early exponential (OD630 0·20±0·02), mid-exponential (OD630 0·50±0·02) and late exponential (OD630 0·70±0·02). After harvesting, the cells were washed in the appropriate buffer and resuspended to a standardized optical density. Efficacy of washing treatment to remove the excess salts was verified by AAS. No significant differences in the amounts of calcium and magnesium in the washed cell suspensions were observed (<0·04 mM and 0·04 mM respectively). Further details of cell treatments are given below under various experimental settings.
Permeability assays.
Two methods were utilized to determine permeability properties of the OM: (i) NPN uptake and (ii) sensitization to bacteriolysis induced by detergents or lysozyme.
(i) NPN uptake by bacterial suspensions was measured using black fluorotitre plates (cat. no. 9502 867, LabSystems) and the automated fluorometer Fluoroskan Ascent FL (LabSystems) as described earlier (Alakomi et al., 2000; Helander & Mattila-Sandholm, 2000
). Briefly, cells grown to different growth phases were deposited by centrifugation at room temperature for 10 min at 1000 g, washed with 5 mM HEPES buffer (pH 7·2), and the suspension's optical density was adjusted to OD630 0·5±0·02 with the same buffer. After centrifugation as above the cells were suspended into 0·5 vol. 5 mM HEPES buffer. Aliquots (100 µl) of this cell suspension were pipetted into fluoroplate wells containing NPN (10 µM), and as test substances either EDTA (1·0 and 0·1 mM) or HEPES buffer (control) to make up a total volume of 200 µl. If desired, MgCl2 was added to the cell suspension before addition of NPN. Fluorescence was monitored within 3 min from four parallel wells per sample (excitation, 355 nm, half bandwidth 38±3 nm; emission, 402 nm, half bandwidth 50±5 nm). Each assay was performed at least three times.
(ii) Sensitization of target cells to the lytic action of lysozyme and the detergents SDS and Triton X-100 by EDTA was investigated according to the method described in detail by Helander et al. (1997a). Briefly, bacteria at standardized OD630 0·5±0·02 were subjected to treatments with EDTA (0·1 and 1·0 mM) for 10 min at room temperature and added to microtitre plate wells which already contained either lysozyme (10 µg ml-1), SDS (0·05 and 0·1 %), Triton X-100 (0·1 and 1·0 %) or buffer only. Turbidity of the cell suspensions was then monitored with the Multiskan MCC/340 spectrophotometer (LabSystems).
Results from the permeability assays were analysed statistically using two-tailed unpaired Student's t-test to determine differences.
LPS release.
EDTA-induced LPS/lipid release was studied in different growth phases and measured by two methods.
(i) Release of LPS and glycerophospholipids. The release of lipid components, including LPS, was assayed by fatty acid analysis (gas chromatography of fatty acid methyl esters) of cell-free supernatants after treatment of the bacterial suspensions with EDTA. Cells in different growth phases were collected by centrifugation, washed with 10 mM Tris/HCl (pH 7·2) and resuspended to OD630 0·5±0·02 in the same buffer containing either 0·1 or 1·0 mM EDTA (total volume, 10 ml). The control sample was suspended in buffer only. After a 10 min incubation at 37 °C with shaking (150 r.p.m.) the samples were distributed into Eppendorf tubes and centrifuged (13 000 g) in an Eppendorf microfuge for 1 min at room temperature. A total of 9·1 ml of cell-free supernatants per sample was collected, freeze-dried and processed for fatty acid analysis by saponification and methylation as described by Helander et al. (1997a).
(ii) Radiolabelling of lipopolysaccharide, release of [14C]Gal-LPS. Smooth S. enterica Typhimurium E- 95582T cells were grown in LB at 37 °C with shaking (200 r.p.m.) to the desired growth phase and supplemented with [14C]galactose (0·1 µCi ml-1) in order to label their LPS (Hukari et al., 1986). Labelling with [14C]galactose was performed for 5 min at 37 °C with shaking (200 r.p.m.). A 1 ml aliquot was then removed for checking the level of incorporation of radiolabel (Wallac 1410 Liquid Scintillation Counter, Pharmacia). The remaining cells were collected by centrifugation (1000 g, 10 min, 25 °C), and washed with 10 mM Tris/HCl buffer (pH 7·2) at room temperature. After centrifugation the cells were resuspended in the same buffer to OD630 0·5±0·02, divided into three portions and supplemented with either 1·0 or 0·1 mM EDTA; buffer was used as control. After adding the test substance, the assay suspensions were incubated at 37 °C for 10 min with shaking (100 r.p.m.). Samples (2x20 µl) were taken for radioactivity measurements and remaining cells were centrifuged twice (1500 g) at room temperature. After centrifugation, samples from the cell-free supernatants were taken for radioactivity measurements. The amount of radioactivity in the cell-free supernatant was taken as the measure of liberated LPS and the percentage value for LPS release was calculated by comparison to the radioactivity of a similar volume of untreated and uncentrifuged bacterial suspension. Whereas it is known that the buffer used, Tris, a bulky primary amine, alone at high concentrations (50 mM or higher, pH 7·2) can cause release of some LPS and to make bacterial cells somewhat more sensitive to various agents (Vaara, 1992
), its concentration in these experiments was only 10 mM, and we observed no additional sensitivity due to the buffer.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fig. 1 shows results obtained in the NPN assay with cells grown in LB with salts and without salt additions. Fig. 1(a)
shows that the NPN uptake by cells grown in LB did not differ significantly at different growth phases (fluorescence levels of control cells from 100 to 200 units). Upon treatment with EDTA, however, the cells showed increased NPN uptake, but this phenomenon was significant at the early exponential phase of growth only. Addition of 2 mM CaCl2 to the growth medium stabilized the OM, as indicated by the lower NPN uptake of the control cells (Fig. 1b
, fluorescence levels of less than 100 units). In general, supplementation of cells with Ca2+ during growth considerably increased their NPN uptake induced by EDTA, especially in the mid- and late exponential phases. Accordingly, significant increases in NPN uptake by 1·0 mM EDTA were observed at all growth phases, cells in the earlier phases again exhibiting higher uptakes. An EDTA concentration of 0·1 mM was obviously not sufficient to destabilize and increase the permeability of the OM in early exponential cells (OD630 0·2) grown in LB supplemented with 2 mM CaCl2, indicating the presence of massive ionic interactions within the OM (Fig. 1b
). In the presence of 2 mM added Mg2+ the results with 1 mM EDTA (Fig. 1c
) were similar but not as high as with Ca2+ addition. However, the presence of 2 mM Mg2+ during cultivation was sufficient to destabilize and increase the permeability of the target cells by 0·1 mM EDTA already in the early growth phase. Addition of 1 mM MgCl2 to the buffer used in the NPN assay abolished the permeabilizing activity of EDTA (data not shown) and the NPN uptake of target cells was at the same level as in the corresponding control cells. The conclusion from these experiments was that the growth phase has a profound effect on the bacterium's sensitivity to EDTA as assayed by NPN uptake, with early exponential phase cells exhibiting particularly high sensitivity.
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanism underlying the sensitization to EDTA in the early exponential growth phase remains unknown at present, but its existence opens up interesting possibilities concerning stability-affecting properties of OM components. Since NPN fluorescence is associated with the presence of this hydrophobic probe in a glycerophospholipid environment (Träuble & Overath, 1973), it is evident that early exponential phase cells are very susceptible to the effect of EDTA in allowing access of NPN to glycerophospholipids either directly on the OM surface or via the periplasm. If differences in the stability properties of OM components are responsible for the effects described, there should be demonstrable differences in the structure of OM components as a function of the growth phase. Such differences could be expected to be found in the fine structure of LPS, especially in the degree of substitution of phosphate groups of lipid A and the core oligosaccharide. One could postulate, in addition to the EDTA-releasable fraction, the presence of a population of LPS whose interactions with neighbouring components are disturbed by EDTA to an extent that does not, however, result in LPS release. In order to prove this, the LPS obtained from distinct points of the growth phase should be scrutinized for phosphate substituents known to be critical for OM integrity (Helander et al., 1997b
; Raetz & Whitfield, 2002
; Yethon et al., 1998
; Yethon & Whitfield, 2001b
). In addition, LPS fractions which are releasable and non-releasable by EDTA should be studied for similar fine structures. In this context it is interesting to note the recent report of Kanipest et al. (2001)
, who demonstrated in E. coli that the critical parameter determining the presence or absence of phosphoethanolamine was the CaCl2 concentration in the medium. They observed a novel CaCl2-induced enzyme that modifies the outer Kdo moiety of E. coli LPS with a phosphoethanolamine group in the presence of 550 mM CaCl2. Such a modification would increase the average resistance of LPS molecules to the releasing action of EDTA, as molecules capped with phosphoethanolamine at Kdo are less prone to be stabilized by divalent cations. Our finding that the addition of Ca2+ ions to the growth medium in some cases stabilized the OM is in agreement with this. It is equally true, however, that similar addition of Ca2+ can stabilize the OM, as already suggested by Leive (1974)
; in our experiments this was observed in late exponential phase cells by NPN uptake.
Finch & Brown (1975) observed in Pseudomonas aeruginosa with low growth rates increased sensitivity to EDTA (cell lysis) when cells were grown either under carbon limitation or in Ca2+-enriched medium. Furthermore, increased resistance to EDTA in P. aeruginosa was reported when cells were grown in Mg2+-limited medium. According to Finch & Brown (1975)
the removal of cations from the cell membrane is due to the greater affinity of the cations for EDTA than for cell membrane components. In addition, the higher stability constant for EDTA interaction with Ca2+ than with Mg2+ (10·7 and 8·7, respectively) also influences the activity of EDTA in the growth medium.
Studies of Kotra et al. (2000) employing atomic force microscopy demonstrated that the OM surface in E. coli is not uniform; i.e. LPS molecules form distinct patches with depressions in between. Furthermore, a non-uniform distribution of metal ions in the OM was implied, giving rise to local variations in the interactions between OM components. Our findings are in agreement with this view.
Another mechanism could be that efflux pumps that remove substances such as NPN from the periplasm are functionally impaired by EDTA, and that they are present in larger numbers in the early exponential phase of growth as compared to the late exponential phase. In this case the phenomenon is not one of permeabilization but rather a more indirect one.
Finally, our results demonstrate that NPN uptake assays should be carried out with cells that have been cultivated in standardized conditions, especially with respect to their growth phase.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Finch, J. E. & Brown, R. W. (1975). The influence of nutrient limitation in a chemostat on the sensitivity of Pseudomonas aeruginosa to polymyxin and to EDTA. J Antimicrob Chemother 1, 379386.[Medline]
Helander, I. M. & Mattila-Sandholm, T. (2000). Fluorometric assessment of Gram-negative bacterial permeabilization. J Appl Microbiol 88, 213219.[CrossRef][Medline]
Helander, I. M., Mäkelä, P. H., Westphal, O. & Rietschel, E. T. (1996). Lipopolysaccharides. In Encyclopedia of Molecular Biology and Molecular Medicine, vol. 3, pp. 462471. Edited by R. A. Meyers. Weinheim: VCH.
Helander, I. M., Alakomi, H.-L., Latva-Kala, K. & Koski, P. (1997a). Polyethyleneimine is an effective permeabilizer of gram-negative bacteria. Microbiology 143, 31933199.[Abstract]
Helander, I. M., Kilpeläinen, I. & Vaara, M. (1997b). Phosphate groups in lipopolysaccharides of Salmonella typhimurium rfaP mutants. FEBS Lett 409, 457460.[CrossRef][Medline]
Helander, I. M., Alakomi, H.-L., Latva-Kala, K., Mattila-Sandholm, T., Pol, I., Gorris, L. G. M. & von Wright, A. (1998). Characterization of the action of selected essential oil components on gram-negative bacteria. J Agric Food Chem 46, 35903595.[CrossRef]
Hukari, R., Helander, I. M. & Vaara, M. (1986). Chain length heterogeneity of lipopolysaccharide released from Salmonella typhimurium by ethylenediaminetetraacetic acid or polycations. Eur J Biochem 154, 673676.[Abstract]
Kanipest, M. I., Lin, S., Cotters, R. J. & Raetz, C. R. H. (2001). Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. J Biol Chem 276, 11561163.
Kotra, L. P., Amro, N. A., Liu, G.-Y. & Mobashery, S. (2000). Visualizing bacteria at high resolution. ASM News 66, 675681.
Leive, L. (1965). Release of lipopolysaccharide by EDTA treatment of E. coli. Biochem Biophys Res Commun 21, 290296.[Medline]
Leive, L. (1974). The barrier function of gram-negative envelope. Ann N Y Acad Sci 235, 109127.[Medline]
Loh, B., Grant, C. & Hancock, R. E. W. (1984). Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa. Antimicrob Agents Chemother 26, 546551.[Medline]
Nikaido, H. (1989). Outer membrane barrier as a mechanisms of antimicrobial resistance. Antimicrob Agents Chemother 33, 18311836.[Medline]
Nikaido, H. (1996). Outer membrane. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol. 1, pp. 2947. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Raetz, C. R. H. & Whitfield, C. (2002). Lipopolysaccharide endotoxins. Annu Rev Biochem 71, 635700.[CrossRef][Medline]
Träuble, H. & Overath, P. (1973). The structure of Escherichia coli membranes studied by fluorescence measurements of lipid phase transition. Biochim Biophys Acta 307, 491512.[Medline]
Vaara, M. (1992). Agents that increase the permeability of the outer membrane. Microbiol Rev 56, 395411.[Medline]
Vaara, M. (1999). Lipopolysaccharide and the permeability of the bacterial outer membrane. In Endotoxin in Health and Disease, pp. 3138. Edited by H. Brade, S. M. Opal, S. N. Vogel & D. C. Morrison. New York & Basel: Marcel Dekker.
Yethon, J. A. & Whitfield, C. (2001a). Lipopolysaccharide as a target for the development of novel therapeutics in gram-negative bacteria. Curr Drug Targets Infect Disord 1, 91106.[Medline]
Yethon, J. A. & Whitfield, C. (2001b). Purification and characterization of WaaP from Escherichia coli, a lipopolysaccharide kinase essential for outer membrane stability. J Biol Chem 276, 54985504.
Yethon, J. A., Heinrichs, D. E., Monteiro, M. A., Perry, M. B. & Whitfield, C. (1998). Involvement of WaaY, WaaQ, and WaaP in the modification of Escherichia coli lipopolysaccharide and their role in the formation of a stable outer membrane. J Biol Chem 273, 2631026316.
Received 20 February 2003;
revised 16 April 2003;
accepted 28 April 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |