Parallel study of thermal resistance and permeabilitybarrier stability of Enterococcus faecalis as a�ected by salt

composition, growth temperature and pre-incubationtemperature

I.T. Ivanova,*, S. Boytchevab, G. Mihailovab

aDepartment of Physics and Biophysics, Stara Zagora Medical Institute, Thracian University, Armeiska Str. 11, Stara Zagora, 6000,

BulgariabDeparment of Microbiology and Dairy Technology, Agrarian Faculty, Thracian University, Stara Zagora, 6000, Bulgaria

Received 24 October 1998

Abstract

In this study Enterococcus faecalis cells were grown to stationary phase in various conditions resulting in strongbut similar variations in both cellular thermoresistance and permeability barrier stability (the temperature Tm thatinduced rapid dissipation of the ion concentration gradient during constant heating). Cells grown at 17±228C were

heat sensitive and barrier labile whilst cells grown at 10±138C and 42±478C were heat resistant and barrier stable.The thermal resistance and barrier stability in heat-sensitive cells, compared to the same parameters in heat-resistantcells, remained low after an additional culture at 43±478C, indicating a persistent e�ect of culture at 17±228C. Incells grown at 10±138C, these parameters were as low as they were in the heat-sensitive cells, provided the growth

media contained an ammonium salt (1%) which thus abolished the cold acclimation. Both parameters were reducedin cells growth at increased salinity (1±3% Na and K salts) and the reduction was more pronounced during growthat 17±228C. Moreover, cells pre-cultured at 218C with increased salinity (3% NaCl) displayed strong phenotypic

e�ect during subsequent culturing which re¯ected in a 68C decrease in both the optimal temperature and maximaltemperature of growth. Compared to other bacterial strains, only a part of the change in membrane stability couldbe related to the variations in fatty acid composition. The index of unsaturation changed in accordance with the

barrier stability and survival of cells. These ®ndings support the conclusion that stability of permeability barrier asa�ected by the growth temperature, presence of ammonium and cultural conditions of progenitor cells was involvedin thermal sensitivity and temperature-acclimation of E. faecalis. # 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Thermal resistance; Permeability barrier; Temperature acclimation; Salinity; Ammonium; Phenotypic modi®cation;

Enterococcus faecalis

1. Introduction

Heat resistance and temperature-acclimation of bac-teria cells are important to biotechnology and foodsciences (Busta, 1976). In spite of the large body of

work, how bacteria are killed by heat is not under-

Journal of Thermal Biology 24 (1999) 217±227

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* Corresponding author. Fax: +359-042-3-31-98..

E-mail address: medfac-sz@mBox.digsys.6g (I.T. Ivanov)

stood (Teixeira et al., 1997). Membranes, ribosomes,nucleic acids and certain enzymes have been proposed

as cellular sites of primary injury by heat (Gould,1989). Teixeira et al. (1997) have suggested that struc-tures contained in or making up the cell membrane

could be the critical site responsible for loss in viabilityduring heating below 648C. Some reports indicate theimportance of the barrier disturbances during heat

injury of bacteria. The tolerance of plasmalema toward7.5% NaCl was lost in bacteria exposed to heat stressand the consequent reparation of this function corre-

lated the restored viability (Clark et al., 1968). The ionpermeability of liposomes prepared from cytoplasmicmembrane lipids has been found to be related to themaximum growth temperature of bacteria

(VandeVoseenberg et al., 1995).Under constant heating, the membranes of

Enterococcus faecalis cells have been found to sustain

an isothermic breakdown of their permeability barrierabout an inducing temperature Tm which was abovetheir maximal temperature of growth. In cells grown in

conditions that changed their thermal resistance, theTm also changed in correlation with the thermal resist-ance. The Tm coincided with the projected inactivation

temperature of the critical target responsible for thekilling of cells by heat. Thus, the inducing temperatureTm of this membrane event was assumed relevant tothe thermal resistance of E. faecalis (Ivanov and

Boytcheva, 1994).The permeability properties of a cell is a vital requi-

site and the ability to maintain it emphasizes resistance

to deleterious conditions. Generally, the membranes ofcells undergo an ethanol facilitated permeabilization athigh temperatures (Green et al., 1985), which in some

cases has been related to cellular thermoresistance (Wuand Walner, 1984; Ivanov and Boytcheva, 1994). Thismembrane event appears to be a general phenomena.More speci®c data about it have been obtained with

enucleated mammalian erythrocytes and their isolatedmembranes (Ivanov and Benov, 1992). For example,the permeability barrier of these membranes also

underwent breakdown over a narrow temperaturerange about a speci®c inducing temperature Tm.Kinetic data obtained with intact erythrocytes and iso-

lated membranes supported the suggestion of thermallyinduced conformation change of membrane proteins inthis event. The proteins involved were presumably

intrinsic ones inasmuch as their thermal stability,expressed by the value of Tm, was markedly a�ectedby the sphingomyelin content of erythrocyte mem-branes which strongly correlated the thermal resistance

of erythrocytes in di�erent mammal species (Ivanov,1993). A recent study (Ivanov et al., 1999) evidencedthat this change in protein conformation could be of a

pre-denaturational type in respect to the consequentheat-induced unfolding. Studying whole membranes of

Escherichia coli at the pre-denaturational temperatures(above 408C), Stowell et al. (1994) have observed a

new isotropic 31P NMR down®eld component whichapparently arose from conformation change in mem-brane proteins.

In E. faecalis, the barrier breakdown event alsoappeared to be a�ected by membrane modulationssince the Tm was strongly reduced after oleic acid sup-

plementation (Ivanov and Boytcheva, 1994). In ad-dition, the presence of any n-alkanols from themethanol to octanol series decreased the Tm of this

event to a degree depending mainly on the molar con-centration of the alcohol within the plasmalema of E.faecalis cells (Ivanov and Boytcheva, 1994) and mem-brane of enucleated erythrocytes (Ivanov and

Zlatanov, 1995). Based on these data, the barrierbreakdown event in E. faecalis could also be assumedto involve a thermally induced conformation change of

intrinsic membrane proteins, the stability of which issomehow a�ected by the lipid milieu.The data obtained so far indicate that the stability

of permeability barrier, as represented by the tempera-ture Tm, could be important in cellular sensitivity toheat. Taking this into account, we undertook a parallel

study on the changes in cellular thermal resistance andbarrier stability of E. faecalis cells grown within theentire temperature range of growth under various cul-tural conditions. E. faecalis was convenient because

the broad 10±488C temperature range for viability(Niemi et al., 1993) where it displays enormous vari-ations in its barrier stability compared to other bac-

teria strains.

2. Materials and methods

2.1. Preparation of bacteria cells

Several strains of E. faecalis were isolated and ident-i®ed in our laboratory. One of them, which did notmetabolize sorbitol, was studied in recent work. Using

a thermostat, the bacteria cells were grown at the indi-cated temperatures in MRS broth that contained pep-tone 10 g/l; glucose 20 g/l; yeast extract 5 g/l; MgSO4

0.1 g/l; MnSO4 0.05 g/l; diammonium citrate 2 g/l andthe bu�er K2HPO4 2 g/l and Na-acetate 5 g/l. Cellgrowth was followed spectrophotometrically at 600 nm.

To determine the cellular resistance to heat, only cellsharvested from cultures in the early stationary phasewere used. The stationary phase was attained after120 h growth at 118C, 55 h growth at 208C, 30 h

growth at 178C, 15 h growth at 378C and 10 h growthat higher growth temperatures.To study the impact of salt composition and concen-

tration on thermal resistance of cells, a modi®ed MRSbroth was used. It was prepared by substituting the

I.T. Ivanov et al. / Journal of Thermal Biology 24 (1999) 217±227218

original bu�er with an equimolar Tris±HCl bu�er inorder to minimize the content of Na. Salts were added

to a ®nal concentration less than 6.5% (w/v) to avoidplasmophtysis. The addition of salts did not changethe pH of growth media by more than 0.15 units, thus

the observed e�ects on thermostability were not causedby pH changes.

2.2. Preparation of spheroplasts and membrane vesicles

The Gram negative bacteria cells (E. coli andPseudomonas aeroginosa ) were twice washed in 3 mM

NaCl/70 mM sucrose media and incubated at 58C for10 h in a media containing 25% sucrose, 3 mM NaCl,2 mg/ml lysozyme (Sigma, St. Louis, MO) and 15 mM

EDTA. The spheroplasts were washed twice in amedia containing 3 mM NaCl and 25% sucrose andsubjected to a hypotonic lysis in a cold media of100 mM sucrose, 45 mM NaCl and 5 mM phosphate

bu�er, pH 7.0. The membrane vesicles obtained wereincubated at 208C for 15 min to reseal, isolated andwashed in the same media.

2.3. Test of thermal stability of plasma membrane

Thermal stability test was carried out as previouslydescribed (Ivanov and Boytcheva, 1994). Cells or iso-lated membrane vesicles were suspended in a low elec-

trolyte medium and subjected to constant heating.Upon passing through a narrow temperature intervalabout the temperature Tm that induced a loss of per-meability barrier for ions, the imposed outward ion

concentration gradient collapsed. This thermallyinduced dissipation of the ion gradient was detectedconductometrically, allowing a precise determination

of the Tm which represented the stability of mem-branes.Prior to usage, the cells were washed in excess cold

medium of 3 mM NaCl/70 mM sucrose and resus-pended in the same medium to a ®nal concentrationbetween 5 and 30 g (wet weight) cells per litre medium.In other experiments, the NaCl concentration of sus-

pension media was changed in order to impose adi�erent transmembrane gradient of ions. The suspen-sion was immediately heated with constant rate of

Fig. 1. Temperature dependence of the conductivity derivative dKs/dt of a suspension of bacteria cells (A) and their membrane ves-

icles (B). The suspension contained 10 mg/ml (wet weight) of cells (Pseudomonas aeroginosa ) or membrane vesicles and was rapidly

heated with 28C/min heating rate. The suspension conductivity Ks was continuously measured and its ®rst derivative recorded on

chart. The top temperature Tm of the right peak was considered as an indicator of the thermal stability of membranes (resistance

of cellular membranes to a heat-induced barrier impair). (A): The suspension media contained 70 mM sucrose and NaCl at concen-

trations 3 mM (1), 10 mM (2), 25 mM (3) and 50 mM (4). (B): The suspension media contained 200 mM sucrose and NaCl at con-

centrations 3 mM (1), 10 mM (2) and 50 mM (3). The vesicles were resealed with 100 mM sucrose, 45 mM NaCl and 5 mM

phosphate bu�er, pH 7.0.

I.T. Ivanov et al. / Journal of Thermal Biology 24 (1999) 217±227 219

2.08C/min. During the heating, the suspension conduc-tivity was continuously measured, di�erentiated and

the ®rst derivative obtained was recorded on chart(Fig. 1, curve 1 as a typical result). During repeatedexperiments with samples of a same probe of cells, the

reliability of determining Tm was about20.38C.

2.4. Hyperthermic killing of cells and determination of

surviving fraction

One ml of cell culture was placed in a water bath at

528C. During the heating (the warm-up time wasexcluded), aliquots were taken and diluted. Serial di-lutions were made in sterile water with 0.1% peptoneand plated on MRS agar supplemented with 0.04%

cysteine hydrochloride. After 72 h at 378C, the colonieswere counted and the survival fraction determined(Konings et al., 1984).

2.5. Gas±liquid chromatography of the fatty acids

Lipids of bacterial membranes were extracted andtheir fatty acid content analysed according to Barkerand Bowler (1991). Before usage the chloroform

extracts were stored at ÿ68C under nitrogen. Themethyl esters of the fatty acids were formed by treat-ment with 20% boron tri¯ioride/methanol reagent at608C for 1 h. The fatty acid separation was carried out

by a gas±liquid chromatography using a Pye UnicamSeries 304 chromatograph. The FFAP columns wererun from 70 to 2108C with 108C/min heating rate,

using hydrogen as a carrier gas. The peaks obtainedwere identi®ed by comparison of their retention timeswith known standards. Quantitative determinations of

the fatty acid methyl esters was obtained using aTrilab 2 (with graphics) computer integrator.

3. Results

Fig. 1(A) (curve 1) shows the derivatic conductivity

thermogram of one-step washed E. faecalis cells sus-pended in a low electrolyte medium. This shows a typi-cal record of the ®rst derivative of the electrical

conductivity as obtained with heated suspension ofcells under outward ion concentration gradient. Theconductivity thermograms of Gram positive (E. fae-

cium, Bacillus subtilis ) as well as of Gram negative (E.coli, P. aeroginosa, Yersinia entrocolitica ) bacteria cellshad similar form with two separate peaks on it (notshown). A similar thermogram which also contained

two distinct peaks was obtained by heating membranevesicles isolated from the spheroplasts of Gram nega-tive bacteria (Fig. 1(B), curve 1).

The results indicate that either peak representedthermally induced membrane processes, the ®rst one

taking place within the temperature range of growthand the second at the temperature interval where pro-

teins denature. The two peaks, the ®rst one with areduced amplitude, showed up again on the repeatedthermogram of cells that were preheated slightly above

the ®rst peak and completely disappeared when thepreheating was stopped beyond the second peak (notshown). This demonstrated at least partial reversibility

of the ®rst process and complete irreversibility of thehigh temperature process in which a denaturation ofmembrane protein was apparently involved.

According to the method applied both the low- andhigh-temperature peaks indicated an increase in con-ductivity possibly related to separate leakages of cyto-solic ions into the suspension media. The number of

preliminary washings of cells did not a�ected the sec-ond peak but strongly a�ected the amplitude of the®rst one: in twice-washed cells this peak has a reduced

amplitude and in thrice-washed cells this peak wasabsent (not shown). Apparently, the ®rst peak corre-sponded to the known removal of particular cell elec-

trolytes during the washing procedure (Marquis, 1981).The amplitude of the ®rst peak was found to be threeto four times greater in cells harvested at logarithmic

phase while in cells withdrawn at the stationary phaseit was strongly decreased or absent (not shown). Thusthe amplitude of this peak possibly corresponded tothe cytosolic concentration of ions readily permeable

through the plasmalema at the temperatures of growth.The ®rst peak took place at the same temperatureinterval where membrane lipids undergo phase tran-

sition (McElhaney, 1984) and could be ascribed to theconsequent activation of the facilitated transport ofsome electrolytes (Russell, 1989).

The second peak depended on the amplitude anddirection of the ion concentration gradient that wasimposed across the membranes of whole cells (Fig.1(A)) and isolated membrane vesicles (Fig. 1(B)) prior

to heating. Thus, it represented thermally inducedalteration of cellular membranes accompanied by abreakdown of their permeability barrier. According to

previous studies carried out with scanning microcalori-metry, the barrier breakdown event took place at thesame temperature interval where a protein denatura-

tion had been registered in isolated membranes of A.laidlawii (Melchior and Steim, 1976) and E. coli(Mackey et al., 1991). This coincidence possibly associ-

ated the breakdown of permeability barrier with theconcomitant structural changes in membrane proteins.Consequently, the top temperature Tm of the secondpeak was further considered as related to the thermal

stability of permeability barrier and membrane proteininvolved.During growth of E. faecalis in a media containing

depleted NaCl, the thermal stability of plasmalema(Tm ) strongly depended on the growth temperature Tgr

I.T. Ivanov et al. / Journal of Thermal Biology 24 (1999) 217±227220

(Fig. 2). On the other hand, thermal resistance of cellsgrown at di�erent Tgr corresponded to thermal stab-ility of their membranes (Fig. 3(A)). According to

these data, the cells grown at 11±138C and 42±468Cwere thermally resistant and had thermally stable per-meability barrier (Tm correspondingly about 638C and668C) while cells grown at 17±228C were heat sensitive

and had a labile membrane barrier (Tm about 568C).Apparently, the Tm/Tgr curve represented the processof thermal acclimation of permeability barrier and cel-

lular thermoresistance as well. According to this curve,the acclimation of cells to cold and warm environ-ments resulted in markedly increased thermal stability

of their membranes in relation to that of cells grownat 17±228C temperature range which is best consideredto be non-stressful.

Generally, the presence of NaCl in the growthmedia reduced the thermal stability of cellular mem-branes (Fig. 2) and thermal resistance of cells (Fig.3(B)) depending on its concentration. However, it did

not eliminate the particular response of membranesduring the acclimation of cells to cold and warm: at3% and even at 6% NaCl, the cells remained able to

increase membrane stability during growth at the tem-perature extremes in comparison to that found in cells

at the non-stressful temperatures (Fig. 2). The heatsensitizing e�ect of the presence of NaCl was morerestricted in thermally resistant cells than in cells

grown at non-stressful temperatures (Fig. 2). In ad-dition, the interval of non-stressful temperatures wasincreasingly broadened from 208C to 308C in presenceof NaCl (Fig. 2).

Practically the same set of Tm/Tgr curves as thatshown in Fig. 2 was obtained with cells grown in amedia in which NaCl was equimolarly replaced by

KCl, Na2SO4 and KNO3 (not shown). This result indi-cates that the same heat sensitization e�ect was pro-

Fig. 2. Temperature acclimation curve of Enterococcus faecalis

as a�ected by the presence of NaCl. The cells were harvested

from cultures in the early stationary phase, isolated, washed

and the thermal stability of their membranes (Tm ) determined

as described for Fig. 1. The cells were grown at the indicated

growth temperatures Tgr in a Tris-bu�ered MRS broth con-

taining NaCl at the concentrations 0% (q), 1% (_) 3% (r)

and 6% (w). Data for the Tm are the mean of three di�erent

experiments.

Fig. 3. Thermal resistance of Enterococcus faecalis as a�ected

by the growth temperature (A) and the presence of NaCl in

growth media (B). The cells were grown in a NaCl depleted

Tris-bu�ered MRS broth at 458C (r), 178C (q) and 118C (�)(A) and at 308C in a Tris-bu�ered MRS broth containing 0%

(q), 1% (�), 3% (r) and 6% (w) NaCl (B). After growth,

the cells were exposed to 528C for the indicated time intervals

and their colony forming ability counted.

I.T. Ivanov et al. / Journal of Thermal Biology 24 (1999) 217±227 221

duced in cells and their membranes at the same molarconcentration of any salt. This e�ect could be possiblyassociated with the increased salinity and/or osmotic

pressure of growth medium. The thermal resistance ofcells grown at 10±138C and 43±468C was slightlyreduced and that of heat-sensitive cells increased when

the growth medium contained sorbitol in concentrationproducing the same osmotic pressure as that of 3%KCl (Fig. 4). This result showed that the membrane

labilization of heat-sensitive cells that occurred in thepresence of K and Na salts appeared mainly as ane�ect of salinity. Thus, the e�ect that was produced inthe presence of Na and K salts solely consisted in a

more or less uniform decrease in cellular thermoresis-tance throughout the temperature range of growth.However, the ability of cells to respond with an

increase in their thermoresistivity during growth at thetemperature extremes was not a�ected by these salts.In the presence of MgCl2 at the same osmoactivity

as that of 3% KCl, the Tm also decreased in accord-ance to the salinity e�ect (Fig. 4). At this and even atmuch smaller concentrations (0.1%) of Mg, the ther-

mal resistance of cells growing at 43±468C wasstrongly increased (about 38C increase in Tm ) (Fig. 4).

The presence of other bivalent cations (Mn, Co, Cu) atsuch concentrations also resulted in a similar increase

in membrane stability and, as a rule, in the growthrate of cells at 42±468C (not shown). Apparently, thesedivalent cations supported the growth of cells and

increased their thermoresistance. This result was in ac-cordance with the reports that some bivalent cations(Cu, Cd, Zn) increased the thermal resistance of plant(Bonham-Smith et al., 1987) and animal (Li et al.,

1982) cells.Above 158C, the growth of cells in the presence of

ammonium salts (1% (NH4)2SO4 or NH4Cl) was ac-

companied by a membrane labilization in accordancewith the salinity e�ect and the membrane response towarm acclimation remained preserved (Fig. 4). In the

presence of ammonium ions, the growth rate at 10±138C was increased by a factor of two to three (notshown). Although ammonium supported the growth at10±138C, the membranes of cells remained as labile

during growth at 118C as during growth at 208C (theTm equals 538C in both cases) (Fig. 4). This ®ndingindicated that the membrane response that was speci®c

for cold-acclimated cells was abolished during growthwith ammonium. Apparently, ammonium displayed an

Fig. 4. Temperature acclimation curves of Enterococcus faeca-

lis as e�ected by the media composition. The cells were har-

vested from cultures in the early stationary phase washed and

the thermal stability of their membranes (Tm ) determined as

described for Fig. 1. The cells were grown at the indicated

growth temperatures in a Tris-bu�ered MRS broth containing

160 g/l sorbit (r), 2% (w/v) MgCl2 (q) and 1% (w/v) NH4Cl

(w). Data for the Tm are the mean of three di�erent exper-

iments.

Fig. 5. Temperature acclimation curves of Enterococcus faeca-

lis as a�ected by the pre-cultivation temperature. The cells

were pre-cultivated to the stationary phase at di�erent tem-

peratures: 118C (r), 188C (�), 238C (q), 378C (w) and 428C(+). These progenitor cells were inoculated and grown to the

early stationary phase at the indicated growth temperatures.

The obtained progeny cells were harvested, washed, and used

to determine the thermal stability of their membranes. Data

for the Tm are the mean of three di�erent experiments.

I.T. Ivanov et al. / Journal of Thermal Biology 24 (1999) 217±227222

inhibitory e�ect on the mechanism for temperature ac-

climation of cells growing at cold.The temperature acclimation of E. faecalis, as rep-

resented by the Tm/Tgr curves, was markedly a�ected

by the temperature of pre-cultivation (Fig. 5). The cellswere ®rst cultivated to stationay phase at the indicatedtemperature (parent cells). The parent cells were re-

inoculated (dilution 1:50) into the same growth med-ium and cultured at di�erent temperatures to ascertainthe Tm/Tgr dependence of the progeny cells. Fig. 5demonstrates a lasting impact of pre-incubation tem-

perature on the ability of cells for temperature acclim-ation during subsequent culture. Cells pre-incubated atboth the cold and warm ends of the temperature span

of growth remained adaptable and increased their ther-moresistance during the next growth in similar con-ditions. In contrast, cells pre-incubated at a non-stress

temperature (218C) demonstrated impaired ability forwarm acclimation during next culturing (Fig. 5). It isquite surprising that at the same time the latter cells

remained able to acclimate during growth at cold en-vironment (Fig. 5).These ®ndings were supported by measurements of

colony forming ability of heat-shocked cells grown at

equal but pre-cultivated at di�erent temperatures (Fig.

6). Even though the cells were pre-cultivated at verydi�erent temperatures, 208C and 408C, they had anequal thermal resistance after consequent growth atcold (118C). However, cells grown at warm (458C) dis-played markedly di�erent resistance to heat, dependingon the temperature of pre-cultivation. The cells pre-cultivated at 408C acquired high thermal resistance

during next growth at 458C, while cells pre-cultivatedat the non-stress temperatures (208C) remained heatsensitive after second culturing at 458C. Apparently

the mechanism for temperature acclimation wasa�ected by the prehistory of cells. Thus, the growth ofE. faecalis at non-stress temperatures a�ect the abilityof the next generation for warm acclimation while at

the same time preserved their ability for cold acclim-ation.Compared to the warm- and cold-acclimated cells,

the cells grown at the non-stress temperatures appar-ently undergo a phenotypic modi®cation, because theywere more sensitive to heat and salinity and were

poorly adaptable to warmth. This phenotypic modi®-cation was even more pronounced after growth underthe combined impact of both the non-stress tempera-

ture and increased salinity. This was well expressed

Fig. 6. Thermal resistance of Enterococcus faecalis cells as

a�ected by the temperature of pre-cultivation. Pairs of cell

cultures were cultivated at 208C (®rst pair) and 408C (second

pair) to stationary phase. These progenitor cells were re-

inoculated for a ®nal growth at 118C (20/11 cells and 40/11

cells) and 458C (20/45 and 40/45 cells). The progeny cells

obtained after the second growth were exposed to a heat

shock at 528C for the time intervals indicated and their colony

forming ability counted. The survival curves indicate: (q) 20/

11 cells pre-cultivated at 208C and grown at 118C; (�) 40/11cells pre-cultivated at 408C and grown at 118C; (r) 20/45

cells pre-cultivated at 208C and grown at 458C; (w) 40/45 cells

pre-cultivated at 408C and grown at 458C.

Fig. 7. Temperature dependence of growth in phenotypically

changed Enterococcus faecalis cells (w) as compared to con-

trol cells (q). The phenotypic modi®cation was produced by

pre-cultivation of cells at the non-stress temperature (218C) ina Tris-bu�ered MRS broth containing 3% NaCl. The control

cells were pre-incubated at 378C in MRS broth. The maximal

cell yield in a single culture was about 5 mg cells/ml at the

stationary phase.

I.T. Ivanov et al. / Journal of Thermal Biology 24 (1999) 217±227 223

with cells pre-cultivated at 218C in the presence of 3%NaCl (Fig. 7). In such cells, the optimal growth tem-perature and the maximal temperature of growth wereboth strongly decreased (by about 68C) compared to

control, non-modi®ed cells, pre-cultivated at 378C in amedia with low salinity. This data also substantiatedthe occurrence of phenotypic modi®cation that was

preserved across several dozen of cell generations.Possibly, a lasting change in gene expression tookplace during growth of cells at the non-stress tempera-

tures as a temperature e�ect on gene activation.Generally, the thermal resistance of bacteria has

been found related to the lipid composition and fatty

acid content of membranes (Russell and Fukunaga,1990; Sajbidor, 1997). Using gas chromatography, thefatty acid content of E. faecalis was found to varydepending on the growth temperature (Table 1). The

content of long-chained fatty acids decreased and theindex of unsaturation increased with lowering thegrowth temperature from the maximal temperature of

growth to the non-stress one. Similar changes in fattyacid composition have been reported for E. coli(Sinensky, 1974) and for B. subtilis (Herman et al.,

1994), the thermal resistance of which also depends ongrowth temperature. Despite the common pattern of

changes in the fatty acid composition of these strains,the maximal variations in Tm were, however, stronglydi�erent by amplitude: 1.5±28C in B. subtilis, 58C in E.coli and about 108C in E. faecalis (Fig. 8). These data

possibly showed that the variations in membrane stab-ility (Tm ) could be only partially related to the changesin fatty acid content of these cells.

Fig. 8 also shows that in contrast to many otherbacteria strains (B. subtilits, E. coli, P. aeroginosa andY. entrocolitica ), only in enterococci (E. faecalis and

E. faecium ) the membrane stability (Tm ) was increasednear the minimal temperature of growth. In E. faecalisgrown near the minimal temperature of growth, a

sharp rise in the content of branched-chain fatty acidswas established (Table 1). This is, however, a commonphenomena in bacteria that grow at temperaturebeneath the temperature of phase transition (Russell

and Fukunaga, 1990) when a signi®cant portion oftheir lipids are in the gel state. Consequently, theincrease in membrane stability during growth at cold,

as found in E. faecalis in particular, could not bedirectly ascribed to the concomitant alteration of fattyacid composition.

Apparently, the strong variations in membrane stab-ility of E. faecalis that took place within the entire

Table 1

Fatty acid composition and index of unsaturation in Str. faecalis cells grown at the indicated temperatures. The cells were washed

in growth media and the lipids were extracted and esteri®ed. The fatty acid esters were separated by gas±liquid chromatography

and identi®ed using known standards. The amount of di�erent fatty acids is presented in relative %

Growth conditions 10.58C MRS broth 208C MRS broth containing 3% NaCl 408C MRS broth

Index of unsaturation 0.3866 0.486 0.2275

C7(Cx) 22.6 24.7 25.8

iso C8 2.35 ± ±

C8:0 7.4 9.0 11.1

C8:1 9.4 4.49 0.74

iso C10 4.07 ± ±

C10:0 11.28 11.79 12.0

C10:1 4.12 2.06 0.97

C10:2 5.17 7.49 1.15

C11:0 6.27 8.23 2.76

C12:0 2.51 1.87 7.18

C12:1 1.57 7.02 1.52

C12:2 0.47 0.75 0.14

C13:0 3.14 ± 5.25

C14:0 1.41 1.03 0.46

C14:1 0.70 0.19 6.0

C14:2 0.47 0.75 0.14

C15:0 0.63 0.28 0.14

iso C16 4.39 1.96 0.69

C16:0 ± ± 13.86

C16:1 ± ± 0.46

C18:0 0.78 1.68 3.59

C18:1 7.21 9.83 0.97

C18:2 1.25 0.47 4.14

Total 100% 100% 100%

I.T. Ivanov et al. / Journal of Thermal Biology 24 (1999) 217±227224

temperature range of growth could hardly be associ-

ated with the concomitant changes in fatty acid com-position alone. However, another parameter ofmembrane lipids, the index of unsaturation, was foundto be closely related to the changes in membrane stab-

ility. The index of unsaturation (Table 1) also changedstrongly during growth at di�erent temperatures butalways remained strictly related to the membrane stab-

ility (Fig. 2) and cellular thermoresistance (Fig. 3(A)).Since the Tm was related to the thermal stability ofmembrane proteins, this result showed that the double

bond index, respectively, the membrane ¯uidity couldplay a substantial role in determining the thermal stab-ility of membrane proteins and thermal resistance ofthese cells.

4. Discussion

The ability to avoid loss of the selective permeabilitybarrier under thermal stress was studied in E. faecaliscells grown at di�erent environmental conditions. The

thermal stability of membranes was represented bytemperature Tm inducing a rapid dissipation of initiallyimposed transmembrane ion gradient. A technique sen-

sitive to the loss of cellular ion gradient during con-stant heating was applied that allowed precise andreliable determination of Tm. The Tm was related to

the thermal stability of membrane proteins and, as

con®rmed by the data on colony forming ability ofheat-tested cells, to the thermal resistance of cells.Compared to other bacteria strains, the thermal

stability of E. faecalis membranes demonstrated astrong and biphasic dependence on the growth tem-

perature Tgr. This outcome appears to be speci®c forsome enterococci such as E. faecalis and E. faecium.These cells cannot synthesize aminoacids, vitamins,

purynes, pyrimidines and sometimes even fatty acids.In addition, they grow in a broad temperature spanthat includes separate temperature ranges (10±158Cand 42±468C) correspondingly characteristic for phsy-crophiles and thermophiles. This should bring about

restrictions on both the uptake of nutrients and metab-olism of cells, possibly resulting in such a complicatedtype of relationship between cellular thermoresistance

and rear conditions.Apparently, the Tm/Tgr dependence demonstrated

the temperature acclimation of E. faecalis. Cells grown

at the non-stressful temperatures (17±208C) were heatsensitive, while cells acclimated to the temperature

extremes of growth were heat resistant. Recently,Flahaut et al. (1997) have demonstrated that heat waspotent inductor of stress protein synthesis in E. faecalis

strain and emphasized the role of de novo synthesizedproteins in acquired thermotolerance. In this study, thegrowing conditions at 10±138C and 42±478C could

constitute a thermal stress resulting in a production ofstress proteins. The presence of these proteins could

impact both thermal stability of membranes and survi-val of cells as well.This acclimation response of cells was, however,

speci®cally a�ected by di�erent factors. High concen-trations of ammonium (1%) abolished the response in

cold-acclimated cells, while pre-cultivation of cells atthe non-stress temperatures prevented the response inwarm-acclimated cells. These ®ndings are in compli-

ance with the view that di�erent molecular mechanismsgovern the cellular sensitivity to heat and cold. Aprime candidate for the factor determining the minimal

temperature of growth is the loss of membrane ¯uidityduring the liquid crystalline to gel state transition

which inhibits the membrane transport and cell growth(Pledger et al., 1994). Relatedly, the ability of psychro-philes to take up nutrients e�ciently at low tempera-

tures has often been mooted as a key element ofpsychrophili (Russell, 1989a). On the other hand,membrane hyper¯uidity and the loss of selective per-

meability at high temperature could be the cause ofthe upper limit of growth (Russell and Fukunaga,

1990).The inhibition of E. faecalis response to cold acclim-

ation under ammonium shock should be studied

further to obtain an insight into the molecular mechan-ism of this acclimation. It could be possibly related to

Fig. 8. Temperature acclimation curves for membrane stab-

ility in di�erent strains: Enterococcus faecalis (+);

Enterococcus faecium (q); Escherichia coli (w) and Baccilus

subtilis (r). The Tm/Tgr curve in Pseudomonas aeroginosa and

Yersinia enterococcus was similar to that in Baccilus subtilis.

The experimental variations in determining Tm were within

20.38C for each cells sample.

I.T. Ivanov et al. / Journal of Thermal Biology 24 (1999) 217±227 225

glutamine synthetase, a key enzyme in the utilizationof nitrogen in enteric bacteria, which is inhibited at

high external concentration of ammonium (Stadtmanet al., 1976).An augmented supercoiling of DNA and strongly

increased rate of protein synthesis have been reportedin E. faecalis during growth under increased salinity(6.5% NaCl) (Flahaut et al., 1996). This can possibly

be related to the ®nding that both membrane stabilityand thermal resistance of E. faecalis cells were stronglyreduced during growth under salt stress. This possibly

emphasized the role of de novo synthesized proteins inthermal stability of membranes and thermal resistanceof cells. Another ®nding also indicated the role of pro-teins in determining thermal properties of cells and

membranes. The acclimation of cells at non-stressfultemperatures under increased salinity induced phenoty-pic modi®cation that persisted in progeny cells during

next culturing. In addition to the impaired adaptationof modi®ed cells to warm (Figs. 5 and 6), their optimaland maximal temperatures of growth were strongly

reduced (Fig. 7). This could not be due to a lastingmodi®cation of lipids since the cells very rapidly read-just their lipid content in response to shift in growth

temperature by using the existing pool of enzymes.Presumably, the programme of gene expression chan-ged at particular conditions (growth at non-stressfultemperatures under increased salinity) leading to a last-

ing synthesis of less stable membrane proteins in pro-geny cells. A future study on this change of proteinsynthesis can be helpful for elucidating which proteins

are important in determining the thermal properties ofbacteria.

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