Changes in Listeria monocytogenes Membrane Fluidity in Response to Temperature Stress

Listeria monocytogenes is a food-borne pathogen that has beenimplicated in many outbreaks associated with ready-to-eat products.Listeria adjusts to various stresses by adjusting its membranefluidity, increasing the uptake of osmoprotectants and cryoprotectants,and activating the ${sigma}$ ^B stress factor. The present work examinesthe regulation of membrane fluidity through direct measurementbased on fluorescent anisotropy. The membrane fluidities ofL. monocytogenes Scott A, NR30, wt10403S, and cld1 cells culturedat 15 and 30°C were measured at 15 and 30°C. The membraneof the cold-sensitive mutant (cld1) was more rigid than themembranes of the other strains when grown at 30°C, but whengrown at 15°C, it was able to adjust its membrane to approachthe rigidity of the other strains. The difference in rigidities,as determined at 15 and 30°C, was greater in liposomes thanin whole cells. The rates of fluidity adjustment and times requiredfor whole cells to adjust to a different temperature were similaramong strains but different from those of liposomes. This suggeststhat the cells had a mechanism for homeoviscous adaptation thatwas absent in liposomes.

INTRODUCTION

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INTRODUCTION

Listeria monocytogenes is a eurythermal, osmotolerant, and pathogenicgram-positive rod. Its ability to grow at refrigeration temperaturesis a constant challenge for food preservation (9, 14, 22). Listeriaresists salt, nisin, refrigeration, and low pH after previousexposure, but it can gain cross-resistance to various stressors(2, 5, 6, 16, 21). The pathogen has been associated with a varietyof products, including milk and dairy products and ready-to-eatmeat and fish, as well as cabbage, radishes, pâté,and soft cheeses (9, 26). The Centers for Disease Control andPrevention estimates that food-related illnesses result in 325,000hospitalizations and 5,000 deaths in the United States yearly;a significant part of this is caused by L. monocytogenes (22).Due to its high mortality rate, especially among the immunocompromised,elderly people, and infants (26), the FDA set a "zero tolerance"status for L. monocytogenes in ready-to-eat foods.

Listeria monocytogenes’ ability to adjust to a wide rangeof environmental stresses has been extensively studied and reviewedin an attempt to identify and understand the factors behindthis efficient adaptation (3, 7, 11, 14). When L. monocytogenesis grown at low temperatures, the phospholipids of the cellmembrane become more rigid and can interfere with normal functionsof the membrane and membrane-associated proteins (23, 29, 32,34). To counteract this phenomenon, L. monocytogenes modifiesits plasma membrane to maintain its fluidity at low temperature(32); this is collectively termed homeoviscous adaptation (30).Low-temperature growth induces several levels of adjustmentsat the cell membrane; there are rapid enzyme-mediated adjustmentsand slower adjustments that require de novo protein synthesis.These cause an increase in the amount of odd-numbered fattyacids and branched-chain fatty acids, a switch from iso to anteisobranching patterns, and, later, a shortening of the length ofthe branched-chain fatty acids (1, 19, 20, 25). These changeslead to maintenance of constant membrane fluidity at low temperaturesby including fatty acids with lower phase transition temperatures(Tc) (12, 18, 20). Several studies have used cold-sensitive(1, 8) and nisin-resistant (7, 33) mutants to better understandthe changes that lead to their phenotypes. The cold-sensitivemutant's cell membrane is deficient in branched fatty acids,odd-numbered fatty acids, and anteiso fatty acids. The membraneof the nisin-resistant mutant has more straight chains, lessbranching, and higher Tc compared to the wild type. This furtheremphasizes the major effect of fatty acid composition on membranefluidity and cell viability at low temperatures.

Fluorescence techniques are becoming increasingly popular dueto their high sensitivity and favorable time scale and are usedto measure a variety of cell parameters, including membranefluidity (15, 31). Anisotropy measurements are used for quantizationof membrane fluidity. The hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene(DPH) is inserted in the phospholipid bilayer and moves as themembrane moves. A fluid membrane allows greater probe mobility,leading to interference with its emitted signal. By measuringthe emitted light, one can assess the rigidity of the cell membrane,which is directly proportional to the emitted intensity (21,31). In order to assess membrane properties, either liposomesor whole cells can be used. Liposomes are the most commonlyused system when studying biological membranes (10, 24, 35)as they offer the benefit of examining phospholipid membranefluidity in a system having a fixed composition. In contrast,measurement using whole cells allows the assessment of membranefluidity while taking into consideration the contribution ofphysiological changes.

Several authors have examined changes in phospholipid compositionto evaluate membrane fluidity at various temperatures (18, 20,25, 33). The present research uses static and real-time measurementsto better understand the role of membrane fluidity in the stressresistance and sensitivity phenotypes of the two mutants. Inthis study, liposome, whole-cell, and real-time whole-cell anisotropywere performed on the four L. monocytogenes variants Scott A(the wild type from which NR30 is derived), NR30 (nisin resistantat 30°C), wt10304S (a wild-type strain), and cld1 (cold-sensitivemutant derived from 10304S) to better understand the dynamicsof membrane fluidity alterations at different growth temperaturesand the effect of the nisin-resistant, cold-sensitive phenotypeson membrane fluidity compared to that of the wild-type strains.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Culture and culture conditions.
L. monocytogenes Scott A (wild type), NR30 (ATCC 700302; nisinresistant at 30°C derived from Scott A) (20), wt10403S (wildtype), and cld1 which is a cold-sensitive mutant of the wildtype with a transposon (Tn917) inserted into its branch fattyacid synthesis sequence (37) were maintained in Trypticase soybroth slants (Difco, MI) supplemented with 2.5 g/liter glucoseand 6 g/liter yeast extract. cld1 slants were supplemented with1 µg/ml erythromycin.

Lipid extraction.
Brain heart infusion broth (BHI [Difco, MI]) was inoculatedwith overnight L. monocytogenes cells and incubated at the desiredtemperature (15 or 30°C) until mid-log phase (A₆₀₀ = 0.6to 0.8). The cells were harvested by centrifugation (10 min,4°C, 9,000 x g), washed with 0.1% buffered peptone water(Difco, MI), and then pelleted by centrifugation. The extractionmethod described by Bligh and Dyer (4), modified for Listeria(36), was used for lipid extraction. In a 50-ml centrifuge tube,6 g of glass beads (<106 µm in diameter) and 0.1 NHCl-methanol-chloroform were added to the cell pellet in a ratioof 0.5:1:0.4 by volume. Mixtures were vortexed every 10 minfor 1 h. Chloroform and HCl were added to the tubes to achievea ratio of 1:1:1. Tubes were centrifuged (3,500 x g, 8 min),and the chloroform layer (below the solid layer) was collected.The pellets in the centrifuge tubes were washed with 5 ml chloroformand centrifuged, and the chloroform layer was collected again.The combined chloroform layers were neutralized with 1 N NH₄OHin methanol and dried under nitrogen. Lipids were resuspendedin chlolorform-methanol (9:1) in siliconized microcentrifugetubes, flushed with nitrogen, and stored at –20°Cfor up to 2 weeks.

Liposome construction.
The method described by Hromy and Carman (13), with modifications(36), was used for liposome preparation. A lipid aliquot ina siliconized tube was dried under nitrogen and further driedin a Speedvac (Savant, MN) for 2 h. Dry lipids were resuspendedin 50 µM 2-(N-morpholino)-ethansulfonic acid (MES [Sigma,St. Louis, MO]), and n-octyl-ß-D-glucopyranoside (Sigma)was added to achieve a 1:14 lipid/detergent ratio. Lipids weregently scraped off the sides of the tubes, which were then vortexedfor 1 h. In the meantime, 1 g Sephadex G-50 (Sigma) was hydratedfor 1 h with 40 ml of MES. Microcolumns (Poly-prep columns [Bio-Rad,CA]) were loaded with Sephadex resin and equilibrated with MES,and then the lipids were gently deposited on top of the Sephadexbed. The column was gently eluted with MES. Liposomes were formedin the void volume as the detergent was trapped in the column.Cloudy drops containing liposomes were collected in siliconizedmicrocentrifuge tubes and stored on ice for same-day use.

Steady-state fluorescent anisotropy.
Anisotropy measurement, also known as membrane rigidity measurement,is a fluorometric method to measure membrane fluidity. Anisotropyvalues were based on the emitted intensity of DPH using a Perkin-Elmerfluorimeter (PE LS 50B, CT). The lower the fluidity of the lipids,the less the membrane moves and subsequently the less the probemoves. This leads to less distortion of the emitted signal andhigher rigidity values recorded by the fluorimeter (21).

The fluorimeter's vertically polarized light was set at 360nm to excite the probe. The emitted light was measured at 430nm through a polarizer vertically (I_vv) and horizontally (I_vh)compared to the excited light. The grating factor (G) used was1.27. The slit width for excitation and emission was set at10 nm.

For anisotropy measurement, 50 µl of liposomes was addedto 2 ml 50 µM MES in a quartz cuvette (four clear sides)and placed in the fluorimenter chamber for 5 min to achievetemperature equilibration. Then 10 readings were obtained andaveraged. Afterwards, DPH in tetrahydrofuran (Sigma) was addedto the cuvette to a final concentration of 10^–6 M, mixedby pipetting, and read after exactly 4 min for 1 min, and readingswere averaged. The anisotropy (r) was calculated using the intensityrecorded before (I°_vv and I°_vh) and after (I'_vv andI'_vh) DPH according to the equation

Formula

Whole-cell anisotropy.
The protocol for whole cells was similar to the procedures followedfor the liposomes. BHI was inoculated with L. monocytogenesand incubated at the desired temperature (15 or 30°C) untilmid-log phase (A₆₀₀ ${approx}$ 0.7). Afterwards, cells were harvestedby centrifugation and then washed with phosphate-buffered saline(Sigma), centrifuged, and then resuspended to an A₄₅₀ of 0.28in phosphate-buffered saline. DPH was added to the cells toachieve a final concentration of 10^–6 M, and the tubeswere incubated for 45 min at the same temperature used to growthe cells. For anisotropy measurement, 2 ml of unlabeled cellswas measured to establish baseline values.

Real-time anisotropy measurements.
L. monocytogenes cells used for real-time anisotropy were grownand equilibrated under the same conditions as those used forstatic measurements. DPH-labeled cells were preequilibratedat the growth temperature (15 or 30°C). While continuousmeasurements were recorded, the temperature was steadily increased(5°C every 5 min) until the target temperature was reached.The change in rigidity per temperature ( ${Delta}$ r/ ${Delta}$ T) was plotted.

Measurement of cell adjustment to temperature shift.
To determine the ability of cells to adjust to temperature changes,cells grown at one temperature were equilibrated to a secondtemperature and then returned to the original temperature. Theanisotropy of Listeria monocytogenes strains (cells or liposomes)grown at 30°C was measured at 30°C for 5 min to establisha baseline. The temperature was shifted to 15°C and heldfor 1.5 h with anisotropy measurements taken every 15 min. Thetemperature was shifted back to 30°C, and the anisotropywas measured every 10 min for 30 min. The reverse experiment(cells grown at 15°C, equilibrated at 30°C, and shiftedback to 15°C) was also conducted.

Nisin MIC.
Nisin MICs were determined for the four Listeria monocytogenesstrains. The assay was performed in a 96-well plate using aDynex MRX plate reader (Dynex, VA) at 30°C with nisin (2.5%nisin [Sigma]) concentrations ranging from 5 IU/ml to 1,000IU/ml. Overnight cultures of L. monocytogenes were diluted inBHI (1:100) and added to the wells. Nisin was later added toachieve the desired concentrations. The plate was read (A₆₃₀)every half hour for 24 h, and the results were duplicated.

RESULTS AND DISCUSSION

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RESULTS AND DISCUSSION

Liposome and whole-cell static anisotropy.
Listeria monocytogenes strains (Scott A, NR30, wt10403S, andcld1) were grown at 15 and 30°C, followed by lipid extractionand liposome construction (as described earlier), and theirmembrane fluidity was measured at 30 and 15°C. These normaland stress growth temperatures were used to examine the influenceof temperature on membrane fluidity in different genetic backgrounds.The rigidities of liposomes measured at 15°C were almostdouble (84% increase) the rigidities of liposomes grown at 30°C,regardless of the growth temperature (Fig. 1a). However, cld1had a different response. When grown and measured at 30°C,cld1 displayed a 73% higher rigidity than the other strains,but when grown at 15°C and measured at 30°C, cld1 hadless difference in rigidity than the others.

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FIG. 1. Growth and measurement temperatures (°C) impact membrane rigidity (anisotropy) of Listeria monocytogenes Scott A (hatched bars), NR30 (checkered bars), wt10403S (horizontally striped bars), cld1 (vertically striped bars), and 2-methylbutyrate-treated cld1 (stippled bars) liposomes (a) and whole cells (b). The x axis displays the growth temperature followed by the measurement temperature. Significant differences (P < 0.05) are indicated by solid stars.

To elucidate the behavior of membrane lipids in the native state,while accounting for the effect of membrane proteins and otherconstituents on membrane fluidity, whole-cell anisotropy wasmonitored under various conditions (Fig. 1b). Irrespective ofthe growth temperature, whole cells measured at 15°C hadmembranes that were more (37%) rigid than those measured at30°C. The rigidity of cld1 whole cells was similar to thatof liposomes. When grown at 30°C, cld1 had significantlyhigher rigidity (t test; P < 0.05) than other strains, irrespectiveof the measurement temperature. The addition of 2-methylbutyrate(Sigma) to cld1 grown at 30°C restored rigidity values backto wt10403S levels (Fig. 1b). However, when cld1 was grown at15°C, its rigidity was similar to those of the other strains(P > 0.05). This is consistent with changes in fluidity asa function of temperature that were observed in Bacillus andPseudomonas sp. using liposomes and whole cells (17, 34). Theadjustment of cellular membranes in response to multiple factorswas reported (1, 10, 28). Whole cells, but not liposomes, adjusttheir fluidity in response to temperature. This adjustment isbelieved to account for the smaller difference in the measurementbetween 15 and 30°C compared to the difference observedin liposomes (37% and 87%, respectively).

Real-time whole-cell anisotropy.
To further elucidate the kinetics of membrane rigidity adjustmentin response to temperature, real-time anisotropy measurementswere performed for the four strains grown at 30 and 15°C(Fig. 2 and 3). Longer measurement times were needed, thus lengtheningthe exposure of the probe (DPH) to excitation light. This mightlead to photobleaching. To investigate this possibility, sampleswere exposed to excitation light for 20 min. The differencein the intensities emitted by the probe in the first and lastminutes of the assay was negligible (data not shown). The real-timemeasurement was started at the temperature used to grow andequilibrate the cells (15 or 30°C) and then changed by 5°Cevery 5 min until the target temperature was reached. For thecells grown at 30°C, as the temperature decreased to 15°C,the rigidity increased at rates ( ${Delta}$ r/ ${Delta}$ T derived from the linearregression line [Fig. 2]) that are similar for the four strains(Table 1). The whole cells adjusted their fluidity on a continuousbasis. The rigidity values at the start and the end of the experimentwere similar to the values obtained in static measurement atthe same temperature (Fig. 1). This further confirms the absenceof photobleaching. The slopes from the linear regression linesfor the four strains were compared to determine if there wasany difference in their speeds of adjustment (anisotropy change/°C)to changes in temperature. There was, however, no significantdifference in the rates of adjustment between strains (t test;P > 0.05). These results demonstrate that L. monocytogenesresponds to temperature fluctuation with a continuous and rapidadjustment of membrane fluidity.

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FIG. 2. Real-time whole-cell anisotropy measurement examining the effect of temperature decrease (5°C every 5 min) for Listeria monocytogenes Scott A (a), NR30 (b), wt10403S (c), and cld1 (d) grown at 30°C. Dashed black lines show the linear regression of the data.

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FIG. 3. Real-time whole-cell anisotropy measurements during temperature increases (5°C every 5 min) for Listeria monocytogenes Scott A (a), NR30 (b), wt10403S (c), and cld1 (d) grown at 15°C. Dashed black lines show the linear regression fit performed on the data.

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TABLE 1. Rate of adjustment of whole cells and liposomes of the four strains grown at 15 and 30°C

Whole-cell adaptation kinetics.
Adjustment of cell membrane fluidity as a function of temperaturechange is known as homeoviscous adaptation (30). This occursto maintain constant membrane fluidity. As the temperature decreases,the lipids found in the cell membrane become more rigid, whichinterferes with the activity of the membrane and membrane-boundcomponents like channels and pumps (27, 29). To the best ofour knowledge, the time required for various Listeria monocytogenesstrains to adjust their membrane fluidity is unknown. To measurethe time needed for adjustment, the anisotropy of cells grownand equilibrated at 15°C was measured for 20 min at 30°Cwithout allowing any time for preequilibration (Fig. 4). Adjustmenttimes for the four strains were 130 to 180 s. These resultsare slightly different from the static measurement values, suggestinga multistage adjustment for the cell membrane. Giotis et al.(12) and Mastronicolis et al. (18) have previously observeda two-stage adjustment of cell membranes to temperature: a fast"emergency" adjustment followed by a slow adjustment consideredas a fine-tuning of the membrane fluidity. These authors’findings reflect a similar response, adopted by Listeria, tomany types of stress (temperature and pH). De novo protein synthesisinhibition, using chloramphenicol, did not stop cell adjustment(data not shown). To further demonstrate the adaptation of wholecells, dynamic anisotropy measurements of liposomes and wholecells were plotted and compared (Fig. 5). cld1 previously showedno adaptation when grown at 30°C (Fig. 1b); this was furthershown by a whole-cell plot parallel to the liposome plot. Whencld1 was grown at 15°C, the whole cells adjusted to changingtemperature, at a lower rate than liposomes (Table 1). The slopesof dynamic anisotropy of all whole-cell strains showed a lowerdegree of change in rigidity compared to liposomes (Table 1).This independently confirms the adjustment of whole cells inresponse to temperature. When cells were measured at 30°Cand then equilibrated at 15°C for 1.5 h, followed by remeasurementat 30°C, they adjusted to 15°C. Their rigidities beforeand after the 15°C equilibration were different, in contrastto the liposomes, which had the same rigidity before and afterthe equilibration (Fig. 6).

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FIG. 4. Time required for homeoviscous adaptation of Listeria monocytogenes. The graph presents the anisotropy (continuous line) of Scott A (a), NR30 (b), wt10403S (c), and cld1 (d) grown at 15°C and measured at 30°C. The black dotted line represents the average equilibrium value from the static measurement. Replicate experiments yielded consistent results.

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FIG. 5. Real-time dynamic anisotropy of liposomes (squares) and whole cells (triangles) of L. monocytogenes strains wt10403S (a and b) and cld1 grown at 30 and 15°C (c and d) showing the physiological adjustment of whole cells to temperature compared to the solely physical adjustment of liposomes. An average of multiple data is shown.

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FIG. 6. Change of anisotropy of whole cells (triangles) measured at 30°C as a result of equilibration at 15°C. The liposomes (squares) have similar values at 30°C pre- and postequilibration at 15°C. The average of multiple data is shown.

These results further demonstrated the active adjustment ofL. monocytogenes in response to temperature.

MICs of nisin.
Nisin induces temperature-independent changes to the membraneof strain NR30 (20). These changes include more straight-chainfatty acids, less branched fatty acids, lower C₁₅ and C₁₇ andhigher C₁₆ and C₁₈ fatty acids, and a higher phase transitiontemperature (20, 23). A similar profile (high even fatty acids,low odd fatty acids, and low branched fatty acids) is displayedby cld1 (1, 8, 12). To examine if the rigid profile of the cld1membrane confers nisin resistance, the MICs of the four strainswere determined in BHI with nisin concentrations ranging from5 IU/ml to 1,000 IU/ml. Nisin at 1,000 IU/ml was the only concentrationthat inhibited Scott A and wt10403S. Strain NR30 was not inhibitedby any tested concentration. Strain cld1 showed the highestsensitivity to nisin, being inhibited by concentrations as lowas 100 IU/ml. The fact that NR30 and cld1 share similar membranecompositions but show different membrane fluidities and differentsensitivities to membrane-acting components (nisin) suggeststhat fatty acid composition is not the sole determinant of membranefluidity and behavior.

The present work suggests a continuous adjustment of the membranefluidity (average ${Delta}$ r/ ${Delta}$ T = 5.5 x 10^–4) and identifies thetime needed for adjustment. The fluidity and nisin MICs of strainsNR30 and cld1 suggest that fatty acid composition can serveonly as a preliminary tool to make predictions about membranefluidity. Finally, as suggested by the NR30 results, a membranewith low odd and branched fatty acid is not necessarily morerigid than the wild type.

ACKNOWLEDGMENTS

This research is supported by the USDA NRI Competitive GrantsProgram in Food Safety (grant no. 2006-01288).

We thank R. Ludescher for sharing his expertise in fluorospectrophotometry.

FOOTNOTES

* Corresponding author. Mailing address: Rutgers, The State University of New Jersey, Department of Food Science, Cook College, 65 Dudley Rd., New Brunswick, NJ 08901. Phone: (732) 932-9611, ext. 222. Fax: (732) 932-6776. E-mail: montville{at}aesop.rutgers.edu

Published ahead of print on 17 August 2007.

REFERENCES

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