Physiological and morphological responses of the soil bacterium Rhodococcus opacus strain PD630 to water stress

Physiological and morphological responses of the soil bacterium Rhodococcus opacus strain PD630 to water stress

FEMS Microbiology Ecology 50 (2004) 75–86 www.fems-microbiology.org Physiological and morphological responses of the soil bacterium Rhodococcus opacu...

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FEMS Microbiology Ecology 50 (2004) 75–86 www.fems-microbiology.org

Physiological and morphological responses of the soil bacterium Rhodococcus opacus strain PD630 to water stress Hector M. Alvarez a,*, Roxana A. Silva a, Ana C. Cesari a, Ana L. Zamit a, Silvia R. Peressutti a,1, Rudolf Reichelt b, Ulrike Keller b, Ursula Malkus b, Christiane Rasch b, Thomas Maskow c, Frank Mayer d, Alexander Steinb€ uchel

e

a

e

Departamento de Bioquımica, Facultad de Ciencias Naturales, Universidad Nacional de la Patagonia San Juan Bosco, Km 4, (9000) Comodoro Rivadavia, Argentina b Institut f € ur Medizinische Physik und Biophysik, Westf €alische Wilhelms-Universit€at, Robert-Koch-Strasse 31, D-48149 M€unster, Germany c Department of Environmental Microbiology, Centre for Environmental Research Leipzig-Halle GmbH, Permoserstrasse 15, D-04318 Leipzig, Germany d Institut f €ur Mikrobiologie und Genetik, Georg-August-Universit€at, Grisebachstrasse 8, D-37077 G€ottingen, Germany Institut f € ur Molekulare Mikrobiologie und Biotechnologie, Westf €alische Wilhelms-Universit€at, Corrensstrasse 3, D-48149 M€unster, Germany Received 5 January 2004; received in revised form 25 March 2004; accepted 2 June 2004 First published online 22 June 2004

Abstract Rhodococcus opacus PD630 was investigated for physiological and morphological changes under water stress challenge. Gluconate- and hexadecane-grown cells were extremely resistant to these conditions, and survival accounted for up to 300 and 400 days; respectively, when they were subjected to slow air-drying. Results of this study suggest that strain PD630 has specific mechanisms to withstand water stress. Water-stressed cells were sensitive to the application of ethanol, high temperatures and oxidative stress, whereas they exhibited cross-protection solely against osmotic stress during the first hours of application. Results indicate that the resistance programme for water stress in R. opacus PD630 includes the following physiological and morphological changes, among others: (1) energetic adjustments with drastic reduction of the metabolic activity (39% decrease during the first 24 h and about 90% after 190 days under dehydration), (2) endogenous metabolism using intracellular triacylglycerols for generating energy and precursors, (3) biosynthesis of different osmolytes such as trehalose, ectoine and hydroxyectoine, which may achieve a water balance through osmotic adjustment and may explain the overlap between water and osmotic stress, (4) adjustments of the cell-wall through the turnover of mycolic acid species, as preliminary experiments revealed no evident changes in the thickness of the cell envelope, (5) formation of short fragmenting-cells as probable resistance forms, (6) production of an extracellular slime covering the surface of colonies, which probably regulates internal and external changes in water potential, and (7) formation of compact masses of cells. This contributes to understanding the water stress resistance processes in the soil bacterium R. opacus PD630. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Rhodococcus opacus PD630; Water stress; Physiology; Morphology

1. Introduction

*

Corresponding author. Tel./fax: +54-297-4550-339. E-mail address: [email protected] (H.M. Alvarez). 1 Instituto Nacional de Investigaci on y Desarrollo Pesquero (INIDEP), Paseo Victoria Ocampo No. 1, Mar del Plata, (7600) Buenos Aires, Argentina.

Members of the genus Rhodococcus, which are nonsporulating actinomycetes, possess a broad metabolic spectrum, with capabilities to degrade a variety of pollutants, such as hydrocarbons, herbicides and other xenobiotic compounds [1–3]. Recent studies demonstrated that Rhodococcus spp. are able to transform different

0168-6496/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.06.002

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hydrocarbons into storage lipids, such as triacylglycerols, during cultivation under nitrogen limiting conditions, which are often found in soil environments [4–7]. The accumulated lipids are then utilized as endogenous carbon and energy sources for maintaining viability during nutrient starvation conditions [8]. Due to these properties, several reports considered the potential of these bacteria for in situ bioremediation of contaminated environments [9–12]. Another interesting feature of actinomycetes is their dominant occurrence in arid environments [13]. These microorganisms may contribute up to 50% of the total microbial population in desert soils. Previous studies revealed that species of the genera Rhodococcus and Gordonia belong to the autochthonous population in pristine and crude oil-contaminated soils in semiarid Patagonia (Argentina), exhibiting high persistence in these environments [12–14]. In this context, Warton et al. [15] reported a case of enhanced biodegradation of metham sodium soil fumigant on a farm located in Western Australia. They identified 11 isolates as Rhodococcus spp. among a total of 18 gram-positive bacteria, which were responsible for the biodegradation of the fumigant in this soil. These strains were able to resist dry heat treatments and to recover their degrading ability following dehydration [15]. The frequent occurrence of non-sporulating actinomycetes, such as Rhodococcus spp., in arid sites around the world may reflect adaptation to and natural selection in these environments. In general, processes and mechanisms for adaptation to arid environments have been poorly studied. Until now, desiccation tolerance has been investigated in relatively few genera of bacteria, and most studies have focused on cyanobacteria, such as Nostoc commune and Chroococcidiopsis spp. [16]. This is a physiological investigation of the ability of Rhodococcus opacus PD630 to survive under water stress conditions and the mechanisms involved in meeting this environmental challenge. R. opacus PD630 is an oleaginous soil bacterium, and its ability to synthesize and accumulate triacylglycerols from a wide variety of carbon sources has been intensively studied [4,6,8,17–20]. The understanding of the mechanisms of resistance to arid environments in soil bacteria could be important not only for bioremediation processes but also for establishment of biotechnological applications.

2. Materials and methods 2.1. Bacterial strain, media, growth and water stress conditions R. opacus strain PD630 (DSM 44193) was isolated and characterized in a previous study [4]. Cells were

grown aerobically at 28 °C in nutrient broth (NB) medium (0.8%, w/v) or in mineral salts medium (MSM) with sodium gluconate (1%, w/v) as carbon source, according to Schlegel et al. [21]. To allow lipid accumulation, the concentration of ammonium chloride in MSM was reduced to 0.1 g/l (MSM0.1); otherwise, the ammonium chloride concentration was 1.0 g/l (MSM1). To solidify media, 1.4% (w/v) agar was added. Hexadecane was not directly included in the solid medium; rather, the hydrocarbon was provided on a filter paper disc in the lid of the Petri dish. For dehydration experiments, cells were incubated at 28 °C and allowed to dry at a relative humidity of 20%, measured with a digital hygrometer HT 06/3306 (TFA, Germany). 2.2. Survival of R. opacus PD630 on dry surfaces and experiments with inhibitors Survival of R. opacus PD630 under water stress conditions was investigated by a modification of the method described by Ophir and Gutnick [22]. Cells were grown aerobically at 28 °C overnight in 10 ml nutrient broth medium on a rotary shaker. After growth, cells were harvested, washed once with sterile NaCl solution (0.85%, w/v), and resuspended at an OD436 nm of 1.0 in sterile NaCl. Approximately 20 aliquots (7 ll each) from this cell suspension were spotted onto 0.45 lm filters (Sartorius, G€ ottingen, Germany). These filters were placed onto MSM0.1 agar plates containing either 1% (w/v) sodium gluconate or 150 ll hexadecane as carbon sources, and were incubated at 28 °C. After growth, the colonies were allowed to desiccate under passive conditions by removing the filters from the agar plates and incubating them in empty sterile plates at 28 °C and 20% relative humidity. The filters, on which the colonies were grown, were cut into small pieces containing only one single colony and were added to sterile Eppendorf microcentrifuge tubes. The cells from each colony were suspended separately, and the viable count (CFU) was determined. The survival rate was calculated as CFUafter drying /CFUbefore drying  100. All CFU determinations were done in triplicate experiments. The data were recorded as means and standard deviations. Acrylic acid (Merck, Darmstadt, Germany) and isoniazid (Sigma, St. Louis, MO) were applied for inhibition of the b-oxidation pathway and fatty acid elongation during mycolic acid biosynthesis, respectively, in order to determine effects of those processes on the survival of strain PD630 under water stress conditions. Cells were grown on 0.45 lm filters placed on MSM0.1 agar plates containing 1% (w/v) sodium gluconate as carbon source, and incubated at 28 °C. After growth of the colonies, the filters were removed from the agar plates and impregnated with solutions of 0.3 or 0.6 mg/ml of acrylic acid or 40 lg/ml of isoniazid, respec-

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tively, for 1 h prior to drying. After incubation with inhibitors, the colonies were dried by incubation in empty sterile plates at 28 °C and 20% relative humidity, and the viable count (CFU) was determined at different times as described above. CFU determinations were done in triplicate experiments. 2.3. Water loss assay To investigate the water loss of the inert support used for the survival assay described above, the following method was applied. Cellulose nitrate filters (0.45 lm) (Sartorius, G€ ottingen, Germany) were placed onto MSM0.1 agar plates containing 1% (w/v) sodium gluconate and incubated at 28 °C for five days. After this period, filters were air dried by removing them from the agar plates and incubating them in empty sterile plates at 28 °C and 20% relative humidity. The progressive water loss from filters was determined gravimetrically. Water content of filters decreased continuously during the first 9 h of incubation and after that, their weight was essentially constant during the next 130 h (0.0832  0.00065 g). 2.4. Treatment of cells with stress conditions To investigate the development of stress resistance in R. opacus PD630, a modified methodology according to van Overbeek et al. [23] was used. Three different sets of cells (exponentially grown cells, cells in stationary growth phase and water-stressed cells) were prepared. To study the exponentially grown and stationaryphase cells, the cultures were grown aerobically at 28 °C overnight in 10 ml nutrient broth medium on rotary shaker. After growth, 100 ll of this culture was used to inoculate 20 ml MSM0.1 containing (1%, w/v) sodium gluconate as sole carbon source; these cultures were incubated for 12 h (exponentially grown cells) or three days (stationary-grown cells) at 28 °C on rotary shaker. After this period, cells were harvested, washed once with sterile NaCl solution, and suspended at an OD436 nm of 0.3 in sterile NaCl solution. To study water-stressed cells, cells were grown aerobically at 28 °C overnight in 10 ml nutrient broth medium on a rotary shaker. After growth, 100 ll of this culture were suspended in 20 ml MSM0.1 with sodium gluconate (1%, w/v) as sole carbon source and incubated for 48 h at 28 °C on a rotary shaker. Then, cells were harvested, washed once with sterile NaCl solution, and spread onto sterile glass slides, which were incubated three days in empty plates at 28 °C and at 20% relative humidity. After drying, cells were collected and suspended at an OD436 nm of 0.3 in sterile NaCl solution. For challenges of cells in the exponential and stationary growth phase and water-stressed cells, aliquots (each 10 ll) of these cell suspensions were used to in-

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oculate 100 ml flasks containing 10 ml MSM1 with sodium gluconate (0.5%, w/v) as sole carbon source, and different stresses were applied to these cultures. Ethanol stress was applied by addition of 96% ethanol to a final concentration of 9% (v/v). Oxidative stress was applied by addition of H2 O2 to a final concentration of 360 lM. Osmotic stress was applied by addition of NaCl to a final concentration of 2.7 M. For challenge with high temperatures, flasks were pre-warmed to 47 °C before cell inoculation and were maintained at this temperature after addition of cells. CFU counts during the above stresses were determined by plating 100-ll aliquots of the treated cultures shortly before and during stress application, after appropriate dilutions. All determinations were done in triplicate experiments. 2.5. Determination of metabolic activity Tetrazolium chloride (Sigma, St. Louis, MO) reduction was used to indicate respiration activity in cells according to the method of Roslev and King [24]. Tetrazolium chloride is an artificial electron acceptor, which is reduced to a water-insoluble red formazan. Formazan was dissolved by hexane and measured at 546 nm. Colonies were prepared exactly as described above for survival experiments, using 0.45 lm filters (Sartorius, G€ ottingen, Germany) as support during growth. After growth, the colonies were allowed to dehydrate under passive conditions at 28 °C and 20% relative humidity. The filters, on which the colonies were grown, were cut into small pieces containing only one single colony. Four drops of a tetrazolium chloride solution (0.3% in 100 mM Tris–HCl buffer) were added to each colony. After 1 h incubation in the dark at room temperature, the pieces with the colonies were added to sterile Eppendorf microcentrifuge tubes containing 1.5 ml hexane, cells were suspended and incubated for 2 h in the dark at room temperature. Cells were separated from solution by centrifugation (13,000 rpm for 5 min), and the supernatants were monitored at 546 nm. Determinations were done in triplicate experiments, and data were recorded as means and standard deviations. 2.6. Analysis of compatible solutes Cells were cultivated in agar MSM0.1 containing sodium gluconate or hexadecane as sole carbon sources, harvested, washed with sterile NaCl solution (0.85%, w/v), and spread onto the glass surface of sterile empty Petri dishes. Cells were allowed to dehydrate under passive conditions at 28 °C and 20% relative humidity for seven days. After that, cells were collected and washed with NaCl solution (10%, w/v), in order to avoid the loss of compatible solutes during rehydration, and then lyophylized. The salt content of the samples was

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quantified by titrating the chloride ion with silver nitrate, and using potassium chromate as an indicator [25]. The osmolytes were extracted from 10 to 30 mg of the sample with 1.14 ml methanol/chloroform/water (1:0.5:0.4), and phase separation was achieved by adding 0.34 ml chloroform and 0.34 ml water [26,27]. The water-soluble phase contained the compatible solutes and was analyzed by isocratic HPLC [28]. Eightyfive percent acetonitrile in water with a flow rate of 2 ml/min was used as eluent. Osmolytes were separated on a Nucleosil 100-5 NH2 column (250  3 mm inner diameter, Macherey and Nagel, Germany) and detected with a refractive index monitor (ectoine, hydroxyectoine, glycinebetaine and trehalose) as well as with a UV/VIS photodiode array monitor (ectoine, hydroxyectoine and glycinebetaine) at 190 and at 225 nm. 2.7. Analysis of fatty acids Cells of strain PD630 were grown for five days on MSM0.1 agar plates containing 1% (w/v) sodium gluconate, harvested, washed once with sterile NaCl solution, and spread onto glass slides, which were incubated in empty plates at 28 °C and 20% relative humidity for dehydration. At different times, cells were collected and lyophilised for chemical analysis. For qualitative and quantitative determination of fatty acids, 3–5 mg of lyophilised cells was subjected to methanolysis in the presence of 15% (v/v) sulphuric acid, and the acylmethylesters [29] were analysed by gas chromatography (GC) with a Konik HRGC3000 gas chromatograph equipped with a Innowax capillary column (30 m  0.53 mm) and a flame ionization detector. Two microlitres portion of the organic phase were analyzed. Nitrogen (50 ml/min) was used as carrier gas. The temperature of both the injector and detector was 260 °C. A temperature program was used for efficient separation of the methyl esters (150 °C for 1 min, temperature increase of 5 °C/min, 240 °C for 10 min). For quantitative analysis, tridecanoic acid was used as internal standard.

made according to the specific requirements of the microscopic method to be applied for investigation. (i) For transmission electron microscopy (TEM) the samples were embedded in SPURR resin [31] (without propylen oxide). Sections with a thickness of 70–80 nm were made with an Ultracut (LEICA Mikroskopie und Systeme GmbH, Wetzlar, Germany) using a diamond knife and placed on a 200 mesh copper grid. Imaging was performed with a H-500 TEM (Hitachi Ltd., Japan) in the bright-field mode at 80 kV acceleration voltage and at room temperature (RT). Micrographs were recorded using Scientia EM film (No. 23D56P3AH). (ii) For scanning electron microscopy (SEM) the samples were subjected to critical point drying with liquid CO2 according to the standard procedure. Subsequently, the samples were mounted on aluminum specimen stubs with a small amount of electrically conductive carbon (PLANO W. Planet GmbH, Wetzlar, Germany) and sputter coated with gold using argon gas as the ionizing plasma. The average thickness of the gold film applied to the samples was approximately 15 nm. Imaging was performed with a S-450 SEM (Hitachi Ltd., Japan) with secondary electrons (SE) at 20 kV acceleration voltage and at RT. Micrographs were recorded using a negative film (Agfapan, APX 100). (iii) For high-resolution field emission scanning electron microscopy (FESEM) the samples were subjected to critical point drying with liquid CO2 according to the standard procedure. Subsequently, the mounted samples were rotationally shadowed with 2 nm platinum/carbon (Pt/C) at an elevation angle of 65° using a BAF 300 (Balzers Hochvakuum AG, Principality Liechtenstein). Imaging was performed with a S-800 SEM (Hitachi Ltd., Japan) with SE at 20 kV acceleration voltage and at RT. Micrographs were recorded using a negative film (Agfapan, APX 100).

3. Results 3.1. Survival of R. opacus under dehydration conditions

2.8. Preparation of cells for electron microscopy Gluconate- and hexadecane-grown colonies of the strain PD630 were prepared for electron microscopy exactly as described above for survival experiments using 0.45 lm filters (Sartorius, G€ ottingen, Germany) as support during growth. The cells were fixed with 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer according to Sørensen (pH 7.3) for 45 min [30]. After three times washing with this phosphate buffer, cells were post-fixed in 1% (w/v) osmium tetroxide in 0.1 M phosphate buffer (pH 7.3) and washed once with phosphate buffer. Then the water was removed by a graded water-ethanol series (30%, 50%, 70%, 90%, 96%, and absolute ethanol). The following preparation steps were

To study the water stress tolerance of R. opacus strain PD630, cells were grown in mineral medium with gluconate or hexadecane as a sole carbon source and tested for their survival under water stress conditions. As shown in Fig. 1, R. opacus was extremely resistant to this challenge, and a high fraction of the cells survived several months under these conditions. Enhanced survival of hexadecane-grown cells on dry surfaces was observed in comparison with cells grown previously on gluconate (Fig. 1). 3.2. Development of water stress resistance in R. opacus Water stress tolerance in R. opacus may involve a wide range of cellular responses at morphological,

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osmotic stress conditions. These differences in survival were statistically significant as revealed by analysis of variance (P < 0:05) (Table 1). In contrast, cells exposed to water stress had become more sensitive to the application of ethanol, high temperature and oxidative stress after 24 h of stress application than exponential- and stationary-phase cells (data not shown). High temperature was the most restrictive factor for survival of waterstressed cells (Table 1).

100

CFU counts (%)

79

80 60 40 20

3.3. Occurrence of compatible solutes in water-stressed cells of R. opacus

0 0

50

100

150

200 250 300 Time (days)

350

400

450

500

Fig. 1. Survival of R. opacus PD630 during incubation under water stress conditions. (d) gluconate-grown cells and (m) hexadecanegrown cells.

physiological, biochemical and genetic levels. Survival experiments were performed to elucidate whether specific or general physiological mechanisms are involved in water stress tolerance of R. opacus. To asses the development of cross-protection against various stress conditions in water-stressed cells of R. opacus, the survival of exponential-phase and stationary-phase cells as well as of water-stressed cells of strain PD630 was compared after application of ethanol (9%, v/v), high temperature (47 °C), oxidative stress (360 lM H2 O2 ) and osmotic stress (2.7 M NaCl). Two parameters, which characterize the response of cells to water stress, such as 70% reduction time and survival of CFU after 2 h stress application, are shown in Table 1. Cells exposed to slow air drying developed also resistance against osmotic stress, principally during the first hours of drying, whereas they were more sensitive than untreated cells to the other stresses studied. They displayed higher survival rates than stationaryand exponential-phase cells after 2 h cultivation under

One of the best characterized responses to dehydration as well as to osmotic stress involves synthesis and intracellular accumulation of compatible solutes, such as trehalose and sucrose [32]. Accumulation of such substances could explain the overlap among water stress and osmotic responses in strain PD630. Therefore, the occurrence of compatible solutes in gluconate- and hexadecane-grown cells after dehydration was investigated. Three different compounds, trehalose, ectoine and hydroxyectoine, which were previously reported to be compatible solutes in other bacteria, were detected in both types of cells (Table 2). However, the relative amounts of each compound depended on the substrate used as carbon source for cell cultivation before drying (Table 2). Trehalose was the major compatible solute accumulated by gluconate-grown cells during water stress. In contrast, hydroxyectoine was the main compatible solute produced during water limitation by cells grown on hexadecane. The cells were also analyzed for occurrence of glycerol, glycinebetaine and sucrose; however, these compounds could not be detected. 3.4. Changes in metabolic activity during water stress In addition to other physiological changes, energy generating systems might be adjusted for better survival

Table 1 Development of stress resistance in R. opacus PD630 at different physiological states Stress

2.7 M NaCl 9% Ethanol 360 lm H2 O2 47 °C

Water-stressed cells

Water-stressed cells

Cells from stationary growth phase

Cells from exponential growth phase

70% reduction time (h)A

Survival after 2 h (%)B

Survival after 2 h (%)B

Survival after 2 h (%)B

23 14 2 1

99.0  0.9a 80.5  2.1b 72.7  2.2c 53.5  1.7d

91.2  1.9a 89.4  1.8a 74.3  1.5b 76.1  0.8b

72.5  2.0a 82.9  2.0b 91.1  4.9c 72.2  0.5a

A Seventy percent reduction time of survival after stress application was estimated by regression analysis using six time points for calculation (linear correlation of Pearson, Instat V2.02, GraphPad Software Inc., San Diego, California, USA). B a, b, c, d indicate significant differences between the values of each column (P < 0:05). Differences in survival after application of each stress condition were determined by analysis of variance (Mann–Whitney test, Instat V2.02, GraphPad Software Inc., San Diego, California, USA).

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Table 2 Accumulation of compatible solutes in gluconate- and hexadecane-grown cells of R. opacus PD630 during water stress conditions Compatible solute

Concentration of compatible solute (%, CDW)

Trehalose Ectoine Hydroxyectoine

Gluconate-grown cells

Hexadecane-grown cells

1.63 0.11 0.48

0.48 0.49 1.74

Cells were cultivated in MSM0.1 agar with gluconate or hexadecane as sole carbon sources, harvested, washed with sterile NaCl solution, spread onto the glass surface of sterile Petri dishes and incubated at 28 °C and 20% relative humidity during seven days for dehydration. After that, cells were collected with NaCl solution (10%, w/v), lyophilized and analyzed for the occurrence of compatible solutes by HPLC. The data correspond to a single determination. CDW, cellular dry weight.

under water stress conditions. The metabolic activity change in cells subjected to dehydration was investigated using tetrazolium chloride as colorimetric indicator of the respiratory activity. A drastic decrease of the metabolic activity in cells subjected to water stress occurred during the first 24 h of stress (Table 3). Thereafter, the decline in the cellular metabolic activity slowed down. After approximately six months of incubation of cells under dehydration conditions, the metabolic activity was maintained at around 10% of the original level (Table 3). 3.5. Endogenous metabolism in R. opacus under water stress conditions Cells of R. opacus PD630 were able to produce CO2 during water stress conditions although no external carbon source was available (data not shown). This indicated that the cells utilized an endogenous carbon source for maintenance, for energy generation and to induce the necessary adaptations and survival processes. In a previous study, we reported that strain PD630 was able to utilize triacylglycerols as carbon and energy source for maintaining viability during starvation con-

ditions [8]. For this reason, the total fatty acid content in strain PD630 under dehydration conditions was investigated. The total amount of cellular fatty acids in the cells decreased under this stress conditions indicating their utilization as endogenous carbon and energy source (Table 4). In order to confirm this, cells of strain PD630 were subjected to water stress after treatment with different concentrations of acrylic acid, which is an inhibitor of the b-ketothiolase enzyme involved in the b-oxidation pathway [33]. No surviving cells were observed after only 24 h dehydration, when the b-oxidation was inhibited in cells of R. opacus by addition of either 0.3 or 0.6 mg/ml of acrylic acid. 3.6. Cellular envelope morphology of R. opacus under water stress In addition to the physiological changes studied above, other changes may occur in cells of strain PD630 during water stress. Although the measure of the thickness of envelope layers depends on the angle of sectioning, analysis of a large number of micrographs did not provide evidence for systematically varied thicknesses nor structures of envelope layers of cells of

Table 3 Metabolic activity variation of gluconate-grown cells of R. opacus PD630 during water stress Water stress (days)

Absorbance (546 nm)

Log CFU/ml in the colony

Rate of metabolic activity

0 1 3 7 20 30 88 135 190

0.108  0.002 0.072  0.002 0.060  0.005 0.048  0.007 0.037  0.005 0.032  0.006 0.025  0.005 0.008  0.003 0.007  0.003

8.13  0.11 8.13  0.08 8.09  0.05 7.91  0.12 7.78  0.07 6.86  0.11 6.38  0.02 4.97  0.07 4.54  0.19

0.13 0.08 0.07 0.06 0.04 0.04 0.039 0.016 0.015

a

Rate of metabolic activity (%) b

Survival c

100 61.5 53.8 46.1 30.7 30.7 30.0 12.3 11.5

100 100 99.5 97.3 95.7 84.4 78.5 61.1 55.8

Metabolic activity changes of cells under water stress conditions were determined using tetrazolium chloride as colorimetric indicator of the respiratory activity, as described in Section 2. a Correction factor of the metabolic activity considering the viable counts, which was calculated as absorbance/log CFU  10. b Percent of the metabolic activity rate considering the value at the beginning of the experiment (0 days) as 100%. c Survival percentage of cells subjected to dehydration was calculated as CFUafter drying /CFUbefore drying  100. Data for log CFU/ml and absorbance were recorded as means and standard deviations (%).

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Table 4 Mobilization of the stored fatty acids by R. opacus PD630 during drying Incubation time (days)

Fatty acid content (% CDW, w/w)

0 1 10

19.5 18.5 13.6

Cells were cultivated in MSM0.1 agar with gluconate for five days, harvested, washed once with sterile NaCl solution, and spread onto glass slides, which were incubated in empty plates at 28 °C and 20% relative humidity for water stress. At different times, cells were collected, lyophilized and subjected to chemical analysis. The data are means of two separate determinations. CDW, cellular dry weight.

R. opacus upon dehydration. Electron microscopy analysis (thin sections of 29 cells) revealed similar values for the envelope thicknesses of cells after seven days of water stress (17.3  1.5 nm) in comparison with cells cultivated under optimal conditions (18.7  1.5 nm). In addition, the same layered profile of the cellular envelope was maintained during water stress. Fig. 2 shows an area where the envelope layers were not separated between two cells. The following layers could be detected: (i) in the center a faint dark line was observed, which probably corresponds to amphiphilic material according to Sutcliffe [34] (W1), (ii) an electron transparent layer containing mycolic acids (W2), (iii) an electron-dense layer representing the cell wall (W3), and finally, the cytoplasmic membrane (Fig. 2). Since no significant changes in the ultrastructure of the cellular envelope could be detected, the adaptation of its fluidity and permeability may be the result of the variation of the lipid content in response to water stress by a controlled turnover of mycolic acids. Since mycolic acids are produced by elongation of fatty acids by the type II fatty acid synthase complex (FAS II), we studied the effect of isoniazid, which is an inhibitor of the FAS II-system, on the survival of water-stressed cells. Cells pre-treated with isoniazid (40 lg/ml) exhibited lower survival percentages, which were approximately 18% less than those of non-treated cells after 22 days under dehydration conditions. Altogether, these results suggested that R. opacus PD630 modulates its lipid content to adapt to water stress conditions. 3.7. Ultrastructure of cells and colonies of R. opacus PD630 subjected to dehydration The ultrastructure of individual cells and colonies of R. opacus PD630 was analyzed by transmission and scanning electron microscopy over a period of seven months under water stress conditions, and in total 554 SEM and 228 TEM micrographs were analyzed in this study. Low-magnification SEM micrographs of R. opacus PD630 colonies grown on gluconate or hexadecane showed the appearance of a matrix of extracellular

Fig. 2. Cell envelope ultrastructure of cells of R. opacus after seven days drying as revealed by electron microscopy. (a) Schematic representation of the cell envelope of R. opacus PD630 grown on gluconate after seven days of water stress. (b) The TEM micrograph shows an area where the envelope layers have not separated between two cells attached to each other. This photograph has been digitally processed to increase magnification (Original magnification: 25,000). W1, W2, W3: wall layers of different electron transparency; CM, cytoplasmic membrane.

polymeric substances (EPS) at the surfaces of colonies after water stress (Fig. 3(b), (c), (e) and (f)). These EPS were absent in the non-water-stressed colonies, which were investigated as control (Figs. 3(a) and (d) and 4(a)). The EPS matrix formed a rather compact slime at the surface of the colonies after seven months of drying (Fig. 3(c) and (f)). High-magnification SEM micrographs showed that cells of strain PD630 exposed to water limitation were frequently located close together, forming compact aggregates surrounded by the EPS (Fig. 4(b)). This was also observed after examination of colonies by TEM (Fig. 5(b)).

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Fig. 3. Low-magnification SEM micrographs of gluconate- and hexadecane-grown colonies of R. opacus PD630 after water stress. Low magnification view of a colony after growth on MSM0.1 with gluconate for five days (a). View of gluconate-grown colonies after one day (b) and seven months (c) of water stress. Low magnification view of a colony after growth on MSM0.1 with hexadecane for seven days (d). Views of hexadecane-grown colonies after one day (e) and seven months (f) of drying. The scale bars correspond to 10 lm.

Otherwise, no major variations in the cell morphology were observed after water stress. Filamentous cells predominated throughout the study, although fragmented cells appeared frequently in colonies subjected to water stress (Fig. 4(a) and (c)). Fragmented cells were more frequently observed in old water-stressed-colonies than in colonies at the beginning of exposure. Another change in the appearance of cells subjected to dehydration is shown in Fig. 4(d). After seven months of incubation, water-stressed cells appeared to shorten in length and widen in cell diameter, and blebs appeared at the cell surface (Fig. 4(d). Similar changes in cell morphology were reported for Mycobacterium smegmatis treated with isoniazid [35]. Thin sections of R. opacus colonies examined by TEM revealed the occurrence of triacylglycerol inclusion bodies at the initial times of the study (Fig. 5(a). Inclusion bodies were also present in cells after seven months of dehydration (Fig. 5(c) suggesting that the storage lipids may play a key role in strain PD630 not

only under water stress conditions but also during the re-wetting of cells.

4. Discussion R. opacus PD630 exhibited an exceptional ability to tolerate water stress, as shown by high survival percentages over periods of 300–400 days on dry surfaces. Some bacteria, such as Bacillus spp. produce spores that are capable of surviving stress conditions [32]. However, Rhodococcus spp. are non-spore-forming actinomycetes; they must therefore possess mechanisms of water stress tolerance, which permit the survival of vegetative cells under these extreme conditions for prolonged times. The results of this study suggest that water-stressed cells of R. opacus PD630 have mechanisms specifically to withstand water limitation, since the same cells are more sensitive to the exposure of other stresses, such as high concentrations of ethanol, high temperatures and oxi-

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Fig. 4. High-magnification SEM micrographs of gluconate-grown colonies of R. opacus PD630 after water stress. (a) High-magnification view of cells grown on gluconate after five incubation days (control); (b) view of compact masses of cells occurring into the colony after 60 days dehydration; (c) view of coccoidal cells occurring in the colony after 60 days dehydration; (d) view of cells of strain PD630 after seven months under water stress conditions. The scale bars correspond to 5 lm (a and b) and to 1 lm (c and d), respectively.

dative stress. The physiological adjustments of waterstressed cells seem to be less effective against the other applied stresses. On the other hand, water-stressed cells of strain PD630 exhibited cross-protection against osmotic stress, principally during the first hours of application. This is predictable, since during the initial stages of slow air drying, when water activity is sufficient to allow a certain degree of growth, cells may achieve a water balance through osmotic adjustment [16]. Thus, cells possess enough time to allow synthesis and accumulation of compatible solutes. The accumulation of osmolytes, such as proline, glutamate, glycerol, trehalose or glycine betaine, during osmotic stress occurs in many prokaryotes [36]. These compounds protect and stabilize macromolecules and cellular structures, such as the membranes of cells, during osmotic stress and desiccation [37]. One of the expected osmolytes in strain PD630 during water stress challenge was glycerol, since R. opacus PD630 utilized triacylglycerols as endogenous carbon and energy sources under these conditions. Thus, glycerol residues can be generated at the expense of triacylglycerol degradation, and in this way, this compound would be an accessible osmoprotectant in cells of R. opacus PD630 during dehydration. However, glycerol was not detected among the compatible solutes identified in this study. Strain PD630 seems to possess a more complex and sophisticated mechanism of water stress tolerance, since cells synthesize and accumulate at least three different osmolytes, trehalose, ectoine and hydroxyectoine, as

revealed this study. Trehalose often occurs in marine and non-halophilic organisms [38]. This disaccharide alone has only a limited potential as osmoprotectant; although, it is frequently found in truly halophilic microorganisms in combination with other more potent compatible solutes such as betaine, ectoine or hydroxyectoine [39]. Hydroxyectoine is a common compatible solute in halophilic microorganisms [39]. In addition, some authors have reported that hydroxyectoine is more effective in stabilizing dried bacteria than trehalose and ectoine [40]. Water stress tolerance of R. opacus may involve a wide range of cellular responses at different levels. Major reorganizations probably occur in the cell during the first hours of stress. The cells appear to regulate their energy generating system under these conditions, as indicated by the drastic decrease of the metabolic activity of cells during the initial period. After the first phase of reorganization, the endogenous respiration reached a minimum necessary for maintaining vital processes. Similar responses have been reported by M arden et al. [41] for a marine isolate DW101 subjected to short-term starvation. Results of this study also demonstrate that strain PD630 utilized storage lipids as endogenous carbon and energy source under water stress conditions. The b-oxidation pathway remains active during dehydration and probably plays a key role in the physiology of Rhodococcus spp. under these conditions. This metabolic route serves not only as a catabolic pathway but also as a source of fatty acids which are required for a

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Fig. 5. TEM micrographs of thin sections of gluconate-grown colonies of R. opacus PD630 during water stress. (a) High-magnification views of cells of colonies grown on gluconate. (b) Micrographs showing cells surrounded by the EPS in gluconate-grown colonies after 25 days dehydration. (c) Views of fragmented cells of strain PD630 after seven months under drying conditions. The scale bars correspond to 0.1 lm.

large variety of lipids in actinomycetes [7]. In this context, fatty acids released from triacylglycerols may serve not only for generating energy but also as precursors for biosynthesis of different mycolic acids. Mycolic acid turnover may contribute to cell envelope adaptation under water stress conditions in strain PD630, as suggested by the inhibitory effect of isoniazid on cell sur-

vival. A variation of cell envelope lipid content according to the environmental conditions has been reported for Mycobacterium strains [42,43]. Whereas desiccation resulted in a significant increase in the cell wall thickness in different Acinetobacter species [44], no evidence for this was found in R. opacus PD630 after water stress challenge. The exact role of cell-wall component variation in R. opacus PD630 under dehydration deserves further study. Scanning electron microscopy demonstrated that cells of strain PD630 produce an amorphous EPS matrix at the surface of colonies when they are subjected to water stress. The accumulation of EPS by cells seems to be progressive over time. It has been suggested that EPS function in protection against desiccation [45]. EPS hold a reservoir of water in the microenvironment surrounding the bacteria, and it may reduce the water loss of the cells, so EPS act like a protective sponge, buffering bacteria against external changes in water potential [46]. In addition, production of EPS may provide the cells additional time for physiological adjustments, which allow bacteria to survive dehydration [46]. The EPS matrix results in a strong association of cells of strain PD630, probably providing additional protection and preventing the population from becoming dispersed. The survival of cells in arid soil environments may require the cooperative effort of more than a single bacterial cell. Such social interactions enable myxobacteria to withstand desiccation, as has been described by O‘Connor and Zusman [47]. Ultrastructural studies of R. opacus cells demonstrated no significant changes in the cell morphology under water stress conditions. Two types of cell morphologies have been detected: (i) filamentous cells predominated in colonies throughout the study, and (ii) fragmented cells were frequently observed after dehydration. Whether these fragmented cells are forms of R. opacus PD630 resistant to environmental stress remains to be investigated. This study aimed to understand the responses of a soil bacterium to a critical environmental factor found normally in semiarid Patagonia (Argentina). The moderately severe water stress imposed in this study may reflect conditions found in natural environments more accurately than conditions used in other studies. The results of this study suggest that R. opacus PD630 possesses the necessary adaptation mechanisms to withstand the water limitation periods of East Patagonia. In addition, this study showed overlap among water stress, starvation and osmotic stress responses. Further studies are necessary to determine the molecular mechanisms involved in water stress tolerance in Rhodococcus spp. and to distinguish what responses are specific for each of these situations. The understanding of biological processes of soil bacteria under water stress conditions is of

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great importance for predicting and manipulating microbial activities in arid environments.

Acknowledgements H.M. Alvarez is indebted to the Deutsche Akademischer Austauschdienst (DAAD) for the award of a postdoctoral scholarship. The authors are grateful to Dr. Rainer Kroppenstedt for preliminary mycolic acid analysis of strain PD630 and to Mrs. G. Kiefermann (Institut f€ ur Medizinische Physik und Biophysik) for excellent photographic work. We thank Rainer Kalscheuer and Edgardo Saavedra for technical assistance in the preparation of the manuscript and W.W. Mohn for reviewing the manuscript. The authors acknowledge the helpful comments of the reviewers.

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