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Keywords:

  • otsA and otsB genes;
  • Pseudomonas sp.;
  • toluene tolerance;
  • trehalose;
  • trehalose biosynthetic gene

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Aim:  The objective of this study was to investigate toluene-induced accumulation mechanism of trehalose in a toluene-tolerant bacterium Pseudomonas sp. BCNU 106.

Methods and Results:  The accumulation of trehalose by a toluene-tolerant bacterium Pseudomonas sp. BCNU 106 was examined at various cultivation time by measuring the total intracellular trehalose content, trehalase activity and mRNA levels of the trehalose-biosynthetic genes. The pattern of trehalose accumulation corresponded to the mRNA expression pattern of the trehalose-biosynthetic genes with the maximum level at 12 h or 4 h of cultivation with 10% (v/v) toluene, respectively. The trehalose-biosynthetic genes were also cloned and sequenced. Furthermore, the effects of toluene addition on the intracellular osmotic pressure and pH were investigated. It was shown that homeostasis was maintained in the bacterial cells.

Conclusions:  In a toluene-tolerant bacterium Pseudomonas sp. BCNU 106, a significant amount of trehalose was accumulated through the toluene-induced expression of the trehalose-biosynthetic genes after the exposure to toluene.

Significance and Impact of the Study:  The accumulation of the high level of intracellular trehalose was preceded by the expression of otsA/B genes in toluene-tolerant bacteria, contributing to the elucidation of the tolerance mechanism.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The organic solvents including toluene are extremely toxic to microbial cells, even at the very low concentration of 0·1% (v/v). The solvents disrupt the cell membrane and get into the cell, resulting in impairing the structural and functional integrity of the cell (Sikkema et al. 1995). However, despite the extreme toxicity of organic solvents, the micro-organism that can actively grow and multiply in the presence of 10% (v/v) toluene was first reported by Inoue and Horikoshi (1989). Since then, many organic solvent-tolerant micro-organisms that are tolerant to organic solvents such as toluene, ethylbenzene, xylene and styrene have been isolated (Cruden et al. 1992; Weber et al. 1993; Ramos et al. 1995). These organisms are tolerant to the solvents even when they are present in a liquid medium at supersaturating concentrations. These micro-organisms are of particularly interest in that they can derive the potential applications in the bioremediation of sites that are heavily polluted with organic solvents, and in the biotransformation of low-solubility chemicals in water (Segura et al. 1999; Sardessai and Bhosle 2004).

The accumulation of organic solvents by solvent-tolerant bacteria results in several response mechanisms at the level of the cytoplasmic membrane, including the changes in the lipid composition of the membrane such as the isomerization of cis-unsaturated fatty acids into trans-isomers (Weber et al. 1994). Moreover, the headgroup composition of membrane lipids may be altered (Weber and de Bont 1996), and there may be changes to a series of energy-dependent solvent efflux pumps (Kieboom et al. 1998; Ramos et al. 1998). In addition, it was assumed that the several compatible solutes such as trehalose, betaine and proline may be accumulated in these solvent-resistant micro-organisms (Weber and de Bont 1996). Recently, we found that the trehalose levels in solvent-tolerant Pseudomonas sp. BCNU 171 increased significantly in response to the organic solvents (Joo et al. 2000). However, whether the solvent exposure induces the expression of the genes involved in the trehalose synthesis existing in solvent-tolerant bacteria has not been clear.

In order to investigate whether the accumulation of trehalose is common in solvent-tolerant Pseudomonas, we used a different strain Pseudomonas sp. BCNU 106 in this study. We have identified two trehalose-biosynthetic otsA and otsB genes from Pseudomonas sp. BCNU 106 and investigated the intracellular trehalose levels in parallel with biosynthetic otsA and otsB gene expression.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Bacterial strain, culture conditions and determination of intracellular trehalose levels

Pseudomonas sp. BCNU 106 was recently isolated as a BTEX-degrading micro-organism tolerating to the high concentrations of organic solvents. The strain was identified as Pseudomonas putida by 16S rDNA sequence analysis (homology 98%, Genbank accession numberDQ229315) and phylogenetic analysis based on 16S rDNA sequences in addition to a standard biochemical test. The bacterial cells were grown at 30°C by shaking at 130 rev min−1 in 500-ml Erlenmeyer flasks containing 100 ml of a Luria–Bertani (LB) medium supplemented with 10 mmol l−1 MgCl2 in the presence or lack of the toluene. Trehalose was extracted from 40 mg (wet wt) bacterial samples in 1 ml of boiling water, as described previously by Kienle et al. (1993) and assayed using commercial trehalase (Sigma, St Louis, MO, USA), according to the procedure described by Blazquez et al. (1994) except that glucose was estimated by the glucose oxidase-peroxidase method.

Assay of trehalase activity

The cells were collected by centrifugation, washed twice, resuspended in 0·5 ml of 10 mmol l−1 MES buffer (pH 6·0), disrupted by sonication and centrifuged to remove the cellular debris for 5 min at 10 000 g. The supernatant was directly used to measure trehalase activity. Trehalase activity was determined by incubating 50 μl of the cell-free extracts with 200 μl of 200 mmol l−1 trehalose in 100 mmol l−1 sodium acetate (pH 5·6) at 37°C for 30 min. The reaction was stopped by heating at 100°C for 5 min. One unit of trehalase was defined as the amount of enzyme that produces 1 mol l−1 glucose per min. The specific activity was expressed as mU mg−1 wet weight. Protein concentrations were measured, as described before (Bradford 1976).

Cloning and nucleotide sequencing of the otsA and otsB genes from Pseudomonas sp. BCNU 106

Total genomic DNA of Pseudomonas sp. BCNU 106 was purified by the instructions of the supplier (Qiagen, Valencia, CA, USA). Plasmid DNA was prepared, as described previously by Sambrook et al. (1989). The otsA (1·5 kb) and otsB (0·8 kb) genes were obtained from Pseudomonas sp. BCNU 106 genomic DNA by amplification with Taq PCR using the following primers: otsA, upstream primer 5′-ACATGAGTCGTTTAGTCGTAGTATCT-3′ and downstream primer 5′-TTGCTACGCAAGCTTTGGAAAGGTAGC-3′; otsB, upstream primer 5′-AACATGACAGAACCGTTAACCGAAACC-3′ and downstream primer 5′-TTGTTAGATACTACGACTAAACGACTC-3′. The respective translation initiation and termination codons of the Escherichia coliotsA and otsB genes (Kaasen et al. 1994) in these primers are in bold. After a standard PCR employing 30 cycles, the reaction aliquots were run on an agarose gel. The fragments of the genes were cloned into a pGEM-T Easy vector (Promega, Madison, WI, USA) and sequenced by an automatic DNA sequencer (ABI 373A; Applied Biosystems, Foster City, CA, USA).

Reverse transcriptase (RT)-PCR analysis

For the RT-PCR experiments, RNA was extracted by using the hot phenol method. The purified RNA samples were treated with DNase I and incubated with the downstream primers described above and the Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) at 42°C for 60 min. This reaction mixture (2 μl) was used as the substrate for PCR (25 cycles of 94°C for 2 min, 50°C for 1 min and 72°C for 2 min in a 50 μl reaction mixture with 2 mmol l−1 MgCl2) employing the AmpliTaq DNA polymerase (Perkin-Elmer, Wellesley, MA, USA). For PCR, the upstream primers described above were used together with the primers employed in the RT reaction. The sizes of the products of these reactions were 1·5 and 0·8 kb, respectively.

Determination of intracellular osmotic pressure and pH

The cells collected by centrifugation were suspended in 50 ml of 10 mmol l−1 Tris saline buffer (pH 7·0) and again pelleted by centrifugation. This washing procedure was repeated twice, and the only cells were finally disrupted by sonication and centrifuged to remove the cellular debris for 5 min at 10 000 g. The supernatant was directly used to measure the intracellular osmotic pressure and pH with an osmometer (M3300, Advanced Instruments, Norwood, MA, USA) and a pH meter (520A, Orion, Boston, MA, USA), respectively.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Accumulation of intracellular trehalose by toluene-treated Pseudomonas sp. BCNU 106

Solvent-tolerant Pseudomonas sp. BCNU 106 was able to grow in a culture medium that contains high concentration of toluene (50% v/v). However, the growth of this strain was retarded for the first 12 h after the toluene was added to the culture medium at concentrations of 10%, 20%, 30% and 50%, respectively; after this period of adaptation, the bacteria grew strongly despite the presence of the various concentrations of toluene (Fig. 1). Indeed, for 20 h after the addition of the toluene, the bacterial growth became more rapid in the presence of high concentration of the toluene, compared with the low concentrations of the toluene. We examined the concentration of intracellular trehalose in Pseudomonas sp. BCNU 106 cells that had been cultured with or without 10% (v/v) toluene. Very low levels of the disaccharide were observed in the untreated cells but 10% (v/v) toluene treatment caused the cells to gradually accumulate a large amount of trehalose. The trehalose levels peaked at 12 h after the incubation with toluene was commenced, after which the levels declined slightly (Fig. 2). The addition of toluene caused an approx. eightfold increase in the trehalose levels, compared with those generated when toluene lacked.

image

Figure 1.  Growth of Pseudomonas sp. BCNU 106 in the presence of toluene at various concentrations (v/v). Toluene is present at 0% (bsl00000), 10% (bsl00001), 20% (bsl00084), 30% (bsl00066), or 50% (bsl00067). Each value shows the mean of the triplicate cultures.

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image

Figure 2.  Effect of toluene on trehalose levels (circles) and trehalase activity (squares) in Pseudomonas sp. BCNU 106 cells. The intracellular trehalose levels (•, ○) and trehalase activity (bsl00001, bsl00000) in 40 mg of cells grown on LB with or without 10% (v/v) toluene were estimated every 4 h for 20 h. The trehalose levels and trehalase activities that are shown are the averages from the three independent experiments. Symbols: with toluene (•, bsl00001); without toluene (○, bsl00000).

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Enzymatic activity of trehalase in toluene-treated Pseudomonas sp. BCNU 106

We estimated the activity of trehalase as this enzyme was potentially involved in the degradation of trehalose. Trehalase activity was determined over time while the bacteria were grown with or without 10% (v/v) toluene. No significant differences were observed in trehalase activity between the toluene-untreated cells and 10% toluene-treated cells (Fig. 2).

Isolation and sequence analysis of the otsA and otsB genes from Pseudomonas sp. BCNU 106

To analyse the mechanism by which Pseudomonas sp. BCNU 106 accumulates trehalose in response to the toluene, we isolated the homologues of the trehalose biosynthetic otsA and otsB genes from Pseudomonas sp. BCNU 106 genomic DNA. The nucleotide sequences of otsA and otsB genes have been registered at the GenBank nucleotide sequence databases under the accession number AY308798 and AY308799, respectively. Based on the BLAST analysis, the deduced amino acid sequence of otsA gene showed a calculated molecular mass of 53·57 kDa and significant similarity to several OtsA proteins from other organisms, including the 91% identity with the E. coli OtsA. The deduced amino acid sequence of otsB, which had a calculated molecular mass of 29·2 kDa, also showed a significant similarity to the several OtsB proteins.

The mRNA expression of the otsA and otsB genes in response to toluene

We speculated that the accumulation of trehalose was likely to be due to the toluene-induced activation of otsA and otsB. Thus, we examined if the expression of these genes was enhanced by 10% (v/v) toluene treatment. Northern blotting analysis of the treated cells did not yield a clear image and thus we addressed this question by real time RT-PCR. We found that the expression of the genes increased over time during the toluene treatment (Fig. 3). The mRNA levels of the two genes peaked after 4 h and were maintained at these levels before decreasing for 24 h after the treatment commenced. These expression patterns were in a good agreement with the toluene-induced increase in the internal trehalose levels, as shown in Fig. 2.

image

Figure 3.  Expression of the otsA (open bar) and otsB (filled bar) genes in response to toluene. (a) otsA and otsB mRNA levels over time during the toluene treatment. The mRNAs of these genes were amplified by RT-PCR and the products were run on an ethidium bromide-stained agarose gel (1%). (b) Quantitative analysis of the otsA and otsB transcripts. The relative transcript levels of otsA and otsB shown in panel a were calculated by using a scanning densitometry.

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Intracellular osmotic pressure and pH in toluene-treated Pseudomonas sp. BCNU 106

Figure 4 shows the effect of toluene addition on the intracellular osmotic pressure and intracellular pH. Intracellular osmotic pressure was measured as about 331 mOsm kg−1 by adding 10% toluene to the culture medium, while taking the medium without toluene addition as a control (at an intracellular osmotic pressure of 323·5 mOsm kg−1). The intracellular osmotic pressure of Pseudomonas sp. BCNU 106 decreased from approx. 330–300 mOsm kg−1 within 4 h perfusion with 10% toluene. After 16 h, the Pseudomonas sp. BCNU 106 cells had regained an intracellular osmotic pressure of approx. 310 mOsm kg−1. Intracellular pH of the cells was constant around 7·5 at a 10% toluene treatment (Fig. 4).

image

Figure 4.  Effect of toluene on intracellular osmotic pressure (circles) and intracellular pH (squares) in Pseudomonas sp. BCNU 106 cells. The intracellular osmotic pressure (•, ○) and intracellular pH (bsl00001, bsl00000) in 40 mg of cells grown on LB with or without 10% (v/v) toluene were estimated every 4 h during 20 h. The intracellular osmotic pressure and intracellular pH are the averages from the three independent experiments. Symbols: with 10% toluene (•, bsl00001), without toluene (○, bsl00000).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

From the results presented in this work as well as those published by Joo et al. (2000), it is obvious that there is a considerable distribution of trehalose accumulation to the adaptive responses of bacterial cells to the toxic organic solvents.

The organic solvents are known to normally damage the cells by impairing functions such as the loss of ions, metabolites, lipids and proteins, dissipation of the pH gradient and electrical potential or by inhibiting membrane protein functions (Sikkema et al. 1995). However, some micro-organisms were tolerant to the different organic solvents inducing the several adaptive mechanisms (Weber et al. 1994; Weber and de Bont 1996; Kieboom et al. 1998; Ramos et al. 1998).

The unusual effectiveness of trehalose in preserving a biological function under the diverse stress conditions may be attributed to its unique physical properties as a nonreducing sugar (Crowe 2002). The inertness of this disaccharide is presumed to allow the accumulation of high intracellular concentrations without disturbing the biochemical processes. Trehalose has been reported to protect the cells by stabilizing proteins and phospholipids of the membrane (Singer and Lindquist 1998) and takes a central role on tolerance against the various environmental stresses including heat shock, dehydration, oxidative stress, toluene shocks or hydrostatic pressure shocks (Iwahashi et al. 1997; Argüelles 2000; Joo et al. 2000; Fernandes et al. 2001). It is assumed that there are at least three different pathways described for the biosynthesis of trehalose (Giaever et al. 1988; Maruta et al. 1995, 1996; Tsusaki et al. 1996, 1997; De Smet et al. 2000). The best known pathway in an E. coli model involves two enzymes, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, which are encoded by otsA and otsB, respectively.

Our data showed that the peaking of trehalose levels was preceded by the expression of the otsA and otsB genes. The results thus suggest that the trehalose biosynthetic pathway was essential for the adaptation to the solvents in the solvent-tolerant bacteria and for their survival. In contrast, the level of trehalase, which degrades trehalose, was not affected by the toluene exposure. A similar observation has also been reported in a thermo-tolerant strain of Candida albicans after the heat treatment (Argüelles 1997), suggesting that this enzyme is important to lower trehalose concentrations once the stress is alleviated.

Homeostasis of the intracellular pH and osmolarity is important for the proper function of a microbe metabolism. Thus, the fact that the BCNU 106 strain was able to maintain homeostasis of intracellular pH and osmolarity upon the toluene exposure, may be an important physiological event governing its ability to tolerate high concentrations of toluene. Furthermore, the response of trehalose biosynthesis by the toluene-tolerant bacteria was rather related to the only osmotic stress generated by the organic solvent than found principally related to the toluene stress.

In conclusion, our results suggest that the toluene-induced effects on the accumulation of trehalose in Pseudomonas sp. BCNU 106 may be primarily caused by a toluene stress. In addition, these findings may indicate that the accumulation of trehalose plays a role in the ability of toluene-tolerant Pseudomonas sp. BCNU 106 to tolerate the toluene stress.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This study was supported by a Korea Research Foundation Grant (KRF 200-005-D20020).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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