Response to (chloro)biphenyls of the polychlorobiphenyl-degrader Burkholderia xenovorans LB400 involves stress proteins also induced by heat shock and oxidative stress

Authors

  • Loreine Agulló,

    1. Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química and Millennium Nucleus of Microbial Ecology and Environmental Microbiology and Biotechnology, Universidad Técnica Federico Santa María, Valparaíso, Chile; and
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  • Beatriz Cámara,

    1. Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química and Millennium Nucleus of Microbial Ecology and Environmental Microbiology and Biotechnology, Universidad Técnica Federico Santa María, Valparaíso, Chile; and
    2. Division of Microbiology, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany
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  • Paula Martínez,

    1. Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química and Millennium Nucleus of Microbial Ecology and Environmental Microbiology and Biotechnology, Universidad Técnica Federico Santa María, Valparaíso, Chile; and
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  • Valeria Latorre,

    1. Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química and Millennium Nucleus of Microbial Ecology and Environmental Microbiology and Biotechnology, Universidad Técnica Federico Santa María, Valparaíso, Chile; and
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  • Michael Seeger

    1. Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química and Millennium Nucleus of Microbial Ecology and Environmental Microbiology and Biotechnology, Universidad Técnica Federico Santa María, Valparaíso, Chile; and
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  • Editor: Skorn Mongkolsuk

Correspondence: Michael Seeger, Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química, Universidad Técnica Federico Santa María, Avenida España 1680, Valparaíso, Chile. Tel.: +56 322 654236; fax: +56 322 654782; e-mail: michael.seeger@usm.cl

Abstract

We report the effects of 4-chlorobiphenyl and biphenyl on the physiology, morphology and proteome of the polychlorobiphenyl-degrader Burkholderia xenovorans LB400. The exposure to 4-chlorobiphenyl decreases the growth of LB400 on glucose, and cells exhibit irregular outer membranes, a larger periplasmic space and electron-dense granules in the cytoplasm. Additionally, lysis of cells was observed during incubation with 4-chlorobiphenyl or biphenyl. Proteome of B. xenovorans LB400 exposed to biphenyl and 4-chlorobiphenyl were analysed by two-dimensional gel electrophoresis. Besides induction of the Bph enzymes of biphenyl catabolic pathways, incubation with 4-chlorobiphenyl or biphenyl results in the induction of the molecular chaperones DnaK and GroEL. Induction of these chaperones, which were also induced during heat shock, strongly suggests that exposure to (chloro)biphenyls constitutes stress conditions for LB400. During growth of LB400 on biphenyl, oxidative stress was evidenced by the induction of alkyl hydroperoxide reductase AhpC, which was also induced during exposure to H2O2. 4-chlorobiphenyl and biphenyl induced catechol 1,2-dioxygenase, as well as polypeptides involved in energy production, amino acid metabolism and transport.

Introduction

Polychlorobiphenyls are widespread aromatic pollutants that persist in the environment. Diverse polychlorobiphenyl-degrading bacteria have been characterized. Burkholderia xenovorans strain LB400 is able to degrade a wide range of polychlorobiphenyls and related compounds (Bopp, 1986; Haddock et al., 1995; Seeger et al., 1995, 1999, 2001; Goris et al., 2004) and has become a model microorganism for research of polychlorobiphenyl degradation. Its genome has recently been sequenced (http://www.jgi.doe.gov), and comprises two circular chromosomes and a megaplasmid. Strain LB400 transforms polychlorobiphenyls, via enzymes of the ‘upper’ biphenyl catabolic pathway located on the megaplasmid, into 2-hydroxypenta-2,4-dienoate and chlorobenzoates (Seeger et al., 1995, 1997, 1999). Chlorobenzoates are ‘dead-end’ metabolites for this bacterium. The performances of polychlorobiphenyl-degrading bacteria are recognized to be affected by the toxicities of polychlorobiphenyls. 2-Chlorobiphenyl, 2,3-chlorobiphenyl and 2,3,5-chlorobiphenyl reduce the viability of Escherichia coli (Cámara et al., 2004). 4-chlorobiphenyl decreases the survival of Pseudomonas sp. DJ-12 cells (Park et al., 2001).

A study of the responses by bacteria to the presence of aromatic compounds is required in order to understand the complexity of the catabolic process. Proteome analysis allows a detailed study of the bacterial response to different environmental signals (Seeger & Jerez, 1993; Seeger et al., 1996; Neidhardt & VanBogelen, 2000; Duchéet al., 2002; Heim et al., 2002). Aromatic compounds induce catabolic enzymes and also other polypeptides (Blom et al., 1992; Gage & Neidhardt, 1993; Lambert et al., 1997; Cho et al., 2000). Stress proteins DnaK and GroEL are induced by 2,4-dichlorophenoxyacetic acid (2,4-D) in Burkholderia sp. YK-2 (Cho et al., 2000). Phenol and benzoate induce stress proteins and some Krebs cycle enzymes in Acinetobacter radioresistens (Giuffrida et al., 2001).

In the present study, we describe changes in global protein patterns during long-term exposure of B. xenovorans LB400 to biphenyl or 4-chlorobiphenyl, substrates of the (chloro)biphenyl catabolic pathway. We found that, in addition to expected catabolic enzymes, molecular chaperones (DnaK and GroEL), alkyl hydroperoxide reductase (AhpC) and other proteins of energy production, general metabolism and transport are induced during exposure to 4-chlorobiphenyl or biphenyl.

Materials and methods

Chemicals

Polychlorobiphenyls (98% purity) and chlorobenzoates (98% purity) were obtained from Lancaster Synthesis (Morecambe, England) and Fluka AG (Buchs, Switzerland), respectively.

Bacterial strain and culture conditions

Burkholderia xenovorans strain LB400 was cultivated at 30°C in minimal M9 medium with trace solution and glucose (5 mM) or biphenyl (5 mM), as the sole carbon and energy source (Bopp, 1986; Goris et al., 2004). Growth was determined by measuring turbidity at 525 nm and by counting colony-forming units (CFU). LB400 cells were incubated without or with 4-chlorobiphenyl (0.5 or 2 mM) for 24 h at 30°C. Aliquots taken from cultures were appropriately diluted and plated on Luria–Bertani agar medium. CFU per millilitre values were calculated as the mean±SDs of, at least, three independent experiments.

Analysis of (chloro)biphenyl degradation products

4-Chlorobenzoate, benzoate, dihydrodiols and dihydroxybiphenyls were analysed by HPLC (Seeger et al., 1995, 1999; Cámara et al., 2004). 4-Chlorobenzoate and benzoate were quantified by comparison with authentic standards. Formation of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) was monitored by visible spectral scanning, with a spectrophotometer (Seeger et al., 1995).

Electron microscopy

Bacterial cells grown to the exponential phase in M9 mineral medium and glucose (5 mM) as the sole carbon source were incubated further for 24 h in the absence or presence of biphenyl (2 mM) or 4-chlorobiphenyl (2 mM). Cells were treated and observed with a Zeiss EM900 electron microscope as described before (Cámara et al., 2004). The micrographs shown were representative and selected from, at least, 10 fields.

Two-dimensional (2-D) gel electrophoresis

2-D gel electrophoresis with nonequilibrium pH gradient electrophoresis was performed as described previously (Seeger et al., 1996). For exposure experiments, cultures grown with glucose to the exponential phase (OD525 nm=0.40–0.45) were harvested and resuspended in 1/25 volume of minimal M9 medium with glucose (5 mM). Cells were incubated further without or with biphenyl or 4-chlorobiphenyl (nominal concentration of 2 mM) for 24 h at 30°C. For heat shock, the cells were incubated for 1 h at 41°C. Additionally, the protein patterns of LB400 grown on glucose (5 mM) or biphenyl (5 mM) to the exponential phase (OD525 nm=0.5–0.6) were determined. Cells were harvested by centrifugation. For oxidative stress, cells grown on glucose were incubated for 2 h in the presence of H2O2 (0.6 mM). The proteins were quantified by the Bradford method (Bradford, 1976). Proteins were visualized by staining with Coomassie brilliant blue R-250. The volume (Intensity × mm2) of each spot was analysed using the Quantity-one image analysis software (Bio-Rad Laboratories). Spot volumes were determined and normalized on, at least, three gel images of independent assays. Proteins were numbered arbitrarily from top to bottom and Bph proteins were designated by capital letters.

Protein sequencing and identification

The protein spots separated on 2-D gels were transferred by an electro-blotting system to a polyvinyldene difluoride membrane. N-terminal amino acid sequencing was performed with an Applied Biosystem model 494A Procise HT sequencer. To perform the mass spectrometric analysis, the protein spots were recovered from gels and prepared as described (Heim et al., 2002). The digestion of proteins was performed with sequencing-grade trypsin (2 μg mL−1). Peptide extracts were eluted directly from the ZipTip microcolumns with saturated α-cyano-4-hydroxycinnamic acid directly onto the MALDI target and analysed with a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) Ultraflex apparatus (Bruker, Bremen, Germany).

Partial protein sequences obtained by Edman degradation were used for searches, using the blast ornl tool in the genome of B. xenovorans LB400 (http://www.jgi.doe.gov). The peptide fingerprints obtained by MS MALDI-TOF were used for searches in the NCBI protein database with the Matrix science MASCOT search tool. Complete sequences of each protein were obtained and searches were executed with the fasta tool (http://www.ebi.ac.uk), to obtain the similarities and identifications.

Results

The response of B. xenovorans LB400 to 4-chlorobiphenyl and biphenyl was analysed. To determine whether B. xenovorans LB400 was metabolizing 4-chlorobiphenyl and biphenyl to their respective 4-chlorobenzoate and benzoate during exposure experiments, the concentrations of these products were monitored. After 24 h incubations with 4-chlorobiphenyl or biphenyl, the concentrations of 4-chlorobenzoate and benzoate were 135 μM and 35 μM, respectively. Therefore, under these conditions, strain LB400 actively degraded 4-chlorobiphenyl to 4-chlorobenzoate and mineralized biphenyl via benzoate. 4-chlorobenzoate is not degraded further by strain LB400 (Maltseva et al., 1999). Genomic analysis revealed that LB400 possesses three pathways for benzoate degradation: the hydroxylation pathway (through catechol 1,2-dioxygenase) and two aerobic pathways via coenzyme A activation (Denef et al., 2006). Neither biphenyl 2,3-dihydrodiol nor 2,3-dihydroxybiphenyl or HOPDA were detected during incubation, ruling out the possibility of accumulation of these metabolic intermediates. Therefore, the effects of 4-chlorobiphenyl and biphenyl on B. xenovorans LB400 are due to these substrates, the degradation of these biphenyls and accumulation of 4-chlorobenzoate and benzoate.

Effects of exposure to (chloro)biphenyls on growth and cell morphology

To study the effect of exposure to 4-chlorobiphenyl on the growth of B. xenovorans LB400, cultures grown to the exponential phase with glucose as the sole carbon source were further incubated in the absence or presence of 0.5 and 2 mM 4-chlorobiphenyl. At both concentrations, 4-chlorobiphenyl decreased the growth of LB400 (Fig. 1), although 4-chlorobiphenyl at a concentration of 2 mM had a stronger inhibitory effect on the growth of LB400. Therefore, this concentration was chosen for further studies. Cells of LB400 exposed to (chloro)biphenyls were analysed by electron microscopy (Fig. 2), to evaluate changes in cell morphology. Burkholderia xenovorans LB400 grown on glucose and exposed to 4-chlorobiphenyl or biphenyl exhibited changes in cell morphology. LB400 cells exposed to 4-chlorobiphenyl showed an irregular outer membrane and large numbers of dense granules in the cytoplasm. At the poles, a fuzzy outer membrane was observed and the periplasmic space was usually larger than in cells not exposed to 4-chlorobiphenyl. Additionally, during exposure to 4-chlorobiphenyl, lysis of some cells was observed. Exposure to biphenyl caused higher degrees of cell lysis, resulting in cell debris and some empty cells from which the cytoplasm had leaked out.

Figure 1.

 Effect of 4-chlorobiphenyl on the growth of Burkholderia xenovorans LB400. Cells grown in M9 medium with glucose as the carbon source were incubated further in the absence or presence of 4-chlorobiphenyl. The exposure to 4-chlorobiphenyl started at the point indicated with an arrow. Each point is an average of results from, at least, three independent assays.

Figure 2.

 Ultrathin sections of Burkholderia xenovorans LB400 exposed to biphenyl or 4-chlorobiphenyl. (a) Control cells; (b) cells exposed to biphenyl; (c) cells exposed to 4-chlorobiphenyl; (d) a detailed view of cells exposed to 4-chlorobiphenyl. Arrows and arrowheads indicate the outer and the inner membrane, respectively. The bar represents 0.6 μm.

Proteomic analysis of the response of B. xenovorans LB400 to (chloro)biphenyls

The proteome of B. xenovorans strain LB400 was analysed after exposure to biphenyl or 4-chlorobiphenyl by 2-D gel electrophoresis. Biphenyl and 4-chlorobiphenyl induced similar changes in the proteome of strain LB400 (Fig. 3a). The soluble protein patterns of strain LB400 exhibited eleven proteins that were induced by biphenyl and by 4-chlorobiphenyl (Fig. 3). The expression of many of the proteins did not change, with respect to exposure of biphenyl or 4-chlorobiphenyl. Table 1 summarizes the proteins of strain LB400 induced by exposure to 4-chlorobiphenyl or biphenyl. As expected, proteins of the biphenyl ‘upper’ pathway (Seeger et al., 1997) were observed to be induced by 4-chlorobiphenyl and biphenyl. Proteins A1 and A2 were identified as the large subunit (BphA1) and the small subunit (BphA2) of the iron-sulphur component of biphenyl-2,3-dioxygenase. Proteins B and D were identified as biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD), respectively. The induction of the enzymes of the biphenyl catabolic pathways is consistent with the degradation of 4-chlorobiphenyl and biphenyl observed under these conditions. Two molecular chaperones, DnaK and GroEL (protein 1 and 2, respectively), were induced in B. xenovorans LB400 under these conditions (Figs 3 and 4a). The dnaK (BxeA3961) and groEL (BxeA3815) genes encoding these chaperones are located in the major chromosome. The chaperones DnaK and GroEL increased more than two-fold during heat shock (Fig. 4a). During heat shock, one additional GroEL chaperone of B. xenovorans LB400 was induced, which is encoded by the groEL gene (BxeA2609), also located in the major chromosome. In LB400 as in other bacteria, groEL genes are clustered with groES genes, and dnak gene is clustered with dnaJ and grpE genes. Proteins 3, 4, 5 and 7 were identified as enzymes associated with energy production and amino acid metabolism. Protein 4 was identified as the β chain of ATP synthase, the catalytic subunit of the enzyme involved in ATP synthesis in the presence of a proton gradient across the membrane. Enolase (protein 5) is involved in glycolysis, transforming 2-phospho-D-glycerate to phosphoenolpyruvate. Protein 3 corresponds to a threonine synthase. Protein 7 was identified as 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyl transferase, an enzyme involved in lysine biosynthesis.

Figure 3.

 (a) Protein pattern of Burkholderia xenovorans LB400 exposed to biphenyl and 4-chlorobiphenyl. Proteins were separated by 2-D gel electrophoresis and stained with Coomassie blue. The figures show a segment of the 2-D gels. Proteins induced by biphenyl and 4-chlorobiphenyl in, at least, three independent experiments are boxed. Molecular mass standards in kDa are shown on the left-hand side of the gels. (b) Induction of proteins in B. xenovorans LB400 by exposure to biphenyl and 4-chlorobiphenyl.

Table 1.   Identification of proteins induced by exposure to (chloro)biphenyls in Burkholderia xenovorans LB400
ProteinPeptide sequenceMethodIdentification*Estimated,Similarity* (%)
MM
(kDa)
pI
  • *

    Based on searches (fasta) with the complete amino acid sequences in the databases of EBI.

  • Calculated from the complete amino acid sequence (JGI) of LB400 (by the compute pI/MW tool of Expasy).

  • Based on 2-D gel migration.

  • NI, Not identified.

1GKIIGIDLGTTNSCVAIMEGNSVN-terminalDnaK705.0Burkholderia malleii ATCC 23344 (92)
2AAKDVVFGDSARAKMVEGVNN-terminalGroEL575.1Burkholderia vietnamiensis (97)
3GAGAGEHHTFSDILLGGLAK
HGQTLNILGATSGDTGSAAEYA
MALDI-TOFThreonine synthase535.5Burkholderia pseudomalleii K96243 (85)
4NTGKPISVPVGKPTLGR
IMDVLGRPIDEAGPINSDVVR
MALDI-TOFATP synthase β chain515.2Burkholderia pseudomalleii K96243 (97)
5KVQLVGDDLFVTNTR
VQLVGDDLFVTNTR
MALDI-TOFEnolase464.8Ralstonia solanacearum (91)
6MALDI-TOFNI405.8
7SQQLQQIIDTAXENRN-terminal2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyl transferase305.5Burkholderia pseudomalleii (95)
A1SSAIKEVQGAPVKWVTNWTPEN-terminalBiphenyl-2,3-dioxygenase large subunit (BphA1)516.0Pseudomonas pseudoalcaligenes (97)
A2TNPSPHFFKTFEWPSKAAGLELN-terminalBiphenyl-2,3-dioxygenase small subunit (BphA2)225.5Pseudomonas sp. B4 (100)
BLKGEAVLITGGASGLGR
LAELETDHGDNVLGIVGDVR
MALDI-TOFcis-2,3-dihydroxy-2,3-dihydrobiphenyl dehydrogenase (BphB)295.3Pseudomonas sp. Cam-1 (100)
DTALTESSTSKFVKINNEKGFSDFN-terminal2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase (BphD)326.0Pseudomonaspseudoalcaligenes (97)
Figure 4.

 Induction of stress proteins in Burkholderia xenovorans LB400 by (chloro)biphenyls. Segments of 2D gels. (a) Induction of molecular chaperones DnaK and GroEL by heat shock and (C)Bs. 1, control cells; 2, cells exposed to heat shock (41°C); 3, cells exposed to biphenyl (2 mM); 4, cells exposed to 4-chlorobiphenyl (2 mM). (b) Induction of AhpC protein during exposure to H2O2 and growth in biphenyl. 1, control cells; 2, cells exposed to H2O2 (0.6 mM); 3, cells grown on biphenyl (5 mM).

In order to analyse the effect of growth on biphenyl as the only carbon source on the proteome of strain LB400, additional experiments were performed. During the exponential phase of growth of LB400 on glucose or biphenyl, several differences in the protein pattern were observed. Table 2 summarizes additional proteins induced in strain LB400 during growth on biphenyl that are not listed in Table 1. Several proteins belonging to the biphenyl-degradative pathways were strongly induced, including BphA1, BphA2, BphA4, BphB, BphC, BphD and BphJ. One of the proteins induced by growth on biphenyl was identified as a catechol-1,2-dioxygenase (C1,2O). This enzyme, which is encoded by the catA gene (BxeA2109) of the major chromosome, belongs to the 3-oxoadipate pathway for the degradation of catechols, the subsequent degradative pathway for benzoate. The molecular chaperone DnaK, which was induced during exposure to biphenyl or 4-chlorobiphenyl, was also induced during growth on biphenyl, suggesting that growth on this compound constitutes a stressful condition for strain LB400. Another protein induced during growth on biphenyl was identified as AhpC (Fig. 4b, Table 2). During growth on biphenyl, AhpC was induced threefold. AhpC is also induced in strain LB400 during exposure to H2O2 (Fig. 4b). This enzyme is encoded by the ahpC gene (BxeA2911) of the major chromosome. In LB400 the ahpC gene is clustered with the ahpF gene as observed in other bacteria. Protein 8 was identified as an elongation factor G, an enzyme involved in protein biosynthesis. This protein is encoded by a gene (BxeA0311) located in the major chromosome. Proteins 9, 10, 11 and 13 show high similarities to a glutaryl-CoA dehydrogenase, a putative secreted substrate-binding protein, an outer membrane protein and an amino acid ABC transporter, respectively.

Table 2.   Identification of additional proteins from Burkholderia xenovorans LB400 induced by growth on biphenyl
ProteinPeptide sequenceIdentification*Estimated,Similarity* (%)
MM (kDa)pI
  • *

    Based on search with complete amino acidic sequence in the databases of EBI (fasta).

  • Calculated from the complete amino acid sequence (JGI) of strain LB400 (by the Compute pI/MW tool of Expasy).

  • Calculated considering the protein signal peptide sequence.

8ARKTPIERYRNIGISAHIDElongation factor G775.2Burkholderia mallei strain ATCC 23344 (93)
9AAQFHWEDPLLXDQQXTGlutaryl-CoA dehydrogenase435.9Ralstonia solanacearum strain GMI1000 (92)
10DQVVKIGHVAPLTGGIAHLGKDPutative secreted substrate-binding protein428.8Burkholderia pseudomallei strain K96243 (91)
11DAFNLIGHGPVSIGMGGTAOuter membrane protein477.8Pseudomonas putida strain F1 (62)
12MNRQAIDALLQKINDSAIHAGNCatechol 1,2-dioxygenase335.1Frateuria sp. strain ANA-18 (94)
13KDWSTIRFGVDASYPPFESKGSDAmino acid ABC transporter, periplasmic amino acid-binding protein288.6Burkholderia mallei strain ATCC 23344 (81)
14IINSQVKPFKAQAYHNGEFVTAlkyl hydroperoxide reductase (AhpC)215.1Burkholderia pseudomallei strain K96243 (90)
A4MIDTIAIIGAGLAGSTAARABiphenyl-2,3-dioxygenase subunit (BphA4)435.9Pseudomonas pseudoalcaligenes KF707 (100)
CMSIRSLGYMGFAVSDVAAWRSBiphenyl-2,3-diol-1,2-dioxygenase (BphC)325.7Pseudomonas pseudoalcaligenes (99)
JMTKKIKCALIGPGNIGTDLLAAcetaldehyde dehydrogenase (BphJ)326.1Pseudomonas pseudoalcaligenes KF707 (100)

Discussion

Mono-chlorinated biphenyls, which are the polychlorobiphenyls with the lowest hydrophobicity, have a greater negative effect on bacterial viability than do the higher chlorinated congeners (Cámara et al., 2004). Burkholderia xenovorans LB400 converts 4-chlorobiphenyl via the ‘upper’ biphenyl pathway to 2-hydroxypenta-2,4-dienoate and 4-chlorobenzoate (Seeger et al., 1995), which accumulates as a dead-end metabolite. Therefore, 4-chlorobiphenyl was selected as a model compound for this study. Additionally, we selected biphenyl, its nonchlorinated ‘parental’ compound, which could be used by LB400 as a sole carbon source for growth.

At contaminated sites, elevated concentrations of polychlorobiphenyls, as high as 28 g kg−1soil, have been observed (Lünsdorf et al., 2000). This indicates that in the environment, bacteria could be exposed to the polychlorobiphenyl concentrations used in this study. The present study showed that the presence of 4-chlorobiphenyl decreases LB400 growth on glucose. The decrease in bacterial growth observed during exposure to 4-chlorobiphenyl correlates with changes in cell morphology (Fig. 2). It has been described that 4-chlorobiphenyl affected survival of the polychlorobiphenyl degrader Pseudomonas sp. DJ-12 (Park et al., 2001). We observed an irregular outer membrane, enlargement of the periplasmic space at the poles and dense granules in the cytoplasm during exposure of B. xenovorans cells to 4-chlorobiphenyl. During conversion of polychlorobiphenyls to chlorinated 2,3-dihydroxybiphenyls in E. coli, an irregular outer membrane protruding at apical ends has been described (Cámara et al., 2004). The dense granules could correspond to polyphosphates, which are accumulated by bacteria during stress conditions (Kulaev & Kulakovskaya, 2000; Chavez et al., 2004). 4-Chlorobiphenyl also caused minor cell lysis. However, during incubation with biphenyl, more significant lysis of bacterial cells was observed, suggesting that damage occurs at the level of the cytoplasmic membrane. Owing to the hydrophobic character of these compounds, accumulation within the membranes is expected and, as in this study, changes in membrane structure and function have been described previously (Sikkema et al., 1994; Schweigert et al., 2001; Cámara et al., 2004). Ramos et al. (1995) reported that the presence of toluene damaged the inner and outer membrane in Pseudomonas putida DOT-T1 (Ramos et al., 1995). Hydroxylated polychlorobiphenyl metabolites produced during the degradation of biphenyl or polychlorobiphenyls affected the DNA content of Comamonas testosteroni TK102 and inhibited cell separation (Hiraoka et al., 2002).

Exposures to 4-chlorobiphenyl or biphenyl provoke similar changes in protein patterns and the induction of diverse proteins in B. xenovorans LB400. Induction of proteins by 4-chlorobiphenyl is, in general, less pronounced than induction by biphenyl, probably due to the lower aqueous solubility of 4-chlorobiphenyl (Cámara et al., 2004), which decreases the rate of its dissolution and its bioavailability. Different enzymes of the biphenyl ‘upper’ pathway of B. xenovorans LB400 growing on glucose were induced during exposure to 4-chlorobiphenyl or biphenyl. Additionally, during growth of B. xenovorans LB400 on biphenyl as the sole carbon source, main Bph proteins of the ‘upper’ pathway and one of the ‘lower’ pathway were induced. In accordance with this study, transcription of the bph locus of B. xenovorans LB400 has been described previously to be up-regulated by biphenyl (Beltrametti et al., 2001). However, it was also reported that bphA of B. xenovorans LB400 is constitutively expressed and not up-regulated by biphenyl (Master & Mohn, 2001). Another of the catabolic proteins induced during growth on biphenyl corresponds to C1,2O, the first enzyme of the 3-oxoadipate pathway for the degradation of catechols. Induction of C1,2O during growth of strain LB400 on biphenyl suggests that benzoate generated from biphenyl is catabolized via the hydroxylation pathway.

Protein patterns of (chloro)biphenyl-exposed cells or cells growing on biphenyl show that under both conditions, molecular chaperones are induced. The induction of molecular chaperones DnaK and GroEL suggests that the presence of 4-chlorobiphenyl and biphenyl constitutes a stressful condition for B. xenovorans LB400. These stress proteins are induced during heat shock of B. xenovorans LB400 (Fig. 4a), and under several stress conditions in other bacteria (Cho et al., 2000; Giuffrida et al., 2001; Park et al., 2001). Moreover, the induction of these polypeptides could be related to an increase of unfolded or precursor proteins, due to the presence of 4-chlorobiphenyl or biphenyl. Aromatic compounds can perturb the membranes, generate unfolded envelope proteins and activate the envelope stress response (Alba & Gross, 2004; Mascher et al., 2004). Biphenyl also induced an EF-G in B. xenovorans strain LB400. EF-G is induced under different stress conditions (Bébien et al., 2002; Duchéet al., 2002) and is able to interact with unfolded proteins as a classic molecular chaperone (Caldas et al., 2000). A different EF-G, encoded by the gene BxeA0793 of the major chromosome, is induced in B. xenovorans LB400 during exposure to 4-chlorobenzoate (P. Martínez, pers. commun.).

During growth of B. xenovorans LB400 on biphenyl, the alkyl hydroperoxide reductase AhpC was induced. This enzyme is responsible for detoxification of organic hydroperoxides and hydrogen peroxides during oxidative stress response (Niimura et al., 2000; Seaver & Imlay, 2001; Chauhan & Mande, 2002). AhpC is induced during exposure of strain LB400 to H2O2 (Fig. 4b). Therefore, induction of AhpC strongly suggests that growth on biphenyl generates oxidative stress in B. xenovorans LB400. In fact, we observed that LB400 cells grown on glucose and exposed to biphenyl showed increased levels of reactive oxygen species (ROS). During biotransformation of (chloro)biphenyls, ROS will be formed and may be harmful to cells (Schweigert et al., 2001; Seaver & Imlay, 2001). Higher levels of ROS were produced in cells of the polychlorobiphenyl-degrading strain C. testosteroni TK102 exposed to monohydroxybiphenyls, which may inhibit cell separation (Yamada et al., 2006). While this work was in preparation, the induction by biphenyl of proteins related to oxidative stress in strain LB400 was described (Denef et al., 2005).

The ATP synthase β subunit and enolase are also induced by 4-chlorobiphenyl and biphenyl. The induction of these proteins involved in the energy production of cells is probably required for the high energy demand for adaptation of strain LB400 to (chloro)biphenyls. Other polypeptides induced in the presence of 4-chlorobiphenyl and biphenyl are proteins of amino acid metabolism and involved in transport.

This study helps to characterize the responses of bacteria to xenobiotics. Proteomic analysis of B. xenovorans LB400 is useful to understand the complex regulation during exposure to (chloro)biphenyls. The regulation of protein synthesis is of critical importance for the adequate expression of polychlorobiphenyl-degradative pathways. In order to establish the optimized bioremediation processes for polychlorobiphenyls, the activities of bacterial enzymes need to be balanced in order to avoid accumulation of toxic metabolites or by-products as ROS. To address open questions concerning the xenobiotic metabolism of B. xenovorans LB400, further comprehensive studies are necessary, including genomic and proteomic analysis.

Acknowledgements

The authors thank Rita Getzlaff and Manfred Nimtz for protein sequencing, and Dietmar Pieper and Edward Moore for helpful discussions. M.S. gratefully acknowledges support from the grants: FONDECYT 1020221 and 7020221, USM 130322 and 130522, MILENIO P04/007-F (MIDEPLAN) and CONICYT-BMBF.

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