Abstract
Abbreviations: CDW, cell dry weight; mclPHA, medium-chain-length polyhydroxyalkanoate; nano-LC MS/MS, nano-electrospray liquid chromatography MS; OMP, outer membrane protein; PA, phenylacetic acid, PHA, polyhydroxyalkanoate; PHB, polyhydroxybutyrate
Two supplementary tables, listing proteins and peptides identified in the study and spectral count abundance measurements, are available with the online version of this paper.
Molecular investigations of the styrene degradation pathway in this organism have been reported (Mooney et al., 2006b; O'Connor et al., 2001). Styrene metabolism in P. putida CA-3 proceeds via initial side chain oxidation and involves an upper pathway converting styrene to phenylacetic acid (PA) (O'Connor et al., 1995), and a lower pathway initiated via activation of PA to phenylacetyl-CoA. The further metabolism of phenylacetyl-CoA involves oxidation of the aromatic nucleus, followed by ring cleavage and oxidation of the alicyclic compound, eventually yielding TCA cycle intermediates (Martinez-Blanco et al., 1990; Mooney et al., 2006b; O'Leary et al., 2002b; Olivera et al., 1998). The pha operon in P. putida CA-3 consists of two class II MCL-PHA synthases (phaC1 and phaC2) flanking the PHA depolymerase-encoding phaZ gene (O'Leary et al., 2005).
While traditional metabolic studies have provided information on single or a small number of molecular targets, high-throughput experimental technologies such as proteomics can generate comprehensive datasets that facilitate understanding of the wider metabolic network of the organism (Park & Lee, 2005). Such system-level analyses are currently changing the strategy of engineering. Bacterial proteome profiling enables elucidation of metabolic networks in bacteria grown under relevant conditions (Krayl et al., 2003; VerBerkmoes et al., 2006). We performed this study with the view to gaining a broader and deeper insight into the response of P. putida CA-3 to N limitation when grown on styrene. We compared the proteome of P. putida CA-3 grown on styrene under mclPHA accumulating and non-accumulating conditions, expecting that the broad overviews of the protein expression patterns will facilitate identification of proteins and/or pathways possibly affecting styrene metabolism and PHA accumulation. A number of tests were performed with whole cells and extracts of bacterial cells to confirm certain observations arising from the proteomic data and to quantify those responses biochemically.
P. putida CA-3 growth.P. putida CA-3 (NCIMB 41162) had been isolated previously from a bioreactor containing styrene (O'Connor et al., 1995). Cultures were grown in a 5 l stirred tank reactor (Electrolab) with the styrene supplied continuously as a vapour, as described previously (Goff et al., 2007). The growth medium used was minimal mineral salts (MSM) containing, per litre: 9 g Na2HPO4.12H2O, 1.5 g KH2PO4, 0.2 g MgSO4.7H2O, 0.002 g CaCl2 and 1 ml trace element solution (Schlegel et al., 1961). The N source was NH4Cl (Sigma-Aldrich). For mclPHA accumulation, N was limited with a starting concentration of 65 mg l–1 and a feeding rate of 1.5 mg l–1 h–1 (Goff et al., 2007). To prevent mclPHA accumulation, N was supplied at an initial concentration of 525 mg l–1 and maintained above 400 mg l–1 using a N feeding rate of 45 mg l–1 h–1. Fermentations were performed over a 48 h period at 30 °C, at an agitation rate of 500 r.p.m. and a controlled pH of 6.8. Samples (40 ml) were taken for analysis at 6, 20, 30 and 48 h (T6, T20, T30 and T48, respectively). From each sample, 10 ml was used for protein preparation, another 10 ml was used for the determination of amino acid uptake rate, and the remainder (20 ml) for mclPHA analysis.
Cell growth was monitored spectrophotometrically (OD540; Unicam Helios δ UV/VIS spectrophotometer) and cell dry weight (CDW) was obtained via a calibration curve of OD540 versus dry weight, where cell suspensions with optical densities ranging from 0.1 to 0.8 were filtered through previously weighed glass fibre membranes (Whatman) and dried at 90 °C until a constant weight was achieved.
Protein sample preparation.
Cells from 10 ml culture aliquots taken from the fermenter at different time points over the 48 h period of incubation were collected by centrifugation (5000 g for 10 min at 4 °C) and washed twice with an equal volume of phosphate buffer (50 mM, pH 7). Cells were then resuspended in phosphate buffer to an equal concentration of CDW of 1.2 g l–1 and centrifuged again. Cell pellets were then resuspended in 500 µl BugBuster (primary amine-free, Novagen). Benzonase nuclease (Novagen) and PMSF (Sigma) were added to the suspension at 2 U ml–1 and 0.3 mg ml–1, respectively. Cell suspensions were incubated at 30 °C with gentle shaking (50 r.p.m.) and the cell debris and unbroken cells removed by centrifugation (5000 g for 10 min at 4 °C). The protein concentration of the supernatant was determined by the Lowry method using BSA as a standard. Protein samples (30 µl) were resuspended in double the volume of 2x non-reduced SDS-PAGE sample buffer (80 mM Tris/HCl, pH 6.8, 10 % β-mercaptoethanol, 2 % SDS, 10 %, v/v, glycerol, 0.1 % Bromophenol Blue) and heated at 100 °C for 5 min. Denatured proteins were separated on a 10 % (w/v) polyacrylamide SDS gel and stained with Coomassie Brilliant Blue (Sigma-Aldrich).
Mass spectrometric peptide analysis.
SDS-PAGE gels were cut into bands (16 bands per time point) and the proteins digested in-gel with trypsin according to the method of Shevchenko et al. (1996). The resulting peptide mixtures were resuspended in 1 % formic acid and analysed by nano-electrospray liquid chromatography MS (nano-LC MS/MS). An HPLC instrument (Dionex) was interfaced with an LTQ ion trap mass spectrometer (ThermoFinnigan). Chromatography buffer solutions (Buffer A, 5 % acetonitrile and 0.1 % formic acid; Buffer B, 80 % acetonitrile and 0.1 % formic acid) were used to deliver a 60 min gradient (35 min to 45 % Buffer B, 10 min to 90 %, hold 10 min, 3 min to 5 %, hold for 15 min). A flow rate of 2 µl min–1 was used at the electrospray source. Spectra were searched using the Sequest algorithm (Craig & Beavis, 2004) against the UniProt database restricted to P. putida entries (downloaded January 7, 2007). Proteins with (a) a Peptide Prophet probability score greater than 0.99 (Keller et al., 2002), and (b) identified by a minimum of two different peptide spectra were automatically accepted, while spectra for the minority of proteins identified by single spectra were manually checked for quality. Spectral counts were automatically calculated for each protein across different fractions using in-house scripts.
mclPHA extraction and analysis.
Cells collected at different time points of fermentation were harvested by centrifugation (5000 g for 10 min at 4 °C) and washed twice with an equal volume of phosphate buffer (50 mM, pH 7). Cells were centrifuged again (5000 g for 10 min at 4 °C) and freeze-dried. The polymer content was determined by subjecting 5–10 mg lyophilized whole cells to acidic methanolysis according to published protocols (Brandl et al., 1988; Lageveen et al., 1988). This method degrades the intracellular PHA to methyl esters of its constituent 3-hydroxyalkanoic acids. The 3-hydroxyalkanoic acid methyl esters were assayed by GC using a Hewlett Packard HP6890 chromatograph equipped with a BP-20 capillary column (30 m by 0.25 mm, 0.25 µm film thickness; J & W Scientific) and a flame-ionization detector (FID). A temperature programme of 60 °C for 3 min, temperature ramp of 5 °C per min, 200 °C for 1 min was used. For the peak identification, methyl esters of 3-hydroxyalkanoic acid were prepared in a similar manner and PHA standards from P. putida CA-3 were used (Ward et al., 2005).
Nitrogen assay.
The nitrogen concentration in the growth medium was determined by the indophenol method, as described by Scheiner (1976).
Phenylacyl-CoA ligase enzyme assay.
The phenylacetyl-CoA ligase activity was determined by measuring the rate of formation of phenylacetylhydroxamate in the presence of cell lysates (200 µl; prepared as described in the protein sample preparation section), ATP, CoA, PA and neutral hydroxylamine, as previously described (Martinez-Blanco et al., 1990; Ward & O'Connor, 2005). Phenylacyl-CoA ligase activities were measured at 30 °C over a 30 min period. The molar extinction coefficient of phenylacetylhydroxamate in the presence of ferric chloride under these conditions is 0.9 mM cm–1 (Martinez-Blanco et al., 1990; Ward & O'Connor, 2005).
Branched chain amino acid uptake.
For the measurement of leucine and valine uptake rates, 10 ml samples of cells taken from the fermenter at various time points were harvested by centrifugation (5000 g for 10 min at 4 °C), washed and resuspended in deionized H2O to OD540 4. Cell suspensions (2 ml) were then supplemented with 1 mM leucine and/or 1 mM valine and incubated at 30 °C with shaking (200 r.p.m.). Samples (50 µl) were removed every 30 min over a 2 h period. The amino acid detection was based on derivatization with isobutyl chloroformate followed by GC–MS, as described elsewhere (Sobolevsky et al., 2004). Briefly, reaction mixture (50 µl) was mixed well with deionized H2O (50 µl), isobutanol (30 µl), pyridine (10 µl) and isobutyl chloroformate (30 µl). The derivatives were extracted into chloroform (500 µl) by vigorous shaking and subsequent centrifugation (10 000 g for 1 min). A 1 µl volume of the chloroform layer was injected in the splitless mode onto an HP-5 column (20 m by 0.25 mm, 0.33 µm film thickness; J & W Scientific) using an Agilent 6890 gas chromatograph coupled to a 5973 mass-selective detector. The column was held at 50 °C for 2 min then heated to 300 °C at a rate of 10 °C min–1. Under the above conditions, N-isobutoxycarbonyl isobutyl esters of leucine and valine had retention times of 14.37 and 15.12 min, respectively.
Immunoblotting of outer membrane protein (OMP).
Crude cell lysates, prepared as described in Methods, Protein sample preparation, and used for the mass peptide analysis were separated by SDS-PAGE (10 %, w/v) and transferred to a nitrocellulose membrane (Hybond ECL, Amersham Biosciences). For Western blotting, the membrane was probed with mAb 7.3 [mouse antibodies against P. putida KT2440 outer membrane lipoprotein as described by Ramos-Gonzalez et al. (1992)]. Peroxidase-conjugated goat anti-mouse immunoglobulins (Merck) were used for the chemiluminescent detection. The blots were developed using Immobilon Western chemiluminescent HRP substrate (Millipore) according to the manufacturer's protocol. Immunoluminescence was detected and evaluated using the chemiluminescence-compatible FluorChem imaging system equipped with AlphaEase FC2 software (Alpha Innotech).
Growth characteristics of P. putida CA-3
P. putida CA-3 was grown in the stirred tank bioreactor as previously described (Goff et al., 2007) using styrene as the sole carbon and energy source over 48 h, during which time biomass, N utilization and PHA production were monitored (Fig. 1). After a 48 h fermentation, during which the nitrogen (supplied as NH4Cl) concentration was kept above 400 mg l–1, a final biomass of 7.2 g l–1 (total 35.8 g from 5 l culture) was obtained (Fig. 1). No PHA was accumulated under these growth conditions. When the N supply was limited (65 mg N l–1 at the start of fermentation), a final CDW of 6.5 g l–1 was achieved (total 30.3 g CDW). However, this weight consisted of 52 % PHA (w/w), thus total biomass was 15 g (a total of 10.2 g of mclPHA was obtained from 5 l culture). Thus, N limitation resulted in a 2.4-fold decrease in biomass yield. During the exponential phase (T6–T25), the growth rates for non-limiting and limiting N growth conditions were 0.37 and 0.11 g CDW l–1 h–1, respectively. Therefore, we expected that the different growth rates and concentrations of N source during fermentation would most likely affect the protein expression pattern (Ferenci, 1999).
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Shotgun proteomics of P. putida CA-3 grown in a bioreactor on styrene under N limitation (permissive for PHA accumulation) and under N excess (not permissive for PHA accumulation)
P. putida CA-3 cultures were grown as described above and harvested at four time points: 6, 20, 30 and 48 h post-inoculation (Fig. 1). In order to produce an extensive dataset of protein expression, the lysate was fractionated by SDS-PAGE and analysed by LC-MS. Over 75 000 peptide tandem mass spectra were used to identify 9033 unique peptides, in turn generating 1761 high-confidence (≥0.99 Protein Prophet score) protein identifications (Supplementary Table S1). This represents a substantial proportion (33 %) of the predicted P. putida proteome, and reflects the extensive MS analysis at different time points under different growth conditions. Moreover, the proteins exhibited a wide range of annotated biophysical (molecular mass, isoelectric point), biochemical (functional annotations) and structural (domains) properties, suggesting that the analysis was not strongly biased in favour of or against any protein class.
Spectral counts were used as a semiquantitative measure of protein abundance (Cagney et al., 2005; Liu et al., 2004; Washburn et al., 2003). Using this analysis, it was possible to follow the expression of individual proteins in the different samples (Supplementary Table S2, Fig. 2). Comparison of our results with a preliminary experiment using P. putida CA-3 cultures suggested that the percentage of proteins detected in two MS analyses of independently grown cultures is 70–80 % (data not shown). Analysis of 20 ribosomal proteins, whose expression might be assumed to remain relatively independent of the time points and experimental treatment, showed that all 20 were detected in all fractions (% CV for spectra counts=0.34). Identified proteins were next organized into functional pathway groups (e.g. nitrogen assimilation, amino acid uptake) using annotations from the Gene Ontology database (Raghava, 2006). High-throughput protein annotation and metabolic pathway prediction were enabled by the availability of the genome sequence of P. putida KT2440 (Nelson et al., 2002). The spectral counts, reflecting the relative abundance of all the proteins engaged in the respective pathways, were then totalled. In this way, potentially up- or downregulated pathways could be observed (Table 1). Changes in the relative abundance of individual proteins or whole pathways are discussed below.
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Table 1. Proteins upregulated in P. putida CA-3 grown on styrene under N limitation
Styrene degradation proteins
P. putida CA-3 is a styrene-degrading bacterium that metabolizes styrene aerobically, employing enzymes organized in two distinct catabolic pathways (O'Leary et al., 2002b). An upper pathway involves oxidation of the styrene side chain, leading to epoxystyrene, which is subsequently isomerized to phenylacetaldehyde and then oxidized to PA. The lower pathway involves the conversion of the PA through CoA intermediates into aliphatic compounds that can enter the Krebs cycle (Fig. 2a).
The genes encoding the upper pathway are organized on the styABCD operon, which is regulated at the transcriptional level by the StySR two-component sensory apparatus in this strain (O'Leary et al., 2001) and other species such as Pseudomonas fluorescens ST (Beltrametti et al., 1997) and Pseudomonas sp. Y2 (Bartolomé-Martin et al., 2004; Velasco et al., 1998). The genes encoding the lower pathway (PACoA catabolon) are typically organized in five contiguous operons: paaABCEF, paaGHIJK, paaLMN, paaX and paaY (Luengo et al., 2001). Proteins encoded by these genes can be classified into functional units: a transport system (PaaL and PaaM); a phenylacetyl-CoA-activating enzyme (PaaF); a ring-hydroxylating enzymic complex (PaaGHIJK); a ring-fission protein (PaaN); a β-oxidation system (PaaABCE); and two regulatory proteins, a repressor (PaaX) and a putative regulator (PaaY) (Arias et al., 2008; Olivera et al., 1998).
While the typical PACoA catabolon has been suggested to be present in P. putida CA-3 (O'Leary et al., 2005), this is the first time, to our knowledge, that all of the enzymes were detected and their relative amounts observed over time when this strain was grown on styrene (Fig. 2b). All of the enzymes of the upper and lower pathways could be detected throughout the 48 h fermentation under both N-limiting and non-limiting conditions. However, the relative amounts of the styrene degradation enzymes ranged from 18–22 % under N excess to 11–18 % under N-limited conditions with respect to the total proteome. StyA and StyD were the most abundant proteins expressed over time in the upper pathway. StyA was expressed to higher levels at time 6 h under N limitation but to lower levels after this time point (Fig. 2). This observation is in agreement with our previous finding that the catabolic efficiency of styrene degradation dramatically decreases under PHA-accumulating (N-limiting) conditions (Ward et al., 2005).
With the exception of PaaE, proteins of the lower pathway were abundant under both N-limiting and non-limiting growth conditions (Fig. 2b). PACoA ligase (PaaF) enzyme activity was detected at similar levels throughout the growth cycle in crude extracts of cells supplied with styrene under N limitation (Fig. 3). The current observation supports an earlier observation that N limitation has little effect on the consumption of PA by whole cells grown on styrene (O'Connor et al., 1996). Under full N growth conditions, extracts of P. putida CA-3 cells grown on styrene showed a sharp decrease in both the peptide score for PaaF and PACoA ligase activity at T20 and T48, while the peptide score and enzyme activity at T6 and T30 were higher (Figs 2 and 3). These sharp fluctuations at specific time points raise the possibility that post-translational control of PaaF is occurring.
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While Mooney and co-workers demonstrated that StyE, a membrane-associated enzyme, is involved in the facilitated uptake of styrene in P. putida CA-3 (Mooney et al., 2006a), we detected only minor levels of StyE under both N-limiting and non-limiting growth conditions with styrene as the growth substrate (Fig. 2b). These findings suggest that other transporters as well as diffusion play a role in addition to StyE-mediated active transport in the uptake of styrene in P. putida CA-3.
Recently, Nogales and co-workers have established that the final product of aerobic PA degradation in P. putida KT2440 is succinyl-CoA (Luengo et al., 2004; Nogales et al., 2007). They also demonstrated, through generation of random insertion mutants and biochemical analysis, that this last step of PA degradation is catalysed by β-ketoadypil-CoA thiolase (PaaE). PaaE was also detected in P. putida CA-3 when grown on styrene under both N-limiting and non-limiting growth conditions (Fig. 2b).
The regulatory proteins StyS, StyR, PaaX and PaaY were detected at low levels, while PA specific permease (PaaL) was not observed in the proteome of P. putida CA-3 grown on styrene. O'Leary and co-workers observed that the transcription of the styS gene (upper pathway regulatory sensor kinase) was growth condition-dependent (O'Leary et al., 2002a). Indeed, we observed higher amounts of StyS present under N-limiting conditions (Supplementary Table S1). Del Peso-Santos and co-workers have demonstrated a role for PaaX as a major regulatory protein in the phenylacetyl-CoA catabolon (del Peso-Santos et al., 2006). They recently revealed that phenylacetyl-CoA binds to PaaX, and inactivates PaaX-mediated repression of both the paa genes and the styABCD operon in P. putida Y2 (del Peso-Santos et al., 2008). In agreement with this, PaaX was the most abundant of the regulators detected at T6 after inoculation under non-limiting N conditions (data not shown).
Proteins upregulated under N limitation
The expression of proteins involved in energy/metabolism, amino acid biosynthesis, DNA/RNA biosynthesis and processing, transcription and translation was quite similar under both N excess and N limited conditions (Supplementary Table S1) in P. putida CA-3 over time. Most of the N (NH4Cl) supplied was consumed within 20 h of inoculation under N limitation. Under N limitation (2 mg N l–1) the biomass (CDW) yield was 2.4 g l–1 lower than under N excess conditions (Fig. 1). The lack of any essential nutrient, including N, causes growth limitation (Ferenci, 1999) and physiological changes, whereby cells attempt to optimize nutrient scavenging and growth. We observed differentially expressed proteins belonging to the pathway groups of N assimilation, amino acid transport and uptake, outer membrane/lipoproteins, PHA synthesis, motility/chemotaxis and iron assimilation (Fig. 4). Major representative proteins for each of these functional groups with their respective spectral counts are given in Table 1. In addition, two stress-related proteins (peroxidase/catalase HPI and ATP-dependent Clp protease) were observed to be slightly upregulated under N limitation (Table 1).
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Upregulation of N assimilation
As expected, one of the most strongly affected functional groups under N-limited growth were the nitrogen assimilation proteins (Fig. 4). This group is represented by arginine deiminase, N regulatory protein P-II and proline iminopeptidase (Table 1), but also contains proteins such as N utilization substance protein A, nitrate binding protein NasS, nitrite reductase, nitroreductase and aminotransferases. N assimilation proteins represented 8 % of total detected proteins at 30 h of fermentation, while at the same time point of fermentation when N was supplied in excess 4.4 % of total proteins detected were associated with N assimilation. Arginine deiminase (EC 3.5.3.6) together with proline deaminase (EC 3.4.11.5) was the most abundant protein in this group. Arginine deaminase from Pseudomonas aeruginosa (PaADI) catalyses the hydrolysis of arginine to citrulline and ammonium ion, which is the first step of the microbial L-arginine degradation pathway (Lu et al., 2006). Proline iminopeptidase is also a hydrolytic enzyme involved in proteolysis as one of the ways to assimilate N when its supply is limited.
In poor growth conditions, the metabolism of cells is oriented towards an economical use of substrates by utilization of the available metabolites for the synthesis of proteins and nucleic acids (Lavallee et al., 2005). Here, we observed increased levels of amino acid transport proteins under N-limiting conditions (Table 1). This was confirmed in branched chain amino acid uptake experiments, where samples taken from the bioreactor at T48 grown under N-limiting conditions showed 2.2-fold higher leucine uptake compared with cells cultured under non-limiting growth conditions (Fig. 5). All bacterial cell samples were normalized so that they had the same biomass (g CDW l–1) in the amino acid uptake assay. Cells harvested from the N-limited fermentation at T48 consumed 1.5-fold more leucine than cells harvested at T6 over the 2 h test period (Fig. 5). In all samples tested N limited cells had a higher rate of leucine uptake than cells grown under non-limiting conditions (Fig. 5). Similar results were obtained when the amino acid used was valine (data not shown). These data were in agreement with the amounts of branched chain amino acid transporters detected by MS analysis (Table 1).
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Proteins involved in polyamine (putrescine) metabolism were also upregulated under N-limiting growth conditions (Supplementary Table S1). Polyamines (including putrescine and spermidine) are a group of ubiquitous polycations necessary for cell growth (Lu et al., 2002). In P. aeruginosa, putrescine can be synthesized from arginine through the arginine decarboxylase pathway (Patel et al., 2006). Through this pathway, arginine is sequentially converted and channelled into the tricarboxylic acid cycle (Patel et al., 2006). In cells grown under N limitation, the polyamine ABC transporters were particularly abundant, reflecting the cellular mechanisms used to deal with the N limitation. Other transport proteins were also upregulated, providing a range of means for scavenging N-based compounds. Santos et al. (2004) suggested that the differential expression of ABC transporters serves as a global signal for response to the concentration and the quality of the carbon source, starvation conditions and toxic stress. N limitation also stimulated upregulation of the proteolytic enzymes (Hsl protease, ATP-dependent La protease and SohB).
mclPHA synthesis occurs only under N limitation
One of the metabolic adaptations to suboptimal N levels during P. putida CA-3 growth on styrene is the accumulation of an intracellular carbon and energy storage material in the form of mclPHA granules (Ward et al., 2006). This has also been observed in many other micro-organisms as a response to stress imposed on cells during imbalanced growth, when essential nutrients are limited and carbon is in abundance (Hoffmann & Rehm, 2005; Rehm, 2007; Wältermann & Steinbüchel, 2005).
PHA accumulation was observed under N-limiting growth conditions when the N levels reached a concentration of 26 mg l–1 (T6). PHA accumulation continued throughout the fermentation, during which time N was kept at low levels (2–3 mg l–1) using a feeding strategy described by Goff et al. (2007). PHA levels increased 12-fold over 14 h (from T6 to T20), with a further 1.5- and 2.2-fold increase over the following 10 and 28 h, respectively (Fig. 1). No PHA was detected in cells grown with N excess.
PHA biosynthetic proteins were detected in the proteome of P. putida CA-3 cells grown under N limitation (Fig. 4). We observed poly(3-hydroxyalkanoate) polymerase 1 (PhaC1), poly(3-hydroxyalkanoate) polymerase 2 (PhaC2), 3-hydroxyacyl-CoA-acyl carrier protein transferase (PhaG), and granule-associated proteins 1 and 2 (Table 1). The PHA biosynthetic enzymes were detected at relatively low levels, while the depolymerase was not detected in proteomes of P. putida CA-3 during the course of this study. It has been documented that in PHA-producing organisms the polymerase and depolymerase, as well as phasin enzymes, are associated with PHA granules and form a distinct protein surface network (Pötter et al., 2004; Pötter & Steinbüchel, 2005; Sandoval et al., 2007). PHA granules are synthesized by PHA polymerases that utilize CoA thioesters of the respective 3-hydroxy fatty acids as substrates. Granules are mobilized (degraded) by PHA depolymerases, and formation of the granules depends on the activity of several granule-associated proteins (phasins).
Lipoproteins, chemotaxis-related proteins and iron ion transport proteins are upregulated under N limitation
In cells accumulating PHA (grown under N-limiting conditions) we observed upregulation of other OMPs, such as lipoproteins and lipoprotein-releasing proteins (Lol proteins), that target and anchor lipoproteins to the periplasmic surface of either the inner or the outer membrane (Miyamoto & Tokuda, 2007; Narita & Tokuda, 2006). It has been shown that nutrient limitation induces the expression of proteins in the outer membrane of bacterial cells such as Pseudomonas species under carbon, N and phosphorus limitation (Kragelund & Nybroe, 1994). Lipoproteins have been shown to have emulsifying properties that increase surface area and hence enhance the bioavailability of hydrophobic substrates such as styrene (Kuiper et al., 2004; Whang et al., 2008). They are also known to play a role in the bacterial adaptation to changes in environmental conditions such as osmotic stress (Guyard-Nicodème et al., 2008). Among OMPs, OmpA, G, H, E3, F and I were detected in the current study, but only OmpF and OmpA porins were twofold to fourfold upregulated by N limitation (Table 1, Supplementary Table S1). OmpF is a major membrane protein in P. aeruginosa, where it has a non-specific porin function (Hancock & Brinkman, 2002). In fact, Li et al. (1995) have demonstrated that an ompF– mutant of P. aeruginosa was toluene-tolerant, and therefore it was proposed that toluene enters the cell through the OmpF channel in wild-type cells. In contrast, Volkers and co-workers found that OmpF is downregulated in the presence of toluene excess in a chemostat-based culture of P. putida S12 and proposed that this strain is capable of shutting down this channel upon toluene stress (Volkers et al., 2006). In the same study, OmpH was dramatically upregulated (12-fold), while we observed more moderate (twofold) upregulation of this OMP. The higher levels of OmpA observed in this study are in agreement with the findings of Benndorf and co-workers, who identified OmpA as being upregulated in P. putida KT2440 in response to lipophilic herbicides (Benndorf et al., 2006).
Proteomic data were confirmed through Western blotting using a highly specific monoclonal antibody (mAb 7.3) which was previously raised against surface determinants of P. putida 2440 (LPS and membrane proteins) (Ramos-Gonzalez et al., 1992). Approximately 1.7-fold higher amounts of outer membrane lipoproteins containing O-antigen were detected in cells grown under N limitation compared with cells grown under N excess at T48, as determined by densitometry (Fig. 6). Interestingly, two bands were observed when mAb 7.3 was used. This suggests that P. putida CA-3 has more than one unit of O-antigen in its LPS (Fig. 6). Cowell et al. (1999) suggested that the increased adhesion to surfaces exhibited by P. aeruginosa grown under N limitation is due to an increase in OMP expression.
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Under N-limiting (PHA accumulation) conditions, a number of motility-associated proteins were upregulated (Fig. 4, Table 1). The majority of these proteins (∼80 %) were chemotaxis-related proteins, including chemotaxis histidine kinase CheA, chemotaxis proteins CheV, Y and Z, and a variety of methyl-accepting chemotaxis transducers. These methyl-accepting chemotaxis transducers are outer membrane receptors that mediate chemotaxis to diverse signals, responding to changes in the concentration of attractants and repellents in the environment by changing swimming behaviour (Adler, 1966). This has been observed in P. putida in response to various substances, including naphthalene (Grimm & Harwood, 1997; Harwood et al., 1990; Law & Aitken, 2006). The second group of motility-related proteins that was upregulated were flagellar biosynthetic proteins such as FliA, C, F, E, G, K and N. The importance of chemotaxis and flagellins was identified in a study by Raberg and co-workers, who studied flagellation changes in response to nutrient supply and the state of polyhydroxybutyrate (PHB) production in Ralstonia eutropha H16 (Raberg et al., 2008).
We have also identified number of proteins involved in iron ion acquisition and storage that were induced by N-limiting growth conditions (Fig. 4, Table 1). The most differentially expressed was bacterioferritin (Q88NX1), a protein that interacts with siderophores. Ferritins are part of a class of iron binding proteins that reversibly sequester excess iron and can thereby serve to ease iron-related oxidative stress (Smith, 2004). Bacterioferritins whose expression levels vary as a function of environmental and intracellular iron levels have been identified in P. putida KT2440 (Chen et al., 2009). Similarly, bacterioferritins are highly expressed in biofilms of P. aeruginosa (Patrauchan et al., 2007), but they have not been identified as being upregulated under N-limited growth conditions.
Solvent tolerance response by P. putida CA-3 to the presence of styrene
When grown on styrene, P. putida CA-3 cells face a paradox. On the one hand, styrene represents a carbon source that can be metabolized to yield carbon and energy for growth. On the other hand, with an octanol/water partition coefficient [log(POW)] of 3, styrene falls into a category of organic solvents extremely toxic to micro-organisms, due to their ability to bind to the cells, disturbing membranes by removing lipids and proteins and causing cell lysis (Ramos et al., 2001; Taylor et al., 2008). It is well documented that solvent-tolerant pseudomonads possess three membrane-associated solvent tolerance mechanisms: (A) active efflux of organic solvents, (B) outer membrane changes and (C) cytoplasmic membrane changes (Volkers et al., 2006).
(A) Active efflux of organic solvents.
Several laboratories have identified efflux pumps belonging to the resistance–nodulation–cell division (RND) family as being the most important mechanism of solvent tolerance (Kieboom et al., 1998; Ramos et al., 2001). They are energy-dependent active efflux pumps, which export toxic organic solvents to the external medium. This mechanism has also been identified in P. putida KT2440 (Benndorf et al., 2006). These efflux pumps were present throughout the fermentation of P. putida CA-3 under both N-limiting and N-excess growth conditions. (e.g. UniProtKB/TrEMBL Q88DA4, homologous to toluene efflux pump). Nevertheless, these proteins were present at considerably higher levels (by a factor of 10) by T30 under N-limiting conditions (Table 1). It has been determined elsewhere that the operation of these efflux pumps seems to be coupled to the proton motive force, that they have dual pumping capacity, and that specific and global regulators control their expression at the transcriptional level (Kieboom et al., 1998; Ramos et al., 2002).
(B) Outer membrane changes.
An integral part of the efflux systems described above are OMPs, which represent an outer membrane channel by which the pumped molecule is released into the medium (Kieboom & De Bont, 2001). OMPs associated with the efflux pumps (TolC, OmpJ) were detected in the proteome of P. putida CA-3 grown on styrene (Supplementary Table S1). As described above in Results and Discussion, Lipoproteins, chemotaxis-related proteins and iron ion transport proteins upregulated under N limitation, a putative lipoprotein is expressed by cells grown on styrene, but increased outer membrane lipoprotein and bacterial surface antigen expression occurs under N limitation.
(C) Cytoplasmic membrane changes.
An immediate response of micro-organisms to harsh environmental conditions (chemical or physical stresses) is the readjustment of cell-membrane fluidity by the alteration of their phospholipid composition. One of the key elements in this response is the cis to trans isomerization of unsaturated fatty acids by the Cti isomerase, which causes membrane rigidity (Heipieper et al., 1996; von Wallbrunn et al., 2003). However, in this study, Cti isomerase (which is located in the periplasm) was not detected. In agreement with this finding, Junker and co-workers described a P. putida DOT-T1E Cti null mutant that is still able to survive at high toluene concentrations, indicating that although important, cis/trans isomerization is not essential for bacterial solvent resistance (Junker & Ramos, 1999).
Given that the Cti fatty acid isomerase was not detected in this study, it would appear that efflux pumps and outer membrane lipoproteins (i.e. Q88MH2, Table 1) are more likely to be involved in the maintenance and integrity of the cell membrane in P. putida CA-3 grown on styrene, especially under N-limiting conditions.
Stress-related proteins
A number of proteins involved in detoxification, oxidative stress response and protein folding mechanisms were identified with higher spectral counts in cells grown under N limitation, e.g. peroxidase/catalase HPI and ATP-dependent Clp protease (Table 1). It is thought that the catalase/peroxidase subfamily provides protection under oxidative stress in bacteria (Welinder, 1991). Increased catalase levels have been detected in the proteome of Pseudomonas sp. M1 and KT2440 when grown on or in the presence of phenol (Santos et al., 2004, 2007). Multiple stress-related proteins, such as AphC, SodB and other antioxidants, are induced in P. putida KT2440 cells in the presence of a range of aromatic substrates (Kim et al., 2006). The presence of stress-related proteins in cells grown on styrene supports the claim that styrene has a toxic effect on bacteria cells (Ramos et al., 2001). Bacteria accumulating PHB have been previously reported to upregulate heat shock (HspA) proteins in response to stress (Kang et al., 2008; Tessmer et al., 2007). The authors propose that HspA can act like a PHA granule-associated protein (phasin) which affects PHA granule coalescence (Tessmer et al., 2007). In the current study, Clp protease (subunit ClpX) was upregulated in P. putida CA-3 cells under N limitation (Table 1). Recently, we generated a transposon mutant negatively affected in PHA accumulation disrupted in the clpA gene, suggesting that the activity of the ClpP protease is important for PHA accumulation in P. putida CA-3 (Goff et al., 2009). While some members of the Clp family are involved in proteolysis regulation, this family also has many attributes of molecular chaperones (Squires & Squires, 1992). ClpXP is involved in DNA damage repair, stationary-phase gene expression and protein quality control (Neuwald et al., 1999), and to date more than 50 proteins, including transcription factors, metabolic enzymes, and proteins involved in the starvation and oxidative stress responses, have been identified as substrates (Flynn et al., 2003). Clp protease in Bacillus subtilis ClpP is thought to play a role during heat shock as well as oxidative and salt stress (Volker et al., 1994), while in P. fluorescens (O'Toole & Kolter, 1998), it is linked to biofilm formation, which is a bacterial response to specific environmental triggers/stresses.
Conclusions
A wide variety of proteins are expressed in P. putida CA-3 in response to the presence of styrene and limiting concentrations of N in the growth medium. The broad range of these proteins indicates the complex and varied response of this bacterium to the presence of a lipophilic substrate (styrene) and to the decrease in the concentration of an essential inorganic nutrient (N). The internal and surface protein expression response also reveals the multi-faceted nature of the physiological response of P. putida CA-3 to environmental stimuli. The data generated here will allow us to investigate specific molecular targets to examine the effect of gene disruption on styrene metabolism, N assimilation and mclPHA accumulation by P. putida CA-3. The data also form a basis for the design and evaluation of future metabolic engineering efforts, where analysis of multiple pathways and global responses will increase as this field moves towards systems biology methods (Park et al., 2008).
Edited by: M. A. Kertesz
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Received 28 May 2009; revised 8 July 2009; accepted 9 July 2009.
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