1 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
2 Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S1 1WB, UK
Correspondence
Howard Dalton
h.dalton{at}warwick.ac.uk
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ABSTRACT |
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Three figures showing representative 2D gels of the various protein samples are available as supplementary material with the online version of this paper.
Present address: Department of Agro-Industrial Technology, Faculty of Applied Science, King Mongkut's Institute of Technology (KMITNB), Piboonsongkram Rd, Bangsue, Bangkok 10800, Thailand.
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INTRODUCTION |
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The role of formaldehyde in methylotrophic metabolism has been most extensively studied in the pink-pigmented methylotroph Methylobacterium extorquens AM1, which is a facultative methanol utilizer and a member of the -proteobacteria (Peel & Quayle, 1961
; Anthony, 1982
; Lidstrom, 1992
; Bratina et al., 1992
). Here, as in other methylotrophic bacteria, formaldehyde is an important branch point in metabolism, from which carbon can be dissimilated to carbon dioxide to produce energy, or assimilated into biomass (Anthony, 1982
; Lidstrom, 1992
). In M. extorquens AM1, there are at least two pathways for formaldehyde dissimilation, and one for its assimilation. Conjugation of formaldehyde to tetrahydromethanopterin (H4MPT) (Chistoserdova et al., 1998
) and tetrahydrofolate (H4F) (Marison & Attwood, 1982
) in the two major pathways for formaldehyde dissimilation minimizes the cellular concentration of this toxic intermediate, and may trap the otherwise freely diffusible formaldehyde within the cell. The genes and enzymes of the H4MPT- and H4F-dependent pathways are now well characterized, as are most of the steps in the serine cycle for formaldehyde assimilation in M. extorquens AM1 (Chistoserdova et al., 2003
), and a gapped genome sequence is available (http://www.integratedgenomics.com/genomereleases.html).
Previous studies have shown that formaldehyde is toxic to succinate-grown M. extorquens AM1 at 1 mM (Marx et al., 2003), and that this strain is unable to utilize formaldehyde at 21 mM as a growth substrate (Peel & Quayle, 1961
). In order to characterize how methylotrophic metabolism may be exploited to bioremediate formaldehyde, we have investigated two strains of pink-pigmented methylotrophs, BIP and ROS1, which can utilize formaldehyde at high concentrations as a growth substrate. Strain BIP is used commercially by BIP Ltd for remediation of formaldehyde-contaminated effluents that also contain methanol. Here, we present results from a study involving characterization of cell growth and formaldehyde removal in both strains, together with preliminary two-dimensional protein gel analysis of acclimatization to formaldehyde in strain BIP.
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METHODS |
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Strains BIP and ROS1 were grown at 30 °C on nitrate mineral salts (NMS) and ammonium mineral salts (AMS) liquid medium or agar plates (Whittenbury et al., 1970) with added growth substrate, as stated for each experiment. Formaldehyde stock solution (approx. 2 M) was prepared by heating aqueous paraformaldehyde in sealed glass tubes at 115 °C for 60 min; the concentration of formaldehyde was determined accurately before use. Formaldehyde stock solution and methanol were sterilized by filtration through disposable 0·2 µm pore-size disposable sterile filter units (Millipore) before adding to media; other carbon sources were added from stock solutions that had been sterilized by autoclaving. Liquid cultures of 50 or 200 ml were grown in 250 or 1000 ml Quickfit flasks, respectively, sealed with Suba-Seals (W. H. Freeman), and incubated with shaking at 180 r.p.m. Cell growth was monitored by measuring OD550. Strain BIP was also grown in continuous culture in an LH 500 series fermenter (L. H. Engineering), with a working volume of 1·8 l. The pH was maintained at 6·8 by automatic addition of 0·5 M NaOH. At steady state during continuous operation, when the dilution rate was 0·03 h1 and the concentration of formaldehyde in the feedstock was 120 mM, the concentration of formaldehyde in the fermenter vessel (measured from the outflow) was
1 mM. Cells were harvested from the fermenter vessel by removing no more than one quarter of the working volume of the culture, and then the reactor was fed with formaldehyde-containing medium to resume steady state for at least 120 h before further collections.
Formaldehyde and methanol determination.
Cells were removed from culture samples by centrifugation (7500 g, 4 °C, 5 min), and then the formaldehyde concentration in the supernatant was quantified using an assay based on the Hantzsch reaction (Nash, 1953), as follows. A sample (0·5 ml) was mixed with an equal volume of reagent B (2 M ammonium acetate, 0·05 M acetic acid and 0·02 M acetylacetone), and incubated at 58 °C for 5 min. Formaldehyde concentration was then determined colorimetrically at 412 nm as the formaldehyde adduct diacetyldihydrolutidine. The blank contained distilled water in place of the sample. The range of formaldehyde standards was linear from 0 to 1 mM; samples with concentrations beyond this range were diluted as appropriate. Methanol concentrations were estimated by GC analysis of 2 µl samples using a Series 104 gas chromatrograph (Pye Unicam) fitted with a 4 mm i.d. x1 m Porapak Q column (Waters). Nitrogen was used as a carrier gas at a flow rate of 25 ml min1. The column was maintained at 200 °C, the flame-ionization detector temperature was 220 °C, and the injection temperature was 200 °C. The quantification of peak areas was achieved using a 3390A integrator (Hewlett Packard). Methanol concentrations of samples were determined by reference to a standard curve constructed using solutions of methanol in the range 02 g l1.
Molecular genetics methods.
DNA of strains BIP and ROS1 was isolated by a modification of the method of Oakley & Murrell (1988), as follows. Cells harvested from a 50 ml culture were resuspended in 2 ml SET buffer (20 %, w/v, sucrose, 50 mM EDTA and 50 mM Tris/HCl, pH 8·0) plus 100 µl lysozyme solution (5 mg lysozyme ml1, 10 mM NaCl, 50 mM EDTA, 50 mM Tris/HCl, pH 8·0) and incubated at 37 °C for 1 h. A 100 µl volume of 20 % (w/v) SDS, 50 µl 20 mg ml1 proteinase K in 50 mM EDTA, and 50 mM Tris/HCl (pH 8·0) was added, and the mixture was incubated at 5560 °C for 1 h. The lysate was stored at 20 °C until subjected to phenol/chloroform extraction, and nucleic acid was subsequently recovered by ethanol precipitation, as described by Sambrook et al. (1989)
.
Amplification of 16S rRNA genes was achieved using eubacterial universal primers pA (5'-AGAGTTTGATCCTGGCTC-3') and pH (5'-AAGGAGGTGATCCAGCCGCA-3'), designed by Edwards et al. (1989). PCRs were performed using an OmniGene thermal cycler (Hybaid). Each reaction (100 µl) contained 1·5 mM MgCl2, 20 mM Tris/HCl (pH 8·4), 50 mM KCl, 0·05 % (v/v) detergent W-1, 200 µM each dNTP, 1 ng µl1 each primer, 2·5 units Taq DNA polymerase (Gibco-BRL) and 0·1 ng µl1 template DNA. Reaction conditions were as follows: denaturation at 94 °C for 5 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 1 min, polymerization at 72 °C for 1 min 30 s; and a final extension at 72 °C for 5 min.
The 1·5 kb PCR products were cloned into the pCR II vector using the TOPO TA Cloning Kit (Invitrogen), and the resulting plasmids were purified by using QIAprep Spin Miniprep kits (Qiagen). The DNA sequence was determined by cycle sequencing with the Dye Terminator Kit (PE Applied Biosystems).
Two-dimensional electrophoresis and MS analysis.
The sample preparation procedure before two-dimensional electrophoresis (2DE) depended on the gel staining method to be used. Gels that were to be used to compare levels of protein expression, and for MS identification of some protein spots, would eventually be silver stained; the samples to be loaded on such gels were prepared as follows. Cells were harvested, resuspended in 25 mM MOPS (pH 7·5) containing 1 mM benzamidine, broken by passing three times through a French pressure cell (American Instrument Company; 110 MPa, 4 °C) and centrifuged (49 000 g, 30 min, 4 °C) to remove unbroken cells. The supernatant was used as the crude extract, and, where necessary, was separated into soluble and membrane components by centrifugation (150 000 g, 90 min, 4 °C). The new supernatant was kept as the soluble fraction, and the membrane-containing pellet was washed with 25 mM MOPS buffer (pH 7·5) containing 10 mM EDTA and 1 mM benzamidine before centrifuging again (150 000 g, 60 min, 4 °C) to pellet the particulate material. Before 2DE, crude extract and soluble samples were denatured using lysis solution I (9 M urea, 2 M thiourea, 4 % CHAPS and 30 mM Tris base), whereas membrane samples were solubilized and denatured in lysis solution II [5 M urea, 2 M thiourea, 2 % CHAPS, 40 mM Tris base and 60 mM N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB3-10)]. MS identification of some abundant proteins was achieved using two-dimensional gels stained with Coomassie blue; samples were prepared in the same way as for silver-stained gels, except that the cells were suspended in lysis buffer I before breaking in the French pressure cell.
The first-dimension separation (which was IEF) was effected by applying protein samples (50 µg for silver staining, or 500 µg for Coomassie-blue staining) to immobilized pH gradient (IPG) strips (linear pH gradient 47, 18 cm in length; Amersham Pharmacia). The IPG strips were next soaked in rehydration solution [9 M urea, 2 M thiourea, 2 % CHAPS, trace of Bromophenol blue, 2·8 mg DTT ml1 and 0·5 % IPG buffer (Amersham Pharmacia)] for 12 h at 20 °C, according to the manufacturer's instructions. IEF of proteins was carried out at 500 V (1 h), 1000 V (1 h) and then 8000 V (11 h) by means of an IPGphor IEF system (Amersham Pharmacia). After IEF, the IPG strips were equilibrated with 10 ml SDS equilibration buffer [50 mM Tris/HCl (pH 8·8), 6 M urea, 30 % (v/v) glycerol, 2 % (w/v) SDS, and a trace of Bromophenol blue) containing 100 mg DTT for 15 min, and then the buffer was replaced with the same volume of equilibration buffer containing 250 mg iodoacetamide for a further 15 min, according to the manufacturer's instructions. Second-dimension separation was performed in a precast SDS-12·5 % (w/v) PAGE gel at 30 mA (30 min) and then 50 mA (80 min), using a Multiphor II flatbed system (Amersham Pharmacia). Molecular masses were estimated by reference to Dalton Mark VII-L markers (Sigma) run alongside the experimental sample during SDS-PAGE. Proteins were visualized by silver staining according to the method of Heukeshoven & Dernick (1985), or by using Coomassie blue.
Protein spots of interest were excised from the gel, and a blank piece of gel was also excised to serve as a control. The gel pieces were washed with 50 mM ammonium bicarbonate, and subjected to three cycles of shrinking with acetonitrile (10 min), and washing with 50 mM ammonium bicarbonate (10 min). The solution was removed, and the gel pieces were thoroughly dried in a vacuum centrifuge for 10 min, and then incubated for 40 min at room temperature with digestion buffer consisting of 50 mM ammonium bicarbonate and 10 ng µl1 sequencing grade trypsin (Promega). The digestion buffer was removed and replaced with 10 µl 50 mM ammonium bicarbonate, and then the gel pieces were incubated at 37 °C for 20 h. Peptides were extracted from the gel with 3x20 µl 50 % (v/v) acetonitrile containing 5 % (v/v) formic acid (Shevchenko et al., 1996). The extracts were pooled, evaporated to dryness using a vacuum centrifuge, and dissolved in 10 µl 0·1 % (v/v) trifluoroacetic acid in Milli-Q water. The peptide-containing extracts were then concentrated and desalted by reverse-phase chromatography using ZipTiP C18 spin columns (Millipore), as described by Regula et al. (2000)
.
The desalted samples were analysed using a Q-TOF mass spectrometer (Micromass) fitted with a nanoflow infusion capillary inlet. The potentials of the nanoflow capillary and cone were set at 1000 and 40 V, respectively. MS data were acquired over the range of 4002400 m/z during a 2·4 s scan by means of the MassLynx data system version 3.4 (Waters). Ions with multiple charges were subjected to low-energy tandem mass spectrometry (MS/MS). Precursor ions were selected using the first quadrupole, and subjected to collision-induced dissociation with argon gas, and with the collision energy between 18 and 45 eV. MS/MS data were acquired over the range of 502400 m/z during a 2·4 s scan.
MS/MS spectra obtained from the tandem mass spectrometry were exported as sequest files, and were searched against the NCBI non-redundant protein database using the Mascot MS/MS ion search from Matrix Science (http://www.matrixscience.com). Variable amino acid modifications of carbamidomethylcysteine, and oxidation of methionine, were allowed, since these modifications commonly occur as a result of protein separation by 2DE. The peptide mass tolerance was set to ±2·0 Da, and the fragment mass tolerance to ±0·8 Da. Data were reported with individual ion scores. When a peptide ion produced a significant match via a Mascot search, the sequence suggested by Mascot was checked by eye against the MS data to ensure that the assignment of the sequence was an accurate fit to the data. In some cases, short amino acid sequences could be obtained from peptide ions with no significant matches to the database. Such sequences were searched against the translated M. extorquens AM1 ORF database (http://pedant.gsf.de). Any proteins that were not identified after these searches were processed by the MaxEnt 3 algorithm, which converts multiply charged peaks onto a singly charged axis. Amino acid sequences were then manually assigned, and the derived sequences were subjected to the same database searches.
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RESULTS |
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Strains BIP and ROS-1 are facultative methylotrophs that can remove and grow on formaldehyde over a wide range of conditions
BIP and ROS1 formed colonies when cultivated on plates containing methanol as the sole source of carbon and energy, and could also grow using formaldehyde as the growth substrate. As with other -proteobacterial pink-pigmented methylotrophs (Anthony, 1982
), the methylotrophic lifestyle of BIP and ROS1 was facultative, and the strains were not able to use methane as the growth substrate. Growth of both strains was observed on plates containing succinate or pyruvate as the sole carbon and energy source. Growth was also observed on plates containing glucose, sucrose, citrate or acetate, although the colonies were small, showing that these compounds were not good growth substrates for strain BIP; indeed the possibility of growth due to scavenging of nutrients from impurities in the agar cannot be excluded with these poorer growth substrates. These results are consistent with those previously obtained using M. extorquens AM1, except that the latter organism did not grow on formaldehyde or acetate (Peel & Quayle, 1961
).
Optical density and formaldehyde concentrations were monitored during growth of strains BIP and ROS1 in AMS medium containing formaldehyde, at a range of concentrations, as the only source of carbon and energy. Growth and efficient removal of formaldehyde to 10 µM were observed with cultures incubated with starting concentrations of formaldehyde up to 61 mM (Fig. 1
). The growth rate of the cells did not vary greatly with formaldehyde concentrations within the range tested, and there was a positive correlation between the amount of formaldehyde added and the time taken to remove it completely (Fig. 1
).
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Strains BIP and ROS1 show acclimatization to growth at very high formaldehyde concentrations
Direct cultivation of succinate-grown BIP and ROS1 cells was found to be impossible in liquid cultures at concentrations of formaldehyde greater than 58 mM (data not shown). However, stepwise acclimatization of the culture at increasing concentrations of formaldehyde (typically 20, 40, 60, 72 and then 100 mM, with about 7 day intervals between successive increases in concentration) allowed the cells to survive at formaldehyde concentrations up to 100 mM. Upon exposure to the high concentration of formaldehyde, growth continued for about one generation, and then cell density reached a plateau, whilst formaldehyde removal proceeded at a roughly uniform rate until the residual formaldehyde concentration had fallen to about 50 mM, whereafter growth resumed (Fig. 2).
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DISCUSSION |
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Metabolism at high concentrations of formaldehyde showed evidence of the uncoupling of catabolism from anabolism (Tempest & Neijssel, 1981), since no increase in biomass was observed during the plateau period before the acclimatized cells resumed growth (Fig. 2
). Here, it may be to the cells' advantage to sacrifice biomass yield to the removal of a growth substrate that is at a dangerously high concentration. A similar decrease in growth yield at very high methanol concentrations has been observed in M. extorquens (Harrison, 1976
). Enhanced formaldehyde efflux from the cells may also contribute to survival at high concentrations of formaldehyde.
The results from the 2DE protein expression analysis are remarkably consistent with those found in a recent study, published during the preparation of this manuscript, which identified 229 proteins from M. extorquens AM1, 68 of which increased in expression during growth on methanol (Laukel et al., 2004). The multiple isoforms of the methanol dehydrogenase
-subunit and formaldehyde-activating enzyme are similar to multiple forms that were observed after 2DE of M. extorquens AM1 samples (Laukel et al., 2004
). These may be due to in vivo posttranslational changes that alter the pI of the protein; alternatively, they may be due to partial carbamylation of lysyl residues during sample preparation (McCarthy et al., 2003
). There were no formaldehyde-induced proteins identified in strain BIP that were not induced by methanol in M. extorquens AM1, although the 50S ribosomal protein L9 was not identified in the M. extorquens AM1 study (Laukel et al., 2004
), and methenyl H4MPT cyclohydrolase was constitutive in strain BIP, but is known to be increased in level of expression during growth on methanol in M. extorquens AM1 (Laukel et al., 2004
; Chistoserdova et al., 1998
; Vorholt et al., 1999
). It is interesting that methanol dehydrogenase, which is induced by methanol (Laukel et al., 2004
), is also induced during growth of strain BIP on formaldehyde. Whilst this may be incidental to formaldehyde oxidation, and result from the induction during formaldehyde growth of the proteins that are specific to growth on methanol, it is possible that methanol dehydrogenase participates in formaldehyde oxidation, since formaldehyde is a substrate for this enzyme (Heptinstall & Quayle, 1969
; Anthony, 1982
).
The evidence currently available suggests that many of the changes in protein expression that allow strain BIP to grow using formaldehyde at 20 mM are associated with expression of the same formaldehyde oxidation and assimilation enzyme that M. extorquens AM1 possesses to process formaldehyde as a metabolic intermediate during growth on methanol. This is consistent with the observation that strain BIP is itself able to utilize methanol, and that the formaldehyde and methanol resistance of succinate-grown M. extorquens AM1 is diminished in mutants that are defective in the H4MPT pathway (Marx et al., 2003). Nonetheless, the fact that strain BIP is able to grow at concentrations of formaldehyde (
20 mM) that support no growth in M. extorquens AM1 (Peel & Quayle, 1961
) indicates that BIP possesses adaptations to life at high formaldehyde concentrations that M. extorquens AM1 does not.
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ACKNOWLEDGEMENTS |
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Received 25 January 2005;
revised 9 May 2005;
accepted 10 May 2005.
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