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Applied and Environmental Microbiology, October 2005, p. 5850-5857, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5850-5857.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Photoheterotrophic Metabolism of Acrylamide by a Newly Isolated Strain of Rhodopseudomonas palustris

David A. Wampler and Scott A. Ensign*

Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300

Received 14 February 2005/ Accepted 26 April 2005


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ABSTRACT
 
Acrylamide, a neurotoxin and suspected carcinogen, is produced by industrial processes and during the heating of foods. In this study, the microbial diversity of acrylamide metabolism has been expanded through the isolation and characterization of a new strain of Rhodopseudomonas palustris capable of growth with acrylamide under photoheterotrophic conditions. The newly isolated strain grew rapidly with acrylamide under photoheterotrophic conditions (doubling time of 10 to 12 h) but poorly under anaerobic dark or aerobic conditions. Acrylamide was rapidly deamidated to acrylate by strain Ac1, and the subsequent degradation of acrylate was the rate-limiting reaction in cell growth. Acrylamide metabolism by succinate-grown cultures occurred only after a lag period, and the induction of acrylamide-degrading activity was prevented by the presence of protein or RNA synthesis inhibitors. 13C nuclear magnetic resonance studies of [1,2,3-13C]acrylamide metabolism by actively growing cultures confirmed the rapid conversion of acrylamide to acrylate but failed to detect any subsequent intermediates of acrylate degradation. Using concentrated cell suspensions containing natural abundance succinate as an additional carbon source, [13C]acrylate consumption occurred with the production and then degradation of [13C]propionate. Although R. palustris strain Ac1 grew well and with comparable doubling times for each of acrylamide, acrylate, and propionate, R. palustris strain CGA009 was incapable of significant acrylamide- or acrylate-dependent growth over the same time course, but grew comparably with propionate. These results provide the first demonstration of anaerobic photoheterotrophic bacterial acrylamide catabolism and provide evidence for a new pathway for acrylate catabolism involving propionate as an intermediate.


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INTRODUCTION
 
Acrylamide is a toxic three-carbon compound containing an amide group and an {alpha},ß-unsaturated olefin bond. It exerts its toxic effects by forming adducts to nucleophilic moieties such as sulfhydryl groups on proteins (4). Acrylamide is potent neurotoxin (30) and suspected to be a carcinogen (21, 27). Although acrylamide is not typically found in nature, it is widely used in industrial processes as a polymerizing agent, hardener, and as a flocculent in water treatment. Accordingly, acrylamide released from industrial processes has resulted in the contamination of both soils and aquatic environments (6). Recently, it was discovered that acrylamide is formed in starchy foods after being cooked at the high temperatures required for frying and baking (26, 27). These foods, which have undetectable levels before cooking, have acrylamide levels as high as 2.3 mg/kg after cooking (25). The formation of acrylamide is believed to be a product of the reaction between asparagine and glucose during the cooking process (14, 24).

In spite of acrylamide's toxicity in the monomer form, some microorganisms are able to utilize acrylamide as their sole carbon source for growth (16, 17, 22, 32, 33). All studies conducted to date show that there is an initial deamidation step that converts acrylamide to acrylic acid (acrylate) (15, 17, 22, 33). The subsequent fate of acrylate has not been well defined for acrylamide-utilizing bacteria but probably involves pathways and enzymes that have been characterized to various degrees for other bacteria capable of acrylate catabolism. In aerobic acrylate-utilizing bacteria, acrylate metabolism has been shown to proceed via hydroxylation to ß-hydroxypropionate, which is further oxidized to CO2 (1, 2). Highlighting a different fate for acrylate under anaerobic conditions, Clostridium propionicum is capable of fermenting acrylate to acetate and propionate (10). Acrylate has also been shown to serve as a terminal electron acceptor for Desulfovibrio acrylicus, producing propionate which is not further metabolized as a growth substrate (31). Cell extracts of C. propionicum have been shown to dehydrate (R)-lactate to acrylate (20), suggesting that the reverse reaction could be another strategy for acrylate catabolism. Figure 1 summarizes the deamidation reaction that produces acrylic acid from acrylamide and possible subsequent fates of acrylate.



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  		FIG. 1. Possible biological fates of acrylate produced from acrylamide deamidation.

In order to provide additional information on the microbial diversity of acrylamide metabolism and pathways involved in the degradation of this compound, we have focused on a class of bacteria recognized for their high degree of metabolic diversity, i.e., the purple nonsulfur photosynthetic bacteria. In spite of their well-characterized metabolic versatility, no photosynthetic bacterium has been studied with regard to acrylamide metabolism. Accordingly, enrichments using acrylamide as the carbon source and specifically targeting purple nonsulfur photosynthetic bacteria were performed. The results of these studies, presented here, demonstrate that pure culture isolates of purple nonsulfur bacteria are indeed capable of using acrylamide as a carbon source and suggest the involvement of a novel, previously uncharacterized pathway for acrylate metabolism.


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MATERIALS AND METHODS
 
Materials.
[1,2,3-13C]acrylamide was purchased from Cambridge Isotope Laboratories, Andover, MA. Acrylamide (98.5% purity for cell growth and 99.9% purity for analytical procedures) was purchased from Fisher Scientific. Nuclear magnetic resonance (NMR) tubes and stem coaxial inserts were from Wilmad Labglass Buena, NJ. All other chemicals were of analytical grade. Rhodopseudomonas palustris strain CGA009 was kindly provided by Caroline Harwood.

Isolation of phototropic bacteria.
Environmental samples were collected from various locations from the aerobic-anaerobic interface of the effluent from a bovine slaughterhouse in Hyrum, Utah. The growth medium was as described by Tayeh and Madigan (28), except that acrylamide (16 mM) was used in place of malic acid as the carbon source, and 20 mM MOPS [3-(N-morpholino)propanesulfonic acid] was added to buffer the pH at a value of 6.9. Enrichments were incubated in sealed screw top culture tubes (14 ml) filled completely with medium in a controlled temperature floor shaker (100 rpm) at 30°C under illumination from a bank of 60-W incandescent lights. Control enrichments were performed identically but in the absence of an added carbon source. Positive enrichments were characterized by acrylamide-dependent turbidity and the characteristic purple/red pigmentation of the desired photosynthetic bacteria, both in the initial enrichments and after successive subculturing into fresh medium containing acrylamide. Red-pigmented isolates were purified by repeated plating on agar with succinate as the carbon source. The plates were incubated at 30°C in Brewer jars flushed with nitrogen under illumination as described above. Individual red colonies were restreaked on agar containing acrylamide as the sole carbon source. Individual colonies from the acrylamide plates were used to inoculate culture tubes. From these enrichments, a pure culture was obtained that was characterized by a relatively high rate of acrylamide-dependent growth (doubling time of 12 h). This isolate was used for all subsequent studies.

16S RNA analysis.
Genomic material was extracted by standard genomic phenol extraction-ethanol precipitation protocols (3). The 16S rRNA gene was amplified by PCR with the forward primer 5'-TTGGATCCAGAGTTTGACMTGGCTCAG-3' and the reverse primer 5'-GTTGGATCCACGGYTACCTTGTTACGAYT-3'. The PCR product was purified by using a QIAGEN PCR purification kit. The 1.5-kb fragment obtained from the PCR amplification was digested by using BamHI, ligated to pBluescript and transformed into Escherichia coli strain JM109. These cells were then plated on Luria-Bertani (LB) medium with ampicillin at 50 µg/ml, X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) at 80 µg/ml, and IPTG (isopropyl-ß-D-thiogalactopyranoside) at 1 mM. White colonies were selected and grown up in 5 ml of LB medium containing ampicillin (50 µg/ml). Plasmids were isolated by using an Eppendorf Perfectprep Plasmid Minikit. Sequencing with T-7 and T-3 primers was performed by the Utah State University Biotechnology Center.

The data was BLAST searched on NCBI. The organism was named Rhodopseudomonas palustris strain Ac1 based on BLAST analysis and morphology.

Microscopy.
Morphology of the isolated cells was observed after safranin staining under illumination by optical microscopy using a Steindorff Binocular halogen microscope (Mel Sobel Microscopes LTD.) under 1,000x oil immersion.

Small-scale growth.
Cultures of the newly isolated R. palustris strain Ac1 and R. palustris strain CGA009 for which the genome has been sequenced were grown photoheterotrophically in culture tubes (14 ml) with a 20-mm optical density (OD) opening sealed with gray butyl stoppers. The carbon sources were either 10 mM succinate, propionate, acrylamide, or acrylate. The OD of the cultures was determined at the desired time points by placing the tubes in a Klett-Summerson photoelectric colorimeter with filter number 66. The Klett colorimeter was standardized relative to a Shimadzu model UV-160 spectrophotometer for the conversion of Klett readings to absorbance values (i.e., OD) at 600 nm.

Correlation of cell growth with acrylamide degradation and acrylate production and consumption.
Cells were grown in triplicate in 100 ml of mineral salts medium with acrylamide (10 mM) as the carbon source in 125-ml Erlenmeyer flasks modified by the addition of a crimp seal (20 mm) top and side arm and of a Klett side arm for measuring cell growth. The media was degassed through the side arm by repeated evacuation and flushing with sterile nitrogen. Flasks were inoculated with late-logarithmic-phase cells added by using a Hamilton syringe to an initial OD of 0.6. Flasks were incubated on a shaker at 30°C illuminated by 60-W incandescent lights. The OD of the cultures was determined by placing the Klett side arm in the Klett colorimeter. For analysis of acrylamide and acrylate levels, samples (50 µl) were periodically removed from the side arm with a Hamilton microsyringe, and the cells were pelleted by a microcentrifuge at 16,000 x g. The cell-free supernatant was diluted 20-fold with deionized H2O, and acrylamide and acrylate concentrations determined by high-pressure liquid chromatography (HPLC) as described below.

Large-scale growth.
Cells were grown under photoheterotrophic conditions using acrylamide (16 mM) as the carbon source. Cell growth was scaled up from 14-ml capacity culture tubes to a 45-liter capacity photosynthetic fermentor vessel as follows. Cultures that had been grown to logarithmic phase in screw-cap culture tubes (14 ml) were used to inoculate medium bottles (1 liter) that were completely filled with medium and cells. The 1-liter cultures were incubated with illumination as described above. Two 1-liter cultures that had been grown to logarithmic phase were used as the inoculant for a 45-liter capacity glass fermentor vessel modified for photosynthetic growth as described previously (23). Illumination was provided by three 500-W tungsten-halogen "type T" light bulbs connected in series to a 240-V power source and immersed in the culture vessel in a water-jacketed tube to provide cooling. The temperature of the fermentor vessel was maintained at 30°C. Additional acrylamide (10 mM) was added to the culture as necessary. Cells were harvested by using an A/G Technologies polysulfone membrane cartridge filtration system as described previously (5). Cell paste was frozen in liquid nitrogen and stored at –80°C.

Quantitation of acrylamide and acrylate.
Acrylamide and acrylate were quantified by HPLC using a Shimadzu SCL-10 Avp HPLC system equipped with Shimadzu LC10 AT pumps, a Shimadzu SPD-10A UV-visible detector, and a Shimadzu CR501 integrator. Acrylamide and acrylate were separated by using a Phenomenex Hydro-RP column (250 by 4.60 mm) with an isocratic mobile phase of 25 mM sodium phosphate buffer (pH 6.5) and a flow rate of 1.5 ml/min. The absorbance was measured at 210 nm. Acrylamide and acrylate eluted with retention times of 2.3 and 1.7 min, respectively. The concentration of samples was determined by comparing integrated peak areas to standard curves generated using standards of acrylamide and acrylate.

Acrylamide degradation and acrylate production and consumption by resting cell suspensions and cell extracts.
Logarithmic-phase cells were pelleted by centrifugation, washed with 50 mM sodium phosphate buffer (pH 6.9), and resuspended in 5 volumes of the same buffer. Samples (1 ml) were placed in serum vials (9 ml), crimp sealed with gray butyl stoppers, evacuated and flushed with nitrogen, and placed in a shaking water bath at 30°C with illumination. Acrylamide (10 mM) was added to start reactions. At the desired time points, 50-µl samples were removed, the cells were pelleted by centrifugation, and the supernatant was diluted 20-fold with deionized H2O. Acrylamide and acrylate concentrations were determined by HPLC as described above.

Cell suspensions (100 to 300 ml) that had been prepared as described above were treated with 30 µg of DNase I/ml for 15 min, followed by passage through a French pressure cell at 4°C and 125,000 kPa. The insoluble material was pelleted by centrifugation at 180,000 x g for 45 min. The supernatant (soluble cell extract) was then decanted and stored in liquid nitrogen.

Soluble cell extracts were thawed in a 30°C shaking water bath and stored on ice. Assays of acrylamide degradation were performed as described for cell suspensions, except utilizing only ambient light, with 13 mg of protein per assay. At the desired time points, 50-µl samples were removed and combined with 100 µl of a saturated solution of ammonium sulfate to precipitate proteins. After centrifugation at 18,000 x g for 2 min, the supernatant was diluted threefold with deionized H2O and analyzed by HPLC.

Acrylamide deamidase induction.
Cells which had been grown for several generations with either acrylamide or succinate as the sole carbon source were grown in 14-ml culture tubes to an OD of 2.5 (A600). The cells were then pelleted by centrifugation, washed with 50 mM sodium phosphate buffer, and resuspended in the original volume of fresh growth medium. The acrylamide-grown cells were resuspended in medium with no added carbon source, whereas the succinate-grown cells were resuspended in medium containing 10 mM succinate. Then, 1-ml samples of the resuspended cells were transferred to 9-ml serum vials and evacuated and flushed with nitrogen. Where desired, rifampin (0.4 mg) or chloramphenicol (0.2 mg) was added to vials. The vials were preincubated for 15 min in a 30°C shaking water bath with illumination, followed by the addition of acrylamide (3 mM final concentration) to initiate the assays. Acrylamide degradation was monitored over time by HPLC as described above.

13C NMR studies.
For the first analysis, acrylamide-grown cells (0.5 ml of cells at an OD of 1.5) were transferred to an NMR tube. [1,2,3-13C]acrylamide (20 mM) and [13C]NaHCO3 (16 mM) were added to the cell suspension in the tube. The sample was made anoxic under a stream of nitrogen gas, followed by the insertion of a coaxial tube insert containing CDCl3 as a standard. The NMR tube was then sealed with a tight-fitting plastic cap and incubated with illumination and shaking in a 30°C water bath. At the desired times, the tube was removed, and proton-decoupled 13C NMR spectra were recorded by using a Bruker ARX-400 NMR spectrophotometer with a frequency of 100.6 MHz and a sweep width of 333 ppm. Spectra were averaged from 1,428 scans and processed with a 5-Hz line broadening. After NMR data collection for a given time point, the NMR tubes were immediately placed back in the shaking water bath for continuation of the assay time course.

For the second analysis, a more concentrated cell suspension (OD of ~300) was used, and both nonenriched sodium succinate (12 mM) and [13C]NaHCO3 (12 mM) were included as additional components of the assay mixture. Since the concentrated cell suspension consumed acrylamide very rapidly, a control tube was prepared identically but with cells that had been boiled for use as the t = 0 reference point. NMR tubes were also prepared with natural abundance sodium succinate or sodium propionate (12 mM) standards and with boiled cells to which no additional substrate had been added.


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RESULTS
 
Enrichment, isolation, and classification of an organism with the ability to utilize acrylamide as the sole carbon source.
Diverse aerobic bacteria, including those of the genera Rhodococcus, Pseudomonas, and Xanthomonas, are capable of using acrylamide as a source of carbon and energy for growth (16, 17, 33). Given the metabolic versatility of the purple nonsulfur photosynthetic bacteria and their distribution in a wide variety of ecosystems, we set up enrichments with acrylamide as the carbon source under conditions known to select for this class of bacteria.

Within several days of inoculation, enrichments were obtained containing large populations of red-pigmented bacteria. Repeated subculturing of the bacteria confirmed that their growth was dependent on acrylamide, light, and anoxic conditions. Organisms were isolated from these enrichments by alternately streaking onto plates that contained succinate or acrylamide as the carbon source. Small red circular convex colonies formed on the plates, along with numerous nonpigmented colonies. Eventually, pure isolates of red-pigmented bacteria were obtained that grew using acrylamide as the sole carbon source under anoxic phototrophic conditions. One of the most rapidly growing isolates was selected for subsequent characterization.

The 16S rRNA gene was amplified from a chromosomal DNA preparation from this isolate, and 993 bp of the gene were sequenced and analyzed (submitted to the National Center for Biotechnology Information [NCBI] under accession numbers AY919318 and AY919319). BLAST analysis revealed 99.3% identity with the 16S rRNA gene of the reference stain of R. palustris (ATCC 17001, NCBI accession number L11664) (19) and 97.1 to 99.8% identity to other R. palustris strains for which 16S rRNA genes have been fully or partially sequenced (12, 18). Microscopic analysis of the new isolate showed motile, irregularly shaped rods that replicated by budding. Based on these results, the new isolate has been named R. palustris strain Ac1.

Correlation of cell growth with acrylamide depletion and acrylate production and depletion.
R. palustris strain Ac1 grew best with acrylamide as the carbon source under photoheterotrophic conditions: no growth was observed aerobically in the dark, and anaerobic growth in the dark was at least 20 times slower than in the light. It should be noted that acrylamide is sensitive to UV light, which accelerates the free-radical-based polymerization of acrylamide to form polyacrylamide products. In their natural habitat, purple nonsulfur photosynthetic bacteria occupy anaerobic niches where only the longer wavelengths of visible and near-infrared radiation penetrate and thus little exposure to UV light occurs. Since laboratory conditions make use of high-intensity broad spectrum incandescent lights, it is likely that some of the added acrylamide undergoes polymerization and/or abiotic decomposition during the course of cell growth. Polymers of acrylamide would be expected to be more recalcitrant than the monomer form but, regardless, growth with acrylamide needs to be correlated with acrylamide monomer degradation and production of acrylate, the expected deamidation product, to ensure that the cells are indeed growing with the acrylamide monomer by the expected pathway.

As shown in Fig. 2, the growth of R. palustris Ac1 was closely correlated with acrylamide consumption and acrylate production and subsequent consumption. Cultures inoculated to an initial OD of 0.6 completely consumed a 10 mM concentration of added acrylamide in a 3-day period, in the process producing an essentially stoichiometric amount of acrylate (Fig. 2). Cell growth continued until all of the acrylate had been degraded, at which time further cell growth ceased (Fig. 2). After 4 days, additional acrylamide (10 mM) was added, resulting in an immediate growth spurt correlated with acrylamide degradation and acrylate production/degradation. This cycle of acrylamide addition/metabolite analysis was repeated an additional two times with the same result. Thus, acrylate is the stoichiometric product of acrylamide degradation, and acrylate degradation appears to be the rate-limiting reaction for acrylamide-dependent growth.



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  		FIG. 2. Correlation of R. palustris Ac1 cell growth with acrylamide consumption and acrylate formation and consumption. The OD (A600) is presented on the left axis, and acrylamide and acrylate concentrations are on the right axis. The arrows indicate the time points where additional acrylamide (10 mM) was added to the cultures. The datum points represent the average of three cultures grown under identical conditions. Symbols: •, A600 of cell culture; {blacksquare}, acrylic acid; {blacktriangleup}, acrylamide.

Comparison of growth of R. palustris Ac1 and R. palustris CGA 009.
In order to determine whether acrylamide-dependent growth is a unique feature of this new isolate, R. palustris strain CGA009, which has been extensively studied with regard to the metabolism of a range of novel substrates (9, 12), was tested for acrylamide-dependent growth. Both strains Ac1 and CGA009 grew similarly with 10 mM succinate as the carbon source, reaching similar OD values after 5 days of growth. In contrast, strain CGA009 grew very poorly relative to strain Ac1 with acrylamide as the carbon source. The cell density of strain CGA009 increased slowly and linearly over time (doubling time of ca. 5 to 10 days), but the cells never entered the logarithmic phase characteristic of exponential growth. A likely explanation for the slow-growth trend observed is that strain CGA009 is using a spontaneous acrylamide breakdown product for growth and that growth is limited by the rate of formation of this metabolite. To test this, the cells were pelleted after 5 days of growth and resuspended in the same volume of new medium with fresh succinate or acrylamide as the growth substrate. Both strains immediately responded by rapidly growing in the presence of succinate, and strain Ac1 responded similarly to acrylamide. In contrast, the same slow linear growth trend was observed for strain CGA009.

Acrylamide and acrylate degradation by resting state cell suspensions and cell extracts.
As shown in Fig. 3A, resting cell suspensions in buffer rapidly degraded acrylamide to acrylate, which was further degraded at a much slower rate. These results agree with those presented in Fig. 2 for actively growing cells, showing acrylate degradation to be a rate-limiting reaction of acrylamide metabolism by R. palustris Ac1. In order to see if the respective activities could be easily reconstituted in vitro, soluble cell extracts were prepared, and the activity assays were repeated for these. As shown in Fig. 3B, acrylamide was rapidly deamidated to acrylate in cell extracts, in the absence of any exogenous factors, producing acrylate in near stoichiometric amounts. Thus, the acrylamide deamidase is a robust enzyme not requiring additional cofactors, which is consistent with the general properties of this class of enzymes (17). In contrast, little if any degradation of the acrylate produced by the amidase occurred in the soluble extracts over the same time course (Fig. 3B). This could be due to any combination of a number of reasons, including lack of an essential cofactor, reductant, or energy source, the acrylate-utilizing enzyme may be membrane-associated, or the inactivation of the acrylate-utilizing enzyme may have occurred during cell lysis. Some acrylate-degrading activity was observed when membranes were recombined with the soluble extract, suggesting that the acrylate-degrading activity may be membrane bound (data not shown).



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  		FIG. 3. Acrylamide degradation and acrylate consumption and degradation in cell suspensions and extracts of R. palustris strain Ac1. (A) Assay with whole-cell suspension (8.65 mg of protein). The inset shows an expansion of the time points between 0 and 12 min. (B) Cell extract (12.94 mg of protein). Symbols: •, acrylamide; {blacksquare}, acrylate.

The specific activities for acrylamide deamidase observed in cell suspensions and cell extracts were typically in the range of 0.15 to 0.3 µmol of acrylamide degraded/min/mg of protein. These rates are more than sufficient to support the observed growth rates and are much higher than the rate of acrylate consumption, which appears to be rate limiting for growth (Fig. 2). Acrylamide amidases have been purified from a Rhodococcus species (17) and a Brevibacterium species (29). The specific activities of these amidases in cell extracts were 26 and 8.2 µmol of acrylamide degraded/min/mg of protein, respectively, values that are substantially higher than those observed here for R. palustris. With regard to the Rhodococcus species, the observed growth rate (aerobically) with acrylamide was comparable to that observed here for photoheterotrophic growth of R. palustris (doubling time of about 10 h in logarithmic phase) (17). The amidase from Rhodococcus was reported to be expressed constitutively (17), as opposed to the amidase from R. palustris, which is shown below to be inducible. Thus, it appears that the level of expression of the R. palustris amidase is lower than for the two characterized aerobic acrylamide utilizers, which appear to express their respective amidases at levels much higher than that necessary for the observed growth rates.

Acrylamide deamidase is an inducible enzyme not subject to catabolite repression by succinate.
Cultures of R. palustris Ac1 were grown with either succinate or acrylamide as the carbon source for several generations and then assayed for their ability to degrade acrylamide in the absence or presence of chloramphenicol (protein synthesis inhibitor) or rifampin (mRNA synthesis inhibitor). As shown in Fig. 4, cells that were previously grown with acrylamide as the carbon source readily degraded acrylamide from the onset of the assay, and the rate of degradation was not affected by rifampin or chloramphenicol. In contrast, there was an ~3-h lag period prior to the onset of acrylamide degradation in succinate-grown cells, after which acrylamide was degraded at a rate comparable to acrylamide-grown cells (Fig. 4). The presence of either chloramphenicol or rifampin prevented this time-dependent increase in activity in succinate-grown cells, indicating that new protein synthesis is required. Succinate was present during this induction, thus, acrylamide deamidase expression is not inhibited by the presence of a conventional carbon source. These results suggest that acrylamide deamidase is an inducible activity and not subject to catabolite repression.



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  		FIG. 4. Requirement of new protein synthesis for acrylamide degradation by R. palustris strain Ac1 grown with succinate as the carbon source. The datum points represent the average of assays performed in duplicate. The standard deviations for datum points were between 0 to 10%, with average standard deviations of 5%. Closed symbols show acrylamide degradation by acrylamide-grown cells; open symbols show acrylamide degradation by succinate-grown cells. Symbols: circles, no antibiotic added; triangles, 10 mg of chloramphenicol added; squares, 5 mg of rifampin added.

Analysis of acrylamide degradation intermediates using 13C NMR.
The results presented in Fig. 2 and 3 show that acrylate is an intermediate in acrylamide catabolism by R. palustris but do not provide insights into how acrylate is further metabolized. To investigate this, [1,2,3-13C]acrylamide, along with [13C]NaHCO3, was added to actively growing cells, and the fate of the labeled carbon atoms was investigated by 13C NMR. As shown in Fig. 5, the spectrum of [1,2,3-13C]acrylamide contains a multiplet at 128.4 to 130.6 ppm corresponding to the olefin carbon atoms, along with a doublet at 171 and 171.5 ppm that corresponds to the amide carbon. Incubation of labeled acrylamide with the cells resulted in the simultaneous disappearance of these spectral features at the same time new features appeared consistent with the formation of uniformly 13C-labeled acrylate (doublet at 126.5 to 127.1 ppm, multiplet at 133.4 to 134.7 ppm, and doublet at 175.4 to 176 ppm due to the C3, C2, and C1 carbon atoms, respectively). As shown in Fig. 5, after 48 h of incubation, all of the acrylamide had been consumed and a large amount of acrylate had been produced. During this time course there was a slight increase in intensity of a peak centered at about 24 ppm, but no other signals could be detected (the large signal at 160.5 ppm is due to the [13C]NaHCO3 present in the assay). Further incubation resulted in the slow disappearance of the acrylate signal, again with no detectable increase in other signals.



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  		FIG. 5. Proton-decoupled 13C NMR spectra resulting from [1,2,3-13C]acrylamide metabolism by actively growing cells. The assay was conducted in an NMR tube using 0.5 ml of OD 1.5 cells as described in Materials and Methods. The spectra were obtained after incubation for 0 h (A), 14 h (B), 26 h (C), or 48 h (D). The resonances for acrylamide are a multiplet at 128.4 to 130.56 ppm arising from C2 and C3 (olefin C atoms) and a doublet at 171 to 171.5 ppm arising from C1. The resonances for acrylate are a doublet at 126.5 to 127.1 ppm arising from C3, a multiplet at 133.4 to 134.7 ppm arising from C2, and a doublet at 175.4 to 176.0 ppm arising from C1. The resonance at 160.1 ppm is due to the NaH13CO3 present in the sample. The multiplet centered at 77.0 ppm is due to the reference CDCl3 present in the locking tube.

An additional 13C NMR analysis was conducted with a more concentrated suspension of cells to accelerate the rate of acrylate degradation, and a preferred carbon source, succinate (natural abundance), was included in the assays in an attempt to slow the further metabolism of product(s) formed downstream of acrylate. As shown in Fig. 6, under these conditions, acrylamide was quantitatively converted to acrylate within 30 min of incubation. The further disappearance of acrylate was correlated with the appearance of three new signals, visible as a doublet at 10.4 to 10.7 ppm, a multiplet at 30.6 to 31.4 ppm, and a doublet at 185.0 to 185.4 ppm (Fig. 7.). Importantly, these signals must arise from a product of acrylamide and acrylate degradation, since the splitting patterns observed show that they result from a compound that is enriched with 13C label at each carbon atom (the succinate added to the assay was natural abundance in 13C, and so any products of succinate metabolism will yield spectra with singlets rather than multiplets due to the large abundance of 12C carbons on adjacent atoms). Prolonged incubation of concentrated suspensions resulted in the complete disappearance of the acrylate signals, followed by the simultaneous disappearance of the three new features described above.



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  		FIG. 6. Proton-decoupled 13C NMR spectra resulting from [1,2,3-13C]acrylamide metabolism by concentrated cell suspensions in the presence of natural abundance succinate. The spectra are shown for boiled cell control with no substrates added (A), boiled cell control with all substrates present (B), 30 min time point (C), 3-h time point (D), and 14-h time point (E). The positions of resonances are the same as for Fig. 5, with the following additions. The resonances (singlets) at 34.5 and 182 ppm arise from the natural abundance succinate included in the assays. Three new resonances resulting from acrylate degradation appeared during the course of the incubation, highlighted in the rectangular boxes. The new resonances consist of a doublet at 10.3 to 10.7 ppm, a multiplet at 30.6 to 31.4 ppm, and a doublet at 185 to 185.4 ppm. The chemical shifts of these new resonances correspond to the carbonyl (C1), {alpha} (C2), and ß (C3) carbon atoms of propionate (the structure of propionate is shown as an inset). There are several additional singlets, also present in the boiled cell control (without substrates), that are not considered further.



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  		FIG. 7. Comparison of growth of R. palustris strains Ac1 (closed symbols) and CGA009 (open symbols) on C3 metabolites and succinate. Propionate-grown cells were used as the source of the inoculants, as described in Materials and Methods. (A) Acrylamide; (B) acrylate;(C) propionate; (D) succinate.

The chemical shifts and splitting patterns of the new resonances described above are consistent with the spectrum that would be expected for [1,2,3-13C]propionate. As further evidence of this, a spectrum of natural abundance propionate was run under the same conditions and compared to the product formed after the 14-h incubation time point in Fig. 6. The propionate standard exhibited singlets at chemical shifts exactly equal to the chemical shifts of the new product formed (data not shown). Starting with the low field signal and going to higher chemical shift, these resonances are due to the ß, {alpha}, and carbonyl carbons of propionate (Fig. 6). Thus, propionate is a formed as a product of acrylate degradation under the conditions shown in Fig. 6.

Growth of R. palustris on proposed intermediates of acrylamide metabolism.
Purple nonsulfur photosynthetic bacteria in general grow well with organic acids as carbon sources, and thus the two electron reduction of acrylate to propionate should provide a suitable conventional carbon source for supporting growth of R. palustris Ac1. In confirmation of this, acrylamide-grown cultures inoculated into media containing propionate as the sole carbon source rapidly grew with no apparent lag. In order to determine whether propionate metabolism is rapid enough to support the observed rates of acrylamide-dependent growth, we compared growth rates of strain Ac1 with acrylamide, acrylate, and propionate as carbon sources under identical growth conditions. For this experiment, the cultures were initially grown with propionate as the carbon source to see if any lag period is associated with acclimation to the new growth substrate. As shown in Fig. 7, strain Ac1 grew well with each of propionate, acrylamide, and acrylate as carbon sources. No significant lag was associated with growth with either propionate or acrylate, while a slight lag followed by rapid growth was associated with growth with acrylamide. R. palustris strain CGA009 grew well with either propionate or succinate under these conditions, but no significant growth was observed with either acrylamide or acrylate during the time course of these assays.


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DISCUSSION
 
Purple nonsulfur photosynthetic bacteria in general, and R. palustris strains in particular, are recognized for their high degree of metabolic versatility under a wide range of growth conditions. The recently completed genome sequence of R. palustris strain CGA009 highlights the nutritional versatility of this bacterium with diverse compounds of interest to the fields of biodegradation and biotechnology (12). The work described here expands the extensive list of compounds utilized by R. palustris to include acrylamide and acrylate, toxic compounds that are produced by industry and which are of concern as pollutants and health hazards.

In order to obtain a photosynthetic bacterium capable of rapid acrylamide- and acrylate-dependent growth, it was necessary to perform enrichments from a suitable source, in this instance the waste effluent from a bovine slaughterhouse. It is not surprising that an R. palustris strain was obtained from this enrichment, since purple nonsulfur phototrophs are known to flourish in wastewater environments such as these (7, 11). Interestingly, R. palustris CGA009, in spite of its noted metabolic versatility, was unable to effectively metabolize acrylamide and acrylate. Preliminary studies of two other purple nonsulfur photosynthetic bacteria we work with in our laboratory, Rhodospirillum rubrum strain UR1 and Rhodobacter capsulatus strain B10, indicate that neither of these bacteria grew using acrylamide as the carbon source (data not shown). Thus, acrylamide metabolism is not a ubiquitous feature of purple nonsulfur photosynthetic bacteria.

As shown in Fig. 4, acrylamide metabolism is an inducible process in R. palustris Ac1 and not subject to catabolite repression by the presence of succinate. The prevention of induction by RNA and protein synthesis inhibitors demonstrates that new protein synthesis is required for acrylamide metabolism in succinate-grown cells. The key enzyme(s) of acrylamide metabolism are thus specialized enzymes not required during growth with a conventional carbon source and are presumably expressed in response to the inducer acrylamide.

A number of aerobic bacteria are capable of growth with acrylamide, including strains of Pseudomonas, Rhodococcus, and Xanthomonas (16, 17, 22). In all instances acrylamide metabolism has been shown to involve an initial deamidation reaction producing acrylate. The studies described here show the same reaction to occur as the first step in the newly isolated R. palustris Ac1. Diverse bacterial amidases, including those that hydrolyze acrylamide to acrylate, have been purified and characterized (17, 29). Although acrylamide deamidation has received considerable attention, there is very little information available on the subsequent fate of acrylate in acrylamide utilizers and, to our knowledge, no complete pathway has been proposed for an acrylamide-utilizing bacterium. Acrylate metabolism has been studied in bacteria that ferment acrylate (10) and in aerobic bacteria that grow with acrylate or produce acrylate as an intermediate of dimethysulfoniopropionate (DMSP) metabolism (1, 2). While the fermentation of acrylate produces propionate, acrylate formed from DMSP cleavage undergoes hydration to ß-hydroxypropionate, which is further oxidized to CO2 and an additional unidentified product (1, 2). Thus, two fates for acrylate have been demonstrated: reduction to propionate and hydration to ß-hydroxypropionate under anaerobic and aerobic conditions, respectively.

The present study supports a pathway of acrylate metabolism in R. palustris Ac1 involving propionate as an intermediate. This seems to be a logical reaction to us, since, under anaerobic photoheterotrophic conditions, there would be sufficient reductant available for the reaction and because organic acids such as propionate are excellent growth substrates for this class of bacteria. Of possible relevance to this, no growth of R. palustris Ac1 with acrylamide was observed under aerobic conditions. As shown in Fig. 7, both strains Ac1 and CGA009 are capable of growth with propionate, although, under the conditions of this experiment, only strain Ac1 was capable of acrylamide- and acrylate-dependent growth. Propionate was only detected as an intermediate of acrylate degradation when succinate was added to cells as an additional carbon source, a finding consistent with the idea that propionate catabolism is rapid and not rate limiting under normal growth conditions. The presence of succinate may allow observation of propionate accumulation since the pathways of propionate and succinate metabolism converge at succinyl-CoA in the citric acid cycle, assuming that propionate catabolism involves carboxylation to methylmalonyl-CoA followed by rearrangement to succinyl-CoA, the expected pathway in these bacteria. To lend further support to this being a likely pathway, results from the recent genome sequencing of R. palustris strain CGA009 reveal all of the genes necessary for the conversion of propionate to succinyl-CoA (12).

To our knowledge, no acrylate reductase has been purified or characterized to date. However, methacrylate reductases have been characterized from Geobacter sulfurreducens strain AM-1 (13) and Wolinella succinogenes (8). In these organisms, methacrylate can serve as a terminal electron acceptor for other carbon sources, in the process undergoing reduction to isobutyrate. The methacrylate reductases from these organisms were found to be periplasmic, use cytochrome c as a reductant, and were shown to be homologs of fumarate reductase (13). Given the structural similarities between fumarate, methacrylate, and acrylate, it is tempting to speculate that a similar reductase is involved in acrylate metabolism in R. palustris Ac1. The nature of the acrylate-reducing enzyme of R. palustris Ac1 will be the subject of future investigations.


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ACKNOWLEDGMENTS
 
This study was supported by National Institutes of Health grant GM51805.

We thank Ashley Ellsworth and Calli Bishop for technical assistance. We also thank Caroline Harwood for kindly providing R. palustris strain CGA009.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT 84322. Phone: (435) 797-3969. Fax: (435) 797-3390. E-mail: ensigns{at}cc.usu.edu. Back


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Applied and Environmental Microbiology, October 2005, p. 5850-5857, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5850-5857.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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