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Journal of Microbiological Methods, 17 (1993) 273-281 273 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167 - 7012/93/$06.00 MIMET 00568 Direct extraction of proteins from environmental samples O.A. Ogunseitan Laboratory for Molecular Ecology, Environmental Analysis & Design Department, University of California, lrvine, CA 92717, USA (Received 17 August 1992; revision received 23 October 1992; accepted 6 December 1992) Summary Two methods were developed and evaluated for extracting proteins from water, sodiment, and soil samples. In the first method, microbial proteins were extracted from 1 g soil or sediment, or pellet from 10 ml wastewater, by boiling the samples in a solution containing 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% SDS, 10% glycerol, and 0.2% bromophenol blue. In the second method, the same quantities of environmental samples were incubated for 1 h at 0°C in a 2 volume solution containing 50 mM Tris-HC1 (pH 7.6), 1 mM EDTA, 10% sucrose, 1 mM dithiothreitol, 300/tg/ml lysozyme, 0.1% polyoxyethylene 20 cetyl ether. Lysis was completed by four 10-min freeze-thaw cycles (37°C to dry-ice bath). Cellular debris and other particulate matter were removed from the protein preparations by centrifugation. The boiling method recovered high concentrations of proteins from wastewater (10 to 30 gg per ml), but not from soils and sediments. The freeze-thaw method performed better for soils and sediments, yielding 20 to 50/tg protein per g. Protein extracts were resolved by electrophoresis using 15% polyacrylamide gels under denaturing conditions. The sizes of proteins extracted by both methods ranged from less than 14 kDa to greater than 97 kDa. Both methods are simple and rapid, and both have potential applications for direct analysis of microbial community response to changes in environmental conditions, and for direct measurement of gene expression in complex microbial communities. Key words: Protein; Extraction; Environmental sample Introduction Expression of genes in many microorganisms correlate with a variety of environ- mental stimuli, ranging from the presence of particular nutrients to changes in physical-chemical conditions [1]. Although several techniques have been developed for detecting and analyzing the abundance of genes [2,3,4,5], and mRNA transcripts [6] in natural microbial communities, no direct methods were developed until now for analyzing the protein products of gene expression from environmental samples. Correspondence to: O.A. Ogunseitan, Laboratory for Molecular Ecology, Environmental Analysis & Design Department, University of California, Irvine, CA 92717, USA.

Direct extraction of proteins from environmental samples

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Page 1: Direct extraction of proteins from environmental samples

Journal of Microbiological Methods, 17 (1993) 273-281 273 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167 - 7012/93/$06.00

MIMET 00568

Direct extraction of proteins from environmental samples

O.A. Ogunseitan

Laboratory for Molecular Ecology, Environmental Analysis & Design Department, University of California, lrvine, CA 92717, USA

(Received 17 August 1992; revision received 23 October 1992; accepted 6 December 1992)

Summary

Two methods were developed and evaluated for extracting proteins from water, sodiment, and soil samples. In the first method, microbial proteins were extracted from 1 g soil or sediment, or pellet from 10 ml wastewater, by boiling the samples in a solution containing 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% SDS, 10% glycerol, and 0.2% bromophenol blue. In the second method, the same quantities of environmental samples were incubated for 1 h at 0°C in a 2 volume solution containing 50 mM Tris-HC1 (pH 7.6), 1 mM EDTA, 10% sucrose, 1 mM dithiothreitol, 300/tg/ml lysozyme, 0.1% polyoxyethylene 20 cetyl ether. Lysis was completed by four 10-min freeze-thaw cycles (37°C to dry-ice bath). Cellular debris and other particulate matter were removed from the protein preparations by centrifugation. The boiling method recovered high concentrations of proteins from wastewater (10 to 30 gg per ml), but not from soils and sediments. The freeze-thaw method performed better for soils and sediments, yielding 20 to 50/tg protein per g. Protein extracts were resolved by electrophoresis using 15% polyacrylamide gels under denaturing conditions. The sizes of proteins extracted by both methods ranged from less than 14 kDa to greater than 97 kDa. Both methods are simple and rapid, and both have potential applications for direct analysis of microbial community response to changes in environmental conditions, and for direct measurement of gene expression in complex microbial communities.

Key words: Protein; Extraction; Environmental sample

Introduction

Expression of genes in many microorganisms correlate with a variety of environ- mental stimuli, ranging from the presence of particular nutrients to changes in physical-chemical conditions [1]. Although several techniques have been developed for detecting and analyzing the abundance of genes [2,3,4,5], and mRNA transcripts [6] in natural microbial communities, no direct methods were developed until now for analyzing the protein products of gene expression from environmental samples.

Correspondence to: O.A. Ogunseitan, Laboratory for Molecular Ecology, Environmental Analysis & Design Department, University of California, Irvine, CA 92717, USA.

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Much is known about the synthesis, partitioning, and secretion of proteins in axenic cultures of individual microorganisms of ecological importance [7], and about the activities of few ectoenzymes in natural environments [8] but virtually no information exists on the status of cell-associated proteins in microbial consortia indigenous to natural environments. The extraction of proteins directly from environmental sam- ples is desirable for multiple reasons: (1) Analysis of proteins extracted directly from environmental samples may help characterize the response of microbial communities to stressful conditions such as contamination with toxic chemicals [9], starvation [10], heat [11], or oxygen levels [12,13]. (2) Analysis of total proteins extracted from an environmental sample can be employed as a 'fingerprint' to type the diversity in the sample, in a way similar to grouping of bacteria according to enzyme polymorphisms and immunological reactions [14]. Such fingerprints may eventually be used to monitor the deterioration or enrichment of species diversity in microbial commu- nities. (3) The abundance of proteins to which specific antibodies are available can be directly measured in total proteins extracted from complex ecosystems, and used as an index for monitoring the progress of a biocatalytic reaction in situ. This paper describes methods for extracting total proteins from soil, sediment, and sewage, ecosystems of considerable biotic and abiotic heterogeneity.

Materials and Methods

Environmental samples Wastewater samples were collected from the sewage treatment facility operated by

the Irvine Ranch Water District (IRWD). Samples (100 ml) were aseptically collected on three different dates from the influent and pre-chlorination effluent ports at the facility. The samples were kept at 4°C and analyzed within 2 h of collection. Soil samples were collected from a diesel oil-contaminated plot of loamy soil in Parsip- pany, NJ. The soil samples were collected, shipped, stored on ice and processed within 48 h of collection. Sediment samples were collected from six different sites at Convair lagoon in the San Diego Bay. Sediment samples were collected by means of a bottom sampling dredge (LaMotte Chemical, Chestertown, MD) into pre-cooled sterile Whirl-pak bags (Nasco Co., Modesto, CA), and stored at 4°C until processed. The population densities of bacteria in the samples were determined by serial dilution with phosphate buffer and spread plating on Trypticase Soy Agar (Difco, Detroit, MI).

Extraction of proteins from environmental samples Ten milliliters of wastewater, or 1 g each of soil or sediment were used as starting

material for direct extraction of proteins.

The boiling method. For wastewater, microorganisms and other particulate matter were collected from the aqueous phase by centrifugation at 12 000 x g for 10 min. The concentrated particulate matter from wastewater, or 1 g soil or sediment was washed once in 1 ml ice-cold 50 mM Tris-HCl, pH 7.4. After the wash solution was removed, the samples were resuspended by vortexing for 30 s in 500 #1 of water. This was followed by adding 500 #1 of a freshly made solution containing 100 mM Tris- HCI, (pH 6.8), 200 mM dithiothreitol, 4% SDS, 20% glycerol, and 0.2%

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bromophenol blue, and continued vortexing for 2 min. The samples were then placed in a boiling water bath for 10 min. Cellular debris and other particulate matter were removed by centrifugation at 12000 x g for 30 rain at 4°C. The supernatant was collected and repeatedly passed through a 250 /A Gastight (No. 1725) syringe (Hamilton Co., Reno, Nevada), to shear any DNA that may be present. The samples were again centrifuged at 12000 x g for 10 min at room temperature. The supernatants were transferred into fresh tubes and used for protein analysis by electrophoresis (25 #1).

Because of the potential application of this rapid technique for monitoring enzyme activity and microbial integrity in a continuous process such as wastewater remedia- tion, a control experiment was done to apply the method for comparative analysis of proteins extracted from dominant organisms in the wastewater samples. In that experiment, 10 ml each of wastewater samples collected on three separate dates were used to inoculate 100 ml of minimal phosphate medium [2] containing (g/l) sodium acetate (5.67), NHaC1 (1.0), KCI (0.5), Na2SO4 (0.1), NaH2PO4.2H20 (0.509), MgC12.6H20 (1.067), CaC12 (0.132), Tris-base (12.1), trace element solution (1 ml), pH 7.4. A phosphorus-accumulating wastewater bacterial isolate, .4cinetobacter calcoaceticus ATCC 33308 was used as the pure culture control for growth in the medium. The cultures were incubated at 25°C for 2 days, and proteins were extracted from 1 ml aliquots, using the boiling method.

The freeze-thaw method. Initial preparation of samples was as for the boiling method, with particulate matter concentrated from 10 ml wastewater, and 1 g portions of soil or sediment samples washed once in 1 ml cold 50 mM Tris-HCl, pH 7.4. The concentrated particulates from wastewater, or washed 1 g portions of soil or sediment were suspended in 1 ml of a solution containing 50 mM Tris-HC1 (pH 7.6), 10% sucrose, 1 mM dithiothreitol. After gentle mixing, 1 ml of a solution containing lysozyme (600 #g/ml), EDTA (2 mM), and polyoxyethylene 20 cetyl ether (Brij 58) (0.2%) was added. The suspensions were incubated on ice for 1 h with gentle mixing every 15 min. The suspensions were then subjected to four freeze-thaw cycles from a dry-ice ethanol bath to 37°C. Following these cell lysis steps, the lysates were centrifuged at 23 000 x g for 2 h to remove cell debris and other particles. Nucleic acid molecules in the samples were sheared as described above. The supernatants were collected and used for protein concentration determination and electrophoresis.

Determination of protein concentrations The concentrations of proteins in the extracts were determined by using the

Bradford Dye protein determination reagent (USB, Cleveland, OH) according to the manufacturer's protocol. The light absorbance of protein extract-dye complex was determined by scanning aliquots with a UV-Visible spectrophotometer (Beck- man DU-7, Irvine, CA). The concentration of proteins in the experimental extract was determined by interpolating the absorbance at 595 nm against a standard curve generated with bovine serum albumin (Sigma Chemicals, St. Louis, Missouri).

Polyacrylamide gel electrophoresis Typically, 25/~1 of the fresh protein extract from the boiling method was used for

Page 4: Direct extraction of proteins from environmental samples

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electrophoresis. For the freeze-thaw method, 25/~1 of the fresh extract to which 25 #1 of gel loading buffer (100 mM Tris-HC1 (pH 6.8), 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) was added, was used for electrophoretic separation of polypeptides. Polyacrylamide gels (15%), layered with 5% stacking gels, and Tris-glycine buffer were made according to standard methods [15]. The gels

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Wavelength (nm) Fig. 1. (A) Spectrophotometric curve for light absorbance by proteins extracted from wastewater. (B) Spectrophotometric curve showing modified light absorbance by proteins extracted from wastewater, bound to Bradford dye. The concentration of proteins in the extracts was determined by interpolating the absorbance values at 595 nm from a standard curve generated with bovine serum albumen (Sigma, St.

Louis, Missouri).

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were run at 8 V/cm for 2 h, followed by 6 h of 15 V/cm. Resolved bands of proteins in the gels were visualized by silver nitrate staining according to standard procedures [15].

Results and Discussion

The average (n = 3) population density of bacteria in the raw wastewater sample was 3.1 (+ 0.2) x 105 colony forming units (CFU) per ml, and in the partially treated wastewater, 9.6 (+ 0.3) x 10 4 CFU/ml. The average (n = 6) population density of bacteria in the soil sample was 4.9 ( _ 1.2) x 10 7 CFU/g, and in the sediment sample, 5.2 (+ 0.8) x 105. The differences in bacterial population densities did not appear to affect the yield of proteins, especially in wastewater, although the contributions of other microorganisms different from colony-forming bacteria, and extracellular protein molecules in the environmental samples were not determined.

Fig. 1 shows the typical curves for spectrophotometric absorbance patterns of protein macromolecules extracted from the environmental samples using the direct extraction methods. In unstained samples, the maximum absorbance was observed at a wavelength of 220 nm, with a shoulder peak at 280 nm. The latter peak is likely to represent protein molecules (Fig. 1A), whereas the former peak was initially specu- lated to be sheared nucleic acids, but this speculation was not supported by agarose gel analysis and ethidium bromide staining of the extracts (data not shown). When the protein extracts were stained with Bradford dye to determine the concentration, the maximum absorbance shifted to 595 nm as expected, and the absorbance value

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Fig. 2. Silver-stained bands of protein extracted from wastewater with the boiling method, and resolved by polyacrylamide gel electrophoresis (PAGE). Lane M, molecular weight marker proteins (USB Biochemicals, Cleveland, OH). (a, c, e) Proteins extracted from raw wastewater samples collected on three different dates. (b, d, f) Proteins extracted from partially treated wastewater collected on dates corresponding to lanes a, c, e. (g) For comparison; protein band extracted from sediment using the same

method.

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278

was used to interpolate the protein concentration (Fig. 1B). Additionally, there was an absorbance-peak shift at 280 nm which further supported the identity of the macromolecules as protein (Fig. 1). When the boiling method was used, the concen- tration of proteins extracted from the wastewater ranged between 10 and 30 #g per ml, and from the soil and sediment samples, from less than 1 to 5 #g/g. The freeze- thaw method gave similarly high yields of proteins from wastewater but improved the yield of proteins from soil and sediment to 20-50 #g protein/g.

Fig. 2 shows a silver-stained band profile of proteins extracted directly from 10 ml of raw and partially treated wastewater, using the boiling method. Significant differences were observed between the banding patterns of proteins extracted from raw wastewater samples (Fig. 2, lanes a,c,e), and the extracts from partially treated (aerated and settled) wastewater (Fig. 2, lanes b,d,f). These differences were consis- tent on a temporal scale because there were no noticeable changes in banding patterns of proteins in samples collected on different dates (Fig. 2, lanes a,b, compared with lanes c,d, or lanes e,f). However, there were significant differences in the banding patterns of proteins extracted from dominant organisms selected from wastewater samples by cultivation in minimal phosphate medium (Fig. 3). These data indicate that although the diversity of bacteria in the raw wastewater samples are similar (Fig. 2), different proteins are induced when organisms are selected in a defined growth medium (Fig. 3). In all six wastewater, samples examined, there was a constant protein band that migrated to a distance corresponding to approximately 66 kDa molecular size on electrophoresis gels (Figs. 2 and 3).

Fig. 4A shows the electrophoretic separation of proteins extracted from soil, using the boiling method. The yield was poor and the visible protein bands were slightly less than 43 kDa in size. Similarly, the yield of sediment proteins extracted with the boiling method was low, and only a single band co-migrating with the 43 kDa marker protein band was visible (Fig. 4B). Because of the poor yield of proteins from the soil

M

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Fig. 3. Silver-stained bands of protein extracted with the boiling method, from a pure culture of Acinetobacter calcoaceticus ATCC 33308 (a), and cultivated wastewater bacteria (b, c, d, representing

wastewater samples collected on three different dates). M, molecular weight marker proteins.

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Fig. 4. (A) Silver-stained bands of soil proteins extracted with the boiling method showing poor yield and resolution (a, b, c, d represent extracts from different soil samples taken from the same area). M, molecular weight marker proteins. (B) Silver-stained bands of sediment proteins extracted with the boiling method, showing poor yield and better resolution than the soil extract (Fig. 4). Lanes a, b, c, d, e, and f represent sediment samples taken from different locations in the same lagoon. M, molecular weight

marker proteins.

and sediment samples, it was speculated that lysis of microorganisms in these samples was incomplete. Therefore, a more rigorous freeze-thaw technique was developed to increase the yield. When the freeze-thaw method was tested on wastewater samples, the results were not significantly different from the results obtained by the boiling

Fig. 5. (A) Silver-stained bands of soil proteins extracted with the freeze-thaw method. Lanes a, b, c, d, e, and f represent different samples taken from the same site. M, molecular weight marker proteins. (B) Silver-stained bands of sediment proteins extracted with the freeze-thaw method. Lanes a, b, c, and d represent extracts from different sediment samples collected from different locations in the same lagoon.

Lane M, molecular weight marker proteins.

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method (data not shown), indicating that the freeze-thaw process did not damage the integrity of proteins. However, the yield of proteins from soil and sediment samples was higher with the freeze-thaw method, ranging between 20 and 50 #g protein/g. There was also a broader size range of protein bands observed with the freeze-thaw method (Fig. 5A,B for soil and sediment, respectively). The low signal-to-background ratio in protein extracts produced by the freeze-thaw method may be due to co- extraction of humic substances that bind unspecifically to the silver nitrate stain.

In summary, methods now exist for extracting proteins from environmental samples. Further concentration and purification of the proteins may be conducted for certain applications, for example, Western blotting and specific enzyme assays, that may be adversely affected by interfering non-protein molecules. For those purposes, prefabricated protein purification columns (Sigma Immunochemicals, St. Louis, MO) may be employed. Additionally, application of two dimensional poly- acrylamide gels may prove useful for isolating specific proteins, or for 'fingerprint' analysis of different microbial communities.

Acknowledgements

This work was supported in part by a joint grant (RAN 91-05) from the National Water Research Institute (Fountain Valley, CA) and the Irvine Ranch Water District. I thank Dong-Ju Min for general laboratory assistance. I am grateful for the support provided by UC, Irvine Faculty Career Development Award.

References

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196. 11 Neidhardt, F., VanBogelen, R. and Vaughn, V. (1984) The genetics and regulation of heat-shock

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