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Two-dimensional polyacrylamide gel electrophoretic analysis of protein synthesis during aromatic acid… Chow, Kevin Toshio 1996

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TWO-DIMENSIONAL P O L Y A C R Y L A M I D E GEL ELECTROPHORETIC A N A L Y S I S OF PROTEIN SYNTHESIS DURING A R O M A T I C ACID C A T A B O L I S M B Y STREPTOMYCES VIOLACEUSNIGER by K E V I N TOSHIO CHOW B.Sc , The University of British Columbia, 1992 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard THE/UNJVERSITY OF BRIT-f$HCOLUMBIA April 1996 © Kevin Toshio Chow, 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. degree at the University of British Columbia, I agree that the Library shall make it Department The University of British Columbia Vancouver, Canada Date DE-6 (2/88) A B S T R A C T Streptomycetes were isolated from soil samples and screened for biodegradative activity against lignin-related aromatic compounds. One isolate, Streptomyces violaceusniger, efficiently utilized 4-hydroxy-3-methoxybenzoic acid (vanillate) and 4-hydroxybenzoic acid (p-hydroxybenzoate) as sole sources of carbon and energy. S. violaceusniger was able to biotransform indole to the dye indigo, indicative of aromatic dioxygenase activity. Colorimetric Rothera assays demonstrated the induction of aromatic dioxygenase activity in S. violaceusniger in the presence of vanillate and p-hydroxybenzoate. Two-dimensional gel electrophoresis of total cell proteins revealed the synthesis of specific groups of proteins upon growth in the presence of vanillate, p-hydroxybenzoate or 3,4-dihydroxybenzoic acid (protocatechuate). The amino-terminal sequence of a heavily expressed 52 kDa protein, induced by vanillate andp-hydroxybenzoate, was highly similar to the amino-terminus of a hypothetical 55 kDa protein discovered during the course of the Escherichia coli genome sequencing project. The S. violaceusniger sequence aligns with conserved regions adjacent to nucleotide binding domains of the GTP-binding cc-subunit protein family and valyl-tRNA synthetases. Because biodegradative monooxygenases are known to possess nucleotide binding domains near the amino terminus and the protein is synthesized in such abundance, it is suggested that the 52 kDa protein is an enzyme involved in aromatic biodegradation. The results of this work are of interest to the study of lignin biodegradation, and also to the study of the catabolism of phenolic compounds in Streptomyces. TABLE OF CONTENTS A B S T R A C T . i i T A B L E OF CONTENTS i i i LIST OF T A B L E S v LIST OF FIGURES vi A C K N O W L E D G E M E N T S vii INTRODUCTION 1 M A T E R I A L S A N D METHODS 7 Bacterial Isolation and Identification 7 Screening Isolates for Catabolism of Aromatic Acids 7 Assays for Oxygenase Activity 9 Bioconversion of Indole to Indigo 9 Colorimetric Rothera Assay 9 Time Course Experiments for 2D-PAGE 10 One-Point Sampling 10 35 Multi-Point Sampling with S Labeling 11 Preparation of Protein Extracts for Electrophoresis 11 35 Measurement of S-Methionine/Cysteine Protein Labeling 12 Determination of Protein Concentration 12 2D-PAGE Protein Electrophoresis 13 Computer-Aided QUEST Analysis of Autoradiograms 13 Protein Sequencing 14 iv RESULTS A N D DISCUSSION 15 Isolation of Streptomycetes from Soil 15 Biotransformation of Indole to Indigo 19 Evidence of Protocatechuate 3,4-Dioxygenase Activity 20 Rates of Catabolism of Aromatic Acids 21 Gene Expression Induced by Aromatic Acids 23 Detection and Sequencing of a Putative Aromatic Degradative Enzyme 33 LITERATURE CITED 45 LIST OF T A B L E S Table Page 1 Colorimetric Rothera assay results 20 2 Proteins showing significant variation on growth of Streptomyces violaceusniger in aromatic acids 26 vi LIST OF F I G U R E S Figure Page 1 Some common low molecular weight products of partially decayed lignin 2 2 Two pathways of vanillate catabolism 3 3 Bromothymol blue minimal medium agar plate assay 8 4 Streptomyces catabolism of lignin-related aromatic acids 16 5 Cell wall fatty acid methyl ester (FAME) identification of Streptomyces violaceusniger 17 6 16S rRNA sequence confirmation of Streptomyces violaceusniger as a member of the genus Streptomyces 18 7 Time course of catabolism of aromatics acids by S. violaceusniger 22 8 Radioactivity incorporation into cell protein during induction with aromatic acids 24 9A,B 2D-PAGE of aromatic-induced S. violaceusniger cell extracts 27,28 10A,B Expression patterns for the eight major aromatic acid-induced proteins 29,30 11 A , B Proteins whose synthesis is repressed on growth of S. violaceusniger in aromatic acids 31,32 12 ExpressionprofilesofSSP3210 34 13 Expression profiles of SSP 4408 35 14 Expression profiles of SSP 1405 36 15 ExpressionprofilesofSSP3717 38 16 Alignment of S. violaceusniger 52 kDa N-terminal sequence with N-terminal sequence of E. coli hypothetical 55.3 kDa protein 39 17 Alignment of S. violaceusniger 52 kDa N-terminal sequence with a conserved domain in several GTP-binding alpha subunit proteins 40 18 Alignment of S. violaceusniger 52 kDa N-terminal sequence with B. subtilis valyl-tRNA synthetase region adjacent to ATP binding site 41 VII A C K N O W L E D G E M E N T S I would like to thank Prof. Julian Davies for excellent supervision, and Prof. Douglas Kilburn and Prof. William Mohn for useful consultation and guidance. I received invaluable help from all the members of the Davies Laboratory and the West East Centre for Microbial Diversity, especially Sakura Iwagami, who provided enzyme assay expertise and technical consultation. Many thanks to Dr. Jirka Vohradsky and Prof. Charles Thompson for technical assistance with 2D-PAGE analysis techniques. The research divisions of Canadian Forest Products Ltd. and MacMillan Bloedel Ltd. provided much appreciated technical assistance and funding support. This thesis is dedicated to the memory of Prof. Paul Steiner, Department of Wood Science, Faculty of Forestry, U B C , without whom this work would not have been possible. Work for this thesis was funded by a Fellowship from the Science Council of British Columbia G.R.E.A.T. Program, and by an equipment grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). 1 I N T R O D U C T I O N Streptomycetes are gram-positive, filamentous eubacteria well known for their roles as producers of secondary metabolites, namely antibiotics. These prokaryotes also play a major role in global recycling of organic carbon (1). Secondary metabolite production by Streptomyces has been studied intensely for decades; however, biodegradation processes performed by Streptomyces have not been examined in detail. Of the biodegradative pathways studied in Streptomyces, the biodegradation of lignin and lignin-related aromatic compounds has been scrutinized most thoroughly. The biodegradation of lignin is of interest because it presents an economical alternative to, or at least reduces the need for chemical delignification processes in the pulp and paper industry (2). Furthermore, lignin solublizing enzymes are sought after by industry because they may allow delignification by-products to be transformed into useful value-added chemicals (3). Biological activity against lignin-related methoxylated aromatic acids (Figure 1) has long been thought to be an indication of an organisms' potential to be effective against lignin itself, though this remains to be proven (4). Degradation of lignin-related aromatic compounds by prokaryotes has been studied thoroughly, and pathways leading to the catabolism of such compounds through the common intermediates 1,2-benzenediol (catechol), or 3,4-dihydroxybenzoic acid (protocatechuate) have been elucidated. The degradation of 4-hydroxy-3-methoxybenzoic acid (vanillate) by Streptomyces setonii 75Vi2 (Figure 2A) has been shown to proceed by decarboxylation of vanillate to yield 4-hydroxy-3-methoxybenzene (guaiacol) (5). Guaiacol is then demethoxylated to form catechol leading to mineralization via the ortho (3-oxoadipate) pathway. The degradation of vanillate by decarboxylation to guaiacol is unlike the degradation pathway elucidated in Pseudomonas (6), in which vanillate is demethoxylated to form protocatechuate, then mineralized (Figure 2B). While the Pseudomonas vanillate demethylase gene has been cloned and sequenced, information CHO CHO COOH Vanillin Syringaldehyde Vanillic Acid CH I Veratric Acid Acetosyringone Isovanillic Acid Figure 1 Some common low molecular weight products of partially decayed lignin. 3 A . COOH OCH, OChL - • M I N E R A L I Z A T I O N OH OH OH OH B . COOH COOH vanillate d e m e t h y l a ^ OCH, M I N E R A L I Z A T I O N OH OH OH Figure 2 Two pathways of vanillate catabolism. (A) Streptomyces degradation of vanillate via a decarboxylase, leading to catechol formation and mineralization, (from Crawford et ai, 1978) (B) Pseudomonas biodegradation of vanillate via vanillate demethylase, leading to protocatechuate and mineralization, (from Brunei etal., 1988) 4 regarding the molecular genetics of degradation of lignin-related aromatics by Streptomyces remains limited. The Streptomyces vanillate degradation pathway was deduced using spectrophotometric analysis of cell-free extracts and culture supernatant catabolic intermediates. The vanillate andp-hydroxybenzoate catabolic genes of Streptomyces have yet to be isolated, cloned and sequenced. Not only is information regarding the genetics of aromatic acid degradation in Streptomyces limited, the regulation of streptomycete catabolic pathways is also little understood. Regulatory mechanisms of aromatic compound catabolic pathways in Pseudomonas, Rhizobium, Agrobacterium, Alcaligenes, Acinetobacter, Nocardia, and other prokaryotes have been shown to be as diverse as the organisms themselves. It is known that in the actinomycete Nocardia opaca, protocatechuate has no inductive function in its own degradation, B-ketoadipate being the specific inducer (7). p-Hydroxybenzoate hydroxylase, on the other hand, is substrate-induced in N. opaca. By comparison, in Alcaligenes, Acinetobacter and Agrobacterium, both the protocatechuate and p-hydroxybenzoate catabolic pathways are substrate-induced. Catabolic genes are usually organized into operons (8); such clustering allows an entire set of genes encoding biodegradative enzymes to fall under transcriptional control of a single regulatory step. A well characterized example is the sal operon of Pseudomonas putida (9), which consists of catabolic genes specifying the oxidative degradation of salicylic acid, the key intermediate in the pathway of naphthalene catabolism, as well as degradative genes for downstream intermediates such as catechol and cis,cis-mucomc acid. The sal operon is positively regulated by a LysR-like regulatory protein, NahR, which binds a nucleotide motif upstream of the sal operon. LysR-like transcriptional regulators have been shown to be involved in activation of catabolism of a number of aromatic compounds (9). The work presented here examines, using protein two-dimensional gel electrophoresis, gene expression in response to catabolism of lignin-related aromatics by Streptomyces 5 violaceusniger. Proteins expressed during these catabolic processes may be involved in the regulation of associated catabolic pathways (such as the LysR family), or may be enzymes active in degradation of lignin-related aromatic acids. We have isolated streptomycetes from soil which utilize several lignin-related aromatic acids as sole sources of carbon and energy. One isolate, identified by cell wall fatty acid methyl ester (FAME) analysis as Streptomyces violaceusniger, grew well on minimal medium supplemented with vanillate or /7-hydroxybenzoate. Cultures of S. 35 • violaceusniger were pulse-labeled at several time points with S-methionine following the addition of vanillate, />-hydroxybenzoate or protocatechuate to the growth medium. Total cellular proteins were visualized by two-dimensional gel electrophoresis (2D-PAGE) and autoradiography for analysis of the induction or repression of protein synthesis in response to these phenolic compounds. It is shown in this work that specific groups of proteins are induced or repressed in their synthesis in response to growth in the presence of lignin-related aromatic acids. To our knowledge, this is one of the first reported uses of 2D-PAGE in the study of catabolic processes. A study by Lupi et al. highlighted the 2D-PAGE profiles of Pseudomonas putida KT2442 when exposed to 2-chlorophenol (10). P. putida KT2442 was shown to produce a number of proteins when grown in the presence of the aromatic compound; however, the organism was not capable of catabolizing 2-chlorophenol and was instead displaying a stress response. 2D-PAGE was also applied by Matin et al. to the study of xenobiotic-induced stress proteins in Escherichia coli (11). Similar to the work of Lupi et al., this was a stress response study and did not involve an organism that catabolized the xenobiotics. 6 The information presented in this thesis should allow for the future characterization of the streptomycete genes responsible for vanillate and ^ -hydroxybenzoate catabolism. The data accumulated from these 2D-PAGE studies complements work being performed by Thompson et al. (12) on the Streptomyces "proteome" (a term used to describe an organism's entire protein complement) (13). Proteome studies such as the work described here, when combined with evolving knowledge of the Streptomyces genome, will provide a better understanding of this industrially significant organism. 7 M A T E R I A L S A N D M E T H O D S Bacterial Isolation and Identification Soil samples were taken from various sites around the University of British Columbia campus. In addition, borehole soil samples were obtained from sawmill sites in British Columbia that had been evaluated for soil contamination. Soil samples were air-dried for several days, and 1 gram of each sample was resuspended in 4 mL of sterile distilled water. Each sample was vortexed vigorously for ten seconds and then allowed to settle for about fifteen minutes to remove large particulate matter. Each sample was diluted 10" , and 0.1 mL of each diluted sample was spread on ISP4 agar plates containing 75 u, g/ml cycloheximide (Sigma) to suppress fungal growth. Plates were incubated overnight at 30°C lid-side up to allow liquid to soak into the agar. Subsequently plates were incubated lid-side down for 4-7 days. After incubation, sporulating colonies presumed to be streptomycetes were picked by sterile toothpicks and cultivated on ISP4 plates. S. violaceusniger was identified by both cell wall fatty acid methyl ester (FAME) analysis and 16S rRNA analysis by services provided at the West East Centre. Screening of Isolates for Catabolism of Aromatic Acids Screening for the catabolism of aromatic acids was carried out on agar minimal medium containing aromatic acid (0.5-3 g/L), trace elements, phosphate buffer and the pH indicator bromothymol blue, pH 7.2 (14). At pH values 7.2 and below, bromothymol blue is green to yellow. At pH values above 7.2, the indicator is blue. Degradation of the aromatic acid being tested would result in decreased acidity in the medium, thus causing a rise in the pH and a change of the medium colour from green (at pH 7.2) to blue (at pH > 7.2). In most cases, isolates which caused the medium to turn blue were also observed to grow well in the presence of the aromatic acid being tested (Figure 3). Isolates which were positive in the bromothymol blue plate assay were grown in liquid minimal medium in the presence of the aromatic acid for which catabolism was indicated by the plate 8 Figure 3 Bromothymol blue minimal medium agar plate assay. Plate on left contains 1 g/L vanillate, plate on right contains no aromatic carbon source. Both plates have been streaked with Streptomyces violaceusniger and incubated for one week at 30 degrees Celsius. 9 assay. Culture supernatants sampled at various times during time-course studies were analyzed by UV/Vis spectrophotometry. Decrease in absorbance at the A, m a x for the particular aromatic being studied (for example, 250 nm Xmax for vanillate) was evidence that the aromatic was indeed being mineralized. In the case of vanillate catabolism by S. violaceusniger, culture supernatants were injected onto a HPLC C i 8 column, and degradation of the aromatic acid was readily observed when the results were correlated to known vanillate standards. Assays for Oxygenase Activity Bioconversion of Indole to Indigo. Conversion of colourless indole to the blue dye indigo is an indication of the presence of catabolic dioxygenase activity (15). S. violaceusniger was plated on both mineral salts medium with 0.5 g/L yeast extract (MSMYE) agar (5) and tryptic soy agar and incubated at 30°C for two days until mycelial growth was clearly visible. Two 0.25 inch filter paper discs were placed at opposite ends of the plate. 20 uL of 175 m M indole in 50% ethanol was added to one disc; an equivalent volume of 50% ethanol was added to the other disc. Observations were made after approximately four days incubation at 30°C. For a liquid media assay, 100 mL of M S M Y E in a 250 mL baffled erlenmeyer flask was inoculated with one colony of S. violaceusniger from ISP4 agar and incubated for 36 hours. Indole was added to the flask to a final concentration of 2 m M from a stock solution of 175 m M in 50% ethanol. Upon observation of colour change in the medium, the culture was extracted with 10 mL chloroform and analyzed by UV/Vis spectrophotometry. Colorimetric Rothera Assay. The colorimetric Rothera assay (16) determines i f oxidative biodegradation of aromatics is occurring in cell-free extracts, and whether catabolism proceeds via the ortho (intradiol) or meta (extradiol) cleavage mechanisms. S. violaceusniger was grown in tryptic soy broth until the culture reached approximately 10 mid-logarithmic growth phase. Cells were harvested by centrifugation, washed twice in isotonic minimal medium, then resuspended in fresh minimal medium. Cells were incubated for nine hours under the following conditions: bromothymol blue liquid minimal medium + 0.3% vanillate, bromothymol blue medium + 0.3% p-hydroxybenzoate, bromothymol blue medium + 0.3% glucose and TSB + 0.3% glucose. Cells were harvested and washed twice in 50 m M Tris-HCl, pH 8.5, before being frozen at -20°C. Cells were thawed on ice, then resuspended in 2 mL of 50 m M Tris-HCl, pH 8.5, 1 mg/mL lysozyme, 100 u.g/mL DNAse I, and 100 u-g/mL RNAse A . Disruption of the cells was achieved by incubation at 37°C for one hour and homogenizing the suspension on ice. Unlysed cells and cell debris were removed by centrifugation. Assay reactions consisted of 100 u.L cell-free extract, 100 u,L of 100 m M sodium phosphate, pH 7.5, and 10 u M of either protocatechuate, catechol or gentisate as substrate. Reactions were observed at this time for the development of a yellow colour, characteristic of 2-hydroxy muconic semialdehyde, which is indicative of meta cleavage activity. Reaction tubes were then incubated for three hours at 37°C, after which solid ammonium sulfate was added to saturation, as well as one drop of 5N ammonium hydroxide and one drop of 25% sodium nitroprusside. Development of a purple colour indicated a positive reaction for ortho cleavage indicating the presence of B-ketoadipate, a key intermediate in ortho cleavage pathways. Time Course Experiments for 2D-PAGE One-Point Sampling. Preliminary time course experiments involved determining the rates of growth and vanillate catabolism, then harvesting vanillate-degrading cultures in mid-logarithmic phase of degradation for 2D-PAGE analysis. For these experiments, one colony of sporulating S. violaceusniger on ISP4 agar was used to inoculate a seed culture consisting of 10 mL of M S M Y E and vanillate at 7.3 m M (1 g/L) in a 50 mL baffled flask (Bellco). The seed culture was incubated at 30°C, 260 rpm for approximately 21 hours, 11 after which the cells were centrifuged, washed twice in 10 mL sterile distilled water, and resuspended in 10 mL sterile, distilled water. 100 mL preparations of M S M Y E with and without 7.3 m M vanillate in 250 mL baffled flasks were inoculated with 1 mL of the washed seed culture. Vanillate degradation was measured as the decrease in A 2 5 o and cell density was measured as the increase in A 5 7 8 . In replicate experiments, cultures growing in the presence and absence of vanillate were harvested by centrifugation when vanillate degradation was most rapid, for cell lysis and analysis by 2D-PAGE. 35 Multi-Point Sampling with S Labeling. For a more detailed study of the catabolism of aromatic acids by S. violaceusniger, vanillate, p-hydroxybenzoate or protocatechuate was added to a final concentration of 3.6 m M to separate 100 mL cultures of the organism in M S M Y E in 250 mL baffled flasks during the early logarithmic phase of growth. A control culture contained no aromatic acids. Immediately prior to addition of 35 the aromatic acids, 1 mL of the control culture was labeled with 100 p,Ci S methionine/cysteine (DuPont-NEN) at 30°C, shaking at 260 rpm for 30 minutes in a 15 mL polypropylene tube. After incubation, the labeled sample was pelleted, frozen in a dry ice ethanol bath and stored at -70°C. 1 mL aliquots of each induced culture were labeled similarly 1,2, 5,12.5 and 18 hours after addition of the aromatic acids. The concentration of each aromatic in the culture medium was measured as absorbance at the A, m a x for each compound in a UV/Vis spectrophotometer. Preparation of Protein Extracts for Electrophoresis Cell pellets were thawed on ice and washed twice in 2 mL lysis buffer (10 m M Tris-HCl pH 7.5, 5 m M EDTA, 100 ug/mL PMSF (Sigma), 1 ug/mL Pepstatin A), then resuspended in 0.5 mL (radiolabeled cells) or 1 mL (non-labeled cells) of the same buffer. Samples were sonicated on ice with a microtip in three bursts, 15 seconds each, with 15 second delays to allow for cooling. Immediately after sonication, samples were 12 centrifuged at 14,000 x g for 15 minutes. The pellet was discarded, and DNase/RNase solution (24 m M Tris base, 476 m M Tris HC1, 50 m M M g C l 2 , 1 mg/mL DNAse I, 0.25 mg/mL RNAse A) was added to 1/10 the total volume of each supernatant, followed by incubation for 10 minutes on ice. For non-labeled samples from one-point experiments, the protein concentration of the extract was determined and volumes of extract containing 150 u.g total cell protein were concentrated in 10,000 Da cut-off ultracentrifugation devices (Microcon-10, Amicon) and resuspended in 10 uL isoelectric focusing buffer (9.9M urea, 4% NP-40, 2.2% ampholytes pH 3-10, 100 m M DTT) for 2D-PAGE and silver staining. For labeled, multi-point experiment samples, total cell extracts (approximately 1 mL per time-point sample) were concentrated by ultrafiltration at a molecular weight cut-off of 10,000 Da, then resuspended in 20 u.L isoelectric focusing sample buffer. In addition to concentrating the sample, the ultrafiltration step removed 35 unincorporated S-labeled amino acids. Measurement of S-Methionine/Cysteine Protein Labeling To measure relative amounts of protein synthesis upon induction of cultures with aromatics, 1 u.L of each sample protein cell extract was spotted on a Whatman GF/A glass fiber filter disc and placed in a scintillation vial with 5 mL of scintillation fluid. The radioactivity in each vial was counted for 1 minute. Background consisted of 1 u.L of IEF sample buffer treated in the same manner. Determination of Protein Concentration Protein concentration in cell free extracts was measured by the bicinchoninic acid method (17) using a kit from Sigma Chemicals Ltd. Bovine serum albumin was used for preparation of a standard curve. 13 2D-PAGE Protein Electrophoresis Two-dimensional gel electrophoresis was performed according to Garrels (18) using the protocols, chemicals, and equipment of the Investigator system (Oxford Glycosystems). First dimension isoelectric focusing (IEF) tube gels incorporated ampholytes in the pH range 3-10, optimized for analysis of total cell extracts. Second dimension high-tensile strength slab gels contained 12.5% acrylamide and an acrylamide to Ar,JV'-methylene-bisacrylamide ratio of 30:0.65 (Duracryl, Oxford Glycosystems). Ten uL of each sample containing 1 x 106 C P M was applied to each IEF tube and focused for 18,000 V-h" 1. After second dimension electrophoresis, slab gels were agitated in fix solution (40% methanol, 10% glacial acetic acid) for 1 hour, followed by treatment with a fluor solution (Enhance, DuPont-NEN) and dried. Proteins were visualized by exposure of dried gels to Kodak Bio-Max M R film for 7-10 days at -70°C. Computer-Aided QUEST Analysis of Autoradiograms Autoradiograms were scanned with a Howtek scanner controlled by PDQuest 2D analysis software version 5.0 (PDI) for 2D gels. Automated spot detection was performed using the PDQuest standard algorithms for 35S-labeled gels. Spot quantitation was performed by computer generated two-dimensional Gaussian modeling. Gels were compared by a process of landmarking and matching spots among all gels in the experiment. A correction factor for each 2D gel protein spot was calculated as the total OD (optical density units) detected in the standard gel (a master reference gel created by merging all gels in a time course matchset) divided by the total OD detected in the particular gel to which each protein spot belonged. The PDQuest gel analysis software was run on a SparcStation 2 (Sun Microsystems). 14 Protein Sequencing To obtain amino-terminal sequence from 2D gels, 150 u.g of total cell protein from a non-radiolabeled, vanillate-induced sample was run on ten replicate gels and stained post-electrophoretically by the zinc-imidazole method (19). The zinc-imidazole stain results in a white background and clear protein spots that are not fixed in the gel matrix. The clear spots are visualized when the gel is viewed against a dark background or several centimeters from a lightbox. Protein spots of interest were cut from each gel and placed together in a single 1.5 mm thick well of a BioRad Protean II xi 12.5% acrylamide slab gel. Laemmli SDS-PAGE sample buffer (lx) was added on top of the gel pieces in the well, and the protein was electrophoresed into the slab gel. The slab gel was blotted to Immobilon-PS Q PVDF membrane (Millipore) using the semi-dry graphite blotter supplied with the Investigator 2D system. A three buffer protocol was used, in which s-amino-n-caproic acid was substituted for glycine (20). The membrane was stained with a Coomassie Blue R-250 solution for several seconds, destained in 40% methanol, then washed with 18 MQ-cm" 1 distilled water several times. The purified, blotted protein band was excised from the membrane, air-dried, then placed in a 1.5 mL eppendorf tube for delivery to the Nucleic Acid Protein Sequencing (NAPS) Unit at the University of British Columbia. 15 R E S U L T S A N D DISCUSSION Isolation of Streptomycetes from Soil Drying of soil samples for several days prior to suspension and plating greatly enhanced the number of streptomycetes isolated. Soils consisting of sand and/or humic matter seemed to contain the greatest number of streptomycetes. Conversely, soil rich in wet clay contained few, i f any, streptomycetes. The soil sample of most interest was taken from a wood-waste landfill at a sawmill in the British Columbia interior. Consisting of loose humic matter and wood debris (ie. sawdust), this sample produced an abundance of streptomycetes of varying morphologies. When screened for degradation of lignin-related phenolic compounds, many isolates were found to be active. However, each isolate displayed an unique "profile" of degradation capabilities (ie. they did not all degrade the same selection of compounds) (Figure 4). These overlapping catabolic profiles indicate that in a microcosm such as a wood-waste landfill, lignin degradation may not necessarily be attributed to one particular lignin degrading organism, but rather to a consortium of bacteria with varying degrees of catabolic activity against lignin. For the purposes of this study, one isolate was chosen for its ability to degrade vanillate and/7-hydroxybenzoate rapidly. This isolate, on ISP4 agar, had dark gray mycelia, light gray spores, and produced a brillant yellow pigment that later changed to a dull orange colour upon aging. Cell wall fatty acid methyl ester analysis performed by gas chromatography identified this isolate as Streptomyces violaceusniger (Figure 5). 16S rRNA analysis of the isolate confirmed its identity as a member of the genus Streptomyces (Figure 6). S. violaceusniger was routinely cultivated on ISP4 agar or tryptic soy agar (TSA). 16 A B C D E Isolate COOH OH COOH OH COOH T < D C H . OH COOH T OH O C H 3 COOH T OCH3 OCH3 NL14-1A + + + - - - -NL15-2D + + + + + + + + - -NL15-2K + + - + + + + + + + + + NL15-2M + + + + + + + - -NL15-2N + + + - + + + - -NL15-2P + + + + + + + - -NL16-1A + + + + + + + + - -S. violaceusniger + + + + + + + + +++ - -S. rimosus + + + - - - -S. lividans + + + - - - -Figure 4 Streptomyces catabolism of lignin-related aromatic acids. Letter designations of aromatic acids are as follows: (A) p-hydroxy-benzoate (B) protocatechuate (C) vanillate (D) isovanillate (E) veratrate. NL isolates were obatined from a sawmill wood waste landfill in the interior of British Columbia. Streptomyces rimosus and Streptomyces lividans 1326, two commonly used laboratory organisms, are included for comparison purposes. (+) to (++++) indicate degree to which catabolism was qualitatively observed by the bromothymol blue plate assay. (-) indicates no observed catabolic activity. 17 Sherlock Vers ion: 1.06 DATA:C94511475A 11-MAY-94 13:27:12 10: 1831 UN-JED-ACT-D7-B-1 (CREAHY TSB 28 5 DAYS Oate of run: 11-MAY-94 13:02:19 Bot t l e : RT 4 Area SAMPLE Ar/Ht Respon CACTI NO] ECL Name X Comment 1 Comment 2 .530 326791800 0.026 7.030 < min r t .630 17922 0.050 ... 7.233 < min r t 3.802 1266 0.041 ... 11.140 4.225 804 0.029 1.076 11.609 12:0 . 0.17 ECL deviates 0.001 Reference 0.001 5.271 3786 0.030 1.037 12.613 13:0 ISO . 0.78 ECL deviates 0.001 Reference 0.000 5.372 2466 0.031 1.034 12.702 13:0 ANTEI SO . . . . 0.51 ECL deviates 0.001 Reference 0.000 5.706 540 0.036 1.023 12.997 13:0 . 0.11 ECL deviates -0.003 Reference -0.004 6.541 37980 0.033 1.004 13.617 14:0 ISO . 7.59 ECL deviates -0.001 Reference -0.002 6.722 666 0.039 ... 13.752 - • 7.055 3438 0.037 0.993 13.999 14-0 0.68 ECL deviates -0.001 Reference -0.002 8.002 53796 0.035 0.977 14.622 15:0 ISO . 10.46 ECL deviates 0.001 Reference 0.000 8.140 142716 0.035 0.975 14.713 15:0 ANTEISO . . . . 27.70 ECL deviates 0.002 Reference 0.001 8.576 12036 0.036 0.968 15.000 15:0 • • . 2.32 ECL deviates 0.000 Reference -0.001 9.330 6234 0.038 0.959 15.458 16:1 ISO H . . . . 1.19 ECL deviates -0.003 9.608 122376 0.039 0.956 15.627 16:0 ISO , 23.28 ECL deviates 0.001 Reference -0.000 9.919 2520 0.038 0.952 15.816 16:1 CIS 9 . . . . 0.48 ECL deviates -0.001 10.218 32172 0.040 0.949 15.998 16-0 6.08 ECL deviates -0.002 Reference -0.004 10.934 5796 0.042 0.942 16.416 16:0 9? METHYL . . . 1.09 ECL deviates 0.000 11.119 5136 0.041 0.940 16.524 17:1 ANTEISO C . . . 0.96 ECL deviates -0.001 11.298 24930 0.040 0.939 16.629 17:0 ISO . 4.66 ECL deviates 0.000 Reference -0.002 11.458 53718 0.041 0.937 16.722 17:0 ANTEISO . . . . 10.02 ECL deviates 0.000 Reference -0.002 11.577 1452 0.044 0.936 16.792 17:1 CIS 9 . . . . . 0.27 ECL deviates -0.000 11.741 2058 0.050 0.935 16.888 17:0 CYCLO . . . . . 0.38 ECL deviates -0.000 Reference -0.002" 11.932 4968 0.044 0.934 16.999 17-0 0.92 ECL deviates -0.001 Reference -0.003 13.038 1326 0.044 0.926 17.633 18:0 ISO . . . . . . 0.24 ECL deviates 0.001 Reference -0.001 13.677 618 0.038 0.922 17.999 18:0 . 0.11 ECL deviates -0.001 Reference -0.003 16.127 2880 0.111 ... 19.415 > max ar/ht Solvent Ar Total Area Named Area X Named Total Amnt Nbr Ref ECL Deviat ion Ref ECL Shif t 326791800 525678 520866 99.08 502423 17 0.001 0.002 n 647 ACTIN1 CRev 3.80] Streptomyces n'hLl S. violaceusniger n " A A 7 S. v . hygroscopicus n AA7 S. v . h . hygroscopicus ° S. v . violaceusniger ntoA S. exfol ia tus . . n Z, S. h a l s t e d i i J-gJ I: i : o l i v e s - : : : : : : : : : : : : : : : o ^ s . . « * . . ^ 8 , Figure 5 Cell wall fatty acid methyl ester (FAME) identification of Streptomyces violaceusniger. 18 Smallest Sum Sequences producing High-scoring Segment P a i r s : gb|U22972 gb|U22973 emb|X60514 gb|U22974| emb' emb emb emb emb SSU22972 Streptomyces sp., s t r a i n GP 772 1SS... SSU22973 Streptomyces sp., s t r a i n GP 773 16S... |SC16SRNA S . c o e l i c o l o r rRNA 16S gene SSU22974 Streptomyces sp., s t r a i n GP 770 16S... SG16SRR S.galbus gene f o r 16S ribosomal RNA SG16SRRN S.galbus (DSM 40480) 16S rRNA gene. SH16SRR S.hygroscopicus gene f o r 16S riboso... SC16SRDN S . c a e l e s t r i s 16S rRNA gene SL16SRR S . l i n c o l n e n s i s gene f o r 16S ribosom... STMRRND Streptomyces ambifaciens rrnD gene ... X79851 SB16SRRN S . b i k i n i e n s i s gene f o r 16S ribosoma... X79323 SM16SRN S.mashuense (DSM 40221) 16S rRNA gene. X7932S S016SRN S.ornatus (DSM 40307) 16S rRNA gene. Y00484 SLRRNB Streptomyces l i v i d a n s rrnB gene f o r . . . X80825 SSRDNA16S S. subrutilus' 16S rRNA gene X81574 SSPRNA16S Streptomyces sp. 16S rRNA gene X79852 X79325 X79853 X80824 X79854 gb|M27245 emb emb emb emb emb emb High P r o b a b i l i t y Score P(N) N 1170 8 4e-139 3 1170 8 4e-139 3 1170 5 9e-138 3 1156 1 3e-137 3 1152 3 2e-137 3 1152 3 2e-137 3 1152 3 2e-137 3 1152 3 2e-137 3 1152 3 2e-137 3 1170 1 le-136 3 1152 1 8e-136 3 1143 1 8e-136 3 1143 1 8e-136 3 1170 3 3e-136 4 1134 . 1 0e-135 3 855 1 8e-135 4 >gb|U22972|SSU22972 Streptomyces sp., s t r a i n GP 772 16S ribosomal RNA, p a r t i a l sequence. Length = 1481 ^ ^ i n u s Strand HSPs: Score = 1170 (327.2 b i t s ) . Expect = 8.4e-139, Sum P(3)' = 8.4e-139 I d e n t i t i e s =238/243 (9 7 % ) , : P o s i t i v e s = 238/243 (97%), Strand = Minus / Plus Query: 248 Sbjct: 382 Query: 188 Sbjct: 442 Query: 128 Sbjct: 502 Query: 68 Sbjct: 562 Query: . 8 Sbj c t : 622 ! I ! l M I I I I 1 1 1 1 1 1 ! l ! IJ I M 1111111! I ! I I 1 1 I I ! I I111 II i i i i i i u r n m i i i i i i 11 i n ii Figure 6 16S rRNA sequence confirmation of Streptomyces violaceusniger as a member of the genus Streptomyces. BLAST database search results are shown. Although Streptomyces coelicolor is a good match, the result is likely a consequence of limited data in the streptomycete 16S rRNA database. The isolate did not show any of the morphological characteristics of S. coelicolor. 19 Biotransformation of Indole to Indigo Pseudomonas dioxygenases active in the degradation of naphthalene have been shown to oxidize indole, a product of tryptophan catabolism in Escherichia coli (15). The resulting oxidized compound undergoes dimerization via an abiotic process to form indigo, a commercial blue dye used in the colourization of blue jeans. Because enzymatic oxidation is the initial step in the bioconversion process, the transformation of indole to indigo is an indicator of dioxygenase activity. When S. violaceusniger was grown on M S M Y E agar plates in the presence of indole, a dark blue, water-insoluble pigment formed in the medium following several days' growth. Pigment formation was also observed when the organism was grown on tryptic soy agar in the presence of indole, but at a reduced level. S. violaceusniger was grown in M S M Y E to high culture density when indole was added to a final concentration of 2 mM. After overnight incubation, the culture medium appeared blue, and blue pigment specks were observed in the foam covering the medium. Upon extracting the culture with chloroform, the organic phase appeared a rich blue colour while the aqueous phase retained the cell mass and appeared clear, the original colour of M S M Y E . UV/Vis spectrophotometric analysis of the blue chloroform extract confirmed the presence of indigo when compared to a spectrum of commercially produced indigo (Sigma) (data not shown). Ensley et al. reported that indigo formation is a property of dioxygenase systems that form c/s-dihydrodiols from aromatic hydrocarbons (15). While the work performed by that group focused on naphthalene catabolism by Pseudomonas putida PpG7, the production of indigo by S. violaceusniger is indicative of the organism's healthy aromatic catabolic processes. Furthermore, it is possible that the enzymes involved in the production of indigo may be linked to catabolism of the aromatics studied in this thesis. 20 Evidence of Protocatechuate-3.4-Dioxygenase Activity Results for the colorimetric Rothera assay are listed in Table 1. Ortho cleavage activity, indicative of the presence of protocatechuate 3,4-dioxygenase, was observed in the cell-free extracts from 0.3% p-hydroxybenzoate, 03% vanillate and TSB + 0.3% vanillate grown cells. No other ortho cleavage, gentisate 1,2-dioxygenase or meta cleavage activity was detected with this assay. The strongest enzyme activity was observed in the cell-free extract from />hydroxybenzoate-grown cells. The results of this assay suggest that both p-hydroxybenzoate and vanillate are degraded via the intermediate protocatechuate and not catechol as was observed for Streptomyces setonii 75Vi2 (21). Therefore, it is probable that an enzyme with functions similar to the vanillate demethylase found in Pseudomonas (6) is involved in vanillate catabolism in S. violaceusniger. Table 1 Colorimetric Rothera assay results. Purple (and shades of purple) color development indicates positive reaction for ortho cleavage indicating presence of beta-ketoadipate. NT = not tested Growth Substrate(s) Protocat. Catechol Gentisate Extract Alone 0.3% p-hydroxybenzoate purple brown/orange yellow/orange yellow/orange 0.3% vanillate pink/orange brown orange orange TSB + 0.3% vanillate pink/orange brown orange orange 0.3% glucose orange/brown brown brown/yellow brown/yellow no extract orange orange orange NT 21 Rates of Catabolism of Aromatic Acids As shown in Figure 7, vanillate, p-hydroxybenzoate and protocatechuate were not catabolized at the same rates. Vanillate degradation began approximately two hours sooner than p-hydroxybenzoate degradation, accelerated rapidly then gradually slowed down by approximately eight hours after induction. p-Hydroxybenzoate, in contrast, began disappearing from the medium about five hours post-induction, and degradation accelerated rapidly until the compound was almost depleted from the medium at 20 hours post-induction. Protocatechuate did not appear to induce its own degradation, and its concentration remained relatively constant in the medium even at 20 hours post-induction, when vanillate and p-hydroxybenzoate concentrations had fallen by at least 75% of their original levels. However, by 20 hours post-induction the concentration of protocatechuate had gradually fallen, and assays of the culture 35 hours post-induction indicated that the majority of protocatechuate had been depleted. The differences in degradation rates for the aromatic acids is not unexpected. Aromatic catabolism in bacteria is known to consist of several layers of regulatory control (22), and transcriptional activation systems respond differently depending on which catabolite is present in the environment. For example, Nichols et al. demonstrated that Pseudomonas putida degrades benzoate in preference to p-hydroxybenzoate (23). The observation that protocatechuate degradation displayed such a long lag phase may be explained by the fact that protocatechuate, although an intermediate of many aromatic catabolic pathways, was shown not to induce any pea (protocatechuate) gene products in Rhizobium leguminosarum as well as in Agrobacterium tumefaciens (22, 24). In these prokaryotes, P -carboxy-cw.cw-muconate, a degradation intermediate of protocatechuate and other aromatic acids, was shown to induce the pea gene products. It has been demonstrated Figure 7 Time course of catabolism of aromatic acids by Streptomyces violaceusniger. Time zero is the time point at which 3.6 mM of each aromatic was added to separate cultures in early log phase. VA = vanillate, HBA = p-hydroxybenzoate, PCA = protocatechuate. Time points at which cell aliquots were radiolabeled are indicated by the arrows at the top of the graph. 23 that in these organisms, the protocatechuate degradation enzymes are expressed at very low constitutive levels (25, 26). Therefore, a slow accumulation of fi-carboxy-cis,cis-muconate would occur until the concentration of the intermediate would be high enough to induce amplified expression of the pea genes, resulting in rapid protocatechuate catabolism. Such a response could explain the phenomenon observed in S. violaceusniger. Gene Expression Induced by Aromatic Acids Addition of 3.6 m M vanillate,p-hydroxybenzoate or protocatechuate to S. violaceusniger cultures in early exponential phase resulted in a dramatic increase in incorporation of 3 5 S -methionine/cysteine into cell protein versus the uninduced culture, as shown in Figure 8. This period of increased protein synthesis peaked at about two hours post-induction and fell back to the uninduced level at approximately five hours post-induction. The increase in protein synthesis is possibly due to expression of a sensory response elicited by the organism when significant concentrations of aromatics are present in the environment. Interestingly, 2D-PAGE indicated there was no dramatic difference in gene expression patterns of induced cells in comparison to uninduced cells, with the exception of several proteins (as will be outlined below). This observation suggests that the response of the organism to the aromatic acids used in this study is not a global stress response (as seen in the study of Pseudomonas putida KT2442 exposure to 2-chlorophenol (10), when dozens of proteins were induced), but rather a highly specific response characteristic of an organism genetically endowed to catabolize lignin-related aromatic acids. 24 Q_ O 1400000 1200000 1000000 800000 600000 400000 i 200000 0 CTRL VA HBA PCA 5 10 15 20 Time Post-Induction (h) Figure 8 Radioactivity incorporation into cell protein during induction with aromatic acids. Time points are the same as in Figure 5. 1 uL of labeled, crude cell extract for samples at each time point was counted for one minute. Heavy, solid line represents CPM data for the uninduced, control culture. 25 PDQuest 2D-PAGE analysis of radiolabeled time course samples revealed the induction of a specific group of proteins in response to vanillate, p-hydroxybenzoate and protocatechuate. Eight proteins demonstrated significant increases in synthesis versus uninduced cell extracts, as shown in Figure 9 and listed in Table 2. Expression patterns for the proteins are displayed in Figures 10A and 10B. A l l eight proteins were present in p-hydroxybenzoate-induced cell extracts; two of the eight were unique to p-hydroxybenzoate induction. Synthesis of six proteins was detected in response to vanillate induction. Only three of the eight proteins responded significantly in protocatechuate-induced cell extracts; this may be due to the low rate of protocatechuate degradation (compared to the other two aromatic acids) during the course of the experiment. In contrast to other 2D-PAGE xenobiotic stress studies with Pseudomonas and E. coli mentioned in the Introduction, in which many proteins were observed to be repressed upon exposure to xenobiotics, in this study only three proteins were observed to be significantly repressed compared to uninduced cell extracts (Figures 11 A , 1 IB). The seemingly small number of repressed proteins may be indicative of S. violaceusniger's selective adaptation to catabolize the aromatic acids used in this study. By comparison, when in the presence of 3.6 m M catechol, a toxic aromatic diphenol, 2D-PAGE protein patterns of S. violaceusniger appeared dramatically different from those typical of induction by vanillate, p-hydroxybenzoate or protocatechuate (data not shown). Catechol-induced cells displayed numerous repressed and induced proteins, some of the latter were likely to be due to a stress response. The identities of the specific aromatic-induced polypeptides will not be known until they are sequenced and the corresponding genes characterized. One of the eight proteins (52 kDa) was submitted for amino-terminal amino acid sequence analysis as is discussed in the next section. The other proteins have been characterized by molecular mass and charge only. Three of the eight proteins were approximately 30 kDa in mass. It is Table 2 Proteins showing significant variation on growth of Streptomyces violaceusniger in aromatic acids. SSP = sample spot number; SSPs are also shown in Figure 9B. SSP m.w. (kDa) pi Induced 1821 57.4 4.7 3717 51.6 5.0 7622 44.8 5.5 7623 42.0 5.5 4408 35.0 5.1 1309 29.7 4.7 3210 28.7 5.0 7205 28.3 5.5 Repressed 7619 41.8 5.6 1405 34.0 4.7 4208 27.5 5.2 27 A. MW 80 kDa 70 kDa 60 kDa 50 kDa 40 kDa pi 4.5 5.0 5.4 5.8 30 kDa 20 kDa 15 kDa Mr/p I grid • — < • • • m' • • • < • m • • > • * • • • • • f • • i f • t « • J • * • • • • *< I fl • . j • i • • # t • • 1 4 •A i F i g u r e 9 2D-PAGE of aromatic acid induced Streptomyces violaceusniger cell extracts. Shown here is the standard reference gel image used to create the PDQuest database. ( A ) molecular weight and pi grid. ( B , next page) proteins showing induced or repressed synthesis with associated spot database numbers. Induced proteins are marked with squares; proteins showing repressed synthesis are marked with triangles. 28 Q1821 3717 7622 7623 . A1406 •1309 Q4408 1 • M Figure 10A Expression patterns for the eight major aromatic acid induced proteins. (pHBA) p-hydroxybenzoate induction, (VA) vanillate induction, (PCA) protocatechuate induction. Axes: (x) = time post-induction, (y) = protein spot density, (z) = protein spot number in PDQuest database. (Note: z-axis spot numbers are not in the same order for each graph to make data most visible in 3D format) 30 p-Hydroxybenzoate-lnduced Proteins 3210-HBA tS 20 4408-HBA 5 10 15 20 1S 20 Van i I late-Induced Proteins 3717-VA 1821-VA Protocatechuate-lnduced Proteins 3210-PCA 1309-PCA 4408-PCA 5 10 IS 20 F i g u r e 10B Individual graphs for proteins showing induced synthesis in the presence of aromatic acids. Axes: (x) time post-induction in hours; (y) protein spot density in optical density units. 31 4208 Time (h) Figure 11A Proteins whose synthesis is repressed on growth of Streptomyces violaceusniger in aromatic acids. Each graph represents one of the proteins with repressed synthesis. Database spot numbers are at the top of each graph. Axes: (x) = time post induction, (y) = protein spot density, (z) = treatment (aromatic acid inducer). Note the change in the z-axis of the graphs from Figure 10A. 32 4 2 0 8 5 10 Time (h) 7 6 1 9 1 4 0 5 - H B A F i g u r e 1 1 B Proteins showing repressed synthesis in the presence of aromatic acids (same data as Figure 11 A). 33 possible that at least one of these polypeptides could be a subunit of protocatechuate 3,4-dioxygenase (P340). P340 has been purified from Pseudomonas putida, and consists of two subunits, a and (3, with molecular masses of 22.3 kDa and 26.6 kDa respectively (27). Protein 3210 has a mass of 28.7 kDa. The protein is present at very low levels at one and two hours post-induction, but is produced for the remainder of the time course experiment (Figure 12). This observation could be explained i f one considers that in the early stages of degradation, vanillate must first be demethylated and />-hydroxybenzoate must be hydroxylated to form the protocatechuate intermediate before the aromatics can be mineralized. It is also notable that the increase in synthesis of protein 3210 corresponds with the onset of disappearance of protocatechuate after the lengthy lag period (discussed previously). Not all proteins of interest were simply the result of gene expression being completely activated ("turned on") by aromatic acid induction. Instead, some proteins were upregulated in their synthesis versus low-level synthesis in the uninduced state. A n example is protein 4408, which showed increased synthesis under induction conditions, particularly induction by /?-hydroxybenzoate (Figure 13). Certain proteins, discussed above, had their synthesis repressed non-selectively by all three aromatics. A n example of a synthesis-repressed protein, protein 1405, is shown in Figure 14. Repressed proteins could possibly be enzymes needed for catabolic processes in M S M Y E medium deemed unnecessary when "preferred" carbon sources such as lignin-related aromatic acids, requiring other enzymes, are introduced into the culture medium. Detection and Sequencing of a Putative Aromatic Degradative Enzyme Cells harvested during the catabolism of vanillate,reproducibly expressed a 52 kD protein with a pi of 4.9, as revealed by silver-staining on one-point analysis 2D-PAGE gels. This protein, designated 3717 in the PDQuest database, was completely absent in cells 34 SSP 3210 m.w. 28.7 kDa, pi 5.0 • -• e j m t o l j ? H i m Mm • O?mo(_ l v l k'Um liiu< * • • • « n t o ! _ y v l M=m lmi> • •* S • c o m - o M l ? v l M i n i Int V A j i v l ktemtnttgN • • VAJl \ £ H i m Imi' j* • V'A_1£ VI M i l l ! IllC^li • VA_15r M i n i l m i £ t i • • 1 • • # • • * HB& l € v i Mum taug< • *• # l*CA_:5 v» kAlM Imioii • v l M i n i Iltii._; • * * • • l-CA_l<- : v l M i M l i i u J . Figure 12 Expression profiles of SSP 3210. Rows (top to bottom): Control (uninduced), vanillate-induced, p-hydroxybenzoate-induced, protocatechuate-induced. Columns (left to right): pre-induction, +1h, +2h, +5h, +12h, +18h. 35 S S P 4408 m w. 35 kDa, pi 5.1 0 • w I* • • • * X* * *• * • e o n v o U - 1 vl: l A i m hnX] &nre l_ i> \1 IA=ro lnu« c?itscl_ltf v l I .Umliic • —. " • - -* • • • 1 * * • • • * • • « • • • • * — — • > • • • • y i-c.^ _j m H i m taa^l FG^ lC l Wirti l i m a Figure 13 Expression profiles of SSP 4408. See Figure 12 for legend. 36 SSP 140S m.w. 34 kDa, pi 4.7 « i i «o l_0 . \ . f \ t im bmgiS • »• . *• • i-onccl £ Mini Inu'jii * • • * • t * * 4 f t 9**-• t • 41 -. ¥A_1 Vl k-lSlll IlitlS* VA £ Kt lu-><i II-.L.-•. VA tfvl W*m*t|?4 * Y . ^ J l : ...in lu^". • . « 1 • t T t • t HBA 2 v i M&nilnug*! • ML > l_ . i. II (11_• •_ - -\ • • • • > f P O O \^liUiulnui;ti • t • \ i Maim hnnga l-CA_ll5 vl kUmlnu^ii Figure 14 Expression profiles of SSP 1405. See Figure 12 for legend. 37 growing without vanillate. Radiolabeled samples analyzed by 2D-PAGE demonstrated that protein 3717 was induced by both vanillate and/7-hydroxybenzoate during the early stages of the time course experiment (Figure 15). Sequencing of the protein revealed the 22 amino-terminal amino acids, Ala-Tyr-Asp-Asp-Leu-Arg-Tyr-Phe-Leu-Asp-Thr-Leu-Glu-Lys-Glu-Gly-Gln-Leu-Leu-Arg-Ile-Thr ( A Y D D L R Y F L D T L E K E G Q L L R I T ) . Using the BLAST-P protein alignment search utility (28), this protein sequence was found to be most similar to the amino terminus of a hypothetical Escherichia coli 55.3 kDa protein (Figure 16), the function of which is as yet unknown. The nucleotide sequence encoding the 55.3 kDa hypothetical protein lies in the rfaR-rfe intergenic region of the E. coli chromosome. This region is adjacent to the fre gene encoding a flavin reductase and the fadAB operon which encodes a fatty acid-oxidizing multienzyme complex (29). When the streptomycete sequence was input into a database search using B E A U T Y (BLAST Enhanced Alignment Utility), which searches for similarity to highly conserved regions, the amino-terminal sequence aligned with the beginning of a highly conserved nucleotide binding region found in 26 guanine nucleotide (GTP) binding alpha subunit (G a-subunit) proteins (Figure 17). Much of the N-terminal sequence also aligned with a portion of the ATP nucleotide binding domain found in valyl tRNA synthetases (Figure 18). Many flavoprotein monooxygenases involved in biodegradation of aromatics characteristically possess nucleotide (FAD) binding motifs near the amino terminus (30). The 52 kD protein amino terminal sequence obtained in this study, though not possessing a nucleotide binding motif, was similar to regions nearby nucleotide binding motifs in G-proteins and tRNA synthetases, suggesting the possibility that the 52 kD protein is indeed a biodegradative enzyme. It is also encouraging that the sequence of the S. violaceusniger 52 kDa protein aligned with an E. coli protein (albeit hypothetical), which was encoded by an open reading frame in the vicinity of other biodegradative genes, though it is difficult to make parallels between gene organization in Streptomyces and E. coli. It must be noted that several frameshift mutations had to be introduced in the E. coli S S P 3 7 1 7 •wirae l_0 v i k U m Inu «M*OI_1 v l k U m l m i c o n e e l j ; t £ H i m (Km o.Mreol_i? v l M in t Imi-• • i V I I l c I j i l.'l - III lllL • 9 I • • V A j l S v l M u n t a o M • • H6&_£ r£ MUM Inu-sis KEAJ>\1 M l w hWlj^i — * * HBvjS vi M t M Inus • ITISA_1S v l k femlmig-• » _ _ reftj: \ i M i n i i r i t i s • • • * i • 1 i PCA_rZ v l M i n i I n i ^ Figure 15 Expression profiles of SSP 3717. See Figure 12 for legend. 39 2 22 S. violaceusniger YDDLRYFLDTLEKEGQLLRIT 1+I I I II I I++I + 1 III E. coli YNDLRDFLTLLEQQGELKRIT 6 26 Figure 16 Alignment of Streptomyces violaceusniger 52 kDa amino terminal sequence with amino terminal sequence of Escherichia coli hypothetical 55.3 kDa protein. (|) indicates exact amino acid match, (+) indicates similar amino acid 40 S. violaceusniger 52 kDa [1-22] spP27600 [175-190] (mouse) spQ03113 [177-192] (human) pirA48071 [151-167] (human) spP27601 [172-185] (mouse) spP16378 [151-164] (drosophila) AY|DDLRYFLDTLEKEGQLLRIT ESVKYFLDNLDRIGQL ESVKYFLDNLDRIGQL DSAKYFLDDLERLGE|AI E S V K Y F L D N L D K L G j D S A K Y F L D D L D R L G Figure 17 Alignment of Streptomyces violaceusniger 52 kDa amino terminal sequence with a conserved domain in several GTP-binding alpha subunit proteins. The amino acids of each sequence shown, relative to the entire sequence of each protein, are listed in square brackets to the right of each accession number. Boxed regions indicate matches between identical or similar amino acids. 41 3 22 S. violaceusniger DDLRYFLDTLEKEGQLLRIT I I I I I I I I I I I + B. subtilis DSLRYFLATGSSPGQDLRFS 546 565 Figure 18 Alignment of Streptomyces violaceusniger 52 kDa amino terminal sequence with Bacillus subtilis region adjacent to valyl-tRNA synthetase ATP binding site. The ATP binding site begins at amino acid 528 of the Bacillus sequence. (|) indicates exact amino acid match, (+) indicates similar amino acid. 42 nucleotide sequence to reveal the hypothetical 55 kDa protein (29), and therefore may not be produced in E. coli. If S. violaceusniger protein 3717 is a biodegradative enzyme synthesized early in the time course, it could be involved in demethoxylation and hydroxylation of vanillate, or meta hydroxylation of p-hydroxybenzoate, since these steps occur early in the catabolic pathways of the aromatics. The similarity between the 52 kDa amino-terminal sequence and the highly conserved G-protein region may suggest that the product identified is a sensory protein involved in activation of biodegradative processes. However, this is unlikely, since the protein was produced in abundance (visible by silver-staining, and slightly by Coomassie R-250 staining) during biodegradation, which would not be expected for a signal transduction protein in unfractionated cell extracts (31). The suggestion that bacteria as diverse as S. violaceusniger and E. coli share a similar catabolic enzyme is not unfounded. Indeed, the similarity of catabolic enzymes amongst Pseudomonas, Alcaligenes, Azotobacter, Acinetobacter and Rhizobium has been documented (32). These studies indicate that while the amino acid sequences of catabolic enzymes are not very similar between different species of prokaryotes, there are enough similarities to suggest common ancestors for some catabolic oxidative enzymes. Furthermore, it should also be noted that the first six amino acids in the amino-terminal sequence of the S. violaceusniger 52 kDa protein (AYDDLR) are only found in one other protein in the database ~ salicylate-1-monooxygenase of Pseudomonas putida (30). This observation is of interest in light of the fact that salicylate is an aromatic acid similar in structure to vanillate and p-hydroxybenzoate. However, in the Pseudomonas enzyme, the sequence is not at the amino terminus but rather towards the carboxy terminus. Nakatsu et al. have shown evidence for rearrangement of catabolic enzyme domains throughout molecular evolution (33). The connection between the streptomycete 52 kDa protein and 43 salicylate-1-monooxygenase is speculative, but will become more clear when the gene encoding the 52 kDa protein is sequenced in its entirety. Catabolic oxidoreductases do not share high overall sequence homology. Rather, sequence homologies are localized in motifs such as F A D binding sites (30). For example, the overall similarity between salicylate hydroxylase and /?-hydroxybenzoate hydroxylase is 25%, and the overall similarity between salicylate hydroxylase and phenol hydroxylase is only 20%. Therefore, the observation that the amino-terminal sequence of the 52 kDa protein does not align with any known catabolic enzymes does not eliminate it from this class. The complete gene sequence will indicate i f the protein is, in fact, a biodegradative enzyme and will reveal whether or not it is a homolog to the 55 kDa E. coli hypothetical protein and/or related to salicylate-1-monooxygenase. The cloned gene will be used to perform gene disruption experiments, which will permit the function of the protein to be determined. Streptomyces, though exploited extensively over the years for its antibiotic production capabilities, has shown potential for use in biotransformation processes such as chloroaromatic bioremediation. In 1966, the dechlorination of polychlorinated aromatic pesticides by several Streptomyces strains was reported (34). The dechlorination activity observed during that study was likely mediated by catabolic oxidoreductases similar to those involved in degradation of lignin-related aromatic acids. This study has shown 20-P A G E to be a powerful tool in the analysis of catabolic processes, and demonstrates that gene expression changes during catabolism of aromatic chemicals can be effectively monitored by this method. By expanding our knowledge of the proteomes of Streptomyces and other organisms, future studies using 2D-PAGE should reveal more enzymes involved in biodegradation of aromatic chemicals, including man-made xenobiotic compounds. Continued studies will permit the characterization of pathways 44 for the degradation of xenobiotic and lignin model compounds, particularly the cloning and sequencing of Streptomyces genes responsible for vanillate and p-hydroxybenzoate catabolism. The results of such work may ultimately lead to the isolation and development of novel microorganisms, ideally suited for use in biotransformation and bioremediation applications. 45 L I T E R A T U R E C I T E D 1. Atlas, R., and R. Bartha. 1993. Survey of Microorganisms, p. 506-532. In Microbial Ecology: Fundamentals and Applications. Benjamin/ Cummings, Redwood City. 2. Zabel, R.A. and J.J. Morrell. 1992. Wood Microbiology. Academic Press, New York. 3. A . N . Glazer and H. Nikaido. 1995. Horizons of Microbial Biotechnology: Feedstock Chemicals, p. 22-26. In Microbial Biotechnology: Fundamentals of Applied Microbiology. W.H. Freeman and Company, New York. 4. Crawford, R.L. 1981. Lignin Biodegradation and Biotransformation. John Wiley, New York. 5. Crawford, R.L. and P.P. Olson. 1978. Microbial catabolism of vanillate: decarboxylation to guaiacol. Appl. Environ. Microbiol. 36:539-543. 6. Brunei, F. and J. Davison. 1988. Cloning and sequencing of Pseudomonas genes encoding vanillate demethylase. J. Bacteriol. 170:4924-4930. 7. Cain, R.B. 1980. The Uptake and Catabolism of Lignin-Related Aromatic Compounds and Their Regulation in Microorganisms, p. 21-60. In Lignin Biodegradation: Microbiology, Chemistry, and Potential Applications. CRC Press, Boca Raton. 8. Harayama, S. and K . N . Timmis. 1989. Catabolism of aromatic hydrocarbons by Pseudomonas, p. 151-174. In Genetics of Bacterial Diversity. Academic Press, London. 9. Schell, M . 1990. Regulation of the Naphthalene Degradation Genes of Plasmid NAH7: Example of a Generalized Positive Control System in Pseudomonas and Related Bacteria, p. 165-176. In Pseudomonas: Biotransformations, Pathogenesis, and Evolving Biotechnology. A S M Press, Washington, D.C. 10. Lupi, C , Colangelo, T. and C.A. Mason. 1995. Two-dimensional gel electrophoresis analysis of the response of Pseudomonas putida KT2442 to 2-chlorophenol. Appl. Environ. Microbiol. 61:2863-2872. 11. Blom, A. , Harder, W. and A . Matin. 1992. Unique and overlapping pollutant stress proteins of Escherichia coli. Appl. Environ. Microbiol. 58:331-334. 12. Puglia, A. , Vohradsky, J. and C.J. Thompson. 1995. Developmental control of the heat-shock stress regulon in Streptomyces coelicolor. Mol . Microbiol. 17:737-746. 46 13. Kahn, P. 1995. From genome to proteome: looking at a cell's proteins. Science 270:369-370. 14. Grund, E., Knorr, C , and R. Eichenlaub. 1990. Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp. Appl. Environ, Microbiol. 56:1459-1464. 15. Ensley, B.D., Ratzkin, B.J., Osslund, T.D., Simon, M . , Wackett, L.P. and D.T. Gibson. 1983. Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science 222:167-169. 16. Stanier, R.Y. , Palleroni, N.J. and M . Doudoroff. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43:159-271. 17. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A . K . , Gartner, F.H., Provenzano, M.D. , Fujimoto, E.K., Goeke, N . M . , Olson, B.J. and D.C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85. 18. Garrels, J.I. 1979. Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines. J. Biol. Chem. 254:7961-7977. 19. Ortiz, M.L . , Calero, M . , Patron, C.F., Castellanos, L . and Mendez, E. 1992. Imidazole-SDS-Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins. FEBS Lett. 296:300-304. 20. Blotting Protocol: Three Buffer System In Investigator 2-D Electrophoresis System Operating and Maintenance Manual. Revision B, 1993. 21. Pometto III, A . L . , Sutherland, J.B. and D.L. Crawford. 1981. Streptomyces setonii: catabolism of vanillic acid via guaiacol and catechol. Can. J. Microbiol. 27:636-638. 22. Parke, D., Rynne, F. and A. Glenn. 1991. Regulation of phenolic catabolism in Rhizobium leguminosarum biovar trifolii. J. Bacteriol. 173:5546-5550. 23. Nichols, N . N . and C.S. Harwood. 1995. Repression of 4-hydroxybenzoate transport and degradation by benzoate: a new layer of regulatory control in the Pseudomonas putida p-ketoadipate pathway. J. Bacteriol. 177:7033-7040. 24. Parke, D. 1993. Positive regulation of phenolic catabolism in Agrobacterium tumefaciens. J. Bacteriol. 175:3529-3535. 25. Parke, D. 1996. Characterization of PcaQ, a LysR-type transcriptional activator required for catabolism of phenolic compounds, from Agrobacterium tumefaciens. J. Bacteriol. 178:266-272. 47 26. Parke, D. personal communication 27. Frazee, R.W., Livingston, D .M. , LaPorte, D.C. and J.D. Lipscomb. 1993. Cloning, sequencing, and expression of the Pseudomonas putida protocatechuate 3,4-dioxygenase genes. J. Bacteriol. 175:6194-6202. 28. Altschul, S.F., Gish, W., Miller, W., Meyers, E.W. and D.J. Lipman. 1990. Basic local alignment search tool. J. Mol . Biol. 215:403-410. 29. Bailey, M.J.A., Koronakis, V . , Schmoll, T. and Hughes, C. 1992. Escherichia coli HlyT protein, a transcriptional activator of haemolysin synthesis and secretion, is encoded by the rfaH (sfrB) locus required for expression of sex factor and lipopolysaccharide genes. Mol . Microbiol. 6:1003-1012. 30. You, I.S., Ghosal, D. and I.C. Gunsalus. 1991. Nucleotide sequence analysis of Pseudomonas putida PpG7 salicylate hydroxylase gene (nahG) and its 3'-flanking region. Biochemistry 30:1635-1641. 31. Vujaklija, D. personal communication 32. Ornston, L . N . , Houghton, J., Neidle, E.L. and L .A . Gregg. 1990. Subtle selection and novel mutation during evolutionary divergence of the p-ketoadipate pathway, p. 207-225. In Pseudomonas: Biotransformations, Pathogenesis, and Evolving Technology. American Society for Microbiology, Washington, D.C. 33. Nakatsu, C.H., Straus, N .A. and R.C. Wyndham. 1995. The nucleotide sequence of the Tn5271 3-chlorobenzoate 3,4-diooxygenase genes (cbaAB) unites the class IA oxygenases in a single lineage. Microbiol. 141:485-495. 34. Chacke, C.I., Lockwood, J.L. and Zabik, M . 1966. Chlorinated hydrocarbon pesticides: degradation by microbes. Science 154:893-895. 


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