common sources include backyard burning , incineration of plastics , and chlorine bleaching of pulp in paper mills .
documented health effects include acute and chronic effects , including chloracne , various types of cancers , reproductive diseases , circulatory and respiratory diseases , and diabetes .
the traditional approach to environmental remediation includes a host of physical and chemical methods , depending on the characteristics of the polluted site and the extent of contamination present .
bioremediation , which is the use of biogenic materials and organisms for environmental cleanup , has also been proposed , including phytoremediation using plants   and microbial degradation using primarily bacteria and fungi .
bioremediation is an attractive strategy , as it can destroy the pollutant rather than transferring it from one environmental compartment to another .
common bioremediation strategies include the addition of nutrients , degradative microorganisms , or both . 

sphingomonas wittichii rw1 is a microorganism of great interest to the bioremediation community for its ability to biotransform a large number of toxic polychlorinated dioxins and to utilize both nonchlorinated dibenzo - p - dioxin and nonchlorinated dibenzofuran as a growth substrate and sole source of carbon and energy .
one of the major challenges in bioaugmentation strategies relying on the addition of nonnative microbes is to ensure their viability and degradative activity toward the target compounds .
monitoring bioremediation is critical to ensure the efficacy of the process and the reduction of contaminant mass to acceptable levels .
traditionally , the most important characteristics investigated for microorganisms used in bioremediation were their ability to transform the substrate , the rate of substrate removal , and the resulting metabolites [ 69 ] . to optimize the bacterial degradation of pollutants , it is important to understand how these organisms function during growth on recalcitrant substrates and which factors influence their degradative abilities .
this includes analyzing not only the degradative pathways [ 1012 ] , but also the peripheral processes and mechanisms that are involved in taxis ( i.e. , directed motion in a chemical gradient ) , uptake , and transport during exposures to specific substrates .
analysis of dna and rna   can shed light on an organism 's metabolic potential ; however , these measurements poorly correlate to actual protein expression profiles .
therefore , global analyses of protein expression profiles may be a more informative tool for understanding the physiological mechanisms of biodegradation .
in addition to identifying important degradative enzymes in a variety of important microbes [ 1517 ] , proteomic studies have opened the door to a better understanding of system - wide changes in response to differing substrates .
the imperative to perform proteomic analyses is particularly true for s. wittichii rw1 because the enzymes in the dioxin degradation pathway are encoded on different loci throughout the genome , certain elements in the pathway are located on a plasmid , and there may be alternative pathways at work .
the present study builds on previous work   and utilized difference gel eletrophoresis ( dige ) coupled with mass spectrometry ( ms ) to exploit recently gathered rw1 genome data . when used together , these tools yield information on the response of cells of s. wittichii rw1 to dioxin exposure and the bacterium 's degradative activity toward this recalcitrant compound .
the aim of this study was to investigate system - wide changes in protein expression during growth on dibenzofuran , a nontoxic surrogate for dibenzo - p - dioxin , as compared to nonselective growth media .
acetate was selected as the nonselective alternate substrate , as growth on this compound was observed to influence expression of select proteins , including the dioxin dioxygenase .
thus , any changes measured in response to cells grown on dibenzofuran should represent cell - wide effects related specifically to the growth substrate and not to unanticipated extraneous effects .
cultures of s. wittichii strain rw1 ( 100  ml to 1.0  l ) were grown to mid log phase at 30c in m9 phosphate - buffered minimal medium ( ph 7.05 ) supplemented with either dibenzofuran crystals or 50  mm acetate as growth substrates .
cells were grown overnight on dibenzofuran and acetate as sole carbon sources to an optical density of 0.40.6 absorbance units ( 560  nm ) . following biomass processing ,
protein levels in the samples were on the order of 75200  g / ml .
protein concentrations were normalized prior to analysis by concentration and resuspension in dige sample preparation buffer .
culture purity was confirmed by the streak plate method using luria bertani medium supplemented with 1.5% agar .
harvested biomass was washed , spun again , and the resultant pellet suspended in a small volume of 100  mm ammonium bicarbonate ( ph ~7.0 ) . this microbial suspension
was then sonicated under cooling with ice , using a microtip sonicator ( fisher scientific , pittsburgh , pa , usa ) in a sequence of three 10-second bursts delivered in thirty - second intervals .
the sonicated cells were then immediately centrifuged at 10,000  xg for 10 minutes at 4c .
briefly , 8 parts of 10% tca in acetone ( 20c ) were added per volume of supernatant and , following mixing on a vortex , the resultant dilution was incubated at 20c overnight .
following centrifugation ( 10,000  xg , 10 minutes , 4c ) , harvested biomass was washed in cold acetone for 10 minutes at 20c .
following a subsequent centrifugation , the pellet was resuspended in sample preparation buffer ( 7  m urea , 2  m thiourea , 2% chaps , 0.2% dtt , 0.02% bromophenol blue ) and stored at 20c until analyzed .
protein concentrations were measured using the bicinchoninic acid assay ( pierce , rockford , il , usa ) following dilution to reduce the concentration of interfering agents .
twenty - five g of crude cell lysates of rw1 biological replicates grown on dibenzofuran ( n = 3 ) and acetate ( n = 3 ) were labeled using cy dyes ( ge healthcare ) as described elsewhere .
briefly , samples were adjusted to 1  g/l using sample preparation buffer and the ph checked .
subsequently , 0.25  l of 1  pmol/l  cy dyes were added to samples for 30 minutes in the dark on ice . to stop the labeling reaction ,
0.5  l of 10  mm lysine was added to the samples , which were mixed and incubated on ice for 10 minutes prior to storage at 20c until analysis .  
unless stated otherwise , all procedures were carried out in the dark or minimal light to protect the integrity of the fluorescent dyes .
samples were randomized to reduce the effect of dye bias and in - gel variations .
a global pool consisting of fractions of each sample was labeled as outlined above using cy 2 and added to each sample as an internal standard .
one cy-3- and one cy-5-labeled sample were added to each gel , as defined by the experimental randomizing procedures . to each sample
, an additional 175  g of unlabeled sample were added ; the volume was increased to a total of 450  l using sample preparation buffer .
the reducing agent dtt ( dithiothreitol ; 1.3  mg per tube ) and ipg ( immobilized ph gradient ) buffer ( 0.5% ) were added , and the samples incubated and mixed in the dark at room temperature for approximately 1  h. samples were then applied to 24  cm ph 47 ipg strips ( ge healthcare ) and focused for 60  kvh using the following protocol : 12  h rehydration at 30  v ; 1  h step and hold at 500  v ; 7 hour gradient to 1,000  v ; step and hold at 1,000  v for 1 hour ; gradient to 8,000  v for 3  h ; step and hold at 8,000  v until 60  kvh .
strips were then reduced and equilibrated using 10  mg / ml dtt ( 15  min ) followed by 25  mg / ml iodoacetamide ( 15  min ) .
the ipg strips were overlaid on 24  26  cm 816% gradient tris - hcl ph 8.8 precast gels ( nextgen sciences , ann arbor ,
approximately 1  ml of agarose was applied to fix the gels and a cy-2-labeled molecular weight marker was applied adjacent to the acidic side of the strip .
the gels were then run 1 - 2  w per gel overnight ( ~2224 hours ) at 20c until the marker dye ran off the gel .
gels were then imaged with a typhoon 9400 scanner and processed using decyder v6.5 ( ge healthcare ) bva batch processor tool .
images were uploaded to decyder ( version 6.5 ) and spurious image objects ( water spots , streaks , and mismatches ) were identified and excluded from further analysis .
following allocation of changed proteins , individual spots were manually inspected and excluded from analysis if they fell outside acceptable parameters for peak height , area , and slope .  
pick lists were generated by selecting proteins whose expression was statistically changed in the two growth conditions ( p < 0.05 ) following digital image analysis using decyder , and the corresponding spots were automatically picked using an ettan spot picker ( ge healthcare ) with ettan spot pick software v.1.1
. spots were delivered in 100  mm ammonium bicarbonate to a 96-well plate and digested using established protocols .
briefly , gel pieces were sequentially dried using three exchanges of 100% acetonitrile followed by a 10-minute speedvac ( savant ) drying .
gel pieces were rehydrated in 40  l of 10  ng/l trypsin in 100  mm nh4hco3 on ice for 45 minutes .
the supernatant was removed and replaced with 100  mm nh4hco3 and digested at 37c overnight .
peptides were then extracted using 50% acetonitrile/0.1% tfa ( trifluoroacetic acid ) for 30 minutes at 37c .
the peptides were microextracted using omix c18 tips ( varian , palo alto , ca , usa ) following the manufacturer 's instructions and then deposited on a stainless - steel target plate in a matrix consisting of 10  mg / ml 2,5-dihydroxybenzoic acid
.   mass spectra were acquired using a voyager de - str matrix - assisted laser desorption / ionization time - of - flight ms ( applied biosystems , foster city , ca , usa ) in positive reflector mode with delayed extraction using the following parameters : laser energy , 1400 arbitrary units ; mass range , 5005,000  da ; 120  nsec delay , 100 laser shots per spectrum .
external calibration was conducted using a standard peptide mixture ( bradykinin , insulin b chain , p14r , and acth ) , and internal calibration was carried out using trypsin autolysis peaks .
data were processed in data explorer v1.1 ( applied biosystems , foster city , ca , usa ) using noise reduction ( 2 standard deviations ) and peak deisotoping .
peak masses were searched using the mascot online search engine ( http://www.matrixscience.com/ ) with the following settings : database , ncbi entire database ( 5.6 million entries ) ; no missed cleavages ; monoisotopic peaks ; no fixed modifications ; variable modification of methionine oxidation ; error tolerance of 150  ppm .
database and literature searches were used to further characterize and classify the proteins identified by maldi - tof ms . where ambiguous names were encountered , blastp searches   were used to identify homologous proteins from orthologous species .
image analysis of 24  cm 2d - dige gels loaded with protein of s. wittichii rw1 cells grown on either dibenzofuran or acetate revealed 937 unique spots .
differential in - gel analysis of individual gels determined gel - specific parameters for selection criteria and allowed visual examination of changes between growth on the two substrates ( figure 1 ) . of the 937 identified spots ,
595 were matched between all the gels used to statistically compare the quantitative abundance of proteins .
statistical analysis compared triplicate biological observations for each condition , normalized to the internal pooled standard ( figure 2 ) .  
crude cell lysates from s. wittichii rw1 grown on dibenzofuran showed that , of all proteins observed , 22 proteins were modulated in response to changes in culture conditions .
these candidate biomarkers of metabolic activity and phenotype were observed in at least 6 of 9 dige images and were modulated as follows : 16 showed an apparent increase and 6 an apparent decrease ( figure 2 ) .
these proteins , along with 22 proteins selected due to their high abundance in both growth conditions , were further analyzed and identified using mass spectrometry ( figure 3 ) .
a mascot search of the entire ncbi database using mass spectral data generated by peptide mass fingerprinting identified 23 of the 44 proteins ( 52% ) .
all protein identifications corresponded to the genome of s. wittichii strain rw1 . among the 16 proteins upregulated during growth on dibenzofuran ,
7 were successfully identified ( table 1 ) . among the 6 proteins downregulated during growth on dibenzofuran ,
an additional 13 proteins were identified whose expression level remained unchanged regardless of culture conditions ( table 3 ) .  
of the 7 identified proteins increased during growth on dibenzofuran , 2 were directly related to the dibenzofuran degradation pathway ( figure 4 ) ; the others were involved in downstream metabolic processes ( catechol 1,2-dioxygenase , adenosylhomocysteinase ) , cell growth ( elongation factor ts ) , and cell protection ( cold shock dna - binding domain protein , alkyl hydroperoxide reductase ) .
the three identified proteins whose expression was decreased ( fumarylacetoacetate hydrolase , tonb - dependent receptor , and acyl - coa dehydrogenase ) are involved in biosynthesis , catabolism , and transport .
the unchanged proteins represented basic cell functions , although biosynthesis , catabolism , and transport proteins dominated the identities .
the alpha subunit of the dioxin dioxygenase , the first step in the dioxin degradation pathway , was also identified as unchanged .
dige and 2d electrophoresis are an accepted strategy for mining microbial proteomes for biomarkers related to a number of processes [ 2830 ] .
the complete protein content of s. wittichii rw1 consists of approximately 5,000 putative proteins from the bacterial chromosome and two megaplasmids . using simple extraction and purification techniques followed by dige
, over 500 protein spots were resolved on a large ( 24  cm ) 2-dimensional gel and matched between the three biological replicates , representing approximately 10% of the entire protein content .
of these 500 proteins , 22 were found to be regulated in response to growth condition changes .  
of the genes they identified as being changed in their experiments , only two overlapped with the proteins identified in our study : swit_3144 ( tonb - dependent receptor , downregulated ) and swit_3376 ( chaperonin groel , upregulated ) .
both of these gene levels responded to short - term perturbation with peg8000 but not sodium chloride in the transcriptomic study and were unchanged in our study . in our study
, we also identified a number of proteins that are related to dioxin / dibenzofuran degradation ( e.g. , dioxin dioxygenase , meta - cleavage product hydrolase , and 2,3-dihydroxybiphenyl 1,2-dioxygenase ) .
other proteins were identified that showed increases in abundance but whose role was not directly related to the dibenzofuran degradation pathway . the increase in the presence of antioxidants such as alkyl
hydroperoxide reductase suggests that there is an increasing stress upon the bacterial cell during growth on dibenzofuran , perhaps due to a change in catabolism resulting in an increase in endogenous peroxide generation .
increases in a cold - shock dna - binding protein may be further evidence of an increased cellular stress .
however , proteins of the cold shock family and related ones are also known to have transport and protein processing roles .   among the proteins in the dioxin degradation pathway , the most prominent on the gel was the meta - cleavage product hydrolase .
this identification was produced from two adjacent spots , likely representing an artifact due to the protein 's extremely high expression or a reflection of the presence of multiple isoforms or a modified enzyme .
the one identified in the present study is the product of the swit_3055 locus , a gene also known as dxnb2 .
the glyoxalase / bleomycin resistance protein / dioxygenase identified in this study is also annotated as a 2,3-dihydroxybiphenyl-1,2-dioxygenase located on the chromosome at the swit_3046 locus .
again , there are multiple isoforms of this enzyme found both on the chromosome and the megaplasmids .
the kegg dioxin degradation pathway identifies swit_4182 as the dihydroxybiphenyl dioxygenase involved in biphenyl metabolism and swit_4902 as the trihydroxybiphenyl dioxygenase in both dioxin and dibenzofuran metabolism .
the increased expression in response to dibenzofuran suggests that the swit_3046 dioxygenase plays a more important role in dibenzofuran degradation in vivo .
the high degree of redundancy in the dioxin and dibenzofuran degradation pathways , that is , the presence of multiple ring - hydroxylating alpha and beta subunits , glyoxalase / bleomycin resistance protein / dioxygenases , and meta - cleavage product hydrolases , remains to be explained .
one possibility is that the various isoforms have different affinities for chlorinated metabolites that would result from chlorinated dioxins and furans .
further experiments are needed to fully distinguish the roles of these enzymes in s. wittichii rw1 degradation pathways .
although not directly implicated in dioxin degradation , the fumarylacetoacetate hydrolase is also of interest because the gene encoding this protein ( swit_5089 ) flanks the ferredoxin fdx1 ( swit_5088 ) that has been identified as part of the electron supply chain supporting dioxin dioxygenase activity . of the multiple isoforms of this enzyme ,
the electron supply chain also contains two isofunctional reductases . neither the ferredoxin itself nor the reductases could be identified . in previous studies ,
the reductase was present as a much smaller fraction of the soluble cell proteome than either the ferredoxin or the dioxin dioxygenase , so gel - based methods may not be sensitive enough to detect this protein .
if transcription of the ferredoxin is linked to the other genes at that locus , as is predicted , the decreased expression in response to dibenzofuran suggests that another ferredoxin is more important in the dioxin degradation pathway in vivo .
the detection of the dioxin dioxygenase alpha subunit and related enzymes in both acetate- and dibenzofuran - grown cells is potentially of importance for the field of bioremediation because it suggests an avenue of biostimulation .
when utilizing s. wittichii rw1 as a bioremediation agent , it may be possible to induce the expression of the dioxin degradation pathway using acetate .
induction of the dioxin degradation pathway has not been observed when s. wittichii rw1 is grown on glucose or rich medium , and growth in a complex environmental medium ( landfill leachate ) was correlated with a decrease in copy number of the gene encoding the dioxin dioxygenase alpha subunit .
previous studies using s. wittichii rw1 to transform chlorinated dioxins in soil or fly ash have observed a progressive decrease in degradative activity   or viable cells [ 38 , 39 ] , respectively .
the addition of acetate may generate sufficient relevant protein biomass to catalyze the successful degradation of dioxin and dioxin - like compounds in environments bioaugmented with s. wittichii rw1 .
proteomic technology has emerged in microbiology more rapidly than in other fields for several reasons .
the relatively small genomes code for relatively limited proteomes featuring no or very limited posttranslational modifications compared to higher organisms .
furthermore , microbes are easily controlled and manipulated in the laboratory , both during growth and gene expression .
these factors will continue to drive biomarker discovery in microbial proteomes , including phenotypic biomarkers informing on the degradative activity of biomass produced for bioaugmentation of contaminated environments .
furthermore , the field of bioremediation can benefit from methods suitable for monitoring microbial biomarkers in field samples to inform on progress in site bioremediation .
this study highlights a number of proteins that were changed in response to dibenzofuran exposure , opens the door to a greater understanding of how s. wittichii rw1 performs and regulates the degradation of dioxins , and suggests ways to enhance the biodegradation of dioxins .
 
 sphingomonas wittichii rw1 is a bacterium of interest due to its ability to degrade polychlorinated dioxins , which represent priority pollutants in the usa and worldwide . although its genome has been fully sequenced , many questions exist regarding changes in protein expression of s. wittichii rw1 in response to dioxin metabolism . 
 we used difference gel electrophoresis ( dige ) and matrix - assisted laser desorption / ionization mass spectrometry ( maldi - ms ) to identify proteomic changes induced by growth on dibenzofuran , a surrogate for dioxin , as compared to acetate . 
 approximately 10% of the entire putative proteome of rw1 could be observed . 
 several components of the dioxin and dibenzofuran degradation pathway were shown to be upregulated , thereby highlighting the utility of using proteomic analyses for studying bioremediation agents . 
 this is the first global protein analysis of a microorganism capable of utilizing the carbon backbone of both polychlorinated dioxins and dibenzofurans as the sole source for carbon and energy . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion