Proteomic Strategy for the Analysis of the Polychlorobiphenyl-Degrading Cyanobacterium Anabaena PD-1 Exposed to Aroclor 1254

The cyanobacterium Anabaena PD-1, which was originally isolated from polychlorobiphenyl (PCB)-contaminated paddy soils, has capabilities for dechlorinatin and for degrading the commercial PCB mixture Aroclor 1254. In this study, 25 upregulated proteins were identified using 2D electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). These proteins were involved in (i) PCB degradation (i.e., 3-chlorobenzoate-3,4-dioxygenase); (ii) transport processes [e.g., ATP-binding cassette (ABC) transporter substrate-binding protein, amino acid ABC transporter substrate-binding protein, peptide ABC transporter substrate-binding protein, putrescine-binding protein, periplasmic solute-binding protein, branched-chain amino acid uptake periplasmic solute-binding protein, periplasmic phosphate-binding protein, phosphonate ABC transporter substrate-binding protein, and xylose ABC transporter substrate-binding protein]; (iii) energetic metabolism (e.g., methanol/ethanol family pyrroloquinoline quinone (PQQ)-dependent dehydrogenase, malate-CoA ligase subunit beta, enolase, ATP synthase β subunit, FOF1 ATP synthase subunit beta, ATP synthase α subunit, and IMP cyclohydrolase); (iv) electron transport (cytochrome b6f complex Fe-S protein); (v) general stress response (e.g., molecular chaperone DnaK, elongation factor G, and translation elongation factor thermostable); (vi) carbon metabolism (methanol dehydrogenase and malate-CoA ligase subunit beta); and (vii) nitrogen reductase (nitrous oxide reductase). The results of real-time polymerase chain reaction showed that the genes encoding for dioxygenase, ABC transporters, transmembrane proteins, electron transporter, and energetic metabolism proteins were significantly upregulated during PCB degradation. These genes upregulated by 1.26- to 8.98-fold. These findings reveal the resistance and adaptation of cyanobacterium to the presence of PCBs, shedding light on the complexity of PCB catabolism by Anabaena PD-1.


Introduction
Persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs) and organochlorine pesticides, are ubiquitous chloroorganic chemicals in the environment. PCBs were first manufactured in the United States in 1929 [1]. They are a complex class of hydrophobic, lipophilic chemicals that often slowly decompose and metabolize in natural systems [2]. Although PCB production has been banned in 1979, they still pose environmental and human risks in areas of hotspot contamination because of their stable physicochemical properties, hydrophobic properties, and high toxicity [3,4]. Therefore, cleaning residual PCB-contaminated environments has elicited significant research attention in the last few decades.
Microalgae and cyanobacteria are common species in the natural environment. They can act as distinctive biological agents for organic pollutant degradation [12] and can be used to degrade organic pollutants. The use of microalgae and cyanobacteria has become a new method for POP degradation in recent years. Kotzabasis et al. [13,14] have reported that Scenedesmus obliquus can biodegrade dichlorophenols. Chlorella fusca var. vacuolata can remove 23% of 2,4-dichlorophenol after 4 d [15]. The microalga Cyclotella caspia can degrade the aromatic pollutant nonylphenol [16]. The Anabaena flos-aquae strain 4054 can decompose endocrine-disrupting pollutants, such as phthalate esters [17]. Anabaena azotica, another common cyanobacterium, can effectively degrade the organochlorine pesticide c-hexachlorocyclohexane (lindane) [18]. Therefore, cyanobacterial species with degradation functions may be a potential choice for PCB degradation.
Our previous study produced encouraging results in PCB biodegradation by cyanobacteria. The cyanobacterium Anabaena PD-1 was originally isolated from PCB-contaminated paddy soil and exhibited strong ability to degrade PCB congeners (data not shown). However, proteomic analyses of cyanobacterial responses to stressors have mainly focused on salt [29], acid [30], and arsenic [31]. Limited information is available on the cyanobacterial catabolism of PCBs and the responses of cyanobacteria to PCBs [12]. Therefore, the key enzymes involved in PCB degradation need to be identified and the degradation mechanism should be explored to gain important information on the construction of genetically engineered cyanobacteria. Such genetically engineered cyanobacteria may achieve the same or higher PCB degradation efficiency compared with laboratory conditions.
In this study, we separated differentially expressed proteins through 2-DE and identified polypeptides through MALDI-TOF tandem mass spectrometry (MS/MS). Protein information was obtained from the NCBInr database through its Mascot search engine. Real-time PCR was utilized to analyze the genes encoding for highly expressed key proteins during PCB degradation in Anabaena PD-1 cells. The present contribution can provide new insights into the biodegradation of PCBs by Anabaena PD-1 and into the construction of genetically engineered PCB-degrading cyanobacterial species.

Materials and Methods
Strain Anabaena PD-1 and culture conditions Anabaena PD-1, a PCB-tolerant strain, was isolated from PCBcontaminated paddy soil in Taizhou, Zhejiang, China (No specific permissions were required for the sampling locations and activities. The field studies did not involve endangered and protected species. The sampling site in the study is located at Latitude 28u329N Longitude 121u279E.). Anabaena PD-1 cells were grown at 25uC, 998 lux, in BG-11 medium [32], under discontinuous illumination (light : dark = 12 h : 12 h). Cyanobacterial cells in their exponential phase were cultivated with and without Aroclor 1254 (2 mg/L) for 30 d for the PCB-degrading experiment.

Preparation of cellular proteins
The cells were cultured for 30 d and ground to powder with liquid nitrogen. Subsequently, 10 mL of cooled acetone containing 10% trichloroacetic acid and 0.07% DTT was added to 1 g of sample powder at 220uC for 1 h. The deposit was collected after centrifugation at 15000 g for 15 min at 4uC. Cooled acetone containing 0.07% DTT was then added to the deposit at 220uC for 1 h. After another centrifugation at 15000 g for 15 min at 4uC, the deposit was collected and dried with a vacuum freeze dryer. The powder was dissolved in a lysis solution [9 M urea, 4% CHAPS, 1% DTT, 1% IPG buffer (GE Healthcare)] at 50 mL: 1 mg, dissolved at 30uC for 1 h, and then centrifuged again at 15000 g for 15 min at room temperature. The concentrations of the protein extracts were determined with the Bradford method [33]. The extracts were then stored at 280uC for isoelectric focusing electrophoresis (I FE).

2-DE
IFE. Samples containing 200 mg of proteins were mixed with a fresh rehydration buffer [9 M urea, 4% CHAPS, 1% DTT, 1% IPG buffer (GE Healthcare), trace amount of bromophenol blue] to form a 450 mL mixture. DryStrip (GE Healthcare, 24 cm, pH 3 to 10, NL) was obtained from a 220uC freezer, placed at room temperature for 10 min, added to the protein sample in the strip holder, and then subjected to IEF according to the following protocol: rehydration at 50 V (12 h), 500 V (1 h), 1000 V (1 h), 10000 V (1 h), and 10000 V (10 h). All steps were controlled at 50 mA/gel at 20uC.

Equilibration and SDS-PAGE
After IEF, the strip removed from the strip holder was incubated in equilibration buffer 1 [6 M urea, 30% glycerol, 2% SDS, 50 mM Tris-HCl (pH 8.8), 1% DTT, and trace amount of bromophenol blue] for 15 min and then in equilibration buffer 2 [6 M urea, 30% glycerol, 2% SDS, 50 mM Tris-HCl (pH 8.8), 2.5% iodoacetamide, and trace amount of bromophenol blue] for 15 min. After the strip was rinsed with SDS-PAGE buffer for 10 s, a sealing solution was added to the surface of the SDS-PAGE gel. The gel was then moved to the electrophoresis apparatus for electrophoresis at the following parameters: 100 V, 15uC, 45 min, followed by 200 V for 6 h to 8 h (Ettan DALTsix system). The gels were then stained with silver nitrate according to the method described by Shevchenk et al. [34].

Gel visualization, scanning, and analysis
The gels were visualized by silver staining for analysis. The stained gels were scanned by an image scanner (GE Healthcare, USA) at a resolution of 300 dots per inch. All gel images were processed by spot detection, volumetric quantification, and matching with PDQuest 8.0 software. The differences in protein content between the treatment and control groups were calculated as fold ratios. A fold change $2.0 or #0.5 was utilized to differentially select protein spots.

In-gel digestion and MS analysis
The proteins were digested by 50% ceric ammonium nitrate for 5 min, followed by 100% ACN for 5 min, and then rehydrated in 2 mL to 4 mL trypsin (Promega, Madison, USA) solution (20 mg/ mL in 25 mM NH 4 HCO 3 ) for 30 min. A 20 mL cover solution (25 mM NH 4 HCO 3 ) was then added for 16 h of digestion at 37uC. Afterward, the supernatants were transferred into another tube, and the gels were extracted once with a 50 mL extraction buffer (67% ACN and 5% TFA). The peptide extracts and supernatant of the gel spot were combined and completely dried. The samples were analyzed with an ABI 4800 MALDI-TOF/ TOF Plus mass spectrometer (Applied Biosystems, Foster City, USA). Data were obtained with a positive MS reflector. CalMix5 standard was utilized to calibrate the instrument (ABI4800 calibration mixture). MS and MS/MS data were integrated and processed with GPS Explorer V3.6 (Applied Biosystems, USA) with default parameters. According to combined MS and MS/MS spectra, the proteins were successfully identified at 95% or higher confidence interval of their scores in the MASCOT V2.1 search engine (Matrix Science, London, UK). The following search parameters were used: NCBInr database; trypsin as the digestion enzyme, one missed cleavage site, fixed modifications of carbamidomethyl (C), partial modifications of acetyl (protein Nterm), and oxidization (M); 200 ppm for precursor ion tolerance and 0.5 Da for fragment ion tolerance.

RNA preparation and real time-PCR
Total RNA was extracted from 50 mg to 80 mg of cyanobacterial cell pellets (OD 680 = 0.38) using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). RNA was then purified by removal of genomic DNA contaminants using an RNase-free DNase I kit (Invitrogen) and verified by determination of 260/280 nm ratios and 1% agarose-formaldehyde gel electrophoresis with ethidium bromide staining. Total RNA was subjected to cDNA synthesis using a NuGEN OvationW Prokaryotic RNA-Seq System according to the manufacturer's instructions (Haoji Biotechonlogy Hangzhou, China). RT-PCR was carried out with a multiplex real-time PCR detector (BioRad, USA). The reaction mixture included a Power Master Mix (Invitrogen), 0.5 mM of the primers, MilliQ water, and 1 mL of cDNA. The thermal cycling program was as follows: predegeneration at 95uC for 1 min, followed by 40 cycles of denaturation at 95uC for 10 s, and then annealing at 62uC for 25 s. Primer sequences for 3-chlorobenzoate-3,4dioxygenase, cytochrome b 6 f complex Fe-S protein, transporter proteins, and energetic metabolism proteins are shown in Table 1.

Statistical analysis
The statistical differences of the experimental data were determined using one-way ANOVA followed by two-sided Dunnett's t-test. Statistical tests were conducted using SPSS11.0, and the statistical significance values were defined as *P,0.05 and **P,0.01. All data were expressed as mean6standard deviation (S.D.).

Results
Protein expression patterns of Anabaena PD-1 exposed to Aroclor 1254 Total proteins extracted from the PCB-treated and reference cyanobacterial samples were separated through 2-DE. Most proteins were located between pH 4.0 and 6.8 and then weighed 17 kDa to 96 kDa ( Figure 1 and Figure 2). Exposure to Aroclor 1254 diversified the overall protein expression patterns of Anabaena PD-1. A total of 25 protein spots were up-regulated (multiple changes were twice greater; Student's t-test, P,0.05). Twenty-six differentially expressed proteins were identified through MALDI-TOF MS/MS. Detailed information on these proteins is summarized in Table 2. The proteins involved in PCB-degradation, transport processes, energy metabolism, and electron transport are shown in Figure 3.
Real time-PCR analysis of upregulated protein-encoding genes in Anabaena PD-1 cells The expression levels of eight genes in the PCB-degradation groups were significantly upregulated compared with those in the control groups (P,0.01). The results are presented in Figure 4. Genes encoding for dioxygenase were upregulated by 1.26-fold. The cytochrome b 6 f complex Fe-S protein-encoding gene upregulated by 2.64 fold. The ABC transporter substrate-binding protein gene expression level in the PCB-treated groups was 9.98fold of that in the control groups. Transmembrane proteinencoding genes (i.e., heterocyst to vegetative cell connection protein and porin genes) were upregulated by 2.66-and 3.19-fold, respectively. Enolase gene expression level was upregulated by 2.88-fold compared with the control groups. The upregulation levels of the malate-CoA ligase subunit beta gene and methanol dehydrogenase gene were 3.40 and 5.22, respectively.

Discussion
Anabaena PD-1 isolated from PCB-contaminated paddy soil in South Zhejiang, China, efficiently degrades PCBs. Rodrigues et al. [36] suggested that Burkholderia xenovorans LB400, one of the most extensively studied PCB-degrading bacteria, can degrade 57% of    Aroclor 1242 in 30 d. Singer et al. [35] combined Arthrobacter sp. strain B1B with Ralstonia eutrophua H850 to degrade PCB mixtures and achieved a maximum degradation rate of 59% in over 18 weeks. These bacterial species are typical PCB degraders. However, the application of these species to the bioremediation of PCB-contaminated paddy soils is limited because of their specific living conditions. By contrast, Anabaena PD-1, an associated cyanobacterial species in paddy soils, adapts to the condition in paddy soils very well. This condition is one of the essential requirements for this functional species to degrade PCBs in contaminated paddy soils. Thus, Anabaena PD-1 may be an excellent choice for the remediation of PCB-contaminated paddy soils. This study is the first to report on the proteome files for Anabeana PD-1 in the PCB degradation and control groups and the RT-PCR gene data of upregulated proteins during PCB degradation. We synthesized the information from the protein and gene levels of the PCB-degrading cyanobacterial species Anabaena PD-1 and proposed a putative scheme to demonstrate the possible biodegradation mechanism of PCBs by Anabaena PD-1 ( Figure 5). We assumed that 3-chlorobenzate-3,4-dioxygenase, transporter proteins, electron transport proteins, transmembrane proteins, and energetic metabolism proteins and genes encoding for the said proteins have important functions during PCB dechlorination by Anabaena PD-1. As a complex bioreaction, PCB degradation is involved with the transport system, energy system, and photosynthetic system in Anabaena PD-1 cells.
Organic pollutant stressors can generally change the cell membrane structure and function [36] and exert general stress on cells. The different (chlorinated) aromatic compounds induced the overexpression of different proteins (Table 3). Stressors with chlorinated structures can enhance the expression of stress protein DnaK, ABC transporter, and enolase, whereas pollutants without chlorinated structures can increase the expression of branchedchain amino acid uptake ABC transporter periplasmic solutebinding proteins and elongation factor G. This difference in protein expression may be attributed to the various structures of the organic stressors. The upregulated proteins with different functions can provide new insights into the adaptation of Anabaena PD-1 to the presence of PCBs.
In this study, 3-chlorobenzoate-3,4-dioxygenase, a new enzyme that may be involved in PCB degradation by Anabaena PD-1, belongs to the family of Rieske protein family (http://pfam.janelia. org/family/PF00355.). Thus, this dioxygenase is closely related to cytochrome b 6 f complex Fe-S protein, another typical Rieske protein [44]. The gene encoding for 3-chlorobenzoate-3,4dioxygenase is named cbaA [45]. This enzyme has an important function in the degradation of pollutants belonging to the toluene/ biphenyl family [46]. Thus, we believe that 3-chlorobenzoate-3,4dioxygenase may be closely related to the direct biodegradation of Aroclor 1254 by Anabaena PD-1. Nevertheless, the mechanism by which 3-chlorobenzoate-3,4-dioxygenase participates in PCB dechlorination in Anabaena PD-1 cells remains intriguing.
The PCB-treated and reference gels indicated that protein spots (4015, 4310) are significantly upregulated. These spots are petC gene products or PetC proteins that were first discovered and isolated by Rieske et al. [47]. Thus, the proteins are also called Rieske proteins. PetC protein is a subunit of cytochrome bc1 and cytochrome b 6 f complexes. Two to four genes of the petC gene family are found in the cyanobacterial cells of Nostoc sp. PCC 7120 [48]. PetC protein is an essential protein for the functioning of the cytochrome b 6 f complex. Cytochrome b 6 f complex has an important function in the aerobic photosynthetic electron transport chain reaction center [49]. The reaction center is involved in energy metabolism and electron transfer. Organic pollutants, such as PCBs, usually act as electron acceptors. Thus, as a key enzymemediating electron transporter, PetC protein upregulation may influence the biodegradation of PCBs in cyanobacterial cells. The changes in the expression levels of cytochrome b 6 f complex Fe-S protein confirmed this inference (Figure 4b). Comparing Figure 1 and Figure 2, the expression of proteins 0409, 1212, 1712, 0316, 1213, 1517, 2508, 0322, and 0211 were significantly enhanced. These proteins belong to the ABC transporter family and are ABC transporter substrate-binding proteins (0409, 1212, and 1712), ABC transporter periplasmic putrescine-binding protein (0316), phosphonate ABC transporter substrate-binding protein (1213, 0322), urea short-chain amide or branched-chain amino acid uptake periplasmic solute-binding protein (1517), and xylose ABC transporter substrate-binding protein (0211). The ABC transporter family comprises proteins that can transport ions, saccharides, lipids, and heavy metals across membranes [42][43]50]. The upregulation of these transport proteins indicates that they are likely to participate in PCB uptake or metabolite efflux. The upregulation of ABC transport proteins in Pseudomonas putida P8 suggests that the increased transport of amino acids is a cellular response to external stress [27]. The degradation of PCBs by the nitrogen-fixing species Anabaena PD-1 consumes energy. Thus, cyanobacterial cells bind to synthesize large amounts of ATP, thereby increasing the substrates required for ATP synthesis. The upregulated expression of transport proteins in algal cells may be due to the increased need for transporting ATP synthesis substrates. Several other studies have detected the efflux of organic solvents induced by transporters in Pseudomonas putida in response to aromatic and aliphatic solvents and alcohols [51].
The upregulation of enzymes involved in energetic metabolism have also been observed and found to be consistent with the extra-energetic requirements of cells that trigger several energetically expensive short-term adaptation mechanisms to survive and adapt to the toxicity of PCBs. Enolase (2508) and ATP synthase b subunit (8812) are enzymes involved in energy synthesis and metabolism. ATP synthase b subunit is involved in ATP synthesis under the conditions of a transmembrane proton gradient. The upregulation of this enzyme benefits the cells exposed to toxic organic compounds that can cause membrane lesions [52].
Enolase participates in glycolysis, which converts 2-phosphoglycerate to phosphoenolpyruvate [26]. Enolase consumes energy for cells to actively transport organic compounds to the intracellular space or transport such compounds from the intracellular space to the extracellular space [53]. Pérez-Pantoja [54] reported that the enolase superfamily does not participate in aromatic pollutant degradation by Mycobacterium smegmatis. Nevertheless, we still believe that enolase may be indirectly involved in PCB degrada- tion by Anabaena PD-1 because of the significant upregulation in both protein ( Figure 3) and gene levels ( Figure 4). The upregulation of ATP synthase b subunit and F O F 1 -ATP synthase b subunit (3619) indicates that the ATP consumption of nitrogenfixing cyanobacterial cells exposed to Aroclor 1254 significantly increases because cyanobacterial cells actively transport PCB molecules to the extracellular space to protect themselves from the toxicity of PCBs. Another possibility is that cyanobacterial cells consume energy to transport PCB molecules to the intracellular space for degradation. Stress proteins are consistently upregulated  Table 2 to protect cells from organic pollutant stressors. Therefore, the upregulation of several proteins involved in polypeptide folding and synthesis is expected [55]. Molecular chaperone DnaK (3814) is a member of the HSP 70 (heat shock protein weighing 70 kDa) family. HSPs are highly conversed proteins that can assist in the refolding or hydrolysis of abnormal proteins [56]. The expression of these proteins in cells is usually upregulated under several stress conditions to protect cells [57][58]. Aromatic compounds can disrupt the synthesis of cell membranes, produce unfolded membrane proteins, and activate the stress response of membrane proteins [59]. Thus, the presence of PCBs causes stress to nitrogen-fixing cyanobacteria. The upregulated expression of chaperone proteins indicates that Anabaena PD-1 cells produce several stress responses to PCBs probably to protect themselves from the toxicity of PCBs.
In summary, changes in the proteome of Anabaena PD-1 cells during PCB degradation, gene encoding, and upregulation of proteins were observed for the first time. Twenty-five upregulated proteins were successfully identified. The cbaA gene encoding for 3-chlorobenzoate-3,4-dioxygenase was upregulated. Electron transport protein petC gene product was upregulated as well. The largest group of proteins enhanced by PCBs consists of the substrate-binding proteins of ABC transporters and proteins involved in energy metabolism. Although stress protein DnaK and several other proteins, such as elongation factors, compose a small portion of the upregulated proteins, they may still have important functions in the adaptation of Anabaena PD-1 to PCB and the degradation of PCBs by Anabaena PD-1. Thus, more studies should be conducted to identify the metabolites of PCBs and explore the PCB degradation pathway by Anabaena PD-1.