Proteomics analysis of pyrene biodegradation using strain Mycobacterium sp. 16F

ABSTRACT Mycobacterium sp. 16 F can degrade 94% of pyrene (20 ppm) in 4 days. To investigate its pyrene degradation mechanism, proteomic changes were analyzed using two-dimensional differential gel electrophoresis (2DE-DIGE). Comparative analysis of differential proteins revealed 91 differentially expressed protein spots after pyrene exposure. Among these, 65 spots were identified as 57 proteins. Further analysis revealed that 13 spots were involved in the pyrene degradation pathway, and most of these were dioxygenases and dehydrogenases. Further, 16 up-regulated expression protein spots were associated with four pathways that may be related to pyrene degradation. Bioinformatics analysis further revealed that the pentose phosphate and glycolytic pathways led to the production of amino acids and nucleotide precursors in pyrene-induced cells. The metabolites from these processes then entered the shikimate pathway via the β-ketoadipate pathway in conjunction with the pyrene degradation pathway. This study provides a new model for the pyrene degradation pathway in Mycobacteria. Abbreviations: PPP, pentose phosphate pathway; ED-EMP, Entner-Doudoroff pathway and Embden-Meyerhof-Parnas pathway; E4P, erythrose-4-phosphate; PEP, phosphoenol-pyruvate; PAL, phenylalanine ammonia-lyase; SucCoA, succinyl-CoA; AcCoA, acetyl-CoA; TCA, tricarboxylic acid cycle.


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are persistent organic compounds that are widely distributed in natural environments. PAHs are primarily produced during fossil fuel combustion and industrial processing, but are potentially genotoxic, carcinogenic, and are ubiquitous environmental pollutants [1]. Bioremediation is an efficient, economic, and versatile alternative to mitigate PAH contamination compared to physico-chemical treatment. Among bioremediation treatments, microbial biodegradation plays an important role in removing PAHs from polluted soils [2]. PAHs with two or three aromatic rings (e.g. low molecular weight (LMW) PAHs, naphthalene, phenanthrene, and anthracene) are easily degraded by many bacteria that use these substances as their carbon and energy sources [3][4][5]. Higher numbers of fused rings lead to higher hydrophobicity and recalcitrance to microbial degradation. Pyrenes (high molecular weight (HMW) PAHs) are PAHs containing four aromatic rings that are commonly used as a proxy for PAH degradation. Many bacteria with the ability to degrade pyrene have been isolated [6][7][8][9]. Nevertheless, additional investigation of the metabolites and pathways associated with PAH biodegradation in bacteria will significantly expand our understanding of the process.
Proteomics is a powerful technique that can be used to investigate proteins expressed under certain conditions and further explore their roles in PAH catabolism. Consequently, proteomics have been increasingly used to study bacterial PAH degradation [10][11][12], significantly promoting the construction of functional proteome databases. Some of these studies have investigated proteome changes that occur during pyrene degradation by different bacteria, leading to the identification of a few pyrene-induced proteins, including catalase-peroxidase, putative monooxygenase YcdM, and the dioxygenase small subunit NidB [10]. For example, the pathways and enzymes involved in Mycobacterium sp. KMS pyrene degradation have been investigated [13]. A pyrene degradation pathway in M. vanbaalenii PYR-1 has been proposed from genomics and proteomics analyses, only parts of the key enzymes (NidAB2 and PhtAaAb) in the pathway have been identified [11,14].
The PAH-metabolizing bacterium Mycobacterium sp. 16 F was isolated from soils heavily polluted with PAHs near a Coking Plant in Beijing, China, and the strain exhibited 99.8% similarity in 16S rRNA gene sequence to Mycobacterium vanbaalenii PYR-1. Here, pathways directly or indirectly related to pyrene degradation were investigated for Mycobacterium sp. 16 F in order to promote the efficient design of bioremediation procedures. Specifically, 2D-DIGE was used to investigate the mechanism of Mycobacterium sp. 16 F pyrene degradation. These new data provide insights into the potential application of this strain for improving PAH bioremediation.

Microorganisms and growth conditions
Mycobacterium sp. 16 F (GenBank accession number JN966739, deposit number 6367 at the China General Microbiological Culture Collection Center (CGMCC), http://www.cgmcc.net/) was isolated from PAHpolluted soil and can use pyrene as a carbon and energy source. A basal salt medium (BSM) mixed with Luria broth (LB) (4:1 BSM: LB) was used as the culture media for all experiments. Inoculated plates were placed in an incubator at 30°C, and plates with visible colonies and clear zones in the plaque layer were photographed after 9 and 26 days, respectively. Pyrene degradation by Mycobacterium sp. 16 F was determined as follows. About 100 µL of inoculum was added to 25 mL of BSM containing pyrene at 20 ppm to obtain an initial cell concentration of 10 5 CFU/mL. The cultures were incubated at 30°C with shaking at 120 rpm at 30°C until 168 h. Three replicate tubes were then collected every 24 h for residual pyrene analysis with an Agilent 7890A gas chromatograph coupled with a 7000A triple quadrupole mass spectrometer GC/MS (Agilent Technologies Inc, Santa Clara, CA, USA). A standard peak for pyrene was obtained by injecting known concentrations of pure pyrene, followed by GC/MS using the same procedure as for the samples. Pyrene loss by dissipation was also assessed with a control.
For proteomic analysis, eight 1 L flasks were used that each contained 250 mL of BSM plus media and 3 mL of Mycobacterium sp. 16 F inoculum. Cultures were grown in the dark at 30°C with shaking at 120 rpm until the optical density (600 nm) of the culture reached approximately 1.0. Next, 100 µL of pyrene (final concentration of 20 ppm) in DMF was added to each of four flasks that were randomly selected from the eight flasks. The other four flasks were amended with 100 µL of dimethylformamide (DMF) and used as controls. After 24 h of further incubation, cells were collected for protein extraction by centrifugation.

Measurement of pyrene metabolism
The ability of Mycobacterium sp. 16 F to degrade pyrene from the culture medium was monitored spectrophotometrically. Complete solubilization of PAHs was accomplished by mixing 1 volume of culture aliquot with 1 volume of dichloromethane. After vortexing, samples were mixed with dichloromethane and incubated for 30 min. Pyrene extraction was then conducted by mixing 10 mL of dichloromethane with 10 mL of culture solution, followed by vortexing the samples for 30 min. The samples were subsequently centrifuged at 10,000 g for 10 min to remove cell debris and separate aqueous and organic phases. Pyrene content was then determined on a UV-2550 UV-VIS spectrophotometer (Shimadzu Corp., Japan) at 335 nm.

Protein extraction
Protein extraction was performed according to Carpentier et al. [15] with some modifications. Briefly, cell pellets were washed twice with 50 mM Tris-HCl at pH 7.5, and then resuspended in extraction buffer containing 20 mM Tris-HCl (pH 7.5), 250 mM sucrose, 10 mM ethylene glycol tetraacetic acid (EGTA), 1 mM phenylmethanesulfonyl Fluoride (PMSF), 20 mM dithiothreitol (DTT), and 1% Triton X-100. Cells were subsequently ultrasonicated in an ice bath for 20 min at a 3 s intervals every 5 s at a power of 100 W. The homogenates were then centrifuged at 15,000 g for 10 min at 4°C, followed by transfer of the supernatant to a new tube. The proteins in the supernatant were then precipitated by adding 25% volume of cold trichloroacetic acid (TCA), incubating on ice for 30 min, then centrifuging at 15,000 g for 10 min at 4°C. After rinsing three times with ice-cold acetone containing 0.2% DTT, the protein pellets were air-dried for 30 min and resuspended in lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS). The samples were then centrifuged at 15,000 g for 20 min at 4°C, followed by collection of supernatants. Protein concentrations were determined using the Bradford method [16], with BSA used as the standard.

Dye labelling and 2D electrophoresis
Protein samples were labelled using CyDyes DIGE Fluors minimal dyes (GE Healthcare, Bio-Sciences Corp., Piscataway, NJ, USA), according to the manufacturer's instructions. Protein extracts (50 mg) in lysis buffer were mixed with Cy2, Cy3, or Cy5 dyes (400 pmol). Briefly, two samples from the control and pyrene-induced group were dyed with Cy3, while the other four samples in the two groups were dyed with Cy5. An internal standard was also established by pooling mixtures of equal amounts (25 μg) from the eight samples and labelling them with Cy2. The labelled mixtures were subsequently incubated in an ice bath in the dark for 30 min, followed by reaction termination via addition of 1 μL of 10 mM lysine. Cy3-, Cy5-, and Cy2-labelled samples were then mixed together, followed by addition of an equal volume of 2× sample buffer (lysis buffer supplemented with 130 mM DTT and 4% Immobilized pH Gradient (IPG) buffer). Each mixed sample was brought up to 450 μL with 1× sample buffer (lysis buffer supplemented with 65 mM DTT and 2% IPG buffer) and then loaded on 4-7 24 cm linear gradient IPG strips and passively rehydrated overnight at room temperature. Isoelectric focusing (IEF) was performed on an IPGphor3 unit (GE Healthcare, Bio-sciences AB, Uppsala, Sweden) at 20°C with a 50 mA current limit per strip under the following conditions: 1 h at 300 V, 1 h at 600 V, 1 h at 1,000 V gradients, 2 h at 8,000 V gradient, and 40,000 Vhr at an 8,000 V step, followed by a hold. Prior to running second dimension gels, the IPG strips were incubated in 10 mL of equilibration buffer (6 M urea, 30% glycerol, 2% SDS, traces of bromophenol blue, 50 mM Tris, pH 8.8) containing 1% DTT for 15 min. The strips were subsequently incubated in 10 mL of the same buffer containing 2.5% iodoacetamide for 15 min. Second dimension electrophoresis was conducted on 15% polyacrylamide gels using an Ettan DALT six unit (GE Healthcare, Bio-sciences AB, Uppsala, Sweden) by applying 1 W/gel for 30 min and 15 W/gel for the remaining 5 h. Preparative gels for spot picking were prepared as described above, except that each IPG strip was loaded with 360 µg of non-labelled protein and stained with colloidal Coomassie Blue (R-250) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Image acquisition and analysis
Labelled proteins were visualized by scanning with a Typhoon TRIO variable mode imager (GE Healthcare, Bio-sciences AB, Uppsala, Sweden) at the appropriate wavelengths for each dye. All gels were scanned at 100 μm resolution and the photomultiplier tube was set between 400 and 500 V. DIGE images were then analyzed using the Decyder 2D V7.0 software (GE Healthcare). One-way analysis of variance (ANOVA) tests were used to identify significant differences in spot intensity between the average values observed in the control and pyrene-induced samples. Differentially expressed spots were identified by adjusted p < 0.05 and fold change threshold >1.2.

MALDI-TOF-TOF analysis
Differentially expressed spots were excised from gels and destained in 50% acetonitrile (ACN) and 25 mM ammonium bicarbonate. Proteins in the gel pieces were then reduced with 10 mM DTT in 100 mM NH 4 HCO 3 for 1 h at 60°C and incubated with 40 mM iodoacetamide in 100 mM NH 4 HCO 3 for 30 min at room temperature in the dark. The gel fragments were subsequently minced and lyophilized and then rehydrated in 100 mM NH 4 HCO 3 with 10 ng trypsin overnight at 37°C. Peptides were extracted by washing the gel pieces with 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile three times. The supernatants were then collected and vacuum-dried to final volumes of approximately 10 μL. MALDI-TOF-TOF analysis was then performed after desalination. Peptides (0.3 μL) were mixed with an equal volume of a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile containing 0.1% TFA that was spotted onto the target wells of sample plates, dried at room temperature, and then analyzed using a 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA, USA). Protein spots were identified as differentially expressed proteins (DEPs) by automated peptide mass fingerprinting using the Global Proteome Server Explorer software 3.0 (Applied Biosystems, Foster City, CA, USA). Previously described search parameters were used for protein analysis [17].

Bioinformatics analyses
DEPs were identified by BLAST searches against the whole genome sequence of Mycolicibacterium vanbaalenii PYR-1 within the NCBI database (Assembly: ASM1530v1, RefSeq assembly accession: GCF_000015305.1). Functional enrichment analysis of DEPs was performed using the STRING program (https://string-db.org/).

Biodegradation of pyrene by Mycobacterium sp. 16 F
Mycobacterium sp. 16 F grew rapidly on BSM plus agar plates supplemented with pyrene, forming circular, smooth, and yellow colonies with convex surfaces. Clear zones corresponding to pyrene degradation were visible after about 1 week of incubation, followed by expansion to 4 mm diameters after 26 days (Figure 1). The pyrene degradation rate of Mycobacterium sp. 16 F is shown in Figure 2. In BSM plus medium, Mycobacterium sp. 16 F degraded about 94% of pyrene (20 ppm) in 4 days. No pyrene degradation was observed in controls with heat-inactivated cells. The degradation rate was high in the first 2 days but slowed in the last 3 days, after which only 6% of the added pyrene was degraded. The decreased rate may have been caused by the accumulation of pyrene metabolic intermediates.

Proteomic analysis of pyrene-induced proteins
To investigate proteins involved in pyrene degradation by Mycobacterium sp. 16 F, 2DE-DIGE was performed using 24 cm IPG strips to separate proteins in the pH range of 4-7. Approximately 1,600 clearly separated spots were detected with good resolution on each gel (Figure 3), and the spot patterns on the four gels in the same group were highly reproducible. Overall, 91 spots were differentially expressed after pyrene treatment for 1 day, with 48 up-regulated, 29 down-regulated, and 14 newly appearing spots (without corresponding signals on the control) ( Figure 4A and Fig. S1). Of the 91 spots, 57 were identified as DEPs by MALDI-TOF-TOF analysis ( Figure 4B, Table 1). Most of the DEPs were most closely related to those of Mycobacterium PYR-1 based on homology searches.
Most DEPs were significantly or not significantly enriched based on pathway enrichment analysis with the STRING program ( Figure 4C). Among these, 24 DEPs were significantly enriched for five pathways, including microbial metabolism in diverse environments, metabolic pathways, polycyclic aromatic hydrocarbon degradation, degradation of aromatic compounds, and carbon   metabolism ( Figure 4D and Table S1). A total of 10 DEPs were significantly enriched in the polycyclic aromatic hydrocarbon degradation pathway, with eight of these were significantly enriched in the degradation of aromatic compounds pathway (Table S1). The 10 DEPs related to aromatic compound degradation corresponded to the 13 pyrene-induced protein spots (Table S2).

Analysis of key proteins within the pyrene degradation pathway
Several spots were identified as dioxygenases, although the exact functions of these proteins could not be inferred from their annotations alone. The best matches for some of these proteins in the NCBI database were not very specific. Thus, the match results that were not closely related but still exhibited significant (p < 0.05) scores were used as references ( Table 1). The 10 identified proteins exhibited high sequence similarity to enzymes confirmed to operate in the pyrene degradation pathway. In particular, spot 50 was associated with NidB2 proteins that are subunits of aromatic-ring-hydroxylating dioxygenase that are the first proteins involved in the catabolic pathway for oxidizing pyrene to cis-4,5-pyrene-dihydrodiol. Spot 43 was most closely associated with PhdF, that catalyzes steps 3 and 7 in the ortho-and meta-cleavage pathway of the pyrene degradation pathway. Spot 27 was associated with PhdI that catalyzes the reaction between 1-hydroxy-2-naphthoate and trans-2-carboxybenzalpyruvate in the pathway. Spot 19 and 20 corresponded to PhtAa, which adds two oxygen atoms to phthalate to generate phthalate 3,4-dihydrodiol. Spots 23 and 32 were related to a Rieske (2Fe-2S) domaincontaining protein that is the alpha subunit of the putative ring-hydroxylating dioxygenase in Mycobacterium sp. CH-1. Spot 34 corresponded to PhdG that catalyzes the cleavage of C-C, C-O, C-N, and other bonds by mechanisms other than hydrolysis or oxidation, or by adding a group to a double bond. Spot 37 was similar to PhdJ that catalyzes the transformation of trans-2'-carboxbenzalpyruvate to 2-formylbenzoate and pyruvate. Two proteins (spots 44 and 46) were newly detected in pyrene-induced cells. Spot 46 exhibited 98% homology to PhtAb and 96% homology to HcaA (3-phenylpropionate/cinnamic acid dioxygenase subunit beta) in M. vanbaalenii [18]. Finally, spot 44 exhibited 99% homology to PhtB and 98% homology to the HcaB (3-(cis-5,6-dihydroxycyclohexa-1,3-dien-1-yl) propanoate dehydrogenase) of M. vanbaalenii.
Twelve genes were previously identified that are related to pyrene degradation, including PhtAb and PhtB that were speculated to be involved in phenanthrene degradation based on sequence comparisons [18]. However, most genes were annotated in previous studies without any evidence of protein expression or function, and thus, it is possible that some genes suspected to be involved in these pathways are not active in these pathways. HcaA oxidizes cinnamic acid to cis-  3-(3-carboxyethenyl)-3,5-cyclohexadiene-1,2-diol in E. coli and is further metabolized to 2,3-dihydroxycinnamic acid by HcaB [19]. However, the HcaA and HcaB proteins have not previously been reported to be involved in pyrene degradation. Two of the detected aldehyde dehydrogenases (spots 17 and 18) dehydrogenize 1-hydroxy-2-naphthaldehyde to generate 1-hydroxy-2-naphthoate. These enzymes are involved in steps 1, 3,7,8,9,10,11,13, and 14 of the proposed pyrene degradation pathways in M. vanbaalenii PYR-1 [11] (Table S2), suggesting that this pathway is also expressed in Mycobacterium sp. 16 F ( Figure 5). Overall, the proteins identified as pyrene degrading enzymes were mostly dioxygenases or dehydrogenases.

Analysis of proteins involved in pyrene degradation related pathways
Many proteins were identified in this study as being upregulated in pyrene-induced cells, including glycolytic pathway-related proteins, phosphoenolpyruvate synthases (PEPs) (spot 2), and pyruvate dehydrogenases (PDHs) (spot 57). The above two proteins are involved in gluconeogenesis, which is involved in carbohydrate biosynthesis. In addition, the pentose phosphate pathwayrelated proteins glucose-6-phosphate dehydrogenase (coenzyme-F420) (Fgd) (spot 36) and LLM class F420dependent oxidoreductase (spot 45) (Fgd) were upregulated in pyrene-induced cells. G6P is a substrate for the enzyme Fgd in Mycobacteria and is encoded by only  a few bacterial genera. Fgd is an unusual glucose-6-phosphate dehydrogenase that transfers electrons from glucose 6-phosphate (G6P) to F420 instead of NADP [20].
The protocatechuate pathway is a central catabolic pathway of aromatic compounds and is widely distributed in numerous bacterial and fungal taxa [21]. Protocatechuate is converted into vanillin via of SAMdependent methyltransferases [22]. In this study, SAMdependent methyltransferases were significantly expressed in pyrene-induced cells (spots 39 and 41).

Analysis of pyrene degradation-related proteins using 2D-DIGE
More than 20 types of pyrene-degrading mycobacteria have been isolated in the last 10 years, of which M. vanbaalenii PYR-1 is the most widely studied [7]. Pyrene-induced M. vanbaalenii PYR-1 can mineralize over 50% of added pyrene after 1 day of incubation [23]. In addition, many other mycobacteria have been reported to exhibit high pyrene degradation rates, including Achromobacter xylosoxidans strain PY4 [8], Brevibacillus brevis [24], and Pseudomonas aeruginosa strain ASP-53 [25]. Nevertheless, the molecular mechanism underlying pyrene degradation remains unclear, and the pyrene degradation rates are difficult to compare among strains due to differing culture media and incubation conditions among studies.
Here, 2D-DIGE technology was used to identify key proteins involved in pyrene degradation. The identified proteins exhibited various functions, demonstrating the complexity of pyrene degradation in Mycobacterium sp. 16 F. The combined use of proteomics and genomics has led to the identification of several proteins and genes involved in pyrene degradation [11,26,27]. However, many questions remain regarding the pyrene-degrading mechanism. Numerous intermediates are involved in pathways, but many of these have not been identified, and it is not clear how some of the identified proteins are produced or degraded including 4-hydroxyperinaphthenone and cinnamic acid [23,28]. Moreover, M. vanbaalenii PYR-1 can mineralize pyrene [23]. Eight isolated strains of Roseobacter clade bacteria (RCB) have been shown to exhibit low PAHdegrading efficiency when PAHs are used as their sole carbon source [29], implying that RCB degrade PAHs via a co-metabolism pathway. In addition, different types of bacteria may evolve different adaptations to degrade PAHs in different, further contributing to difficulties in elucidating degradation mechanisms.

Proposed pathway model including proteins involved in pyrene degradation
Stoichiometric and flow balance analysis approaches have been used by many researchers to propose reasonable pyrene metabolic pathways in Mycobacterium [21,[30][31][32]. However, to our knowledge, the associated upstream and branching pathways from pyrene degradation to TCA cycle intermediates have not been previously described.
Central carbon metabolism incorporates complex enzymatic steps to convert sugars into metabolic precursors [33] including the glycolytic, the Entner-Doudoroff (ED), the Embden-Meyerhof-Parnas (EMP) and pentose phosphate metabolism (PPP) pathways [34]. Glucose-6-phosphate dehydrogenase (spot 36) and the LLM class F420-dependent oxidoreductase (spot 45) are two enzymes that target the PPP and are production precursors for nucleotides and amino acid metabolism. These enzymes were up-regulated after pyrene exposure, indicating that pyrene may activate a new pathway in Mycobacterium sp. 16 F to adapt to new environments. PEP and E4P are produced in the glycolytic and PPP pathways, respectively. PEPs (spot 2) catalyze the conversion of pyruvate to PEP that is the precursor for in gluconeogenesis [35]. The PDH (spot 57) complex is the main connection between glycolysis and the TCA cycle in bacteria, resulting in oxidative decarboxylation of pyruvate to acetyl CoA [36], under the action of PEP and E4P to produce 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS), which is the initial enzyme of the shikimate pathway [37]. The critical compound chorismate is produced through a seven-step catalytic reaction and is a precursor to numerous secondary metabolites including flavonoids [38]. However, chorismite is the first branch point for these pathways, with one branch leading to phenylalanine. In addition, cinnamic acid is the first molecule in the phenylpropanoid pathway, is synthesized from phenylalanine, and is a precursor of flavonoid biosynthesis [39].
Three proteins (HcaA spot 46, HcaB spot 44, and HcaC) were previously identified to conduct four enzymatic activities on 4-hydroxycinnamic acid derivatives, leading to the metabolism of caffeate, p-coumarate, and ferulate to protocatechuate, p-hydroxybenzoate, and vanillate, respectively [40]. Hca proteins can oxidize 3-phenylpropionic acid (PP) and cinnamic acid to their corresponding derivatives [19]. Consequently, these clusters may play important roles in the catabolism of phenylpropanoid compounds. Benzoate is a model aromatic compound that is also an important intermediate of many aromatic compounds. C2 shortening of the propyl side chain of cinnamic acid produces benzoic acid via one of the three major pathways [41]. It is consequently hypothesized that the metabolic pathways that degrade benzoic acid according to different central metabolites can be divided into catechol, protocatechuate, and gentisate pathways.
The protocatechuate metabolic pathway plays a key role in the biodegradation of aromatic compounds, and protocatechuate is one of the key metabolic intermediates involved in aromatic compound biodegradation [42]. The gentisate (2,5-dihydroxybenzoate) pathway is also an important ring-cleavage pathway involved in the bacterial degradation of various aromatic compounds, including naphthalene [43]. Many aromatic compounds can be converted to the intermediates protocatechuate or catechol that are then metabolized to TCA intermediates via the β-ketoadipate pathway ( Figure 5) [44]. It has been proposed that protocatechuate produced from phthalate is further transformed via the β-ketoadipate pathway to TCA cycle intermediates in Gram-positive bacteria [21,45,46]. This process may be due to shunting of central carbon metabolism intermediates through the shikimic acid pathway, resulting in the production of aromatic precursors that then enter the TCA cycle through the β-ketoadipate pathway.

Conclusions
Proteomic analysis of biochemical processes and mechanisms of pyrene biodegradation in Mycobacterium sp. 16 F indicated that the pentose phosphate and glycolytic pathways led to the production of amino acids and nucleotide precursors in pyrene-induced cells. The metabolites from these processes then entered the shikimate pathway and combined with the pyrene degradation pathway through the β-ketoadipate pathway.