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Isolation of a natural microbial consortium and construction of a synthetic consortium: From a chronically PAH-contaminated soil from Mosconi neighborhood (coordinates S 34° 52´31´ W 57° 55´10´) near La Plata City, Argentina (no specific permissions were required for this field location because it did not involve endangered or protected species), a phenanthrene-degrading bacterial consortium (CON) was obtained by culture enrichment technique in LMM with 2000 mg l-1 of phenanthrene as detailed in previous research [17]. Five different strains were isolated form CON using culture-dependent methods [17] and were used to construct a synthetic consortium (SC); these strains were Sphingobium sp. (AM), Enterobacter sp. (B), Pseudomonas sp. (T and Bc) and Inquilinus limosus (I). Each strain is grown individually in R2 liquid medium for 24 hours at 28°C at 150 rpm and then washed with saline solution and inoculated to form the SC, which was measured at 0.2 absorbance at 580 nm on day 0 of the treatment.
A degradation assay with CON, SC and Sphingobium sp. AM was conducted in triplicate in LMM supplemented with 200 mg.l-1 of sterile phenanthrene as a sole carbon and energy source. Cultures were incubated at 28°C, 150 rpm and monitored at 0, 4, 7 and 15 days. One non-inoculated Erlenmeyer was used as an abiotic control. The chemical extraction of phenanthrene and 1-hydroxy-2-naphthoic acid using ethyl acetate and the analysis of the organic extracts by high-pressure liquid chromatography (HPLC) using a Waters chromatograph with a Symmetry Waters C18 column and a diodearray detector were conducted as described in a previous work [17]. The statistical analysis of the phenanthrene degradation data were performed by a parametric one-way ANOVA test using the SigmaPlot/SigmaStat software program (SPSS Inc., Chicago, Illinois, USA).
Heterotrophic cultivable bacteria and PAH degraders were determined at 0, 4, 7 and 15 days of incubation. The determination of the first group was performed on R2A medium plates according to Reasoner and Geldreich [20], and determination of the latter was performed in sterile polypropylene microplates with a mixture of PAH as a substrate according to Wrenn and Venosa [21]. For a detailed protocol, see a previous work [17]. All of the determinations were performed in triplicate.
Total DNA was extracted from a culture of CON in LMM with 200 mg.l-1 of phenanthrene as a sole carbon and energy source after 2 days of incubation. The protocol was conducted as described by Entcheva and co-workers [22] with the aim of obtaining DNA fragments larger than 23 kb. The DNA concentration was measured using a NanoDrop 2000 (Thermo-Scientific™).
Metagenomic library construction and functional screening: The fosmid clone library was constructed using the CopyControl™ HTP Fosmid Library Production Kit with pCC2FOS™ Vector (Epicenter) according to the manufacturer’s recommendations. Briefly, fragments larger than 23 kb were selected and an end repair reaction was carried out to insert the ends, which were then ligated into the pCC2FOS™ vector, packed with MaxPlax Lambda Packaging Extracts and transfected into EPI300-T1R Phage T1-resistant E. coli. The resulting library was plated in LB medium containing 12.5 μg.ml-1 chloramphenicol, and approximately 8000 clones were replicated and analyzed by functional screening. The first functional screening was developed with an aqueous solution of catechol with a preceding exposure to phenol vapors for 6 h as described by our colleagues [23] with some modifications. Catechol-positive clones were submitted to a second functional screening using a 20 mg.ml-1 2,3-dihydroxybiphenyl solution in acetone as detailed by Ren and co-workers [24]. Both screening methods were performed in LB medium diluted 1/10 with 12.5 μg.ml-1 chloramphenicol. The 18 isolated clones of 3 pools with 6 clones each were formed in order to sequence the insert they contained.
Fosmid extraction, sequencing, phylogenetic affiliation and gene annotation: The Fosmid extraction was conducted using a Fosmid MAX™ DNA Purification Kit (Epicenter) according to the manufacturer’s recommendations. Fosmid DNA reads generated by Illumina and sequence assembling were performed at the Instituto de Agrobiotecnología Rosario (INDEAR, Argentina). Sequence data are available at the NCBI Sequence Read Archive (SRA) under accession number SRP108260. The phylogenetic affiliation of the fosmid insert was evaluated with PhylopythiaS software in the generic mode, which is a previously trained mode with publicly available prokaryotic genomes and a taxonomy available at NCBI and from compositional vectors [25]. Based on a structural support vector machine (SSVM) model, PhylopythiaS permits taxonomic assignment by analyzing the compositional vectors derived from DNA sequence fragments. The open reading frames (ORFs) were identified and automatically annotated using de Rapid Annotation using Subsystem Technology (RAST, http://rast.nmpdr.org/). Automatically predicted genes were verified through a heuristic approach using FGENESB (http://linux1.softberry.com/berry.phtml). Final ORF annotations were performed with Artemis software by applying the criteria proposed by Pallejà and co-workers [26]. Putative functions of predicted ORFs were manually checked by BLASTp searches against all non-redundant protein sequences from the NCBI database and by functional analysis of deduced protein sequences using InterPro web service (http://www.ebi.ac.uk/interpro/), which allowed protein classification into families and predicted the presence of protein domains. To classify RHO into functional classes and predict their potential substrates, the RHOBASE program was used [27].
Total protein extraction was performed with CON cultures in LMM supplied with 200 mg.l-1 of sterile phenanthrene as a sole carbon and energy source after 4 and 15 days of incubation (28°C, 150 rpm). The samples were filtered to discard the remaining phenanthrene crystals. Briefly, the extraction procedure included a first centrifugation for 10 minutes at 6000 rpm; consequently, the pellet was washed twice with MilliQ water and resuspended in MilliQ water to reach a final DO580 nm of approximately 20. A protease inhibitor cocktail and 5 mM phenylmethylsulfonylfluoride (PMSF) protease inhibitor (freshly prepared) were added to each sample, and then the cells were disrupted using a Precellys 24 bead beater (Bertin Technologies) three times for 30 s until clarification. Finally, the samples were centrifuged for 20 minutes at 8000 rpm and the supernatant was retained. Three volumes of solubilization buffer was added (7 M urea, 2 M thiourea, 2% (w/v) Triton X-100, 65 mM DTT, 1% (w/v) and Amberlite 1% (w/v)), and the mixture was shaken for solubilization for 60 minutes. The protein concentration in the supernatant was determined using the Qubit fluorometer (Invitrogen). Two-dimensional electrophoresis was conducted in a PROTEAN II xi 2-D (Bio-Rad, Hercules, CA) connected to a refrigeration bath II Multitemp (GE Healthcare) to detect differential protein expression levels (up- or down-regulated) in microorganisms present in CON. The gels were calibrated with a molecular mass marker PageRuler™ Prestained Protein Ladder (10–170 kDa) (Pierce Endogen), and the spots were visualized with Coomasie Blue G250 stain and documented with Universal Hood II (Bio-Rad, Hercules, CA). Image analysis, including spot detection, matching, abundance quantification, and normalization, were performed using ProteomeWeaver software (Definiens, Munich, Alemania). Each analyzed condition of CON was evaluated in duplicate as independent cultures. The spots of interest were manually excised and sent to the Mass Spectrometry Facility (CEQUIBIEM) at the School of Exact and Natural Sciences, University of Buenos Aires. Tryptic in-gel digestion was performed, and extracted peptides were analyzed in a UV-MALDI-TOF/TOF (UltraflexII BrukerDaltonics). The software used for spectrum visualization and MS/MSMS protein identification was Flex Analysis (v. 3.3) and BioTools (Bruker Daltonics) linked to MASCOT (Matrix Science, Boston, MA 2016 (http://www.matrixscience.com/) to search against the NCBInr protein sequence databases.
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