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Hydrocarbon Biodegradation Potential in Environmental Bacterial Metagenome

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Hydrocarbon Biodegradation Potential in Environmental Bacterial Metagenome Faculty of Biosciences, Fisheries and Economics, Department of Arctic and Marine Biology Hydrocarbon biodegradation potential in environmental bacterial metagenome Alicia Caro Pascual Master’s thesis in Molecular Environmental Biology – BIO3950 – December 2020 Table of Contents 1 Introduction ............................................................................................................................. 1 1.1 Barents Sea and the petroleum industry ...................................................................... 1 1.2 Hydrocarbon pollution in cold marine environments .................................................. 3 1.2.1 The fate of hydrocarbons in the marine environment .......................................... 3 1.3 Hydrocarbon degradation by bacteria .......................................................................... 5 1.3.1 Metabolic pathways of hydrocarbon degradation in bacteria .............................. 5 1.3.2 Hydrocarbon-degrading bacteria in marine environments ................................... 7 1.3.3 Bioremediation ..................................................................................................... 9 1.3.4 Functional metagenomics and fosmid libraries .................................................... 9 1.4 Study background ...................................................................................................... 10 1.5 Objectives .................................................................................................................. 11 2 Material and Methods ....................................................................................................... 12 2.1 Isolation of metagenomic DNA ................................................................................. 12 2.2 Media preparation ...................................................................................................... 12 2.2.1 Minimal media and Luria broth media ............................................................... 12 2.2.2 Diesel and crude oil ............................................................................................ 13 2.3 Metagenomic fosmid library production ................................................................... 14 2.3.1 Ligation of insert DNA into pCC2FOS plasmid vector ..................................... 14 2.3.2 Package of fosmid clones into the lambda phage .............................................. 14 2.3.3 Titer and storage of packaged fosmid clones ..................................................... 15 2.4 Culture of transformed E. coli and selection of clones .............................................. 15 2.5 Fosmid clones extraction and enzyme restriction ...................................................... 16 2.6 Sequencing of fosmid clones ..................................................................................... 17 2.7 Bioinformatic analysis ............................................................................................... 18 2.7.1 treatment of raw data and contig assembly ........................................................ 18 2.7.2 Assignation of taxonomy and functions to ORFs: MEGABLAST, BLASTn and BLASTx searches ............................................................................................................. 22 2.7.3 Determination of the phylogeny ......................................................................... 24 2.7.4 Databases and Servers ........................................................................................ 25 3 Results .............................................................................................................................. 26 3.1 Isolated metagenomic DNA ...................................................................................... 26 3.1.1 Titer of the packaged fosmid clones .................................................................. 27 3.2 Selection of transformed E. coli clones on selective hydrocarbon containing media 28 3.2.1 Fosmid clone DNA extraction and restriction enzyme analysis ........................ 32 3.3 Bioinformatic results ................................................................................................. 35 3.3.1 Preprocessing of raw data .................................................................................. 35 3.3.2 Assembly of contigs ........................................................................................... 35 3.3.3 Gene prediction and analysis .............................................................................. 38 3.3.4 Phylogenetic position of the fosmid clones ........................................................ 41 3.3.5 Analyses by custom BLAST on AromaDeg database and other online databases. 44 4 Discussion ........................................................................................................................ 45 4.1 Selection of fosmid clones ......................................................................................... 45 4.2 Crude oil and diesel cultures ..................................................................................... 45 4.3 Phylogeny .................................................................................................................. 47 4.4 Analysis of ORFs and Bacteria species ..................................................................... 47 4.4.1 Sequences 1, 3, 7 and 10: Phylum Planctomycetes ............................................ 47 4.4.2 Sequence 2: genus Maribacter ........................................................................... 52 4.4.3 Sequences 4 and 6: Synechococcales cyanobacterium ...................................... 54 4.4.4 Sequence 5: family Enterobacteraceae .............................................................. 56 4.4.5 Sequence 8: Azotobacter chroococcum .............................................................. 58 4.4.6 Sequence 9: order Chromatiales ........................................................................ 58 4.5 Bioremediation in Norway and future studies ........................................................... 63 5 Conclusions ...................................................................................................................... 63 Works cited .............................................................................................................................. 64 6 Appendix .......................................................................................................................... 71 Appendix 1: Bacterial genomic DNA isolation using CTAB .............................................. 71 Appendix 2: Current protocols in molecular biology ........................................................... 75 Appendix 3: Copycontrol HTP Fosmid library production kit with pCC2FOS vector ....... 78 Appendix 4: Minipreparation of plasmid DNA (alkaline lysis method) ........................... 113 Appendix 5: Enzyme restriction protocol for XbaI ............................................................ 114 Appendix 6: MEGABLAST and BLASTn searches of ORFs ........................................... 115 Appendix 7: Complete phylogenetic tree ........................................................................... 172 List of Abbreviations ABC transporter ATP-binding cassette HDB Hydrocarbon-degrading bacteria transporters KB Kilobase pair ABS Absorbance LB Luria Broth ATP Adenosine triphosphate Mb Megabase pair BLAST Basic Local Alignment Search Tool NCBI National Center for Biotechnology Information BLASTn Nucleotide BLAST NCS Norwegian Continental Shelf BLASTx Translated BLAST ORF Open Reading Frame BP Base Pair PAHs Polycyclic Aromatic Hydrocarbons BS Biosurfactant PDB Phage Dilution Buffer CG Cytosine Guanine PTS transporter Phosphotransferase CO Crude Oil system transporter CTAB Cetyl Trimethyl Ammonium RNA Ribonucleic acid Bromide RPM Revolutions Per Minute D Diesel rRNA Ribosomal RNA DNA Deoxyribonucleic acid TCA cycle TriCarboxylic Acid cycle DUF Domain of Unknown Function V Volts List of Tables Table 1. Taxonomy of Arctic or Antarctic oil-degrading bacteria [18]. a) An Antarctic, Ar Arctic, S sediment, SI sea ice, SW seawater. ............................................................................. 8 Table 2.Content description of M9 minimal media and hydrocarbon quantities added to the media. ....................................................................................................................................... 13 Table 3. Parameters used in bioinformatic tools BBDuk, Dedupe, BBNorm, BBMerge and Glimmer contained in the program Geneious 2020.2.2 [10]. ................................................... 19 Table 4. Parameters for De novo assembly and Map to reference alignment tools contained in the program Geneious 2020.2.2 [3]. ......................................................................................... 21 Table 5. Parameters for MEGABLAST, BLASTn, BLASTx and custom BLAST searches from the program Geneious 2020.2.2 [3] using NCBI database and tools [59] ....................... 23 Table 6. Nanodrop readings of the total DNA samples: DNA concentration (ng/μl), Absorbance ratio 260/280 (nm), Absorbance ratio 260/230 (nm), total quantity of DNA in samples (ng). ............................................................................................................................ 27 Table 7. Number of colonies grown per plate and CFU/ml in dilutions 1:1 and 1:10 of packaged
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