SCUOLA DI DOTTORATO UNIVERSITÀ DEGLI STUDI DI MILANO-BICOCCA Department of Biotechnology and Biosciences PhD in Biology and Biotechnology XXXI cycle Genomic and functional analysis of Rhodococcus strains to identify genes and degradative functions for soil quality evaluation Di Canito Alessandra 705596 Tutor: Dr. Patrizia Di Gennaro Coordinator: Prof. Paola Branduardi ACADEMIC YEAR 2017/2018 2 Index Abstract ..................................................................................................................................... 8 Riassunto ................................................................................................................................. 10 Chapter 1. Introduction.......................................................................................................... 12 1.1 Preface ............................................................................................................................... 13 1.2 Soil quality ........................................................................................................................ 14 1.3 Soil biodiversity ................................................................................................................ 16 1.3.1 Soil Microorganisms ....................................................................................................... 16 1.4 Rhodococcus genus ........................................................................................................... 19 1.4.1 Rhodococcus genus genomes .......................................................................................... 21 1.4.1.1 Rhodococcus opacus R7...................................................................................... 23 1.4.1.2 Rhodococcus aetherivorans BCP1 ..................................................................... 24 1.4.1.3 Rhodococcus erythropolis MI2 ........................................................................... 24 1.4.2 R. opacus R7 and R. aetherivorans BCP1 comparative analysis .................................... 25 1.5 Scope of the thesis work................................................................................................... 27 1.6 References ......................................................................................................................... 28 Chapter 2. Biodegradative potential of R. opacus R7 and R. aetherivorans BCP1: genome-based analysis for the identification of pathways and gene clusters ................... 35 2.1 Introduction ...................................................................................................................... 36 2.2 Material and methods ...................................................................................................... 37 2.2.1 Identification of genetic aspects for xenobiotic degradation pathways .......................... 37 2.3 Results ............................................................................................................................... 38 2.3.1 Genetic aspects related to the great potential for organic/xenobiotic degradation in R. opacus R7 and R. aetherivorans BCP1 ...................................................................... 38 2.3.2 Aliphatic hydrocarbons and cycloalkanes degradation ................................................... 38 2.3.3 Aromatic hydrocarbons degradation ............................................................................... 39 2.3.4 Polycyclic aromatic hydrocarbons degradation .............................................................. 41 2.3.5 Naphthenic acids degradation ......................................................................................... 43 2.3.6 Genetic aspects related to aromatic peripheral pathways in Rhodococcus opacus R7 and Rhodococcus aetherivorans BCP1 ........................................................................... 45 2.4 Discussion .......................................................................................................................... 48 2.5 References ......................................................................................................................... 49 3 Index Chapter 3. Phenotype microarray analysis may unravel genetic determinants of the stress response by Rhodococcus aetherivorans BCP1 and Rhodococcus opacus R7 .. 52 3.1 Introduction ...................................................................................................................... 54 3.2 Materials and methods .................................................................................................... 55 3.2.1 Statistical analysis of pm data ......................................................................................... 55 3.2.2 Identification of genetic aspects ...................................................................................... 55 3.3 Results ............................................................................................................................... 56 3.3.1 Tolerance to osmotic stress ............................................................................................. 56 3.3.2 Tolerance to acid and alkaline stress ............................................................................... 58 3.3.3. Resistance to antibiotics, metals and other toxic compounds ........................................ 59 3.4 Discussion .......................................................................................................................... 62 3.5 References ......................................................................................................................... 75 Chapter 4. Genome-based analysis for the identification of genes involved in o-xylene degradation in Rhodococcus opacus R7 ............................................................................... 79 4.1 Introduction ...................................................................................................................... 81 4.2 Materials and methods .................................................................................................... 83 4.2.1 Bacterial strain and growth conditions ............................................................................ 83 4.2.2 Bioinformatic analysis: nucleotide sequence determination and protein sequence analysis ............................................................................................................................ 84 4.2.3 Preparation, analysis, and DNA manipulation ................................................................ 85 4.2.4 RNA extraction and RT-PCR, quantitative real-time RT-PCR (qPCR) ......................... 86 4.2.5 Mutant preparation .......................................................................................................... 87 Transposon-induced mutagenesis in R. opacus R7 using IS1415 (pTNR-TA vector) ... 87 Analysis of pTNR-TA insertion sites ............................................................................. 87 4.2.6 Construction of the recombinant strain R. erythropolis AP (pTipQC2-prmACBD-R7) . 88 4.2.7 Bioconversion experiments of the recombinant strain R. erythropolis AP (pTipQC2-prmACBD-R7) in presence of o-xylene ........................................................ 88 4.2.8 Analytical methods ......................................................................................................... 88 4.3 Results ............................................................................................................................... 89 4.3.1 R7 genome sequence analysis ......................................................................................... 89 Analysis and clusterization of dioxygenases .................................................................. 89 Analysis and clusterization of monooxygenases/hydroxylases ....................................... 91 4.3.2 Involvement of the akb genes in o-xylene degradation by RT-PCR experiments .......... 93 4.3.3 Involvement of the akb genes in o-xylene degradation by the identification of R. opacus R7 mutants in this cluster............................................................................... 96 4.3.4 Identification of the involvement of the phe genes and the prm genes in o-xylene degradation by RT-PCR experiments ............................................................................. 97 4 Index 4.3.5 Quantitative real-time RT-PCR (qPCR) analysis ........................................................... 99 4.3.6 Involvement of the prm genes by cloning and expression of the activity in R. erythropolis AP......................................................................................................... 100 4.4 Discussion ........................................................................................................................ 101 4.6 References ....................................................................................................................... 112 Chapter 5. Soil quality assessment by the identified sequences and degradative functions of R. opacus R7 ..................................................................................................................... 117 5.1 Introduction .................................................................................................................... 118 5.2 Materials and Methods .................................................................................................. 120 5.2.1 Characterization