An Insight Into the Aliivibrio Genus a Comparative Study on Relationships and Traits of Species Within the Genus Aliivibrio
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FACULTY OF SCIENCE AND TECHNOLOGY DEPARTMENT OF CHEMISTRY MOLECULAR BIOSYSTEMS RESEARCH GROUP An Insight into the Aliivibrio genus A comparative study on relationships and traits of species within the genus Aliivibrio Terje Klemetsen KJE-3900 Master's Thesis in Chemistry May 2016 Acknowledgements I am grateful for the time I have been granted to work with bioinformatics. I would especially thank my supervisor Nils Peder Willassen for making this masters degree possible, and for all the talks and good suggestions. My work has been both challenging and highly rewarding. Experience on the field has grown in these passing years, and so too, the interest and desire for more knowledge. I also thank my very inspiring office mates Cecilie Bækkedal and Espen Åberg who as well contribute to the field of bioinformatics. Moreover, my family has supported me all the way since I started as a student seven years ago and I am very grateful for all they have done. Terje Klemetsen Tromsø, May 2016 3 4 Abstract Background Few studies have emphasized on the genus Aliivibrio as a whole and lags behind the better known Vibrio. Nevertheless, the Aliivibrio has for several decades been associated with species expressing bioluminescence like the Aliivibrio fischeri, but has also been linked to costly diseases in the fish farming industry such as Aliivibrio salmonicida. In an attempt to gain insight in the genus on a broad level, Aliivibrio genomes were sequenced, assembled and annotated prior to phylogenetic and pan-genome analysis. Additionally, mapping of genes related to quorum sensing and the CRISPR defense system was performed in a comparative manner. Works like this have never been carried out before on this scale for the Aliivibrio and is needed to better understand the complexity of this genus. Results This project found the Aliivibrio to be distinct in the Vibrionaceae family and harbors a diverse range of species with a relatively large set of dispensable genes. Among these appeared five new proposed species, and comparison showed deviations in quorum sensing genes with novel additions to previously described systems. This comes as well as proving the presence of Type I-F CRISPR system in most strains of A. wodanis. Conclusion Aliivibrio was discovered to be a distinct and diverse genus on more than one level, and is likely to harbor additional species and traits that still remain to discover. 5 6 Abbreviations AAI - Average Amino acid Identity AHL - Acyl Homoserine Lactone ANI - Average Nucleotide Identity BIC - Bayesian Information Criterion BLAST - Basic Local Alignment Search cas genes - CRISPR Associated genes CDS - Coding DNA Sequences CLC - CLC genomic workbench COG - Clusters of Orthologous Groups CPU - Central Processing Unit CRISPR - Clustered Regularly Interspaced Short Palindromic Repeat crRNA - CRISPR RNAs, spacer and repeat products DDBJ - DNA Data Bank of Japan DNA - Deoxyribonucleic Acid ENA - European Nucleotide Archive GC - Guanine-Cytosine content GTR - General Time Reversible model G - Gamma distributed I - Invariant positions HGT - Horizontal Gene Transfer IUPAC - International Union of Pure and Applied Chemistry KEGG - Kyoto Encyclopedia of Genes and Genomes MLSA - MultiLocus Sequence Analysis NCBI - National Center for Biotechnology Information NGS - Next Genetation Sequencing PAM - Protospacer Adjacent Motif PCR - Polymerase Chain Reaction QS - Quorum Sensing RNA - Ribonucleic Acid ML - Maximum likelihood 7 8 Table of contents Background .......................................................................................................................................................... 11 The Vibrionaceae and Aliivibrio ........................................................................................................................ 11 Sequencing technology and post processing ...................................................................................................... 12 The Pan-genome era .......................................................................................................................................... 15 Quorum sensing in bacteria ............................................................................................................................... 16 CRISPR – the antiviral defense system in bacteria ............................................................................................ 18 Aims of this project .............................................................................................................................................. 21 Material ................................................................................................................................................................. 23 Genomes and house-keeping genes ................................................................................................................... 23 Software ........................................................................................................................................................ 25 Web-services and databases .......................................................................................................................... 27 Equipment and hardware ............................................................................................................................... 29 Method ................................................................................................................................................................. 31 Assembly and Genome Annotation ................................................................................................................... 31 Inferring the Phylogenetic relationship of Aliivibrio, Vibrio and Photobacteria ............................................... 32 Pan-genome analysis of the genus Aliivibrio ..................................................................................................... 34 Mapping of Quorum Sensing Genes .................................................................................................................. 35 Identification of CRISPR repeats and cas genes ................................................................................................ 36 Results ................................................................................................................................................................... 37 Assembly and annotation overview ................................................................................................................... 37 Phylogenetic relationships based on MLSA ...................................................................................................... 40 Pan-genome analysis of 45 Aliivibrio genomes ................................................................................................. 48 Quorum sensing genes in Aliivibrio genomes .................................................................................................... 53 Detection of the CRISPR defense system ......................................................................................................... 58 Discussion .............................................................................................................................................................. 61 Assembly and Annotation .................................................................................................................................. 61 Draft genome assembly proves the presence of repeat sequences................................................................. 61 Contig mapping explain greater DNA variation among A. wodanis ............................................................. 61 Number of annotated genes varies greatly within the Aliivibrio genus ......................................................... 62 Phylogeny .......................................................................................................................................................... 63 High resolution achieved on the basis on six housekeeping loci ................................................................... 63 Network analysis of Aliivibrio emphasized the existence of novel species................................................... 63 Individual gene loci share unique features .................................................................................................... 64 Pan-genome analysis.......................................................................................................................................... 67 Pan-genome analysis proves Aliivibrio to be a diverse genus with a moderately conserved core ................ 67 The content of the pan-genome was syntenic only at the species level and comprised a wealth of inconsistent and unknown gene products ...................................................................................................... 68 The genome core distances showed favorable resolution in separating species ............................................ 70 Quorum sensing ................................................................................................................................................. 71 The AinR/AinS system is not consistent in the Aliivibrio genera ................................................................. 71 Novel form of lux operon identified in A. modi ............................................................................................ 72 Additional quorum