Characterization of the molecular components and function of BARE-1, Hin-Mu and Mu transposition machineries

Transposable elements, transposons, are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. They are abundant residents in virtually all the genomes studied, for instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Transposons plays essential role in genome evolution, in lateral transfer of antibiotic resistance genes among bacteria and in life cycle of certain viruses such as HIV-1 and bacteriophage Mu. Despite the diversity of transposable elements they all use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. The DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements. This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and Hin-Mu transpositional activity has not been shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The Hin-Mu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates. This flexibility may be evolutionarily and mechanistically important.

Mu in vitro Transposition Technology in Functional Genetics and Genomics: Applications on Mouse and Bacteriophages

Transposons are mobile elements of genetic material that are able to move in the genomes of their host organisms using a special form of recombination called transposition. Bacteriophage Mu was the first transposon for which a cell-free in vitro transposition reaction was developed. Subsequently, the reaction has been refined and the minimal Mu in vitro reaction is useful in the generation of comprehensive libraries of mutant DNA molecules that can be used in a variety of applications. To date, the functional genetics applications of Mu in vitro technology have been subjected to either plasmids or genomic regions and entire genomes of viruses cloned on specific vectors. This study expands the use of Mu in vitro transposition in functional genetics and genomics by describing novel methods applicable to the targeted transgenesis of mouse and the whole-genome analysis of bacteriophages. The methods described here are rapid, efficient, and easily applicable to a wide variety of organisms, demonstrating the potential of the Mu transposition technology in the functional analysis of genes and genomes. First, an easy-to-use, rapid strategy to generate construct for the targeted mutagenesis of mouse genes was developed. To test the strategy, a gene encoding a neuronal K+/Cl- cotransporter was mutagenised. After a highly efficient transpositional mutagenesis, the gene fragments mutagenised were cloned into a vector backbone and transferred into bacterial cells. These constructs were screened with PCR using an effective 3D matrix system. In addition to traditional knock-out constructs, the method developed yields hypomorphic alleles that lead into reduced expression of the target gene in transgenic mice and have since been used in a follow-up study. Moreover, a scheme is devised to rapidly produce conditional alleles from the constructs produced. Next, an efficient strategy for the whole-genome analysis of bacteriophages was developed based on the transpositional mutagenesis of uncloned, infective virus genomes and their subsequent transfer into susceptible host cells. Mutant viruses able to produce viable progeny were collected and their transposon integration sites determined to map genomic regions nonessential to the viral life cycle. This method, applied here to three very different bacteriophages, PRD1, ΦYeO3 12, and PM2, does not require the target genome to be cloned and is directly applicable to all DNA and RNA viruses that have infective genomes. The method developed yielded valuable novel information on the three bacteriophages studied and whole-genome data can be complemented with concomitant studies on individual genes. Moreover, end-modified transposons constructed for this study can be used to manipulate genomes devoid of suitable restriction sites.

Mu in vitro transposition applications in protein engineering

Transposons, mobile genetic elements that are ubiquitous in all living organisms have been used as tools in molecular biology for decades. They have the ability to move into discrete DNA locations with no apparent homology to the target site. The utility of transposons as molecular tools is based on their ability to integrate into various DNA sequences efficiently, producing extensive mutant clone libraries that can be used in various molecular biology applications. Bacteriophage Mu is one of the most useful transposons due to its well-characterized and simple in vitro transposition reaction. This study establishes the properties of the Mu in vitro transposition system as a versatile multipurpose tool in molecular biology. In addition, this study describes Mu-based applications for engineering proteins by random insertional transposon mutagenesis in order to study structure-function relationships in proteins. We initially characterized the properties of the minimal Mu in vitro transposition system. We showed that the Mu transposition system works efficiently and accurately and produces insertions into a wide spectrum of target sites in different DNA molecules. Then, we developed a pentapeptide insertion mutagenesis strategy for inserting random five amino acid cassettes into proteins. These protein variants can be used especially for screening important sites for protein-protein interactions. Also, the system may produce temperature-sensitive variants of the protein of interest. Furthermore, we developed an efficient screening system for high-resolution mapping of protein-protein interfaces with the pentapeptide insertion mutagenesis. This was accomplished by combining the mutagenesis with subsequent yeast two-hybrid screening and PCR-based genetic footprinting. This combination allows the analysis of the whole mutant library en masse, without the need for producing or isolating separate mutant clones, and the protein-protein interfaces can be determined at amino acid accuracy. The system was validated by analysing the interacting region of JFC1 with Rab8A, and we show that the interaction is mediated via the JFC1 Slp homology domain. In addition, we developed a procedure for the production of nested sets of N- and C-terminal deletion variants of proteins with the Mu system. These variants are useful in many functional studies of proteins, especially in mapping regions involved in protein-protein interactions. This methodology was validated by analysing the region in yeast Mso1 involved in an interaction with Sec1. The results of this study show that the Mu in vitro transposition system is versatile for various applicational purposes and can efficiently be adapted to random protein engineering applications for functional studies of proteins.

Plasmids and aromatic degradation in Sphingomonas for bioremediation : Aromatic ring cleavage genes in soil and rhizosphere

Microbial degradation pathways play a key role in the detoxification and the mineralization of polyaromatic hydrocarbons (PAHs), which are widespread pollutants in soil and constituents of petroleum hydrocarbons. In microbiology the aromatic degradation pathways are traditionally studied from single bacterial strains with capacity to degrade certain pollutant. In soil the degradation of aromatics is performed by a diverse community of micro-organisms. The aim of this thesis was to study biodegradation on different levels starting from a versatile aromatic degrader Sphingobium sp. HV3 and its megaplasmid, extending to revelation of diversity of key catabolic enzymes in the environment and finally studying birch rhizoremediation in PAH-polluted soil. To understand biodegradation of aromatics on bacterial species level, the aromatic degradation capacity of Sphingobium sp. HV3 and the role of the plasmid pSKY4, was studied. Toluene, m-xylene, biphenyl, fluorene, phenanthrene were detected as carbon and energy sources of the HV3 strain. Tn5 transposon mutagenesis linked the degradation capacity of toluene, m-xylene, biphenyl and naphthalene to the pSKY4 plasmid and qPCR expression analysis showed that plasmid extradiol dioxygenases genes (bphC and xylE) are inducted by phenanthrene, m-xylene and biphenyl whereas the 2,4-dichlorophenoxyacetic acid herbicide induced the chlorocatechol 1,2-dioxygenase gene (tfdC) from the ortho-pathway. A method to study upper meta-pathway extradiol dioxygenase gene diversity in soil was developed. The extradiol dioxygenases catalyse cleavage of the aromatic ring between a hydroxylated carbon and an adjacent non-hydroxylated carbon (meta-cleavage). A high diversity of extradiol dioxygenases were detected from polluted soils. The detected extradiol dioxygenases showed sequence similarity to known catabolic genes of Alpha-, Beta-, and Gammaproteobacteria. Five groups of extradiol dioxygenases contained sequences with no close homologues in the database, representing novel genes. In rhizoremediation experiment with birch (Betula pendula) treatment specific changes of extradiol dioxygenase communities were shown. PAH pollution changed the bulk soil extradiol dioxygenase community structure and birch rhizosphere contained a more diverse extradiol dioxygenase community than the bulk soil showing a rhizosphere effect. The degradation of pyrene in soil was enhanced with birch seedlings compared to soil without birch. The complete 280,923 kb nucleotide sequence of pSKY4 plasmid was determined. The open reading frames of pSKY4 were divided into putative conjugative transfer, aromatic degradation, replication/maintaining and transposition/integration function-encoding proteins. Aromatic degradation orfs shared high similarity to corresponding genes in pNL1, a plasmid from the deep subsurface strain Novosphingobium aromaticivorans F199. The plasmid backbones were considerably more divergent with lower similarity, which suggests that the aromatic pathway has functioned as a plasmid independent mobile genetic element. The functional diversity of microbial communities in soil is still largely unknown. Several novel clusters of extradiol dioxygenases representing catabolic bacteria, whose function, biodegradation pathways and phylogenetic position is not known were amplified with single primer pair from polluted soils. These extradiol dioxygenase communities were shown to change upon PAH pollution, which indicates that their hosts function in PAH biodegradation in soil. Although the degradation pathways of specific bacterial species are substantially better depicted than pathways in situ, the evolution of degradation pathways for the xenobiotic compounds is largely unknown. The pSKY4 plasmid contains aromatic degradation genes in putative mobile genetic element causing flexibility/instability to the pathway. The localisation of the aromatic biodegradation pathway in mobile genetic elements suggests that gene transfer and rearrangements are a competetive advantage for Sphingomonas bacteria in the environment.