The secret life of conjugative relaxases

Conjugative relaxases are well-characterized proteins responsible for the site- and strand-specific endonucleolytic cleavage and strand transfer reactions taking place at the start and end of the conjugative DNA transfer process. Most of the relaxases characterized biochemically and structurally belong to the HUH family of endonucleases. However, an increasing number of new families of relaxases are revealing a variety of protein folds and catalytic alternatives to accomplish conjugative DNA processing. Relaxases show high specificity for their cognate target DNA sequences, but several recent reports underscore the importance of their activity on secondary targets, leading to widespread mobilization of plasmids containing an oriT-like sequence. Some relaxases perform other functions associated with their nicking and strand transfer ability, such as catalyzing site-specific recombination or initiation of plasmid replication. They perform these roles in the absence of conjugation, and the validation of these functions in several systems strongly suggest that they are not mere artifactual laboratory observations. Other unexpected roles recently assigned to relaxases include controlling plasmid copy number and promoting retrotransposition. Their capacity to mediate promiscuous mobilization and genetic reorganizations can be exploited for a number of imaginative biotechnological applications. Overall, there is increasing evidence that conjugative relaxases are not only key enzymes for horizontal gene transfer, but may have been adapted to perform other roles which contribute to prokaryotic genetic plasticity. Relaxed target specificity may be key to this versatility.Acknowledgements: We are grateful to Mapi Garcillán-Barcia for helpful suggestions. Work in our lab is supported by grants BIO2017-87190-R from the MINECO (Spanish Ministry of Economy and Innovation), and IDEAS211LLOS from the AECC (Spanish Association Against Cancer) to ML. DLG-H is a recipient of a predoctoral appointment from the University of Cantabria

Conjugative relaxases as drivers of protein and DNA translocation through Type IV Secretion Systems: biological and biotechnological implications

Este trabajo se ha centrado en el estudio de diferentes aspectos de la actividad integrasa de las relaxasas conjugativas. Hemos analizado la relación entre la capacidad de oligomerizar de las relaxasas en presencia de su ADN diana y de catalizar la integración del ADN, utilizando como modelo la relaxasa TrwC. También, hemos estudiado el posible papel biológico de esta reacción en la colonización de huéspedes no permisivos. Además, hemos analizado la habilidad de la relaxasa MobA del plásmido RSF1010 en mediar la transferencia de ADN a una célula eucariota a través del SST4 del patógeno B. henselae, así como de promover la integración de este ADN en el genoma eucariota. Finalmente, hemos estudiado el potencial uso biotecnológico de las relaxasas conjugativas como sistemas de envío de ADN y proteínas, fusionando la relaxasa TrwC a la proteína Cas12a. Hemos comprobado que la proteína de fusión es activa y funcional en la célula receptora procariota, tras ser translocada a través del SST4.This work has focused on the study of different aspects of the integrase activity of conjugative relaxases. We have analyzed the relationship between the ability of relaxases to oligomerize in the presence of their target DNA and to catalyze DNA integration, using the TrwC relaxase as a model. Also, we have studied the possible biological role of this reaction in the colonization of non-permissive hosts. Furthermore, we have analyzed the ability of the MobA relaxase from plasmid RSF1010 to mediate the transfer of DNA to a eukaryotic cell through the T4SS of the pathogen B. henselae, as well as to promote the integration of this DNA into the eukaryotic genome. Finally, we have studied the potential biotechnological use of conjugative relaxases as DNA and protein delivery systems, fusing the TrwC relaxase to the Cas12a protein. We have verified that the fusion protein is active and functional in the prokaryotic receptor cell, after being translocated through T4SS.Esta Investigación ha sido financiada por una ayuda para contratos predoctorales en el área de la Biomedicina, Biotecnología y Ciencias de la Salud de la Universidad de Cantabria: 7665391046 Y0SC001170. El trabajo en el laboratorio de Matxalen Llosa Blas ha sido financiado por el Ministerio de Economía, Industria y Competitividad de España: BIO2013-46414-P y BIO2017-87190-R. Las estancias de investigación en el Instituto de Productos Lácteos de Asturias (IPLA) y en el Instituto Pasteur se realizaron gracias a sendas ayudas de la Universidad de Cantabria

An investigation of the replication of the broad-host-range plasmid pTF-FC2

Plasmid pTF-FC2, a 12.4 kilobase pairs (kb) cryptic plasmid, was originally isolated from the acidophilic chemoautotroph Thiobacillus ferrooxidans and subsequently cloned into the pMB1-based vector, pBR325. Deletion of the pBR325 origin of replication revealed that pTF-FC2 was able to replicate autonomously in a number of Gram-negative bacteria besides T. ferrooxidans. Constructs carrying the pTF-FC2 origin were able to replicate independently of DNA polymerase I (Pol I) in Escherichia coli and this feature was used to distinguish between replication from the T. ferrooxidans origin and the pMB1-derived origins of the vectors. A 3.2 kb Sau3A partial fragment was obtained which had retained the ability to replicate in a E. coli polA- mutant and also in Pseudomonas aeruginosa. A series of deletions of this fragment was used to identify the minimal replicon, the vegetative origin of replication (orzV) and the areas determining plasmid incompatibility. The copy number of pTF-FC2 in E. coli was estimated at 12 - 15 copies per chromosome and a deletion plasmid was identi.fied which replicated at a reduced copy number. An area which affected the ability of the replicon to replicate in P. aeruginosa, was identified. The nucleotide sequence of the 3.2 kb minimal replicon of pTF-FC2 was determined from overlapping DNA sequence obtained from a series of sequential deletions from each end of the fragment. Analysis of the orzV sequence revealed three, tandemly repeated 22 base pairs (bp) DNA sequences and two sets of complementary inverted repeats. The 22 bp direct repeats appeared to be essential for replication and also for incompatibility. The role of one of the two sets of complementary inverted repeats was unclear. The deletion plasmid previously shown to replicate at reduced copy number was found to have half of the second set of repeats deleted

Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures

During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.This work was financed by grants BFU2014-55534-C2-1-P from the Spanish Ministry of Economy and Competitiveness and 612146/FP7-ICT- 2013 and 282004/FP7-HEALTH.2011.2.3.1-2 from the European Union Seventh Framework Programme to FC and grant BFU2014-55534-C2-2-P from the Spanish Ministry of Economy and Competitiveness to GM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The use of the broad-host-range plasmid, pTF-FC2, in Agrobacterium tumefaciens mediated plant transformation

Bibliography: leaves 165-195.The transfer of T-DNA from Agrobacterium tumefaciens to eukaryotic cells is on of the most extensively studied examples of interkingdom DNA transfer. This transfer system has revolutionized plant molecular biology to the extent that the introduction of foreign genes into plant genomes is now a basic technique. Apart from the finding that a defined segment (T-DNA) of the A. tumefaciens Ti plasmid can be transferred to plant DNA, the discovery that the mobilization functions of the IncQ plasmid, RSF1010, can mediate its transfer from A. tumefaciens into plant cells as well between Gram negative bacteria has generated considerable interest. The mobilization functions of RSF1010 substitute for the requirement of the 25-bp border T-DNA sequences of A. tumefaciens. In this thesis I investigated the ability of another plasmid, pTF-FC2, to transfer genes to plants

Identification and characterization of agrobacterium tumefaciens virD2-binding protein

Ph.DDOCTOR OF PHILOSOPH

Site-specific relaxase activity of a VirD2-like protein encoded within the tfs4 genomic island of Helicobacter pylori

Four different type IV secretion systems are variously represented in the genomes of different Helicobacter pylori strains. Two of these, encoded by tfs3 and tfs4 gene clusters are contained within self-transmissible genomic islands. Although chromosomal excision of tfs4 circular intermediates is reported to be dependent upon the function of a tfs4-encoded XerD tyrosine-like recombinase, other factors required for transfer to a recipient cell have not been demonstrated. Here, we characterize the functional activity of a putative tfs4-encoded VirD2-like relaxase protein. Tfs4 VirD2 was purified as a fusion to maltose-binding protein and demonstrated to bind and nick both supercoiled duplex DNA and oligonucleotides in vitro in a manner dependent upon the presence of Mg2+ but independently of any auxiliary proteins. Unusually, concentration-dependent nicking of duplex DNA appeared to require only transient protein-DNA interaction. Although phylogenetically distinct from established relaxase families, site-specific cleavage of oligonucleotides by Tfs4 VirD2 required the nick region sequence 5′-ATCCTG-3′ common to transfer origins (oriT) recognized by MOBP conjugative relaxases. Cleavage resulted in covalent attachment of MBP-VirD2 to the 5′-cleaved end, consistent with conventional relaxase activity. Identification of an oriT-like sequence upstream of tfs4 virD2 and demonstration of VirD2 protein-protein interaction with a putative VirC1 relaxosome component indicate that transfer initiation of the tfs4 genomic island is analogous to mechanisms underlying mobilization of other integrated mobile elements, such as integrating conjugative elements, requiring site-specific targeting of relaxase activity to a cognate oriT sequence

Characterisation of plasmid p31T1 isolated from Aeromonas

Thesis (MSc)--Stellenbosch University, 2013.ENGLISH ABSTRACT: Plasmids are an integral part of the horizontal gene pool and, therefore, are the main vectors for the spread of antibiotic and heavy metal resistance genes in the environment. Functional and taxonomic characterization of novel plasmids is, therefore, central to our general understanding of plasmid biology and their contribution to microbial evolution. Two 14-kb mobilizable plasmids, p31T1 and p36T2, conferring resistance to tetracycline were isolated from the opportunistic fish pathogens Aeromonas sobria and Aeromonas hydrophila and were found to have indistinguishable restriction fragment length polymorphism (RFLP) patterns (Marx, MSc Thesis). DNA sequence analysis of the two isogenic plasmids (only p36T2 was sequenced) revealed the presence of 18 putative open reading frames (ORFs), of which the tetAR tetracycline resistance genes, associated with a truncated Tn1721, were the only ORFs with significant similarity to known sequences within the NCBI database. Putative functions were assigned to 10 of the ORFs based on their distant homology with proteins of known function. Six of the 18 ORFs, spanning 5.7-kb, were found to comprise the minimal region required for replication (minimal replicon) by means of deletion analysis using derivatives of p31T1. Of the six ORFs, ORF2 and ORF4 were found to be essential for plasmid replication. Inactivation of ORF3 resulted in an increase of plasmid copy number (PCN) from ~3 to ~7 plasmids per chromosome and a decrease in plasmid stability from ~80 % to 16 % over approximately 127 generations (7 days). Furthermore, by means of β-galactosidase promoter fusion assays it was shown that ORF3 autoregulated its own promoter. These results, therefore, suggested that although ORF3 was not essential for replication, it may be involved in plasmid copy number regulation and control. Host range analysis indicated that p31T1 was able to replicate in two other members of the γ-proteobacteria group (Escherichia coli and Pseudomonas putida) but was unable to do so in an α-proteobacterium strain, thus suggesting a limited host range. Furthermore, p31T1 was mobilized only at low frequencies (5.4 x 10-5 transconjugants per donor) by an IncP-1 conjugative system though it is possible that the mobilization system of these plasmids is adapted to function optimally with alternate conjugative systems. Given the unique PCN, stability, host range and mobilization characteristics determined for p31T1 and that no other plasmid replication and mobilization systems with significant sequence similarity to these plasmids have yet been identified, it is likely that these two plasmids are the first representative members of a new family of plasmids found within aquacultureassociated Aeromonas species and which are involved in the spread of tetracycline resistance.AFRIKAANSE OPSOMMING: Plasmiede vorm ‘n integrale deel van die horisontale geen poel en vorm daarom die hoof vektore vir die verspreiding van antibiotika- en swaarmetaal-weerstandbiedende gene in die omgewing. Funksionele en taksonomiese karakterisering van nuwe plasmiede is belangrik in die begrip van plasmied biologie en hul bydrae tot mikrobiese evolusie. Twee 14-kb mobiliseerbare plasmiedes, p31T1 en p36T2, met tetrasiklien weerstandigheid was vanaf die opportunistiese vis patogene Aeromonas sobria en Aeromonas hydrophila geïsoleer en het identiese restriksie fragment lengte polimorfisme (RFLP) patrone. DNA volgorde analise van die twee isogeniese plasmiede (slegs die volgorde van p36T2 was bepaal) het die teenwoordigheid van 18 moontlike oop leesrame (OLR) getoon. Die tetAR tetrasiklien weerstandbiedende gene, wat met ‘n verkorte Tn1721 transposon geassosieerd is, was die enigste OLR wat beduidende volgorde ooreenkoms met bekende volgordes binne die NCBI databasis getoon het. Moontlike funksies was toegeken aan 10 van die OLRe en was gebasseer op vêrlangse homologie met proteïene met bekende funksies. Ses van die 18 OLRe strek oor ‘n 5.7- kb minimale replikon fragment wat benodig word vir replisering en is deur middel van delesie analises van p31T1 derivate gevind. Van hierdie ses OLRe, word OLR2 en OLR4 benodig vir plasmied replisering. Inaktivering van OLR3 het ‘n toename in plasmied kopiegetal (PKG) vanaf ~3 tot ~7 plasmiede per kromosoom en ‘n afname in stabiliteit vanaf ~80% tot 16% oor 127 generasies (7 dae) tot gevolg gehad. Verder kon daar deur middel van β-galaktosidase fusie analises getoon word dat OLR3 sy eie promotor outoreguleer. Hierdie resultate stel dus voor dat alhoewel OLR3 nie benodig was vir replikasie nie, mag dit dalk by plasmied kopiegetal regulering en beheer betrokke wees. Bakteriële gasheer analises het getoon dat p31T1 in 2 addisionele lede van die γ-proteobakterieë groep (Escherichia coli en Pseudomonas putida) kon repliseer, maar nie in ‘n α-proteobacterium nie. Verder kon p31T1 teen ‘n lae frekwensie (5.4 x 105) gemobiliseer word deur ‘n IncP-1 konjugasie sisteem, maar dit mag wees dat die mobilisering eerder optimaal kan plaasvind met ‘n alternatiewe konjugasie sisteem. Na aanleiding van die unieke PKG, stabiliteit, gasheer en mobilisering eienskappe wat vir p31T1 bepaal is en die feit dat geen ander replisering en mobilisering sisteme met noemenswaardige volgorde homologie tot hierdie plasmiede gevind kon word nie, blyk dit dat hierdie van die eerste lede van ‘n nuwe familie van plasmiede binne die akwakultuur-geassosieerde Aeromonas spesies is, wat betrokke is by die verspreiding van tetrasiklien weerstandbiedendheid