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Find video protocols related to scientific articles indexed in Pubmed.
A translocation motif in relaxase TrwC specifically affects recruitment by its conjugative type IV secretion system.
J. Bacteriol.
PUBLISHED: 08-30-2013
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Type IV secretion system (T4SS) substrates are recruited through a translocation signal that is poorly defined for conjugative relaxases. The relaxase TrwC of plasmid R388 is translocated by its cognate conjugative T4SS, and it can also be translocated by the VirB/D4 T4SS of Bartonella henselae, causing DNA transfer to human cells. In this work, we constructed a series of TrwC variants and assayed them for DNA transfer to bacteria and human cells to compare recruitment requirements by both T4SSs. Comparison with other reported relaxase translocation signals allowed us to determine two putative translocation sequence (TS) motifs, TS1 and TS2. Mutations affecting TS1 drastically affected conjugation frequencies, while mutations affecting either motif had only a mild effect on DNA transfer rates through the VirB/D4 T4SS of B. henselae. These results indicate that a single substrate can be recruited by two different T4SSs through different signals. The C terminus affected DNA transfer rates through both T4SSs tested, but no specific sequence requirement was detected. The addition of a Bartonella intracellular delivery (BID) domain, the translocation signal for the Bartonella VirB/D4 T4SS, increased DNA transfer up to 4% of infected human cells, providing an excellent tool for DNA delivery to specific cell types. We show that the R388 coupling protein TrwB is also required for this high-efficiency TrwC-BID translocation. Other elements apart from the coupling protein may also be involved in substrate recognition by T4SSs.
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HUH site-specific recombinases for targeted modification of the human genome.
Trends Biotechnol.
PUBLISHED: 02-27-2013
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Site-specific recombinases (SSRs) have been crucial in the development of mammalian transgenesis. For gene therapy purposes, this approach remains challenging, because, for example, SSR delivery is largely unresolved and SSR DNA substrates must pre-exist in target cells. In this review, we discuss the potential of His-hydrophobic-His (HUH) recombinases to overcome some of the limitations of conventional SSRs. Members of the HUH protein family cleave single-stranded (ss)DNA, but can mediate site-specific integration with the aid of the host replication machinery. Adeno-associated virus (AAV) Rep remains the only known example to support site-specific integration in human cells, and AAV is an excellent gene delivery vector that can be targeted to specific cells and organelles. Bacterial protein TrwC catalyzes integration into human sequences and can be delivered to human cells covalently linked to DNA, offering attractive new features for targeted genome modification.
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Structural independence of conjugative coupling protein TrwB from its Type IV secretion machinery.
Plasmid
PUBLISHED: 01-04-2013
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The stability of components of multiprotein complexes often relies on the presence of the functional complex. To assess structural dependence among the components of the R388 Type IV secretion system (T4SS), the steady-state level of several Trw proteins was determined in the absence of other Trw components. While several Trw proteins were affected by the lack of others, we found that the coupling protein TrwB is not affected by the absence of other T4SS components, nor did its absence alter significantly the levels of integral components of the complex, underscoring the independent role of the coupling protein on the T4SS architecture. The cytoplasmic ATPases TrwK (VirB4) and TrwD (VirB11) were affected by the absence of several core complex components, while the pilus component TrwJ (VirB5) required the presence of all other Trw proteins (except for TrwB) to be detectable. Overall, the results delineate a possible assembly pathway for the T4SS of R388. We have also tested structural complementation of TrwD (VirB11) and TrwJ (VirB5) by their homologues in the highly related Trw system of Bartonella tribocorum (Bt). The results reveal a correlation with the functional complementation data previously reported.
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Transfer of R388 derivatives by a pathogenesis-associated type IV secretion system into both bacteria and human cells.
J. Bacteriol.
PUBLISHED: 09-09-2011
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Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A ?virB mutant strain was transfer deficient, while a ?trwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs.
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Nuclear targeting of a bacterial integrase that mediates site-specific recombination between bacterial and human target sequences.
Appl. Environ. Microbiol.
PUBLISHED: 10-29-2010
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TrwC is a bacterial protein involved in conjugative transfer of plasmid R388. It is transferred together with the DNA strand into the recipient bacterial cell, where it can integrate the conjugatively transferred DNA strand into its target sequence present in the recipient cell. Considering that bacterial conjugation can occur between bacteria and eukaryotic cells, this protein has great biotechnological potential as a site-specific integrase. We have searched for possible TrwC target sequences in the human genome. Recombination assays showed that TrwC efficiently catalyzes recombination between its natural target sequence and a discrete number of sequences, located in noncoding sites of the human genome, which resemble this target. We have determined the cellular localization of TrwC and derivatives in human cells by immunofluorescence and also by an indirect yeast-based assay to detect both nuclear import and export signals. The results indicate that the recombinase domain of TrwC (N600) has nuclear localization, but full-length TrwC locates in the cytoplasm, apparently due to the presence of a nuclear export signal in its C-terminal domain. The recombinase domain of TrwC can be transported to recipient cells by conjugation in the presence of the helicase domain of TrwC, but with very low efficiency. We mutagenized the trwC gene and selected for mutants with nuclear localization. We obtained one such mutant with a point A904T mutation and an extra peptide at its C terminus, which maintained its functionality in conjugation and recombination. This TrwC mutant could be useful for future TrwC-mediated site-specific integration assays in mammalian cells.
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Functional dissection of the conjugative coupling protein TrwB.
J. Bacteriol.
PUBLISHED: 04-02-2010
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The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwB Delta N70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.
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Bacterial type IV secretion systems in human disease.
Mol. Microbiol.
PUBLISHED: 06-08-2009
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Type IV secretion (T4S) systems are versatile machines involved in many processes relevant to bacterial virulence, such as horizontal DNA transfer and effector translocation into human cells. A recent workshop organized by the International University of Andalousia in Baeza, Spain, covered most aspects of bacterial T4S relevant to human disease, ranging from the structural and mechanistic analysis of the T4S systems to the physiological roles of the translocated effector proteins in subverting cellular functions in infected humans. This review reports the highlights from this workshop, which include the first visualization of a T4S system core complex spanning both membranes of Gram-negative bacteria, the identification of the first host receptors for T4S systems, the identification and characterization of novel T4S effector proteins, the analysis of the molecular function of effector proteins in subverting human cellular functions and an analysis of the role of T4S systems in the evolution of pathogenic bacteria. Our increasing knowledge of the biology of T4S systems improves our ability to exploit them as biotechnological tools or to use them as novel targets for a new generation of antimicrobials.
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Site-specific integration of foreign DNA into minimal bacterial and human target sequences mediated by a conjugative relaxase.
PLoS ONE
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Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.
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New perspectives into bacterial DNA transfer to human cells.
Trends Microbiol.
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The type IV secretion system (T4SS) VirB/D4 of the facultative intracellular pathogen Bartonella henselae is known to translocate bacterial effector proteins into human cells. Two recent reports on DNA transfer into human cells have demonstrated the versatility of this bacterial secretion system for macromolecular substrate transfer. Moreover, these findings have opened the possibility for developing new tools for DNA delivery into specific human cell types. DNA can be introduced into these cells covalently attached to a site-specific integrase with potential target sequences in the human genome. This novel DNA delivery system is discussed in the context of existing methods for genetic modification of human cells.
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