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Fernando De La Cruz - One of the best experts on this subject based on the ideXlab platform.
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Conjugation inhibitors compete with palmitic acid for binding to the conjugative traffic ATPase TrwD, providing a mechanism to inhibit Bacterial Conjugation
The Journal of biological chemistry, 2018Co-Authors: Yolanda García-cazorla, María Getino, David J. Sanabria-ríos, Ignacio Arechaga, Fernando De La Cruz, Néstor M. Carballeira, Elena CabezónAbstract:Bacterial Conjugation is a key mechanism by which bacteria acquire antibiotic resistance. Therefore, Conjugation inhibitors (COINs) are promising compounds in the fight against the spread of antibiotic resistance genes among bacteria. Unsaturated fatty acids (uFAs) and alkynoic fatty acid derivatives, such as 2-hexadecanoic acid (2-HDA), have been reported previously as being effective COINs. The traffic ATPase TrwD, a VirB11 homolog in plasmid R388, is the molecular target of these compounds, which likely affect binding of TrwD to Bacterial membranes. In this work, we demonstrate that COINs are abundantly incorporated into Escherichia coli membranes, replacing palmitic acid as the major component of the membrane. We also show that TrwD binds palmitic acid, thus facilitating its interaction with the membrane. Our findings also suggest that COINs bind TrwD at a site that is otherwise occupied by palmitic acid. Accordingly, molecular docking predictions with palmitic acid indicated that it shares the same binding site as uFAs and 2-HDA, although it differs in the contacts involved in this interaction. We also identified 2-bromopalmitic acid, a palmitate analog that inhibits many membrane-associated enzymes, as a compound that effectively reduces TrwD ATPase activity and Bacterial Conjugation. Moreover, we demonstrate that 2-bromopalmitic and palmitic acids both compete for the same binding site in TrwD. Altogether, these detailed findings open up a new avenue in the search for effective synthetic inhibitors of Bacterial Conjugation, which may be pivotal for combating multidrug-resistant bacteria.
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Type IV traffic ATPase TrwD as molecular target to inhibit Bacterial Conjugation
Molecular Microbiology, 2016Co-Authors: Jorge Ripoll-rozada, Yolanda García-cazorla, María Getino, Cristina Machón, David J. Sanabria-ríos, Elena Cabezón, Fernando De La Cruz, Ignacio ArechagaAbstract:Summary Bacterial Conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific Conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit Bacterial Conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic ATPase, as the molecular target for fatty acid-mediated inhibition of Conjugation. Moreover, 2-alkynoic fatty acids, which are also potent inhibitors of Bacterial Conjugation, are also powerful inhibitors of the ATPase activity of TrwD. Characterization of the kinetic parameters of ATPase inhibition has led us to identify the catalytic mechanism by which fatty acids exert their activity. These results open a new avenue for the rational design of inhibitors of Bacterial Conjugation in the fight against the dissemination of antibiotic resistance genes.
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Tanzawaic Acids, a Chemically Novel Set of Bacterial Conjugation Inhibitors
PloS one, 2016Co-Authors: María Getino, Raul Fernandez-lopez, Carolina Palencia-gándara, Javier Campos-gómez, Jose M. Sánchez-lópez, Marta Martínez, Antonio Fernández, Fernando De La CruzAbstract:Bacterial Conjugation is the main mechanism for the dissemination of multiple antibiotic resistance in human pathogens. This dissemination could be controlled by molecules that interfere with the Conjugation process. A search for Conjugation inhibitors among a collection of 1,632 natural compounds, identified tanzawaic acids A and B as best hits. They specially inhibited IncW and IncFII conjugative systems, including plasmids mobilized by them. Plasmids belonging to IncFI, IncI, IncL/M, IncX and IncH incompatibility groups were targeted to a lesser extent, whereas IncN and IncP plasmids were unaffected. Tanzawaic acids showed reduced toxicity in Bacterial, fungal or human cells, when compared to synthetic Conjugation inhibitors, opening the possibility of their deployment in complex environments, including natural settings relevant for antibiotic resistance dissemination.
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Towards an integrated model of Bacterial Conjugation
FEMS microbiology reviews, 2014Co-Authors: Elena Cabezón, Jorge Ripoll-rozada, Fernando De La Cruz, Alejandro Peña, Ignacio ArechagaAbstract:Bacterial Conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in Bacterial Conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating expansion of antibiotic resistance.
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Changing the recognition site of a conjugative relaxase by rational design.
Biotechnology journal, 2009Co-Authors: Blanca González-pérez, Gabriel Moncalián, José Daniel Carballeira, Fernando De La CruzAbstract:TrwC is a relaxase protein, which starts and finishes DNA processing during Bacterial Conjugation in plasmid R388. TrwC recognizes a specific sequence of DNA (25 nucleotides) in the donor cell: the nic-site. As a model example, a single transversion C24G in nic avoids DNA processing by TrwC. Using this simple model, our objective was to obtain a proof of principle that TrwC specificity can be changed. Several structures of DNA-TrwC complexes were used as reference to design a focused saturation mutagenesis library (NNK) randomizing amino acid Lys262, since its side chain seems to sterically hinder the recognition of the C24G nic mutation by wild-type TrwC. Using Bacterial Conjugation as an in vivo selection system, several TrwC variants were found that show changes in substrate specificity. These variants were also tested in a competitive assay to evaluate their Conjugation efficiency.
Elena Cabezón - One of the best experts on this subject based on the ideXlab platform.
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Conjugation inhibitors compete with palmitic acid for binding to the conjugative traffic ATPase TrwD, providing a mechanism to inhibit Bacterial Conjugation
The Journal of biological chemistry, 2018Co-Authors: Yolanda García-cazorla, María Getino, David J. Sanabria-ríos, Ignacio Arechaga, Fernando De La Cruz, Néstor M. Carballeira, Elena CabezónAbstract:Bacterial Conjugation is a key mechanism by which bacteria acquire antibiotic resistance. Therefore, Conjugation inhibitors (COINs) are promising compounds in the fight against the spread of antibiotic resistance genes among bacteria. Unsaturated fatty acids (uFAs) and alkynoic fatty acid derivatives, such as 2-hexadecanoic acid (2-HDA), have been reported previously as being effective COINs. The traffic ATPase TrwD, a VirB11 homolog in plasmid R388, is the molecular target of these compounds, which likely affect binding of TrwD to Bacterial membranes. In this work, we demonstrate that COINs are abundantly incorporated into Escherichia coli membranes, replacing palmitic acid as the major component of the membrane. We also show that TrwD binds palmitic acid, thus facilitating its interaction with the membrane. Our findings also suggest that COINs bind TrwD at a site that is otherwise occupied by palmitic acid. Accordingly, molecular docking predictions with palmitic acid indicated that it shares the same binding site as uFAs and 2-HDA, although it differs in the contacts involved in this interaction. We also identified 2-bromopalmitic acid, a palmitate analog that inhibits many membrane-associated enzymes, as a compound that effectively reduces TrwD ATPase activity and Bacterial Conjugation. Moreover, we demonstrate that 2-bromopalmitic and palmitic acids both compete for the same binding site in TrwD. Altogether, these detailed findings open up a new avenue in the search for effective synthetic inhibitors of Bacterial Conjugation, which may be pivotal for combating multidrug-resistant bacteria.
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Type IV traffic ATPase TrwD as molecular target to inhibit Bacterial Conjugation
Molecular Microbiology, 2016Co-Authors: Jorge Ripoll-rozada, Yolanda García-cazorla, María Getino, Cristina Machón, David J. Sanabria-ríos, Elena Cabezón, Fernando De La Cruz, Ignacio ArechagaAbstract:Summary Bacterial Conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific Conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit Bacterial Conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic ATPase, as the molecular target for fatty acid-mediated inhibition of Conjugation. Moreover, 2-alkynoic fatty acids, which are also potent inhibitors of Bacterial Conjugation, are also powerful inhibitors of the ATPase activity of TrwD. Characterization of the kinetic parameters of ATPase inhibition has led us to identify the catalytic mechanism by which fatty acids exert their activity. These results open a new avenue for the rational design of inhibitors of Bacterial Conjugation in the fight against the dissemination of antibiotic resistance genes.
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Towards an integrated model of Bacterial Conjugation
FEMS microbiology reviews, 2014Co-Authors: Elena Cabezón, Jorge Ripoll-rozada, Fernando De La Cruz, Alejandro Peña, Ignacio ArechagaAbstract:Bacterial Conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in Bacterial Conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating expansion of antibiotic resistance.
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TrwB: An F1-ATPase-like molecular motor involved in DNA transport during Bacterial Conjugation
Research in microbiology, 2005Co-Authors: Elena Cabezón, Fernando De La CruzAbstract:The mechanism by which TrwB acts as a DNA transporter in Bacterial Conjugation is analyzed. Based on a parallelism between TrwB and F(1)-ATPase, TrwB would use the energy derived from ATP hydrolysis to pump DNA through its central channel, in a manner similar to that used by F(1)-ATPase to produce a rotary movement of the central gamma-subunit.
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TrwB, the coupling protein involved in DNA transport during Bacterial Conjugation, is a DNA-dependent ATPase.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: I. Tato, Sandra Zunzunegui, F. De La Cruz, Elena CabezónAbstract:Bacterial Conjugation is an example of macromolecular trafficking between cells, based on the translocation of single-stranded DNA across membranes through a type IV secretion system. TrwBΔN70 is the soluble domain of TrwB, an essential integral membrane protein that couples the relaxosome (a nucleoprotein complex) to the DNA transport apparatus in plasmid R388 Conjugation. TrwBΔN70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. In this work, we characterize a DNA-dependent ATPase activity for TrwBΔN70. The protein displays positive cooperativity for ATP hydrolysis, with at least three catalytic sites involved. The activity is sensitive to pH and salt concentration, being more active at low pH values. The effective oligonucleotide size required for activation of the ATPase function is between 40 and 45 nucleotides, and the same length is required for the formation of high-molecular-weight TrwBΔN70–DNA complexes, as observed by gel filtration chromatography. A mutation in a tryptophan residue (W216A), placed in the central pore formed by the hexameric structure, resulted in a protein that did not hydrolyze ATP. In addition, it exerted a dominant negative effect, both on R388 Conjugation frequency and ATP hydrolysis, underscoring the multimeric state of the protein. ATP hydrolysis was not coupled to a DNA unwinding activity under the tested conditions, which included forked DNA substrates. These results, together with TrwB structural similarity to F1-ATPase, lead us to propose a mechanism for TrwB as a DNA-translocating motor.
Itziar Alkorta - One of the best experts on this subject based on the ideXlab platform.
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Type IV Coupling Proteins as Potential Targets to Control the Dissemination of Antibiotic Resistance.
Frontiers in molecular biosciences, 2020Co-Authors: Itxaso Álvarez-rodríguez, Lide Arana, Begoña Ugarte-uribe, Elena Gomez-rubio, Sonsoles Martín-santamaría, Carlos Garbisu, Itziar AlkortaAbstract:The increase of infections caused by multidrug-resistant bacteria, together with the loss of effectiveness of currently available antibiotics, represents one of the most serious threats to public health worldwide. The loss of human lives and the economic costs associated to the problem of the dissemination of antibiotic resistance require immediate action. Bacteria, known by their great genetic plasticity, are capable not only of mutating their genes to adapt to disturbances and environmental changes but also of acquiring new genes that allow them to survive in hostile environments, such as in the presence of antibiotics. One of the major mechanisms responsible for the horizontal acquisition of new genes (e.g., antibiotic resistance genes) is Bacterial Conjugation, a process mediated by mobile genetic elements such as conjugative plasmids and integrative conjugative elements. Conjugative plasmids harboring antibiotic resistance genes can be transferred from a donor to a recipient bacterium in a process that requires physical contact. After Conjugation, the recipient bacterium not only harbors the antibiotic resistance genes but it can also transfer the acquired plasmid to other bacteria, thus contributing to the spread of antibiotic resistance. Conjugative plasmids have genes that encode all the proteins necessary for the Conjugation to take place, such as the type IV coupling proteins (T4CPs) present in all conjugative plasmids. Type VI coupling proteins constitute a heterogeneous family of hexameric ATPases that use energy from the ATP hydrolysis for plasmid transfer. Taking into account their essential role in Bacterial Conjugation, T4CPs are attractive targets for the inhibition of Bacterial Conjugation and, concomitantly, the limitation of antibiotic resistance dissemination. This review aims to compile present knowledge on T4CPs as a starting point for delving into their molecular structure and functioning in future studies. Likewise, the scientific literature on Bacterial Conjugation inhibitors has been reviewed here, in an attempt to elucidate the possibility of designing T4CP-inhibitors as a potential solution to the dissemination of multidrug-resistant bacteria.
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The transmembrane domain provides nucleotide binding specificity to the Bacterial Conjugation protein TrwB.
FEBS letters, 2006Co-Authors: Itsaso Hormaeche, Fernando De La Cruz, Rosa L. Segura, Ana J. Vecino, Félix M. Goñi, Itziar AlkortaAbstract:Abstract In order to understand the functional significance of the transmembrane domain of TrwB, an integral membrane protein involved in Bacterial Conjugation, the protein was purified in the native, and also as a truncated soluble form (TrwBΔN70). The intact protein (TrwB) binds preferentially purine over pyrimidine nucleotides, NTPs over NDPs, and ribo- over deoxyribonucleotides. In contrast, TrwBΔN70 binds uniformly all tested nucleotides. The transmembrane domain has the general effect of making the nucleotide binding site(s) less accessible, but more selective. This is in contrast to other membrane proteins in which most of the protein mass, including the catalytic domain, is outside the membrane, but whose activity is not modified by the presence or absence of the transmembrane segment.
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Role of the Transmembrane Domain in the Stability of TrwB, an Integral Protein Involved in Bacterial Conjugation
The Journal of biological chemistry, 2003Co-Authors: Itsaso Hormaeche, Fernando De La Cruz, Félix M. Goñi, Ibon Iloro, José Luis R. Arrondo, Itziar AlkortaAbstract:TrwB is an integral membrane protein encoded by the conjugative plasmid R388. TrwB binds ATP and is essential for R388-directed Bacterial Conjugation. The protein consists of a cytosolic domain, which contains an ATPbinding site, and a transmembrane domain. The complete protein has been purified in the presence of detergents, and in addition, the cytosolic domain has also been isolated in the form of a soluble truncated protein, TrwBN70. The availability of intact and truncated forms of the protein provides a convenient system to study the role of the transmembrane domain in the stability of TrwB. Protein denaturation was achieved by heat, in the presence of guanidinium HCl, or under low salt conditions. In all three cases TrwB was significantly more stable than TrwBN70 with other conditions being the same. IR spectroscopy of the native and truncated forms revealed significant differences between them. In addition, it was found that TrwBN70 was stabilized in dispersions of non-ionic detergent, suggesting the presence of hydrophobic patches on the surface of the truncated protein. IR spectroscopy also confirmed the conformational stability provided by the detergent. These results suggest that in integral membrane proteins consisting of a transmembrane and a cytosolic domain, the transmembrane portion may have a role beyond the mere anchoring of the protein to the cell membrane. In addition, this study indicates that the truncated soluble parts of two-domain membrane proteins may not reflect the physiological conformation of their native counterparts.
Ignacio Arechaga - One of the best experts on this subject based on the ideXlab platform.
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Conjugation inhibitors compete with palmitic acid for binding to the conjugative traffic ATPase TrwD, providing a mechanism to inhibit Bacterial Conjugation
The Journal of biological chemistry, 2018Co-Authors: Yolanda García-cazorla, María Getino, David J. Sanabria-ríos, Ignacio Arechaga, Fernando De La Cruz, Néstor M. Carballeira, Elena CabezónAbstract:Bacterial Conjugation is a key mechanism by which bacteria acquire antibiotic resistance. Therefore, Conjugation inhibitors (COINs) are promising compounds in the fight against the spread of antibiotic resistance genes among bacteria. Unsaturated fatty acids (uFAs) and alkynoic fatty acid derivatives, such as 2-hexadecanoic acid (2-HDA), have been reported previously as being effective COINs. The traffic ATPase TrwD, a VirB11 homolog in plasmid R388, is the molecular target of these compounds, which likely affect binding of TrwD to Bacterial membranes. In this work, we demonstrate that COINs are abundantly incorporated into Escherichia coli membranes, replacing palmitic acid as the major component of the membrane. We also show that TrwD binds palmitic acid, thus facilitating its interaction with the membrane. Our findings also suggest that COINs bind TrwD at a site that is otherwise occupied by palmitic acid. Accordingly, molecular docking predictions with palmitic acid indicated that it shares the same binding site as uFAs and 2-HDA, although it differs in the contacts involved in this interaction. We also identified 2-bromopalmitic acid, a palmitate analog that inhibits many membrane-associated enzymes, as a compound that effectively reduces TrwD ATPase activity and Bacterial Conjugation. Moreover, we demonstrate that 2-bromopalmitic and palmitic acids both compete for the same binding site in TrwD. Altogether, these detailed findings open up a new avenue in the search for effective synthetic inhibitors of Bacterial Conjugation, which may be pivotal for combating multidrug-resistant bacteria.
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Type IV traffic ATPase TrwD as molecular target to inhibit Bacterial Conjugation
Molecular Microbiology, 2016Co-Authors: Jorge Ripoll-rozada, Yolanda García-cazorla, María Getino, Cristina Machón, David J. Sanabria-ríos, Elena Cabezón, Fernando De La Cruz, Ignacio ArechagaAbstract:Summary Bacterial Conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific Conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit Bacterial Conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic ATPase, as the molecular target for fatty acid-mediated inhibition of Conjugation. Moreover, 2-alkynoic fatty acids, which are also potent inhibitors of Bacterial Conjugation, are also powerful inhibitors of the ATPase activity of TrwD. Characterization of the kinetic parameters of ATPase inhibition has led us to identify the catalytic mechanism by which fatty acids exert their activity. These results open a new avenue for the rational design of inhibitors of Bacterial Conjugation in the fight against the dissemination of antibiotic resistance genes.
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Towards an integrated model of Bacterial Conjugation
FEMS microbiology reviews, 2014Co-Authors: Elena Cabezón, Jorge Ripoll-rozada, Fernando De La Cruz, Alejandro Peña, Ignacio ArechagaAbstract:Bacterial Conjugation is one of the main mechanisms for horizontal gene transfer. It constitutes a key element in the dissemination of antibiotic resistance and virulence genes to human pathogenic bacteria. DNA transfer is mediated by a membrane-associated macromolecular machinery called Type IV secretion system (T4SS). T4SSs are involved not only in Bacterial Conjugation but also in the transport of virulence factors by pathogenic bacteria. Thus, the search for specific inhibitors of different T4SS components opens a novel approach to restrict plasmid dissemination. This review highlights recent biochemical and structural findings that shed new light on the molecular mechanisms of DNA and protein transport by T4SS. Based on these data, a model for pilus biogenesis and substrate transfer in conjugative systems is proposed. This model provides a renewed view of the mechanism that might help to envisage new strategies to curb the threating expansion of antibiotic resistance.
Miquel Coll - One of the best experts on this subject based on the ideXlab platform.
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Cut and move: protein machinery for DNA processing in Bacterial Conjugation
Current opinion in structural biology, 2006Co-Authors: F. Xavier Gomis-rüth, Miquel CollAbstract:Conjugation is a paradigmatic example of horizontal or lateral gene transfer, whereby DNA is translocated between Bacterial cells. It provides a route for the rapid acquisition of new genetic information. Increased antibiotic resistance among pathogens is a troubling consequence of this microbial capacity. DNA transfer across cell membranes requires a sophisticated molecular machinery that involves the participation of several proteins in DNA processing and replication, cell recruitment, and the transport of DNA and proteins from donor to recipient cells. Although Bacterial Conjugation was first reported in the 1940s, only now are we beginning to unravel the molecular mechanisms behind this process. In particular, structural biology is revealing the detailed molecular architecture of several of the pieces involved.
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Bacterial Conjugation a two step mechanism for dna transport
Molecular Microbiology, 2002Co-Authors: Matxalen Llosa, Miquel Coll, Xavier F Gomisruth, Fernando De La CruzAbstract:Summary Bacterial Conjugation is a promiscuous DNA trans- port mechanism. Conjugative plasmids transfer them- selves between most bacteria, thus being one of the main causal agents of the spread of antibiotic resis- tance among pathogenic bacteria. Moreover, DNA can be transferred conjugatively into eukaryotic host cells. In this review, we aim to address several basic questions regarding the DNA transfer mechanism. Conjugation can be visualized as a DNA rolling-circle replication (RCR) system linked to a type IV secretion system (T4SS), the latter being macromolecular transporters widely involved in pathogenic mecha- nisms. The scheme 'replication + secretion' suggests how the mechanism would work on the DNA sub- strate and at the Bacterial membrane. But, how do these two parts come into contact? Furthermore, how is the DNA transported? T4SS are known to be involved in protein secretion in different organisms, but DNA is a very different macromolecule. The so- called coupling proteins could be the answer to both questions by performing a dual role in Conjugation: coupling the two main components of the machinery (RCR and T4SS) and actively mediating DNA trans- port. We postulate that the T4SS is responsible for transport of the pilot protein (the relaxase) to the recipient. The DNA that is covalently linked to it is initially transported in a passive manner, trailing on the relaxase. We speculate that the pilus appendage could work as a needle, thrusting the substrate pro- teins to cross one or several membrane barriers into
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Bacterial Conjugation: a two‐step mechanism for DNA transport
Molecular microbiology, 2002Co-Authors: Matxalen Llosa, F. Xavier Gomis-rüth, Miquel Coll, Fernando De La CruzAbstract:Summary Bacterial Conjugation is a promiscuous DNA trans- port mechanism. Conjugative plasmids transfer them- selves between most bacteria, thus being one of the main causal agents of the spread of antibiotic resis- tance among pathogenic bacteria. Moreover, DNA can be transferred conjugatively into eukaryotic host cells. In this review, we aim to address several basic questions regarding the DNA transfer mechanism. Conjugation can be visualized as a DNA rolling-circle replication (RCR) system linked to a type IV secretion system (T4SS), the latter being macromolecular transporters widely involved in pathogenic mecha- nisms. The scheme 'replication + secretion' suggests how the mechanism would work on the DNA sub- strate and at the Bacterial membrane. But, how do these two parts come into contact? Furthermore, how is the DNA transported? T4SS are known to be involved in protein secretion in different organisms, but DNA is a very different macromolecule. The so- called coupling proteins could be the answer to both questions by performing a dual role in Conjugation: coupling the two main components of the machinery (RCR and T4SS) and actively mediating DNA trans- port. We postulate that the T4SS is responsible for transport of the pilot protein (the relaxase) to the recipient. The DNA that is covalently linked to it is initially transported in a passive manner, trailing on the relaxase. We speculate that the pilus appendage could work as a needle, thrusting the substrate pro- teins to cross one or several membrane barriers into
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The Bacterial Conjugation protein TrwB resembles ring helicases and F1-ATPase
Nature, 2001Co-Authors: F. Xavier Gomis-rüth, Elena Cabezón, Fernando De La Cruz, Gabriel Moncalián, Rosa Pérez-luque, Ana Gonzalez, Miquel CollAbstract:The transfer of DNA across membranes and between cells is a central biological process; however, its molecular mechanism remains unknown. In prokaryotes, trans-membrane passage by Bacterial Conjugation, is the main route for horizontal gene transfer. It is the means for rapid acquisition of new genetic information, including antibiotic resistance by pathogens. Trans-kingdom gene transfer from bacteria to plants1 or fungi2 and even Bacterial sporulation3 are special cases of Conjugation. An integral membrane DNA-binding protein, called TrwB in the Escherichia coli R388 conjugative system, is essential for the Conjugation process. This large multimeric protein is responsible for recruiting the relaxosome DNA–protein complex, and participates in the transfer of a single DNA strand during cell mating. Here we report the three-dimensional structure of a soluble variant of TrwB. The molecule consists of two domains: a nucleotide-binding domain of α/β topology, reminiscent of RecA and DNA ring helicases, and an all-α domain. Six equivalent protein monomers associate to form an almost spherical quaternary structure that is strikingly similar to F1-ATPase. A central channel, 20 A in width, traverses the hexamer.
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Structure of TrwB, a gatekeeper in Bacterial Conjugation.
The international journal of biochemistry & cell biology, 2001Co-Authors: F. Xavier Gomis-rüth, Miquel CollAbstract:Bacterial Conjugation implies a trans-membrane passage of DNA, mediated by proteins encoded in conjugative plasmids. This results in a spread of genetic information, including antibiotic resistance acquisition by pathogens. Special cases of Conjugation are trans-kingdom gene transfer from bacteria to plants or fungi, and even Bacterial sporulation and cell division. One of the main actors in this process is an integral inner membrane DNA-binding protein, called TrwB in the E. coli R388 conjugative system. It is responsible for coupling the single-strand DNA to be transferred from the donor to the acceptor cell in its complex with other proteins, with a type IV secretion system making up the mating apparatus. The TrwB protomer consists of two domains: a nucleotide-binding domain of alpha/beta topology, similar to RecA and DNA ring helicases, and an all-alpha domain. The quaternary structure reveals an almost spherical homohexamer, strikingly similar to F(1)-ATPase. A central 20 A wide channel traverses the hexamer, thus connecting cytoplasm with periplasm.