Characterization of ATP and DNA binding activities of TrwB, the coupling protein essential in plasmid R388 conjugation

TrwB is the conjugative coupling protein of plasmid R388. TrwBDeltaN70 contains the soluble domain of TrwB. It was constructed by deletion of trwB sequences containing TrwB N-proximal transmembrane segments. Purified TrwBDeltaN70 protein bound tightly the fluorescent ATP analogue TNP-ATP (K(s) = 8.7 microM) but did not show measurable ATPase or GTPase activity. A single ATP binding site was found per TrwB monomer. An intact ATP-binding site was essential for R388 conjugation, since a TrwB mutant with a single amino acid alteration in the ATP-binding signature (K136T) was transfer-deficient. TrwBDeltaN70 also bound DNA nonspecifically. DNA binding enhanced TrwC nic cleavage, providing the first evidence that directly links TrwB with conjugative DNA processing. Since DNA bound by TrwBDeltaN70 also showed increased negative superhelicity (as shown by increased sensitivity to topoisomerase I), nic cleavage enhancement was assumed to be a consequence of the increased single-stranded nature of DNA around nic. The mutant protein TrwB(K136T)DeltaN70 was indistinguishable from TrwBDeltaN70 with respect to the above properties, indicating that TrwB ATP binding activity is not required for them. The reported properties of TrwB suggest potential functions for conjugative coupling proteins, both as triggers of conjugative DNA processing and as motors in the transport process.     

Reconstitution in liposome bilayers enhances nucleotide binding affinity and ATP-specificity of TrwB conjugative coupling protein

Bacterial conjugative systems code for an essential membrane protein that couples the relaxosome to the DNA transport apparatus, called type IV coupling protein (T4CP). TrwB is the T4CP of the conjugative plasmid R388. In earlier work we found that this protein, purified in the presence of detergents, binds preferentially purine nucleotides trisphosphate. In contrast a soluble truncated mutant TrwBΔN70 binds uniformly all nucleotides tested. In this work, TrwB has been successfully reconstituted into liposomes. The non-membranous portion of the protein is almost exclusively oriented towards the outside of the vesicles. Functional analysis of TrwB proteoliposomes demonstrates that when the protein is inserted into the lipid bilayer the affinity for adenine and guanine nucleotides is enhanced as compared to that of the protein purified in detergent or to the soluble deletion mutant, TrwBΔN70. The protein specificity for adenine nucleotides is also increased. No ATPase activity has been found in TrwB reconstituted in proteoliposomes. This result suggests that the N-terminal transmembrane segment of this T4CP interferes with its ATPase activity and can be taken to imply that the TrwB transmembrane domain plays a regulatory role in its biological activity.     

Purification and properties of TrwB,a hexameric,ATP-binding integral membrane protein essential for R388 plasmid conjugation

TrwB is an integral membrane protein linking the relaxosome to the DNA transport apparatus in plasmid R388 conjugation. Native TrwB has been purified in monomeric and hexameric forms, in the presence of dodecylmaltoside from overexpressing bacterial cells. A truncated protein (TrwBDeltaN70) that lacked the transmembrane domain could be purified only in the monomeric form. Electron microscopy images revealed the hexameric structure and were in fact superimposable to the previously published atomic structure for TrwBDeltaN70. In addition, the electron micrographs showed an appendix, approximately 25 A wide, corresponding to the transmembrane region of TrwB. TrwB was located in the bacterial inner membrane in agreement with its proposed coupling role. Purified TrwB hexamers and monomers bound tightly the fluorescent ATP analogue TNP-ATP. A mutant in the Walker A motif, TrwB-K136T, was equally purified and found to bind TNP-ATP with a similar affinity to that of the wild type. However, the TNP-ATP affinity of TrwBDeltaN70 was significantly reduced in comparison with the TrwB hexamers. Competition experiments in which ATP was used to displace TNP-ATP gave an estimate of ATP binding by TrwB (K(d)((ATP)) = 0.48 mm for hexamers). The transmembrane domain appears to be involved in TrwB protein hexamerization and also influences its nucleotide binding properties.     

Role of the transmembrane domain in the stability of TrwB, an integral protein involved in bacterial conjugation

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 ATP-binding 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, TrwBDeltaN70. 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 TrwBDeltaN70 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 TrwBDeltaN70 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.     

Functional Dissection of the Conjugative Coupling Protein TrwB

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 TrwBΔN70, 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.Bacterial conjugation can be viewed mechanistically as a rolling-circle replication system linked to a type IV secretion process. The two processes come into contact through the activity of a protein that couples the plasmid replication machinery to the export system in the membrane, allowing horizontal dissemination of the replicating DNA molecule (35). This key protein is called “coupling protein” (here “T4CP” for “type IV CP”). It is present in all conjugative systems as well as in many type IV secretion systems (T4SS) involved in bacterial virulence (16). The secreted substrate in bacterial conjugation is the relaxase or pilot protein, attached to the DNA strand. The shoot-and-pump model for bacterial conjugation proposes that, after secretion of the protein through the T4SS, the T4CP works as a motor for export of the rest of the DNA molecule (36). In addition to its presumed role as a DNA transporter, TrwB is also required for transport of relaxase TrwC in the absence of DNA transfer (15).In accordance with its proposed coupling activity, early genetic experiments made patent that the function of conjugative T4CPs depended on interactions with both the cytoplasmic substrate complex (the relaxosome) and the T4SS (6, 7). Thus, T4CP interactions with other conjugation proteins are a key aspect of their function. There have been several reports of interactions between T4CPs from conjugative plasmids and either relaxosomal components—such as F-TraD with TraM (14, 38), RP4-TraG with TraI (49), and pCF10-PcfC with PcfF and PcfG (11)—or T4SS components such as R27-TraG with TrhB (17). T4CP-T4SS interactions have also been reported for the VirB/D4 T4SS involved in DNA transfer from Agrobacterium tumefaciens to plant cells (1, 9). Both sets of interactions have only been concurrently shown for TrwB, the T4CP of plasmid R388. TrwB interacts with proteins TrwA and TrwC, which form the R388 relaxosome, and with the R388 T4SS component TrwE (37). While the interaction with the relaxosome is highly specific for its cognate system (24, 37, 48), the interaction between the T4CP and the T4SS is less specific: a single T4CP can interact functionally with several conjugative T4SS. Interestingly, a correlation was observed between the strength of the T4CP-TrwE-like interaction and the efficiency of DNA transfer (37). T4CPs also interact with TrwE-like components of T4SS involved in virulence (13). In the case of the highly related Trw T4SS systems of plasmid R388 and the human pathogen Bartonella, it was further demonstrated that R388 TrwE could be functionally replaced by the Bartonella tribocorum TrwE homolog, TrwE_Bt (13).T4CPs are integral membrane proteins anchored to the inner membrane by an N-terminal transmembrane domain (TMD). The soluble cytoplasmic domain of TrwB (TrwBΔN70), lacking this TMD, has been biochemically and structurally analyzed in detail. It retains the ability to bind NTPs and to unspecifically bind DNA (42). The characterization of its DNA-dependent ATPase activity (53) strengthened the possibility that T4CPs work as DNA motors. This activity is also stimulated by the oriT-binding protein TrwA (52).The determination of the three-dimensional (3D) structure of TrwBΔN70 indicated a quaternary structure consisting of hexamers that form an almost spherical, orange-shaped structure with a 20-Å inner channel (ICH) (18, 19). Each monomer is composed of two main structural domains: the nucleotide-binding domain (NBD) and the all-alpha domain (AAD). The NBD has α/β topology and is reminiscent of RecA and DNA ring helicases. The AAD is facing the cytoplasmic side and bears significant structural similarity to the N-terminal domain of site-specific recombinase XerD and also to a 40-residue segment of the DNA binding domain of protein TraM, the component of the relaxosome of F-like plasmids that interacts with its cognate T4CP, TraD. The structure of the hexamer as a whole resembles that of the F₁-ATPase, raising interesting perspectives into the possible way of action of coupling proteins as molecular motors in conjugation (5).There have been several attempts to functionally dissect T4CPs. In F-TraD, it was determined that its C terminus is essential for relaxosomal specificity, probably through an interaction with TraM (4, 39, 48). The cytoplasmic domain of the related TraD protein of plasmid R1 stimulates both transesterase and helicase activities of its cognate relaxase, TraI (41, 51). A series of random mutations were shown to affect TraD oligomerization (23). In VirD4, the T4CP of the VirB T4SS of A. tumefaciens, both the periplasmic domain plus key residues of the NBD are required for its location at the cell poles (31); its interaction with the T4SS protein substrate VirE2 does not require the N-terminal TMD (2). Mutational analysis of R27 TraG showed that the periplasmic residues are essential for interaction with the T4SS (22). An N-terminal deletion variant of PcfC, the T4CP of the Enterococcus plasmid pCF10, loses its membrane localization but retains its ability to bind relaxosomal components (11). Biochemical analysis of full-length R388 TrwB showed that the N-terminal TMD stabilizes the protein, aids oligomerization, and affects nucleotide selection (25-27). This region is essential for T4SS interaction, but TrwBΔN70 retains the ability to interact with the relaxosomal components TrwA and TrwC (37). Taken together, these analyses suggested that the N-terminal TMD of the T4CPs is necessary for T4SS interaction, oligomerization, and cellular location and that the C-terminal cytoplasmic domain is necessary for relaxosomal interactions and ATPase activity associated with DNA transport.In this study, we set up different assays to search for mutants affecting TrwB function in DNA and protein transfer. We constructed a series of TrwB point mutants based on the 3D structure of TrwBΔN70. Most selected residues were essential for TrwB function in conjugation, especially under conditions where TrwB was in limiting quantities. We analyzed the in vivo properties of selected mutants with a battery of in vivo assays to map functional domains. Also, random mutants in the TMD were screened for improved interactions with the T4SS, which allowed mapping of the TrwB-TrwE interaction domain.     

DNA binding properties of protein TrwA, a possible structural variant of the Arc repressor superfamily

Conjugative DNA processing of plasmid R388 requires the concerted action of two proteins, the relaxase-helicase TrwC and the relaxase enhancer TrwA. TrwA can be aligned with DNA binding proteins belonging to the ribbon-helix-helix (RHH) protein family. To further analyse TrwA function, the structural domains of the protein have been identified and dissected by limited proteolysis. Two stable domains were found that resulted to be, according to DNA binding experiments and oligomerization analysis, an N-terminal DNA binding domain and a C-terminal tetramerization domain. Using the three-dimensional structure of the Arc repressor as a guide, it was possible to model TrwA DNA binding site with atomic detail. As a result, TrwA polar amino acids Q8, R10 and S12, contained in the polar face of a putative N-terminal beta-strand, were found to be directly involved in DNA binding, in a manner analogous to RHH proteins. In this respect, TrwA seemed to be a new member of the RHH family. However, secondary structure analyses underscored the existence of a substantial difference in the architecture of the TrwA-oriT complex when compared to the Arc repressor-operator complex.     

The Hexameric Structure of a Conjugative VirB4 Protein ATPase Provides New Insights for a Functional and Phylogenetic Relationship with DNA Translocases

VirB4 proteins are ATPases essential for pilus biogenesis and protein transport in type IV secretion systems. These proteins contain a motor domain that shares structural similarities with the motor domains of DNA translocases, such as the VirD4/TrwB conjugative coupling proteins and the chromosome segregation pump FtsK. Here, we report the three-dimensional structure of full-length TrwK, the VirB4 homologue in the conjugative plasmid R388, determined by single-particle electron microscopy. The structure consists of a hexameric double ring with a barrel-shaped structure. The C-terminal half of VirB4 proteins shares a striking structural similarity with the DNA translocase TrwB. Docking the atomic coordinates of the crystal structures of TrwB and FtsK into the EM map revealed a better fit for FtsK. Interestingly, we have found that like TrwB, TrwK is able to bind DNA with a higher affinity for G4 quadruplex structures than for single-stranded DNA. Furthermore, TrwK exerts a dominant negative effect on the ATPase activity of TrwB, which reflects an interaction between the two proteins. Our studies provide new insights into the structure-function relationship and the evolution of these DNA and protein translocases.     

Transfer of R388 derivatives by a pathogenesis-associated type IV secretion system into both bacteria and human cells

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 ΔvirB mutant strain was transfer deficient, while a ΔtrwE 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.     

Autoinhibitory regulation of TrwK, an essential VirB4 ATPase in type IV secretion systems

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. In a previous work we demonstrated that TrwK is able to hydrolyze ATP. Here, based on the structural homology of VirB4 proteins with the DNA-pumping ATPase TrwB coupling protein, we generated a series of variants of TrwK where fragments of the C-terminal domain were sequentially truncated. Surprisingly, the in vitro ATPase activity of these TrwK variants was much higher than that of the wild-type enzyme. Moreover, addition of a synthetic peptide containing the amino acid residues comprising this C-terminal region resulted in the specific inhibition of the TrwK variants lacking such domain. These results indicate that the C-terminal end of TrwK plays an important regulatory role in the functioning of the T4SS.     

Common Requirement for the Relaxosome of Plasmid R1 in Multiple Activities of the Conjugative Type IV Secretion System

Macromolecular transport by bacterial type IV secretion systems involves regulated uptake of (nucleo)protein complexes by the cell envelope-spanning transport channel. A coupling protein receptor is believed to recognize the specific proteins destined for transfer, but the steps initiating their translocation remain unknown. Here, we investigate the contribution of a complex of transfer initiation proteins, the relaxosome, of plasmid R1 to translocation of competing transferable substrates from mobilizable plasmids ColE1 and CloDF13 or the bacteriophage R17. We found that not only does the R1 translocation machinery engage the R1 relaxosome during conjugative self-transfer and during infection by R17 phage but it is also activated by its cognate relaxosome to mediate the export of an alternative plasmid. Transporter activity was optimized by the R1 relaxosome even when this complex itself could not be transferred, i.e., when the N-terminal activation domain (amino acids 1 to 992 [N_1-992]) of TraI was present without the C-terminal conjugative helicase domain. We propose that the functional dependence of the transfer machinery on the R1 relaxosome for initiating translocation ensures that dissemination of heterologous plasmids does not occur at the expense of self-transfer.     

Deletion of a single helix from the transmembrane domain causes large changes in membrane insertion properties and secondary structure of the bacterial conjugation protein TrwB

TrwB is an essential protein in the conjugative transfer of plasmid R388. The protein consists of a bulky cytosolic domain containing the catalytic site, and a small transmembrane domain (TMD). Our previous studies support the idea that the TMD plays an essential role in the activity, structure and stability of the protein. We have prepared a mutant, TrwBΔN50 that lacks one of the two α-helices in the TMD. The mutant has been studied both in detergent suspension and reconstituted in lipid vesicles. Deletion of a single helix from the TMD is enough to increase markedly the affinity of TrwB for ATP. The deletion changes the secondary structure of the cytosolic domain, whose infrared spectroscopy (IR) spectra become similar to those of the mutant TrwBΔN70 lacking the whole TMD. Interestingly, when TrwBΔN50 is reconstituted into lipid membranes, the cytosolic domain orients itself towards the vesicle interior, opposite to what happens for wild-type TrwB. In addition, we analyze the secondary structure of the TMD and TMD-lacking mutant TrwBΔN70, and found that the sum IR spectrum of the two protein fragments is different from that of the native protein, indicating the irreversibility of changes caused in TrwB by deletion of the TMD.     

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDΔN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (ΔL_k = −4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDΔN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membrane-spanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7, 8, 10, 30, 41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site- and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5′ end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3′-OH generated at nic and the subsequent DNA unwinding stage remain obscure.Equally little is known about the process of nucleoprotein uptake by the transport channel. DNA-independent translocation of the relaxases TrwC (R388), MobA (RSF1010), and VirD2 (Ti plasmid) has been demonstrated; thus, current models propose that the relaxase component of the protein-DNA adduct is the substrate actively secreted by the transport system after interaction with the T4CP (42, 66). Cotransport of the covalently linked single-stranded T strand occurs concurrently (42). The mechanisms underlying relaxosome recognition by T4CPs are not understood. Direct interactions have been observed biochemically between the RP4 TraG protein and relaxase proteins of the cognate plasmid (65) and heterologous relaxosomes that it mobilizes (73, 76). TrwB of R388 interacts in vitro with relaxase TrwC and an auxiliary component, TrwA (44). TraD proteins of plasmid R1 and F are known to interact with the auxiliary relaxosome protein TraM (20) via a cluster of C-terminal amino acids (3, 62). Extensive mutagenic analyses (45) plus recent three-dimensional structural data for a complex of the TraM tetramerization domain and the C-terminal tail of TraD (46) have provided more detailed models for the intermolecular contacts involved in recognition.Application of the Cre recombinase assay for translocation of conjugative relaxases as well as effector proteins to eukaryotic cells is currently the most promising approach to elucidate protein motifs recognized by T4CPs (56, 68, 78, 79). Despite that progress, the nature of the interactions between a T4CP and its target protein that initiate secretion and the mechanisms controlling this step remain obscure. In contrast to systems dedicated specifically to effector protein translocation, conjugation systems mobilize nucleoprotein complexes that additionally exhibit catalytic activities, which can be readily monitored. These models are therefore particularly well suited to investigate aspects of regulation occurring at the physical interface of a T4CP and its secretion substrate. For this purpose the MOB_F family of DNA-mobilizing systems is additionally advantageous, since DNA processing within this family features the fusion of a dedicated conjugative helicase to the DNA transesterase enzyme within a single bifunctional protein. The TraI protein of F-like plasmids, originally described as Escherichia coli DNA helicase I (1, 2, 23), and the related TrwC protein of plasmid R388 (25) are well characterized (reviewed in reference 18). Early work by Llosa et al. revealed a complex domain arrangement for TrwC (43). Similar analyses with TraI identified nonoverlapping transesterase and helicase domains (6, 77), while the remaining intermediate and C-terminal regions of the protein additionally provide functions essential to effective conjugative transfer (49, 71). The ability to physically separate the catalytic domains of TraI and TrwC has facilitated a detailed biochemical characterization of their DNA transesterase, ATPase, and DNA-unwinding reactions. Nonetheless, failure of the physically disjointed polypeptides to complement efficient conjugative transfer when coexpressed indicates a role(s) for these proteins in the strand transfer process that goes beyond the need for their dual catalytic activities (43, 50). The assignment of additional functional properties to regions within TraI is a focus of current investigation (16, 29, 49).In all systems studied thus far, conditions used to reconstitute relaxosomes on a supercoiled oriT plasmid have not supported the initiation steps necessary to enable duplex unwinding by a conjugative helicase. The question remains open whether additional protein components are required and/or whether the pathway of initiation is subject to specific repression. In the present study, we applied the IncFII plasmid R1 paradigm to investigate the potential for interaction between purified components of the relaxosome and its cognate T4CP, TraD, to exert regulatory effects on relaxosome activities in vitro. In this and in the accompanying report (72), we present evidence for wide-ranging stimulatory effects of the cytoplasmic domain of TraD protein and its interaction partner TraM on multiple aspects of relaxosome function.     

Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.

We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.     

Mechanism of DnaB helicase of Escherichia coli: structural domains involved in ATP hydrolysis, DNA binding, and oligomerization.

We describe the delineation of three distinct structural domains of the DnaB helicase of Escherichia coli: domain alpha, amino acid residues (aa) 1-156; domain beta, aa 157-302; and domain gamma, aa 303-471. Using mutants with deletion in these domains, we have examined their role(s) in hexamer formation, DNA-dependent ATPase, and DNA helicase activities. The mutant DnaBbetagamma protein, in which domain alpha was deleted, formed a hexamer; whereas the mutant DnaBalphabeta, in which domain gamma was deleted, could form only dimers. The dimerization of DnaBalphabeta was Mg(2+) dependent. These data suggest that the oligomerization of DnaB helicase involves at least two distinct protein-protein interaction sites; one of these sites is located primarily within domain beta (site 1), while the other interaction site is located within domain gamma (site 2). The mutant DnaBbeta, a polypeptide of 147 aa, where both domains alpha and gamma were deleted, displayed a completely functional ATPase activity. This domain, thus, constitutes the "central catalytic domain" for ATPase activity. The ATPase activity of DnaBalphabeta was kinetically comparable to that of DnaBbeta, indicating that domain alpha had little or no influence on the ATPase activity. In both cases, the ATPase activities were DNA independent. DnaBbetagamma had a DNA-dependent ATPase activity that was kinetically comparable to the ATPase activity of wild-type DnaB protein (wtDnaB), indicating a specific role for C-terminal domain gamma in enhancement of the ATPase activity of domain beta as well as in DNA binding. Mutant DnaBbetagamma, which lacked domain alpha, was devoid of any helicase activity pointing to a significant role for domain alpha. The major findings of this study are (i) domain beta contained a functional ATPase active site; (ii) domain gamma appeared to be the DNA binding domain and a positive regulator of the ATPase activity of domain beta; (iii) although domain alpha did not have any significant effect on the ATPase, DNA binding activities, or hexamer formation, it definitely plays a pivotal role in transducing the energy of ATP hydrolysis to DNA unwinding by the hexamer; and (iv) all three domains are required for helicase activity.     

Interaction of Bacteroides fragilis pLV22a relaxase and transfer DNA with Escherichia coli RP4-TraG coupling protein

Many Bacteroides transfer factors are mobilizable in Escherichia coli when coresident with the IncP conjugative plasmid RP4, but not F. To begin characterization and potential interaction between Bacteroides mobilizable transfer factors and the RP4 mating channel, both mutants and deletions of the DNA processing (dtr), mating pair formation (mpf) and traG coupling genes of RP4 were tested for mobilization of Bacteroides plasmid pLV22a. All 10 mpf but none of the four dtr genes were required for mobilization of pLV22a. The RP4 TraG coupling protein (CP) was also required for mobilization of pLV22a, but could be substituted by a C-terminal deletion mutant of the F TraD CP. Potential interactions of the TraG CP with relaxase protein(s) and transfer DNA of both RP4 and pLV22a were assessed. Overlay assays identified productive interactions between TraG and the relaxase proteins of both MbpB and TraI from pLV22a and RP4 respectively. The Agrobacterium Transfer-ImmunoPrecipitation (TrIP) assay also identified an interaction between TraG and both RP4 and pLV22a transfer DNA. Thus, mobilization of the Bacteroides pLV22a in E. coli utilizes both RP4 Mpf and CP functions including an interaction between the relaxosome and the RP4 CP similar to that of cognate RP4 plasmid.     

Evidence for the role of DNA strand passage in the mechanism of action of microcin B17 on DNA gyrase

Microcin B17 (MccB17) is a DNA gyrase poison; in previous work, this bacterial toxin was found to slowly and incompletely inhibit the reactions of supercoiling and relaxation of DNA by gyrase and to stabilize the cleavage complex, depending on the presence of ATP and the DNA topology. We now show that the action of MccB17 on the gyrase ATPase reaction and cleavage complex formation requires a linear DNA fragment of more than 150 base pairs. MccB17 is unable to stimulate the ATPase reaction by stabilizing the weak interactions between short linear DNA fragments (70 base pairs or less) and gyrase, in contrast with the quinolone ciprofloxacin. However, MccB17 can affect the ATP-dependent relaxation of DNA by gyrase lacking its DNA-wrapping or ATPase domains. From these findings, we propose a mode of action of MccB17 requiring a DNA molecule long enough to allow the transport of a segment through the DNA gate of the enzyme. Furthermore, we suggest that MccB17 may trap a transient intermediate state of the gyrase reaction present only during DNA strand passage and enzyme turnover. The proteolytic signature of MccB17 from trypsin treatment of the full enzyme requires DNA and ATP and shows a protection of the C-terminal 47-kDa domain of gyrase, indicating the involvement of this domain in the toxin mode of action and consistent with its proposed role in the mechanism of DNA strand passage. We suggest that the binding site of MccB17 is in the C-terminal domain of GyrB.