The 2.5 Å Structure of the Enterococcus Conjugation Protein TraM resembles VirB8 Type IV Secretion Proteins

Goessweiner‐Mohr, Nikolaus; Grumet, Lukas; Arends, Karsten; Pavkov‐Keller, Tea; Gruber, Christian; Gruber, Karl; Birner‐Gruenberger, Ruth; Kropec-Huebner, Andrea; Huebner, Johannes; Grohmann, Elisabeth; Keller, Walter

doi:10.1074/jbc.m112.428847

Cited by 50 publications

(97 citation statements)

References 82 publications

(109 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…6). Similar models have been proposed recently for the conjugative plasmid pCW3 of Clostridium perfringens (10,40,46,48,50,51), the broad-host-range conjugative plasmid pIP501 (12,41,52), and pCF10 of Enterococcus faecalis (11,53,54), indicating that a general consensus is building as to the composition of the Gram-positive T4SS. ConG and ConB may form a major portion of the ICEBs1 DNA translocation channel within the membrane.…”

Section: Discussionmentioning

confidence: 87%

See 1 more Smart Citation

Critical Components of the Conjugation Machinery of the Integrative and Conjugative Element ICE Bs1 of Bacillus subtilis

et al. 2015

View full text Add to dashboard Cite

Conjugation, or mating, plays a profound role in bacterial evolution by spreading genes that allow bacteria to adapt to and colonize new niches. ICEBs1, an integrative and conjugative element of Bacillus subtilis, can transfer itself and mobilize resident plasmids. DNA transfer is mediated by a type IV secretion system (T4SS). Characterized components of the ICEBs1 T4SS include the conserved VirB4-like ATPase ConE, the bifunctional cell wall hydrolase CwlT, and the presumed VirD4-like coupling protein ConQ. A fusion of ConE to green fluorescent protein (GFP) localizes to the membrane preferentially at the cell poles. One or more ICEBs1 proteins are required for ConE's localization at the membrane, as ConE lacks predicted transmembrane segments and ConE-GFP is found dispersed throughout the cytoplasm in cells lacking ICEBs1. Here, we analyzed five ICEBs1 genes to determine if they are required for DNA transfer and/or ConE-GFP localization. We found that conB, conC, conD, and conG, but not yddF, are required for both ICEBs1 transfer and plasmid mobilization. All four required genes encode predicted integral membrane proteins. conB and, to some extent, conD were required for localization of ConE-GFP to the membrane. Using an adenylate cyclase-based bacterial two-hybrid system, we found that ConE interacts with ConB. We propose a model in which the ICEBs1 conjugation machinery is composed of ConB, ConC, ConD, ConE, ConG, CwlT, ConQ, and possibly other ICEBs1 proteins, and that ConB interacts with ConE, helping to recruit and/or maintain ConE at the membrane. IMPORTANCEConjugation is a major form of horizontal gene transfer and has played a profound role in bacterial evolution by moving genes, including those involved in antibiotic resistance, metabolism, symbiosis, and infectious disease. During conjugation, DNA is transferred from cell to cell through the conjugation machinery, a type of secretion system. Relatively little is known about the conjugation machinery of Gram-positive bacteria. Here, we analyzed five genes of the integrative and conjugative element ICEBs1 of Bacillus subtilis. Our research identifies four new components of the ICEBs1 conjugation machinery (ConB, ConC, ConD, and ConG) and shows an interaction between ConB and ConE that is required for ConE to associate with the cell membrane. Conjugation is a major form of horizontal gene transfer and plays a profound role in bacterial evolution and the acquisition of new traits (1-3). Conjugation can spread antibiotic resistance and disseminate genes involved in symbiosis, degradation of pollutants, metabolism, and pathogenesis. Conjugative elements encode specialized DNA translocation channels classified as type IV secretion systems (T4SSs) (4-7). T4SSs are composed of many interacting proteins that span the envelope of the donor cell. In addition to transferring the conjugative DNA element, the conjugation machinery also can mobilize resident plasmids or other DNA elements that do not encode their own machinery.There is a rich body of mechanis...

show abstract

Section: Discussionmentioning

confidence: 87%

“…1a), as each had characteristics that suggested it encoded a component of the ICEBs1 T4SS. ConB is a putative bitopic membrane protein homologous to conjugation proteins that are structurally similar, but not phylogenetically related, to VirB8 (40,41). ConC is a putative integral membrane protein with two predicted transmembrane helices (8).…”

Section: Resultsmentioning

confidence: 99%

Critical Components of the Conjugation Machinery of the Integrative and Conjugative Element ICE Bs1 of Bacillus subtilis

et al. 2015

View full text Add to dashboard Cite

show abstract

“…For conjugation systems in Gram-negative bacteria, structural resolution of the outer membrane core complex and the inner membrane complex of the T4SS, as well as information about the pathway of DNA translocation across the T4SS in the donor cell, has been elucidated (3)(4)(5)(6)(7)(8)(9)(10)(11). Less is known about conjugation systems in Gram-positive bacteria, with our current understanding based primarily on comparisons to those in Gramnegative bacteria and with the systems in Gram-positive bacteria proposed to involve a "minimized" T4SS (3,(12)(13)(14)(15)(16)(17)(18).…”

mentioning

confidence: 99%

Two Novel Membrane Proteins, TcpD and TcpE, Are Essential for Conjugative Transfer of pCW3 in Clostridium perfringens

et al. 2015

View full text Add to dashboard Cite

The anaerobic pathogen Clostridium perfringens encodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, the tcp locus. Studies of the paradigm conjugative plasmid from C. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified several tcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus. In this study, the role of the conserved hypothetical proteins TcpD, TcpE, and TcpJ was examined. Mutation and complementation analyses showed that TcpD and TcpE were essential for the conjugative transfer of pCW3, whereas TcpJ was not required. To analyze the TcpD and TcpE proteins in C. perfringens, functional hemagglutinin (HA)-tagged derivatives were constructed. Western blots showed that TcpD and TcpE localized to the cell envelope fraction independently of the presence of other pCW3-encoded proteins. Finally, examination of the subcellular localization of TcpD and TcpE by immunofluorescence showed that these proteins were concentrated at both poles of C. perfringens donor cells, where they are postulated to form essential components of the multiprotein complex that comprises the transfer apparatus. Conjugative transfer of plasmid DNA is an important process that contributes to bacterial genome plasticity and adaption, particularly the spread of antibiotic resistance and virulence genes (1, 2). The translocation of a conjugative plasmid relies on the coupling protein, the relaxosome complex, and a type IV secretion system (T4SS), known as the mating-pair formation (Mpf) apparatus (1). For conjugation systems in Gram-negative bacteria, structural resolution of the outer membrane core complex and the inner membrane complex of the T4SS, as well as information about the pathway of DNA translocation across the T4SS in the donor cell, has been elucidated (3-11). Less is known about conjugation systems in Gram-positive bacteria, with our current understanding based primarily on comparisons to those in Gramnegative bacteria and with the systems in Gram-positive bacteria proposed to involve a "minimized" T4SS (3, 12-18).The Gram-positive, anaerobic pathogen Clostridium perfringens carries many disease-mediating toxin genes and antibiotic resistance genes on a unique family of large plasmids that have large regions of sequence identity (19)(20)(21)(22). Most of these plasmids carry the novel tcp conjugation locus (20, 23). The tcp locus has been identified on all known conjugative plasmids in C. perfringens, and proteins encoded by this locus have been shown to be required for conjugative transfer of the paradigm C. perfringens conjugative plasmid, pCW3 (20,(23)(24)(25)(26). The presence of the tcp genes on so many of these toxin plasmids suggests that the transfer of toxin genes by a shared conjugation mechanism in C. perfringens is important in disease dissemination. To this effect, several of these toxin plasmids have been shown experimentally to be conjugative (19,27,28).M...

show abstract

“…The resulting model was completed manually and is currently being refined. The final TraMÁ structure has been published (Goessweiner-Mohr et al, 2012 …”

Section: Figurementioning

confidence: 99%

“…So far, no protein-protein interactions of TraM with other pIP501 transfer proteins have been detected (Abajy et al, 2007) and no relations were found on the sequence level either. As the protein localizes to the cell membrane (Goessweiner-Mohr et al, 2012), we suggest a role in the scaffolding of the pIP501 core complex.…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary structure determination of the transfer protein TraM from the Gram-positive conjugative plasmid pIP501

et al. 2013

Self Cite

View full text Add to dashboard Cite

The major means of horizontal gene spread (e.g. of antibiotic resistance) is conjugative plasmid transfer. It presents a serious threat especially for hospitalized and immuno-suppressed patients, as it can lead to the accelerated spread of bacteria with multiple antibiotic resistances. Detailed information about the process is available only for bacteria of Gram-negative (GÀ) origin and little is known about the corresponding mechanisms in Gram-positive (G+) bacteria. Here we present the purification, biophysical characterization, crystallization and preliminary structure determination of the TraM C-terminal domain (TraMÁ, comprising residues 190-322 of the full-length protein), a putative transfer protein from the G+ conjugative model plasmid pIP501. The crystals diffracted to 2.5 Å resolution and belonged to space group P1, with unitcell parameters a = 39.21, b = 54.98, c = 93.47 Å , = 89.91, = 86.44, = 78.63 and six molecules per asymmetric unit. The preliminary structure was solved by selenomethionine single-wavelength anomalous diffraction.

show abstract

The 2.5 Å Structure of the Enterococcus Conjugation Protein TraM resembles VirB8 Type IV Secretion Proteins

Cited by 50 publications

References 82 publications

Critical Components of the Conjugation Machinery of the Integrative and Conjugative Element ICE Bs1 of Bacillus subtilis

Critical Components of the Conjugation Machinery of the Integrative and Conjugative Element ICE Bs1 of Bacillus subtilis

Two Novel Membrane Proteins, TcpD and TcpE, Are Essential for Conjugative Transfer of pCW3 in Clostridium perfringens

Crystallization and preliminary structure determination of the transfer protein TraM from the Gram-positive conjugative plasmid pIP501

Contact Info

Product

Resources

About