Why is conjugation in bacteria important

This would allow the kill switch to be more specific to the microbe, resulting in a better treatment of that infection.

Another benefit of using antimicrobial peptides over antibiotics to attack non-host bacteria reduces the chance of creating antibiotic resistance.

The small-scale DNA mutations that occur in order for bacteria to resist antibiotics are nothing compared to the grand-scale mutations needed to completely and functionally re-structure a bacterial membrane.

Antibiotic-resistant bacteria are incredibly dangerous, such as methicillin-resistant Staphylococcus aureus MRSA which causes upwards of 18, deaths per year in the U. A alone Boyles, The treatment of these bacteria with more aggressive antibiotics only leads to higher and more complicated levels of resistance.

Circumventing this process may actually save lives by reducing the chance of creating another antibiotic-resistant strain. Conjugation presents many dangers as well.

Because the process of in vivo bacterial conjugation cannot be entirely controlled by scientists, accidental horizontal gene transfer is a cause for concern.

If the AMP gene were transferred to natural bacterial flora, they too would be damaged upon promoter activation. How exactly to prevent this from happening is unclear, as the use of conjugation as a method of DNA transfer, while well versed outside the body, has little research pertaining to its induction in vivo. Another problem with conjugation is the release of endotoxins from Gram-negative bacterial membranes upon disruption.

Bose, B. Regulation of horizontal gene transfer in Bacillus subtilis by activation of a conserved site-specific protease. Cascales, E. Science , — Chatterjee, A.

Antagonistic self-sensing and mate-sensing signaling controls antibiotic-resistance transfer. Clarke, M. F-pili dynamics by live-cell imaging. Clewell, D. Tales of conjugation and sex pheromones: a plasmid and enterococcal odyssey.

Elements 1, 38— Dahlberg, C. Amelioration of the cost of conjugative plasmid carriage in Escherichia coli K Genetics , — Pubmed Abstract Pubmed Full Text.

De la Cruz, F. Conjugative DNA metabolism in Gram-negative bacteria. FEMS Microbiol. Dempsey, W. Sense and antisense transcripts of traM , a conjugal transfer gene of the antibiotic resistance plasmid R Dionisio, F. The evolution of a conjugative plasmid and its ability to increase bacterial fitness. Dunny, G. Regulatory circuits controlling enterococcal conjugation: lessons for functional genomics.

Conjugational junctions: morphology of specific contacts in conjugating Escherichia coli bacteria. Eisenbrandt, R. Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits. Frost, L. Analysis of the sequence and gene products of the transfer region of the F sex factor. Regulation of bacterial conjugation: balancing opportunity with adversity. Future Microbiol. Why is entry exclusion an essential feature of conjugative plasmids?

Plasmid 60, 1— Ghigo, J. Natural conjugative plasmids induce bacterial biofilm development. Nature , — Goessweiner-Mohr, N. Conjugative type IV secretion systems in Gram-positive bacteria. Plasmid 70, — Guglielmini, J. Evolution of conjugation and type IV secretion systems. The repertoire of ICE in prokaryotes underscores the unity, diversity, and ubiquity of conjugation. PLoS Genet. Harrison, E. Plasmid-mediated horizontal gene transfer is a coevolutionary process. Trends Microbiol.

Haudecoeur, E. A fine control of quorum-sensing communication in Agrobacterium tumefaciens. Invasion of E. Lambertsen, L. Plasmid 52, — Lang, J.

Concerted transfer of the virulence Ti plasmid and companion At plasmid in the Agrobacterium tumefaciens -induced plant tumour. Lau-Wong, I. Lawley, T. Bacterial conjugative transfer: visualization of successful mating pairs and plasmid establishment in live Escherichia coli. Madsen, J. The interconnection between biofilm formation and horizontal gene transfer. FEMS Immunol. McCool, J. Measurement of SOS expression in individual Escherichia coli K cells using fluorescence microscopy.

Modi, R. Coevolution in bacterial-plasmid populations. Evolution 45, — CrossRef Full Text. Molin, S. Gene transfer occurs with enhanced efficiency in biofilms and induces enhanced stabilisation of the biofilm structure.

Navarre, W. Genes Dev. Norman, A. Conjugative plasmids: vessels of the communal gene pool. B Biol. TraM of plasmid R1 controls transfer gene expression as an integrated control element in a complex regulatory network. Conjugative transfer and cis-mobilization of a genomic island by an integrative and conjugative element of Streptococcus agalactiae. Ramsey, M. The gonococcal genetic island and type IV secretion in the pathogenic Neisseria.

Reisner, A. In situ monitoring of IncF plasmid transfer on semi-solid agar surfaces reveals a limited invasion of plasmids in recipient colonies. Plasmid 1, 1—7. Rivera-Calzada, A. Structure of a bacterial type IV secretion core complex at subnanometre resolution.

Samuels, A. Conjugative junctions in RP4-mediated mating of Escherichia coli. These genes can be encoded by an autonomous replicating plasmid Smillie et al. Conjugation in Gram-negative bacteria is mediated by the Type IV secretion system T4SS , a large macromolecular complex involved in substrate transport and pilus biogenesis.

T4SSs are implicated not only in bacterial conjugation, but also in the secretion of virulence factors to eukaryotic cells. Many effectors secreted by T4SS are virulence factors involved in pathogenic diseases, such as brucellosis, whopping cough, cat scratch disease, pneumonia or gastric ulcer, caused by bacterial infection with Brucella suis , Bordetella pertussis, Bartonella henselae, Legionella pneumophila or Helicobacter pylori , respectively Vogel et al.

Instead, we will focus on the issues that pertain specifically to conjugative systems, analysing recent advances that have increased our understanding on the structure and function of conjugative T4SS. Conjugative systems can be considered as a particular subfamily of T4SS.

This subfamily is unique among bacterial secretion systems as it is not only able to transport protein effectors but also DNA covalently bound to a pilot protein [for recent reviews see de la Cruz et al. DNA transfer during bacterial conjugation provides plasticity to bacterial genomes, constituting one of the main mechanisms of horizontal gene transfer Frank et al.

Conjugative plasmids encode their own set of MPF genes and are, hence, self-transmissible. These 11 proteins, together with the coupling protein VirD4, assemble into a macromolecular complex that spans the inner and outer membrane and the periplasm in between. Figure 1 shows the organization of these genes in R plasmid, which together with F and A. Synteny conservation is also observed in other systems involved in the assembly of large multi-subunit complexes, such as the bacteriophage capsid Brussow and Hendrix, or the archaeal Patenge et al.

Therefore, synteny conservation seems to have an adaptive value, perhaps harnessing information related to the temporal programme of expression of the elements of the complex. Genetic structure of the Mpf and Dtr regions of conjugative plasmid R Genes encoding proteins involved in mating pair formation Mpf are organized in operons which are separated from those involved in DNA processing and transfer Dtr.

Mpf genes encode proteins that assemble in a large macromolecular structure called Type IV secretion system, whereas Dtr genes encode proteins that bind to the DNA at the origin of transfer region, oriT , forming an structure called relaxosome.

This modular gene organization is shared by most conjugative systems, showing a high degree of gene synteny conservation. T4SS architecture is relatively well preserved regardless of its biological function bacterial conjugation, secretion of virulence factors into eukaryotic cells or DNA uptake Waksman and Fronzes, Four protein domains can be distinguished in T4SS: the pilus, the core channel complex, the inner membrane platform and the hexameric ATPases at the base of the channel that supply the energy for pilus biogenesis and substrate transport see Fig.

This new structure has provided new insights into T4SS assembly. Here, in the light of this new structure and the genetic, biochemical and structural information provided by numerous laboratories along the last years, a description of the architecture, biogenesis and function of the four different T4SS domains is provided. Architecture of a conjugative T4SS. Four distinct domains can be distinguished in T4SS: the pilus, the core complex, the inner membrane platform and the cytoplasmic ATPases.

The pilus is formed by a helical assembly of pilin VirB2 molecules red with adhesin molecules VirB5 at the distal end dark green. The core complex can be divided in two subcomplexes: the outer membrane cap, formed by VirB7, VirB9 and the C-terminus of VirB10, with a fold symmetry depicted in deep blue colour ; and the periplasmic domain magenta , formed by most of VirB8, VirB10 and the N-terminus of VirB6.

A prevalent way for bacteria to accomplish adhesion to recipient cells is by the use of external appendages of different sizes but similar helical structure known as pili Bradley and Cohen, ; Achtman et al.

At the distal end of these filamentous structures, there is a molecule called adhesin Aly and Baron, , which plays an essential role in cell-to-cell contact Anthony et al. Although pilus biogenesis in T4SS is an intricate process that is not well understood, a picture is emerging in which different steps leading to pilus formation can be distinguished Fig.

T4SS pilus biogenesis. Step 1 - Pilin molecule precursors VirB2 , containing an N-terminal leader sequence, partition into the inner membrane where they are processed by a signal peptidase, presumably LepB. Pilin molecules accumulate in the inner membrane until a specific signal triggers pilus biogenesis.

Step 2 - Pilin molecules are dislocated from the inner membrane into the periplasm by VirB4 with the assistance of VirB The peptidoglycan layer is digested by the transglycosylase VirB1.

In some systems, like in Bordetella pertussis , this transglycosylase activity is carried out by the VirB8 orthologue PtlE. Step 3 — VirB8 also acts as an assisting factor for the assembly of the core complex.

The channel is not yet fully assembled, and its inner diameter is wide enough to accommodate the emergent pilus. Pilus elongation could occur by two different mechanisms: from the inner membrane platform, as T2SS and T4P, or from the outer membrane ring, as Type I pilus.

Step 4 — Assembly of the core complex is completed, and the channel adopts its final conformation. Substrate secretion would only be possible if the central stalk see Fig. Alternatively, the central stalk might act as a nucleation platform for pilus biogenesis, with pilus polymerization leading to the ejection of the substrate Note — For simplicity, only one of the VirB4 hexamers of the T4SS is shown.

Pilin proteins VirB2 are synthesized as prepilin precursor molecules, which contain an N-terminal leader signal sequence. Once in the inner membrane, the N-terminal leader sequence is processed by a signal peptidase I, presumably LepB Majdalani et al.

In some VirB2 homologues, the N-terminal residue of the cleaved protein binds covalently to the C-terminus, resulting in a mature, cyclic pilin Eisenbrandt et al. However, not all VirB2 homologues adopt this cyclic conformation.

Some VirB2-homologues, such as TraA of plasmid F, retain their linear sequence, probably by modification of the N-terminus by N-acetylation Moore et al. Pilin acetylation appears to ensure the correct assembly of the F-pilus filament Anthony et al. Because of their hydrophobic nature, pilin molecules can partition into the lipid bilayer without the additional help of any other component of the T4SS Paiva et al.

VirB4 seems to act as the primary dislocation motor, whereas VirB11 would act as a modulator of the VirB4 dislocase activity, inducing conformational changes in the pilin. VirB5 stable expression is dependent on the presence of VirB6 Hapfelmeier et al. VirB5 localizes at the pilus distal end Aly and Baron, although it has also been found in the cytoplasm and the inner membrane fractions of pili-producing bacteria Thorstenson et al.

However, in contrast to VirB2, the mature VirB5 is not hydrophobic and, therefore, it is not expected to partition into the inner membrane. Upon cleavage of VirB5 leader sequence, the mature protein would be exported to the periplasm. This step is likely to trigger pilus assembly.

Packing geometry of VirB2 into the assembled pilus would be in such a way that hydrophobic patches of the protein will be buried at the interface between adjacent pilin monomers Marvin and Folkhard, These hydrophobic-driven interactions would promote the oligomerization of pilin molecules into bundles which, in turn, would result in the formation of the pilus, as observed in the structure of F-pili Wang et al.

The mechanism driving conjugative pili assembly remains unclear, and it is unknown whether pili assembly initiates from the inner membrane platform at the base of the T4SS or from the cap complex at the outer membrane. Pilus polymerization from the inner membrane might be driven by a mechanism similar to that of T2SS and Type IV pilus assembly reviewed in Filloux, ; Craig and Li, ; Thanassi et al. This model of pilus growth from an inner membrane base platform is supported by the finding of VirB2 in both the inner and outer membranes in cell fractionation experiments Shirasu and Kado, Interestingly, in Type II secretion systems, the minor pseudopilins nucleate pilus assembly Cisneros et al.

However, this model of pilus elongation from the inner membrane would be incompatible with the recently solved structure of T4SS from conjugative plasmid R Low et al. Little is known about the size and morphology of VirB-like conjugative pili. In contrast, the morphology of F-pili is well characterized Wang et al.

Therefore, VirB-like conjugative pili might have similar outer dimensions. Considering the inner diameter of the T4SS outer membrane ring, which is 2—3 nm, Chandran et al.

Therefore, an alternative scenario in which pilin polymerization occurs at the outer membrane is also plausible. In contrast to the T4P systems, conjugative pilus retraction has been a puzzling issue for a long time. Demonstration that conjugative pilus can retract came from fluorescent experiments in which F-pilus retraction could be visualized in real time Clarke et al.

However, evidence for retraction of short-rigid pili like those of Rlike conjugative systems is still missing. It has been suggested that F-pilus retraction does not require energy Frost et al. In recent years, several advances have allowed to gather a glimpse on the structural architecture of T4SS Chandran et al. This channel is formed by a large structure 1.

Below, some of their most salient properties are described. VirB7 is a small lipoprotein inserted in the outer membrane Fernandez et al. VirB9 is a hydrophilic protein also located at the outer membrane. VirB9 interacts with VirB10 and, hence, contributes to the stabilization of the secretion channel Beaupre et al. VirB8 is another component traditionally associated with the channel complex Das and Xie, ; Kumar et al.

VirB8 is anchored to the inner membrane by its N-terminal region Thorstenson and Zambryski, ; Buhrdorf et al. The three-dimensional structure of a periplasmic fragment residues of the VirB8 protein of B.

VirB8 interacts with VirB9 and VirB10 Das and Xie, which suggests that it plays an essential role in the assembly of the secretion channel Kumar et al. It is possible that this structure represents an intermediate step during T4SS and pilus assembly in which the central stalk plays an essential role. Then, upon completion of T4SS biogenesis, this central stalk could be unplugged from the channel pore, allowing the passage of substrates.

This notion is supported by the fact that VirB8 seems to be loosely attached to VirB9 and VirB10, as reflected by its absence from the EM structure of the core complex, despite of being initially co-expressed Fronzes et al. In summary, VirB8 could be an assembly factor, needed for complex assembly but not a component of the secretion channel.

VirB10 is the largest protein in the core complex, spanning the inner and outer membranes and the periplasmic space Jakubowski et al. VirB10 shares structural similarities with TonB-like proteins Cascales and Christie, a , such as a common bitopic membrane topology and a prolin-rich extended region in the periplasm Evans et al.

These structural features were later confirmed by the crystal structure of the periplasmic domain residues of ComB, the VirB10 homologue in H. In this crystal structure, the VirB10 periplasmic domain was shown to form dimers, confirming data obtained by two-hybrid experiments Ding et al.

Therefore, given that there are 14 copies of VirB10 in the core structure Fronzes et al. However, the subunit packing observed in this structure, with the N-termini of each monomer at opposite ends, is not compatible with that observed in the core complex structure. Such a small size implies that any protein substrate crossing the channel should be in an unfolded state, unless a large aperture of the channel takes place. Interestingly, the crystal structure of the outer membrane part of the core complex, formed by 14 copies of VirB7, VirB9, and the C-terminal domain of VirB10 Chandran et al.

Therefore, the EM and the crystal structures significantly differ in the inner diameter dimensions of the pore. Comparison of the outer membrane cap crystal structure Chandran et al.

This conformational change could be due to the fact that the cap crystal structure was obtained after removal by proteolysis of the N-terminal half of VirB In any case, it seems that VirB10 N-terminus plays an essential role in the transmission of the signal from the inner membrane to the rest of the core complex, which could result in the opening of the secretion channel.

These interactions take place in the inner membrane de Paz et al. Interestingly, heteromeric transmembrane interactions in VirB10 have been found to be essential for T-pilus biogenesis but not for substrate secretion Garza and Christie, VirB10 is also able to interact with VirB4. A low-resolution EM structure of the core complex bound to the VirB4-homologue of the conjugative plasmid pKM revealed an intimate relation between VirB4 and the channel complex Wallden et al.

Instead, an association to the complex through the integral membrane protein VirB3 seems a more plausible explanation. It is enticing to speculate that the ATPase activity by VirB4 induces conformational changes in the core complex during pilus biogenesis whereas VirD4 does it during substrate transport.

The crystallographic structure of the CTD of a VirB4 homologue in Thermoanaerobacter pseudethanolicus has recently been reported Wallden et al. The structure turned out to be strikingly similar to the computer generated models of the CTD of A.

These three motor proteins seem to have evolved from a common ancestor Iyer et al. The IncX subfamily of VirB4 proteins is characterized by the presence at the N-terminus of an extra region corresponding to a fusion with a VirB3 protein Batchelor et al. VirB4 has been reported to be an integral cytoplasmic membrane protein Dang and Christie, However, computer predictions of VirB4 topology are negative for transmembrane spans in most of the VirB4 members, excluding those of the IncX branch, which contain a VirB3-like sequence at the N-termini Arechaga et al.

Moreover, TraB, the VirB4 homologue in the conjugative plasmid pKM, has been isolated both, as a soluble and as a membrane-associated form Durand et al. It is altogether very likely that VirB3 acts as an anchor of VirB4 to the membrane, assisting it in its VirB2 dislocase function Kerr and Christie, This structure was obtained in its hexameric form, which is likely to be the catalytic active conformation of the enzyme Arechaga et al.

The oligomeric state of VirB4 proteins has been largely under dispute. Initial reports on the oligomeric state of A.

On the other hand, the VirB4 homologue in the conjugative plasmid pKM has been reported to be present both as dimer and hexamer, being the hexameric form soluble and catalytically active and the dimeric form inactive and membrane-associated Durand et al. Moreover, this structure shows not only one but two hexamers attached to the inner membrane domain of the T4SS.

The implications of this arrangement in the molecular mechanism are however still unclear. The linker region between both domains has been proposed to play a key role in enzyme catalysis Hare et al. In fact, although the crystallographic structures of two non-conjugative homologues, HP from Helicobacter pylori Yeo et al. These differences are produced by a large domain swap of the central linker, which is much larger in the Brucella homologue than in the Helicobacter counterpart Hare et al.

Based on biochemical and structural evidence, a common model for the mechanism of action of these secretion ATPases has been proposed Yamagata and Tainer, ; Ripoll-Rozada et al. According to this model, the VirB11 NTD of the nucleotide-free form of the enzyme would be pivoting over the flexible linker. Binding of magnesium and ATP would lock the enzyme in a closed conformation.

A specific signal for instance, substrate binding or release would unlock this state, resuming the catalytical cycle by releasing the ADP for the next turnover Ripoll-Rozada et al.