What is the difference between pilus and conjugation

We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection.

Conjugation is the process by which genetic materials, notably plasmid DNAs, are transferred from one bacterium to another Lederberg and Tatum, It is responsible for horizontal gene transfer among bacteria and is the primary means by which antibiotic resistance genes spread among bacterial populations Thomas and Nielsen, Conjugation is mediated by a type IV secretion T4S system, a versatile secretion machine, operating in both Gram-negative and -positive bacteria and capable of secreting not only nucleic acids during conjugation, but also protein effectors and toxins during bacterial pathogenesis Costa et al.

The pilus is a polymer of the VirB2 protein or pilin. The covalent relaxase-DNA complex is then recruited to the T4S system by VirD4, transported through the machinery, and then through the pilus, which forms a tube that can deliver DNA to a recipient cell located at some distance away Babic et al. The pilus is a dynamic structure that can depolymerize to bring donor and recipient cells closer to one another Clarke et al.

The F plasmid has a remarkable status in the history of the fields of molecular biology and genetics. The F plasmid is not only able to conjugate itself from a donor cell to a recipient cell it indeed encodes all the T4S system and relaxosome components Lawley et al.

This property was used to map the entire E. In the electron microscope, the only visible manifestation of the F system has been its pilus Folkhard et al. The pilus of conjugative T4S systems is not only an essential cylindrical conduit for conjugating DNAs, but also is the first point of entry for many phages, which attach to T4S systems pili before injecting their DNA or RNA into bacterial cells Arutyunov and Frost, In this era of widespread antibiotic resistance and regained interest in phage therapy to combat bacterial infections, it is essential to understand phage-pilus interactions.

A crucial step toward elucidating this interaction is the determination of its structural basis. However, while rapid progress in the structural biology of phages has been made, no atomic resolution details are available for T4S system pili. Previous studies have provided some confusing insights into the helical parameters of the F pilus and were of insufficient resolution to derive an atomic model Folkhard et al.

Here, we present structural details for two F family pilus types, the pED and F pili, derived from 3. These structures provide unprecedented details of conjugative pilus architecture and function. The F and the F-like pED plasmids are two plasmids that encode their own T4S systems and thus produce their own pili. The pili were applied to grids and were vitrified for cryo-EM analysis Figure 1 B. For pED, a 3. During the process of helical reconstruction, as the resolution was increased, an additional separate density became clearly visible and readily interpretable as a phospholipid Figure 1 C.

For the F pilus, it became apparent during the process of helical reconstruction that two populations of filaments were present, differing slightly in the rise between subunits see below. Near-atomic resolution for the F pilus was not achieved, presumably because the F pilus might not be as ordered as the pED pilus. Instead, two 5. C Details of two representative regions of the experimentally derived density for the pED pilus.

The electron density map contoured at a 1. The pED TraA model is in stick representation with atoms color-coded light gray, blue, and red for carbons, nitrogens, and oxygens, respectively. The lipid model is PG in stick representation color-coded green, yellow, blue, and red for carbon, phosphorus, nitrogen, and oxygen, respectively. For clarity, two views are provided: one in which the protein structure is clearly apparent left and the other where the lipid structure is clearly apparent right.

Identical and similar amino acids are boxed in red and yellow, respectively. Since the traditional FSC between two half-maps is not a measure of resolution, but rather of self-consistency Egelman, , the model:map FSC provides a better measure of actual resolution. We show, however, in B an FSC between two independent half-maps, which yields a resolution of 3.

In addition, and more importantly, the visual appearance of the map is also consistent with a resolution of 3. Structure of the F Pilus, Related to Figure 1. The region shown is the same as the one presented in Figure 1 C. The map is contoured at the same level. They can be described in two equivalent ways, as illustrated in Figure 2 : 1 as five-start helical filaments Figures 2 A and 2B where the five helical strands are color-coded differently , or 2 as pentamer layers stacked on top of each other, each layer related to the one below or above by a rotation angle and rise Figures 2 C and 2D.

For pED, the angle between adjacent subunits is Thus, the general pED and F pilus architectures can be considered virtually identical. Previous published work has reported different helical parameters for the F pilus Wang et al. At the near-atomic resolution reported here, there can be no ambiguities as to the assessment of the symmetry and, therefore, the parameters reported here are definitive Egelman, A Side view of the pED pilus structure.

The structure is in surface representation. It consists of a five-start helical assembly. Each of the five helical strands is shown in a different color and is labeled 1—5. Each helical strand consists of Thirteen subunits are shown and named A—M from bottom to top. Although the overall orientation of the pilus relative to the membrane is not known, we hypothesize that the membrane-proximal end of the pilus is at the bottom.

Since, in the structure of TraA determined here, the loop is orientated down within the pilus, this would also position the membrane-proximal end of the pilus down. B Bottom view of the pED pilus structure. Representation is as in A , except that the lipid head group atoms represented as spheres color-coded white, yellow, and red for carbon, phosphorus, and oxygen atoms, respectively are visible inside the lumen of the pilus. The external and internal dimensions of the pilus are reported.

C The pentamer unit of the pED pilus. Each subunit and lipid is in surface and sphere representation, respectively. This figure was generated using the TraA molecule labeled I in each helical strand. Color-coding is as in A and B. Top, top view. Bottom, side view. D Two adjacent pentamer units of the pED pilus structure. The pentamer unit at the base is as in C made from the TraA molecules named I, i. The lipids are as in A.

The angle and rise between equivalent subunits in consecutive pentamer stacks are reported. E The PG array in the context of the pilus strand. Pilus strand 2 of A is shown together with bound PG. Representation of the strand is as in A , while representation of the PG is as in C. This image clearly shows the continuous PG array along the pilus strand.

The amino acid sequences of both the orthologous pED and F pilus TraA pilin are similar, except for the F pilin N terminus, which is 4 amino acids aa longer Figure 1 D. This longer N terminus could not be traced in the F pilus density maps and thus must be disordered. Since the structures of the pED and F pili are very similar but much higher resolution was achieved for the pED system, we will focus all subsequent description of pilus architecture and pilin structure on this pilus type, pointing to notable differences with the F system when required.

The pED TraA pilin is a residue protein, 63 of which residues 2—64 were clearly defined in the electron density. This is consistent with previous results suggesting that the N- and C-terminal regions of the F TraA pilin are accessible for phage attachment and thus must be located on the outside Frost and Paranchych, However, the F TraA structure was solved at a lower resolution, and thus, whether these minor differences are significant remains unclear.

Thus, with the structures and helical parameters of the two F pilus forms being so similar, functional differences between them are unlikely to arise but cannot be excluded. A Location of the subunit shown in B within the pilus. For clarity, and in order to maintain the same orientation throughout, a subunit subunit I in helical strand 3 was chosen arbitrarily as the reference subunit.

B Structure of the TraA-phospholipid complex. TraA is in ribbon representation with the N, C terminus, and secondary structures labeled. The lipid is in sphere representation color-coded as in Figure 2 C. Left, orientation of TraA is as in A. Right, orientation of TraA is 90 degrees away from orientation at left. The proteins and lipids are in ribbon and sphere representations, respectively. For the lipid, color-coding is by atom type with oxygen, nitrogen, and phosphorus atoms in red, blue, and pink, respectively, and carbon in magenta and yellow for the lipid bound to pED or F TraA, respectively.

For the pED pili structure, the resolution could be extended to 3. Map representation and models are as in Figure 1 C. This finding was confirmed in experiments Figure 4 in which the purified pED pili were first treated with phospholipase 2 PLA2 and the remaining bound lipids subsequently extracted and analyzed by mass spectrometry MS.

Two main species bound to the pilin were identified by daughter ion fragmentation as phosphatidylglycerol PG species, PG , and PG , Figure 4. These are also major PG species in the whole-cell membrane Figure 4 A. However, selectivity is observed, as there is no presence of the other two major phospholipid classes, phosphatidylethanolamine PE compare Figure 4 B with Figure 4 A and cardiolipin data not shown , in the PLA2-treated pili extracts.

Thus, selective binding of PG to pilins occurs in both F and pED pili, and thus, the F family of pili are polymers of a selective and stoichiometric protein-PG complex unit. The pilus is held together by interactions not only between pilin subunits, but also between lipids and subunits Figures 5 and 6. Each lipid molecule makes extensive contacts with five surrounding TraA subunits Figure 5 A , while each TraA subunit interacts with five lipid molecules Figure 5 B.

Overall, In the lipid, only the head groups are solvent exposed and directed to the lumen of the pilus Figure 5 C. The acyl chains are entirely buried between subunits. Details of residue-specific pilin-lipid interactions are described in Figure S5.

These involve primarily hydrophobic residues interacting with the acyl chains. Only very few but significant contacts with the phospholipid head group are observed between the phosphate and K41 and Y37, for example. A Each lipid interacts with five adjacent TraA subunits. TraA subunits and phospholipids are shown in ribbon and sphere representation, respectively.

Orientation of the reference magenta subunit labeled I is as in the left panel in Figure 3 B. The lipid molecule is at the interface between subunits in strands 2 and 3. B Each TraA subunit interacts with five phospholipid molecules. Representation and color-coding are as in Figure 3 B. C Electrostatic potential of the pilus lumen calculated without left or with right the phospholipids.

A Each subunit interacts with eight others within the pilus. All subunits interacting with the reference subunit in magenta subunit I in strand 3 are shown, as well as their associated phospholipid. All subunits and lipids are in ribbon representation color-coded various shades of green, red, and cyan for subunits in helical strands 4, 3, and 2 as defined in Figure 2 A , respectively. In this nomenclature, each of the 13 subunits in each helical array was labeled A—M, with subunit A at the bottom of the pilus structure model.

B Surface area buried in subunit-subunit interactions. The reference subunit used in these calculations is in magenta in A. Color-coding is as in A : for example, number in cyan indicates surface area buried between subunits in magenta and cyan in A.

C Mapping of subunit-subunit interactions onto the reference subunit. The reference subunit is in magenta in A. Interactions made between the reference subunit and the subunit in red in A are mapped onto the reference subunit surface by color-coding its surface in red. The same is carried out for all other subunits shown in A. The result is the mapping of interactions that each subunit makes with the reference subunit. Interactions with lipids are mapped in white. A Interaction diagram between one phospholipid and five adjacent TraA subunits.

The interactions are between the phospholipid and the subunits shown in Figure 5 A, using the same color-coding and naming for TraA subunits. B—F Detailed side-chain interactions with each subunit. TraA subunits are shown in ribbon color-coded and labeled as in Figure 5 A. Interacting residues are shown in stick representation color-coded with blue and red indicating nitrogen and oxygen atoms, while the carbon atoms are color-coded as in the ribbon. PG lipid is in stick representation with atoms color-coded white, blue, red, and orange for carbon, nitrogen, oxygen, and phosphorus, respectively.

The head group of the phospholipid interacts with Leu44 and Asn42 through hydrophobic and hydrogen bond interaction, respectively. Two hydrogen bond interactions were observed between the head group of PG and the main chain carbonyl group of Lys41 and between PG sn- 2 oxygen with the hydroxyl group of the Tyr37 side chain. The hydrophobic chain of PG interacts with the side chains of Val11 and Phe The hydrophobic tail of PG interacts with Ala35 and Ile38, while the Arg39 main chain carbonyl oxygen makes a hydrogen bond with the PG head group.

The structures presented here locate this loop to the lumen of the pilus, suggesting that indeed the pilus serves as a conduit for ssDNA transfer. Remarkably, integral to the lining of the lumen is the stoichiometric inclusion of phospholipid head groups. To gain further insight into the potential impact that inclusion of PG head groups within the lumen lining might have, the electrostatic potential of the lumen was calculated with or without PG Figure 5 C.

Inclusion of PG has a profound impact on the electrostatic potential of the pilus lumen: without PG, it is overwhelmingly positive, while with PG, it is moderately electronegative. By generating a conduit with a moderately negative inner surface, phospholipids may facilitate transport of the negatively charged ssDNA substrate. All mutations observed to affect pilus biogenesis locate to protein-protein interfaces, while mutations affecting conjugation and phage attachment locate to either the lumen or the periphery of the pilus.

Thus, the structure presented here provides the structural basis for all published F pilus mutations. However, the PG-binding site was never targeted for mutation, as it was unknown. Pilus biogenesis and function were assessed using negative-stain EM for observation of pilus production at the bacterial cell surface or using conjugation and filamentous phage f1 infection for observation of pilus function Figure 7 ; see details in STAR Methods.

Mutating A28 to the bulky residue F is expected to create severe steric clashes and to be disruptive of pilus biogenesis, and this is precisely what is observed. Substitution to N at this position is less drastic and results in minimal disruption of pilus function.

Mutation of Y37 to F is ineffective, demonstrating that the hydrogen bond between Y37 and the lipid is a minor element in PG-TraA interaction but the aromatic side chain is critical, as mutation to V impairs pilus formation and function. As expected, mutation of R39 to A preserves the structural integrity of the pilus, since the main interaction of R39 with the lipid is through its main-chain amide nitrogen. However, inverting the charge at this position appears to decouple conjugation from phage infection: indeed, R39E does not affect pilus biogenesis and has only a small impact on conjugation but completely abrogates phage infection.

During conjugation, the pilus is known to serve as an export conduit for a mixed nucleo-protein complex consisting of the relaxase protein covalently bound to ssDNA, itself possibly coated with single-strand DNA-binding proteins Ilangovan et al. An R39E mutation would strongly increase the electro-negative potential within the lumen of the pilus, thereby giving rise to a strong repulsive force that would prevent the negatively charged DNA of the phage from entering the pilus conduit.

This would not be the case with the more electrostatically neutral protein-DNA complex that serves as a substrate during conjugation. See main text for mutant description. The scale bar represents nm. B Summary of the effect of TraA single point mutations in pili formation, f1 phage sensitivity, and conjugation efficiency. Each TraA molecule makes contact with eight adjacent subunits Figure 6 A. The subunit-subunit interaction networks involved adjacent subunits in the same helical strand for example, in Figure 6 A, subunit labeled I in helical strand 3 makes contacts with the previous and subsequent subunit within the strand, labeled H and J, respectively , but also with three subunits in the strand above helical strand 4, subunits labeled J, K, and L and below helical strand 2, subunits labeled F, G, H; see notation of pilus subunits in legend to Figure 6.

One consequence of this arrangement is that the entire length of each subunit is involved in contact with other subunits either in the same helical strand or in the strands above and below. The only solvent-accessible surfaces are at the periphery, either facing outward for phage attachment or facing inward toward the lumen for DNA transport. Details of subunit-subunit interactions are shown in Figure S6.

A Same as Figure 6 A, repeated here for clarity. B Interactions between chain I and chain H of strand 3 represent the largest set of interactions between two TraA monomers within the pED pilus. From the bottom of the panel, closer to the lumen, the side chains of Met36, Val32, and Ile45 in chain H make contact with Ile38 of chain I. Also, the side chains of residues Lys41 in chain I and Glu29 in chain H make contact through a salt bridge interaction.

Lys64 at the C-terminal end of chain H interacts with the side chain of Asp18 in chain I through a hydrogen bond. Asp2 of chain H and Asp10 of chain I form a stabilizing hydrogen bond through their main chain groups. Starting from the top of the panel, residue Leu4 in chain I of strand 3 interacts with Thr57 and Val54 of chain L in strand 4. Finally, Ile31, Val32, and Ala35 in chain I of strand 3 are in proximity to Leu47 and Leu44 of chain J in strand 4, stabilizing the two chains through hydrophobic interactions.

Starting from top of the panel, residue Phe62 in chain I of strand 3 is in proximity to Gly7 of chain G in strand 2. Val54 of chain I in strand 3 is surrounded by hydrophobic residues Val11, Phe15 of chain G in strand 2, and Leu4 and Ala5 of chain F in strand 2. The residue Val50 of chain I in strand 3 interacts with Phe15 and Val21 of chain G in strand 2 and also with Ala5 of chain F in strand 2. Leu47 in chain I of strand 3 interacts with Ile31 of chain H in strand 2 and Val21 of chain G in strand 2.

The structure of the F pilus reveals a protein-phospholipid complex as the primary unit from which the pilus is assembled.

Prior to assembly, each TraA molecule is embedded in the inner membrane and then extracted from the membrane during pilus biogenesis Paiva et al. The stoichiometric presence of phospholipid within the pilus demonstrates that, as pilins are extracted from the membrane, each remains associated with one phospholipid molecule. Lipids have been observed bound to proteins but often as a result of unspecific binding.

Only lipid metabolizing enzymes form stoichiometric complexes with their substrates. Thus, to our knowledge, our observation of a lipid bound stoichiometrically to a protein polymer is unprecedented. Moreover, the lipid composition of the pilus is different from that of the membrane, suggesting preferential binding of TraA to a subset of phospholipids.

An essential aspect of pilus function is indeed its ability to enter successive cycles of growth and retraction Clarke et al. One can reasonably hypothesize that stripping off all bound lipids from TraA would have an energetic cost, as would the requirement of partitioning a lipid-free TraA back into the lipidic phase of the membrane during pilus retraction.

Testing such a hypothesis is clearly the next step in research in conjugative pilus biogenesis. Moreover, since there are other bacterial filaments that are assembled from subunits that exist at some point as integral membrane proteins e. Bacteriophages have been used in the past and are still used widely in eastern Europe, notably Russia, to combat bacterial infections. In western Europe, their use declined rapidly when effective, cheap, and broad-range antibiotics became available.

However, with antibiotics becoming increasingly ineffective, it has become urgent to explore all possible avenues in the search for novel therapeutic agents: phage therapy is poised to undergo a major revival as one potential weapon in the arsenal of antimicrobials. Effective treatment by bacteriophages will be greatly facilitated by a detailed characterization of the phage-pilus interaction at a molecular level.

They are also fragile and constantly replaced, sometimes with pili of different composition, resulting in altered antigenicity. Specific host responses to old pili structure are not effective on the new structure.

Recombination genes of pili code for variable V and constant C regions of the pili similar to immunoglobulin diversity. Conjugative pili allow the transfer of DNA between bacteria, in the process of bacterial conjugation. Perhaps the most well-studied is the F pilus of Escherichia coli, encoded by the F plasmid or fertility factor.

A pilus is typically 6 to 7 nm in diameter. During conjugation, a pilus emerging from donor bacterium ensnares the recipient bacterium, draws it in close, and eventually triggers the formation of a mating bridge, which establishes direct contact and the formation of a controlled pore that allows transfer of DNA from the donor to the recipient.

Typically, the DNA transferred consists of the genes required to make and transfer pili often encoded on a plasmid , and is a kind of selfish DNA; however, other pieces of DNA often are co-transferred, and this can result in dissemination of genetic traits, such as antibiotic resistance, among a bacterial population. Not all bacteria can make conjugative pili, but conjugation can occur between bacteria of different species.

The external ends of the pili adhere to a solid substrate, either the surface to which the bacteria are attached or to other bacteria, and when the pilus contracts, it pulls the bacteria forward, like a grappling hook. However, some bacteria, for example Myxococcus xanthus, exhibit gliding motility. Bacterial type IV pilins are similar in structure to the component flagellins of Archaeal flagella.

Attachment of bacteria to host surfaces is required for colonization during infection or to initiate formation of a biofilm. A fimbria is a short pilus that is used to attach the bacterium to a surface.