by Christoph
Figure 1. A mating pair consisting of a normal donor and a normal recipient. The donor cell has Type I pili while the recipient cell has no appendages. The F pili have been made more visible by the addition of RNA F pilus phages to the mating mixture. (Scan from the low-res PDF.) Source
If you've ever taken a class on bacterial genetics, either recently or in the last few decades, you've no doubt seen this image: the conjugation of two bacterial cells (Figure 1). And if you found this image so self-explanatory that you didn't bother to ask for the source, you're not alone. The internet is replete with it, in varying orientations and often colorized, but lacking a reference (here's an exception.) The 1982 edition of Lehninger's Principles of Biochemistry gives a reference, Judith Carnahan and Charles C. Brinton Jr., which allowed me to trace the origin of the image to a paper from 1971. To my surprise, the figure legend is incomplete: neither is it stated which bacterial strains were assayed in the conjugation experiment, nor is the magnification given, nor details of the applied electron microscopic technique (see caption to Figure 1.) Anyway, I could learn − or better: deduce − all this from their earlier paper from 1964.
Let me dissect the "self-explanatory" aspects of this iconic image a bit. One needs to distinguish donor and recipient cells of a mating experiment as it was known from the pioneering work of Esther and Joshua Lederberg and Luigi Cavalli-Sforza that conjugation in E. coli is a unidirectional process in which genetic material is transferred from a strain carrying an F plasmid (F+ or, if the F is integrated, an Hfr strain), to an F− strain. It was also known in the early 1960s that F+ and Hfr strains express a type of pilus that is missing in F− strains, but their involvement in conjugation was unclear. Here, Brinton et al. used a heavily piliated donor strain and an un-piliated recipient strain (Figure 1). Yet, how to unambiguously distinguish the various types of pili that come with different average lengths and only slightly different diameters? They relied on a short communication by Crawford and Gesteland who found that ssRNA phage R‑17 binds more readily to certain pili of F+ and Hfr strains than to any of those of F− strains. Brinton et al. used another ssRNA phage, M12, to "decorate" the F pili specifically in their experiments. If you look closely at the F pilus connecting the (upper) donor cell and the (lower) recipient cell you see a chain of "pearls," phage particles, which make the pilus appear much sturdier and larger in diameter than it is.
Figure 2. Electron micrograph of presumed specific pair between an Hfr cell (bottom) and an F− cell. Phage MS-2 was used to "stain" F pili. The specimen was prepared soon after mixing the Hfr and F- cells. Two F pili have been used to make contact with a single F− cell. magnification ×28,000. Source
They mention this pilus "decoration" by attached phages in their figure legend, but they do not mention that this particular electron micrograph was cherry-picked since the conjugations they showed in their earlier paper invariably consisted of groups of aggregated cells. Cells were lying close to each other or even in direct contact, and often with more than one bent F pilus extruding from donor cells (see here.) Such a grouping of "mating pairs" was repeatedly found by other researchers as well and therefore seems more realistic (Figure 2; note the particularly clear F pilus "decoration"). Therefore, the fully stretched-out appearance of the F pilus in Figure 1 is most likely a sample preparation artifact. Because of its known structure and carefully measured dimensions, the inner channel of the F pilus could serve as a conduit for single-stranded DNA that is formed upon rolling-circle replication of the F plasmid − or the chromosome in case of a Hfr donor − and thus delivered to the recipient cell, much like the injection of phage DNA into a host cell. Brinton et al. proposed this model, but it is necessary to point out that their electron micrograph in Figure 1 is not conclusive evidence because they cannot "see" the single-stranded DNA threaded through the pilus (ssDNA is notoriously difficult if impossible to visualize in electron micrographs.)
Figure 3. Serial thin sections through conjugation-specific junctions. Micrographs (a) to (e) are from a horizontally sectioned junction Note the absence of cytoplasmic fusion over the whole junction. Source
The "Brinton model" for DNA transfer during conjugation was immediately questioned, not the least because it was found that F pili, like the pili of other Type IV secretion systems (T4SS), are dynamic, and can be both extended and retracted. Dürrenberger et al. (1991) say:"Genetic studies supported a sequential model for conjugation in which conjugation proceeds through the following sequence of steps: contact formation by the pilus tip, retraction of the pilus, stabilization of wall-to-wall contact, DNA transfer and disaggregation of the two partners" (see here for a diagram.) These authors indeed found this "stabilization of wall-to-wall contact" as electron‑dense 'conjugational junctions' in electron-microscopic studies of mating cells (Figure 3). However, the molecular details of these junctions remained "invisible" and a proteinaceous transfer channel or pore-like cytoplasmic fusions were never reproducibly observed by conventional electron microscopy. A comprehensive description of this model for conjugation is given by Firth et al. in Chapter 126 of the "E. coli bible", 2nd. Ed. (1996).
(Click to enlarge)
Figure 4. A structure-driven assembly pathway for the F transfer system. Step I: Tra subunits assemble initially as a quiescent F1-Channel complex. Step II: The F1 complex transitions to the F2-Channel/Pilus for dynamic extension and retraction of the F pilus in a "mate-seeking" mode. Dynamic F pilus extension and retraction by the F2 structure favors formation of distant cell-cell contacts during low cell density, e.g., planktonic growth. Step III: Assembled F pili are alternatively deposited onto distinct platforms, yielding F3-Stalk/Pilus or F4-OM/Pilus structures. The presumptively static F3 and F4 structures promote nonspecific aggregation and biofilm development; in polymicrobial settings, these dense growth conditions favor formation of mating junctions. Step IV: Recipient cell contact (lightning bolts) triggers F pilus retraction and recruitment of the TraD receptor and F plasmid substrate to activate the DNA transfer or "mating" mode. Step V: Following plasmid transfer, donor and recipient cells disengage and the "mating" channel reverts to the F1 quiescent complex. The relative percentages of each complex visualized on E. coli minicells by in situ CryoET are shown. Source
There is, however, an almost Solomonic judgement on this at times fiercely fought controversy. Hu et al. (2019) have now visualized the F-encoded transfer channel and F pilus-associated platforms in the cell envelope of E. coli minicells by cryoelectron tomography (cryoET). They say:"the channel supports plasmid transfer or assembly of F pili, which remarkably upon synthesis are deposited onto alternative basal structures around the cell surface" (Figure 4). Since the legend to Figure 4 is highly condensed, let me pick two points
For one, it was technically impossible to see with conventional electron microscopy the membrane-embedded 'basal structures', multi-subunit protein complexes forming the ring-shaped 'outer membrane complex' (OMC), a connecting channel, and the ring-shaped 'inner membrane complex' (IMC). And it would have been even less possible to see that the transfer channel and the pilus-attached platform are essentially the same and can mainly be distinguished by association with either the TraD or the TraC subunits. Thus, the results obtained by Hu et al. support the 'sequential conjugation model' outlined above.
Secondly, there is a small but finite probability that a signal for pilus retraction is "ignored" and the affected 'basal structure' associates with a TraD subunit. In this case, DNA transfer can occur through the pilus ('long distance' conjugations have indeed been demonstrated.) This makes the "Brinton model" a special case, and the image shown in Figure 1 is most likely just a snapshot of successful cell‑cell contact before pilus retraction and DNA transfer.
To complete the overall picture, "conjugationists" still need to find out: 1. what the molecular nature of the signal is that triggers, upon recipient cell contact, pilus retraction and the switch to "mating" mode, that is DNA transfer (the 'lightning bolts' in Figure 4), and 2. which molecular structure(s) take up the "incoming" ssDNA and channel it into the cytoplasm of the recipient cell for lagging-strand synthesis and, in case of Hfr crosses, recombination with the resident chromosome
I keep wondering why our Pictures Considered #55 continues to have an almost iconic status. For sure, it triggers this "a picture is worth a thousand words" feeling. I can't shake off the suspicion though that the picture appeals to very conventional notions of "conjugation," which is inconceivable without a straight pilus. I insinuate − because I cannot ask the now deceased authors about it − that the propagation of this image by the authors was intentional, for this reason.
An Aside. If you (still) find our Pictures Considered #55 appealing enough to consider buying it as an art print for your living room, which is possible, be aware that this embellished version by Dennis Kunkel crosses the boundaries of artistic freedom: it's a no-go to replace the straight sex pilus with a photoshopped or hand-drawn wobbly line (even for youth protection reasons.) But perhaps you'll enjoy other artistic approaches to "bacterial conjugation" contributed by several talented science artists to this year's #Microber2021 series on Twitter, which is what prompted me to consider #55 in the first place.
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