Disclaimer: I’m one of the authors on this paper.
There’s a proper orientation to the human body. Imagine if instead of your arms coming out of your shoulders, they extended from your waist, or if your legs grew from your back. Like us, bacteria also have an appropriate orientation and appendages involved in movement have a ‘correct’ place to be assembled. A recent paper from the Burrows Lab at McMaster University by Carter et al. in mBio investigated how an appendage called a type IV pilus (or T4P) is recruited to the cell poles. I’ll give the gist of the paper here, but for a full appreciation of it, read the paper here!
A little bit of background…
Type IV pili (as in more than one type IV pilus) are filament-like appendages that stick out of the cell. They work like little grappling hooks, repeatedly extending out of the cell, attaching to a surface, and retracting, pulling the cell towards the point of attachment. They’re important virulence factors – things that bacteria use that can make people sick.
T4P are made of many different proteins. These proteins make up 4 subcomplexes that together make up the T4P assembly complex. There’s the pilus filament itself, an outer membrane pore (the hole the filament comes out of), a cytoplasmic (inside the cell) motor complex (proteins driving extension and retraction of the filament), and an alignment complex that bridges the inner and outer membrane parts.
The study examined T4P in P. aeruginosa, an opportunistic, rod-shaped bacterium. It’s Gram-negative, so it has two membranes (an inner and outer), that sandwich a peptidoglycan layer. This layer is a major physical barrier for the insertion of large protein complexes like the T4P. So one of the big mysteries surrounding machines like the T4P is how they’re installed. There are two prevailing hypotheses (i) that the peptidoglycan layer is built, then remodeled to incorporate the T4P, or (ii) that the T4P is pre-installed as the peptidoglycan is being built.
Fluorescent proteins were fused to either the outer membrane pore-forming proteins (PilQ), or one of the pieces of the alignment complex found in the inner membrane (PilO). These fluorescent proteins give off light when excited by a laser. Using this method, the researchers could use fluorescence microscopy to determine if the proteins in question still went to the cell poles.
Localization of PilQ and PilO (whether or not they went to cell poles or sites of cell division) was examined in cells treated or not treated with a drug that stops late stage cell division. So instead of a whole bunch of rods, there is a long filament of cells.
Localization was also tested in different mutants using a simple approach – delete a gene, see what effect(s) there are on your protein. This tells you if the deleted gene was important for whatever behavior you were looking at. In this case, it was to determine if that gene was important in PilQ and PilO localization.
So what did they find out?
Researchers saw that PilQ and PilO could be found at the cell poles in normal cells, and at future sites of cell division in drug treated cells. So it was hypothesized that that the T4P are pre-installed during cell division. This means the cell wouldn’t need to make a whole cell pole, and then remodel it all after completion. The pre-installation method is a more energy-efficient way of getting these big machines embedded into the peptidoglycan layer.
The next question was ‘how these proteins get to the sites of cell division’? To do this, researchers deleted genes corresponding to either PilQ, the outermost component of the alignment complex, or the innermost component of the alignment complex. They found that without PilQ, PilO was not targeted to the sites of cell division. So it looked like PilQ targeted the whole machinery to sites of cell division.
But why PilQ? PilQ as a motif in it called AMIN. AMIN preferentially targets the protein to peptidoglycan found at the cell poles/septa (where the cell divides… it eventually becomes a pole when cell division is done). Specifically deleting only the AMIN motif caused PilO to not go to the sites of cell division. So in addition to its likely pre-installation, the T4P machinery is targeted to the sites of cell division by PilQ and its pole-targeting motif. This targeting doesn’t even require PilQ to be in the outer membrane, as the researchers showed that in the absence of a protein that brings individual PilQ proteins from the inner to the outer membrane so they can come together and form a pore, PilO is still found at sites of cell division.
That wasn’t the end of the localization story though. PilO also uses two other proteins to properly localize. A protein called the ‘polar organelle coordinator’, and FimV, a suspected hub protein that gathers a whole bunch of other proteins at the cell pole (probably a story for another day).
Why should I care about all this?
How these big machines get installed despite a physical barrier like peptidoglycan is a mystery. Some machines have dedicated proteins that remodel the peptidoglycan layer, but T4P don’t. A better understanding of how T4P are installed may shed light on how other similar molecular machines are installed.
T4P are also a critical virulence factor for P. aeruginosa. By having a better understanding of how they work, we can learn more about mechanisms of bacterial pathogenesis.
The paper by Carter et al, in mBio (it’s open access!)
One of several wicked reviews on T4P (Disclaimer: I’m also an author on this)
The Burrows Lab (they authored the study)
Carter T, Buensuceso RNC, Tammam S, Lamers RP, Harvey H, Howell PL, Burrows LL. 2017. The type IVa pilus machinery is recruited to sites of future cell division. mBio 8:e02103-16. https://doi.org/10.1128/mBio.02103-16.
Podcasts I’ve been listening to:
Under the Influence by CBC Radio – What a Difference a Difference Makes: This podcast series tells wonderful stories from the world of marketing. In addition to a great narrative, it gets you thinking about advertising differently. This episode talks about distinct branding to stand out from the crowd.
After LifeAfter: If you remember, I was listening to a fiction podcast series from GE called LifeAfter. This is post season episode hosted by Neil deGrasse Tyson discussing the sci in the sci-fi.