Enter the Pirates

by Christoph
 

Violent communication, including mutual killing, is not uncommon among bacteria, but, surprisingly, microbiologists know of only a small number of genuine rapacious bacterial species. In scientific terms, they are referred to as "predatory bacteria." You can find a current list here (Open Ac­cess). Well-known examples are Myxococcus xanthus and its ilk, which practice "wolf pack" swarm hunting, and the more solitary vam­pires from the large Bdellovibrio  tribe. Enter the pirates with Captain Aureispira...
 

The lab of Martin Pilhofer at the ETH, Zurich, Switzerland has been studying the predator/prey relationship of Aureispira sp. CCB-QB1 (Bacteroi­dota) and Vibrio campbellii  (Gamma­proteo­bac­teria) for some time. What they found, the mechanical tools that Aureispira uses to catch its prey before killing and comsuming it, they published in a preprint in January 2024. They announced these results also in a thread on 𝕏, which I present here, with comments and the captions missing from the tweets (skip them if you deem them tl;dr). Don't be put-off by the slew of emojis (🦠) and hashtags (#) deco­rat­ing the tweets in this thread (🧵 ). The tweets are short & snappy with little regard for grammar and syntax as is com­mon on 𝕏. After all, pirates don't sing long poetic odes either...
 

(click to enlarge)
Movie 1. Source. Frontispiece: 200 year-old pirate flag at the Åland Maritime Museum. CC BY 4.0 Anneli Karlsson.

 

Pilhofer Lab  Jan 30, 2024 🧵1/6

Bacteria can be pirates too... 🏴‍☠️ Let us introduce you to Captain Aureispira ⛵️ A bacterial pirate ship loaded with canons 💉 and grappling hooks 🪝🌴predating on other bacteria 🦠 Check the preprint !

#predation #T6SS #T9SS #cryoEM #cryoET #teamtomo
 

In Movie 1, the 13.8 nm thin slices successively superimposed in a cryo-tomogram re­veal the armament of the Aureispira pirate ship as it leaves the harbor. Shortly before, the lookout had sighted ships on the horizon and reported them. A fine haul would be just the thing right now! Numerous grappling hooks (red) are attached outboard, as are the prepared harpoon tips (white), while the harpoon shafts (blue) remain inside the ship for now (I can't see any real "canons").

 

(click to enlarge)
Movie 2.1. Timelapse light microscopy (LM) images show an Aureispira filament approaching V. campbellii  prey cells followed by rapid lysis of the prey. Scale bar, 5 µm. Source

 

Pilhofer Lab  Jan 30, 2024 🧵2/6

Aureispira can glide towards its prey and shows very efficient killing, for example against diverse Vibrio strains. A predatory behavior that has been described as "Ixotrophy" 🫣 Check out the time-stamp in mins!
 

Aureispira cells do not go pirateering solo, but as an armada of 5–10 cells en­veloped by a thin hyaline sheath, a porous gauze bandage made up of glycoprotein (Movie 2.1). Single Aureispira sp. CCB-QB1 cells are rod-shaped (W, 0.8–1.2 μm; L, 1.5–2.5 μm), and sister cells may still share the outer membrane (OM) after cell division while staying connected via a junction between their inner membranes (IM) (see here). Sheathed cells form flexible, helical, unbranched filaments (helix width 1.5–2 µm; pitch 4–9 µm) that are up to 100 µm long (ref.). In the "open sea," in liquid culture, these Au­reispira armadas adopt a helical shape, which can still be seen when they rapidly move "on land," on agarose pads, on which their gliding motility al­lows them speeds of up to ~2 µm/s (a single filament in Movie 2.1, and whole bunches of filaments in Movie 2.2).
 

It is unknown which signals sent out by V. campbellii  and sensed by Aureispira – the chemoreceptor arrays in the cells could play a role here – and which allow the pirates to glide towards their prey head-on. But as the time-lapse videos demonstrate, it works brilliantly: the Vibrios are boarded and literally liquidated in the blink of an eye. Yet, how the pirates achieve their gliding mobility is still a bit of a mystery. Aureispira don't have flagella and also known mechanisms of pili-mediated movement can be excluded. Lapidus & Berg (1982) studied gliding motility in the distantly related bac­terium of the phylum Bacteroidota, Cytophaga sp. U67, and said: "..our work supports a model for gliding motility in bacteria in which ad­sorption sites within the outer membrane of the cell move along tracks fixed to the rigid framework of the cell wall." A candidate for such an "adsorption site" is the large SprB protein (6497 residues) of Flavobacterium johnsoniae (Bacteroidota), a homolog of which is encoded in the Aureispira sp. CCB-QB1 genome (WP_052599872.1).
 

(click to enlarge) Figure 3.

 

Pilhofer Lab  Jan 30, 2024 🧵3/6

Furthermore, Aureispira is equipped with Type9️⃣Secretion System-translocated grappling hooks interacting with prey's flagella. By solving their structure, we could identify that these hooks are assembled by a heptamer of a ~6000 amino acids loooooooooong protein 🪝🌴😱

 

(click to enlarge)
Movie 2.3. Source

Caption for Figure 5. Heptameric grappling hooks interact with prey flagella. (Left) Cryo-ET of cryo-FIB–thinned Aureispira–V. campbellii  prey mixture reveals interactions be­tween Aureispira grappling hooks (brown arrow­head) and prey flagella (magenta arrowhead). Shown are 13.8-nm-thick slices. Scale bars, 100 nm. (Center) Slice through a cryo–electron tomogram showing an Aureispira cell with several homo­gene­ous extracellular grapp­ling hooks (brown arrowhead). Scale bar, 100 nm. Shown is 13.8-nm-thick slice. Magnified view of the distal end of a grappling hook is provided in the inset. Scale bar, 10 nm. (Right) (E) A com­posite density map combining results of subtomogram averaging and singleparticle cryo-EM to show the overall structure of a grappling hook. Density maps obtained by subtomogram aver­aging are shown in light gray, and density maps obtained from single-particle cryo-EM are shown in brown or green. OM, yellow. (F) GhpA schematic represen­tation (left), indication of structural modules (middle). Me­thodologies used to determine the struc­ture of GhpA fragments and their corresponding amino acid residues are indicated next to the schematic. AF2, AlphaFold2 pre­diction; STA, subtomogram averaging; SPA, singleparticle cryo-EM. Source
 

In the open sea, in a liquid medium, the helical Aureispira armadas use their Type IX Se­cretion System (T9SS) as outboard-mounted grappling hooks to efficiently catch the buz­zing Vibrio campbellii  by their flagella (Figure 3, left). Individual Aureispira cells have so many of these ~240 nm­-long grappling hooks anchored in their outer membrane (Fig­ure 3, center) that in the few seconds-long snippet from the complete movie the fila­ments look almost "spiked" with Vibrios. And, once caught, the Vibrios do not get free again and are doomed (Movie 2.3). The researchers found that the successful "hooking" of flagella-less Vibrios (a Δflagellin mutant) by the pirates was drastically reduced in the open sea, while Vibrio hunting on land, by gliding on agar pads, were unaffected. This underscores the importance of the grappling hooks for the capture of the Vibrios by their flagella. I find it intriguing that the protein makeup of the grappling hooks apparently prevents them from "hooking" into each other – at least the researchers have not documented anything like this.

 

(click to enlarge) Figure 4.

 

Pilhofer Lab  Jan 30, 2024 🧵4/6

The actual killing is achieved by firing a unique Type6️⃣Secretion System 💉, having an additional extracellular antenna 📡 Using Raman microspectroscopy in colla­bo­ration with Kang Soo Lee @theStockerLab, we could show that prey cell compo­nents are taken up by the predator🥘

 

Caption for Figure 4. A type VI secretion system with distinctive features kills prey. (E, left) Composite isosurface re­pre­sentation of the subtomogram averages of closed an­ten­na, transenvelope complex, baseplate, and sheath-tube mo­dules. (L, middle) Slice through a cryo-tomogram of a cryo-FIB–thinned Aureispira–V. campbellii  mixture reveals a con­tracted T6SS sheath (blue arrowhead) and an associated expelled inner tube (green arrowhead). The inner tube is seen penetrating the OM of V. campbellii  (yellow arrow­head), apparently resulting in membrane vesiculation. SP, septum. Thickness of the slice: 13.8 nm. Scale bar, 100 nm. (M, right) Segmentation of the tomogram shown in (L). OM, yellow; IM, light brown; contracted T6SS sheath, blue; T6SS inner tube, green; ribosomes, gray. Source
 

Type VI Secretion Systems (T6SS) are often compared to "inverted bacteriophages" due to the phylogenetic relationship with the structural elements of phage tails and a comparable gene order (see diagram). This comparison largely holds for the T6SS^IV of Aureispira, with the special feature that the harpoon tip does not remain in the cytoplasm until ejection but is presented as an "antenna" outboard, anchored in the transmembrane complex of the T6SS and already attached to full-length sheathed harpoon shafts in the cytoplasm (Movie 1). In­trigu­ingly, the Aureispira harpoon tips were found to come in two flavors, "closed" (58%) and "open" (26%), but it is not known what triggers the opening/closing and whether this conformational switch is mechanistically linked to the launching of the harpoon (see here). Launching of the harpoon occurs by contraction of the cover sheath, which drives the ~420 nm-long harpoon shaft outboard through the transmem­brane complex. The contraction of T6SS sheaths has never been observed experimentally, as it takes place in the millisecond range. Note that this sheath contraction requires no energy input, just like the contraction of phage tails, but is caused by a conformational change of the sheath protein oligomers – similar to a wound spring that is suddenly released; it's pure mechanics (which is why I can't see a "cannon" in the T6SS). What has been observed – in Aureispira as in other systems – are fully assembled harpoons in the cytoplasm (Movie 1) and ejected harpoons associated with the contracted sheath remaining in the cytoplasm (Figure 4). For the actual killing, the harpoon tip may contain as "cargo" a fast-acting peptidoglycan-degrading enzyme, an amidase, because punctured prey cells showed signs of vesiculation (Figure 4) or, occasion­ally, rounding before bursting and releasing their contents close to the pirates.

 

Pilhofer Lab  Jan 30, 2024 🧵5/6

The expression of the costly Type6️⃣Secretion System can be switched on or off 🔛, depending on nutrient availability, by the insertion or excision of insertion sequences into essential T6SS genes (🍗 or ❌🍖)
 

A closer look at Aureispira's galley revealed that the pirates serve a special tidbit for con­nois­seurs of bacterial gene regulation, not the con­ventional activator/repressor menu. Under nu­trient-rich conditions, insertion elements (IS) were found to insert into the cis gene cluster en­coding the T6SS components deactivating their expression (and abolishing prey killing). In nutrient-limiting conditions, the re­searchers ob­served reactivation events by IS excisions after prolonged in­cubation with V. campbelii for ~30 d, leading to a fitness advantage through re­sumed ixo­tro­phy (see here). There are – but only a few – known cases of such "trans-generational" genetic switches that work by insertion/excision of IS elements. One example would be the insertion of an IS element upstream of the promoter of the master regulator of E. coli's flagellar genes, flhDC, which leads to an increase in the synthesis of flagella and a mobility boost for the strain (ref.).

 

(click to enlarge) Figure 5.

 

Pilhofer Lab  Jan 30, 2024 🧵6/6

But does this happen in the environment? 🌍 With the help of @NBartlau and @Mar­­tinPolz, we analyzed a time-resolved metagenomic dataset from the ocean 🌊 and indeed found coupled dynamics indicating a predator-prey relationship between Vibrionaceae and ixotrophic predators
 

Caption for Figure 5. Analysis of a time-resolved metagenomic dataset (Nahant time series) shows correlation be­tween the abundance of Vibrionaceae (gray) and ixotrophic predators (Saprospira; orange and yellow) in the environment. The time-lag in the correlation resembles a dynamic predator prey interaction. In contrast, an ixotrophic-negative Bacteroidota (Algoriphagus machipon­go­nensis; black) does not show a correlation to the Vibrio bloom. Source
 

The coupled dynamics found for the relationship between Vibrionaceae and ixotrophic predators is, of course, indirect yet plausible evi­dence for the predator/prey relationship of Aureispira sp. CCB-QB1 and Vibrionaceae in the natural environment. The authors found Au­reispira sp. CCB-QB1 in their data sets but consistently in such small numbers that a comparison with shortly before observed blooms of Vibrionaceae could not be meaningful. However, one of the organisms detected in the metagenomic data set, Sa­pro­spira grandis str. Le­win, is well known for its ixotrophic lifestyle ("ixotrophy-positive"). It gave Ralph Lewin (1921–2008) reason to define ixotrophy in the first place, as Elio had already written in 2016 in his piece Catch as Catch Can (see here a fine YouTube video explaining ixotrophy).
 

(click to enlarge)
Figure 6. See text for details (Figure partly created with BioRender.com). Source

 

Pilhofer Lab  Oct 18, 2024

The peer-reviewed version of the manuscript can be found here (paywall) 🙏 We thank the reviewers for their constructive feedback!
 

Lien et al. (2024) included a graphical abstract in their paper that visually explains the me­chanism of bacterial predation through ixotrophy (Figure 6). Aureispira sp. CCB-QB1 predates on V. campbellii  in a two-step process: Cell-cell contact with prey is established using gliding motility on solid surfaces or T9SS-secreted grappling hooks in liquid environments. This is followed by prey killing by means of a T6SS and uptake of substrates. Please use the click to enlarge option to make the most of this figure.

 

The daring pirates only cruise in this 🧵 thread on 𝕏. In the published paper, the authors abstained from the metaphor with one exception. They refer to the T9SS of Aureispira as "grappling hook" because of the striking similarity in structure and use with the classic tool of seafarers, includ­ing pirates. "Grappling hooks" are not the specialized tools of a few bacterial pirates but an established scientific term. Already in 2007, in Ar­chae­al Ninjas, Elio introduced the "ha­mi" ([ˈhäːmʊs̠] Latin, hook), a variant of this tool from an archaeon belonging to the DPANN superphylum, Ca. Al­tiarchaeum hamiconexum SM1 (see also here in STC).
 

Métaphores...

Figure 7. Boutique 'Métaphores', Place de Furstemberg, Paris, France. ©2024 Christoph Weigel

Pirate legends have always fascinated me with their ambiguity of both heroism and infamy. So when I read about Captain Aureispira on 𝕏, learned about its armament and saw the movies, I immediately thought of Captain Jack Sparrow from Pirates of the Caribbean. The meta­phor made perfect sense to me, it's playful, and I was glad to spin this out further in my comments.
 

The latter also had a more serious reason. I am so weary of the arsenal of war metaphors that biologists habitually use to cover various aspects of the lives of the small things they con­sider. So habitually, in fact, that the metaphors are stripped of their meta- and are taken as ad­e­quate descriptions of reality. And even become accepted scientific terms – read what Wiki­pedia says about 'evolutionary arms race'.
 

Of necessity, biologists cannot do without metaphors when they observe, describe and talk about biological, physical and chemical processes on scales and timescales that lie beyond their own human horizon of perception. They lack the appropriate vocabulary. By playfully introducing metaphors derived from fictional stories, scientists do not undermine their se­riousness. Rather, they reveal their mindset and cultural background so that the audience can judge the extent to which the proposed metaphor is intelligible and apt. Captain Jack Sparrow would have talked less, I'm afraid, and left it to his piercing gaze! Aye, Captain!
 

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Comments on Bluesky
 

‪· Brigitte Nerlich‬ ‪@bnerlich.bsky.social‬
I like that. A lesson in metaphor use, where playfulness breaks down the walls of dull war metaphors that sometimes hold microbiologists captive (or to quote good old Wittgenstein, 'show the fly the way out of the fly bottle'). What do you think @vdlorenzo.bsky.social
 

‪· Victor de Lorenzo (CNB, Madrid)‬ ‪‪@vdlorenzo.bsky.social
↩ Reply to Brigitte Nerlich
‬I love it! First, a metaphor should be acknowledged as such, without pretending that it is the real thing. And second, piracy is a good one, as the same pirates could at times be evil robbers and at other times respectable merchants … in a context-dependent fashion!
 

‪· christoph_STCmicrobeblog @christophstc.bsky.social
↩ Reply to Victor de Lorenzo (CNB, Madrid)
‬"...pirates could at times be evil robbers and at other times respectable merchants"
the "frenemy" relationship of 𝘌𝘮𝘪𝘭𝘪𝘢𝘯𝘢 𝘩𝘶𝘹𝘭𝘦𝘺𝘪 with 𝘚𝘶𝘭𝘧𝘪𝘵𝘰𝘣𝘢𝘤𝘵𝘦𝘳 D7 comes to mind here. see > pubmed.ncbi.nlm.nih.gov/36691727/
 

‪· Alex Merz @merz.bsky.social‬
Amazing and awesome! What a great summary, Christophe. One minor addition — Katy Forest and used the term "grappling hooks" to describe retractile type 4 pili in 2002, and I doubt that we were the first to apply the term to bacterial adhesion and motility.
pubmed.ncbi.nlm.nih.gov/11967173/
 

‪· STCmicrobeblog @stcmicrobeblog.bsky.social‬
↩ Reply to Alex Merz
You're right, Alex, the use of the term “grappling hook” predates Elio's mentioning them in his piece from 2007. Finding out who first used the term is probably as impossible as exploring all its uses among different bacteria and archaea, and its various structures :-)