How a pandemic pickpockets a prophage

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Bacteria exist to compete for food. The cholera-causing bacterium Vibrio cholerae brings a harpoon gun (that my mentor Stefan Pukatzki and John Mekalanos coined the type VI secretion system or T6SS for short) to this food fight. This defense system is a contractile sheath around a spiked tube tipped with toxic effector proteins that are injected into nearby cells. Evolutionarily, the T6SS looks like a headless bacteriophage loaded onto the bacterial inner membrane. Early on, researchers highlighted the T6SS as a co-opted prophage, but these studies didn’t observe the process in action. While the majority of identified T6SS clusters carry phage-like structural genes, they lack the genes that make a phage mobile – integrases, regulators, etc. That is until we looked at Aux3. Our recent study ( shows how a prophage-like element was degraded into a T6SS cluster in pandemic V. cholerae.

When I joined the Pukatzki laboratory in 2016, Stefan posed a question to me. “This T6SS island (Aux3) has a nearby integrase. Is it mobile?” He gave me five strains and 25 genomes. I did a polymerase chain reaction (PCR) to test whether Aux3 could excise from the chromosome. In the first two weeks of my rotation, I found that Aux3 excised from one environmental isolate (AM-19226), but not from pandemic isolates. I spent the rest of my rotation and several I-70 traffic jams banging my head against the wall/steering wheel for ways to induce excision in the pandemics, but nothing worked. In a Hail Mary, I designed new primers. It turned out that I had an efficiency issue. All strains excised Aux3 without induction, but AM-19226 was excising significantly more (Fig. 1)! So why the difference?

Figure 1. The gel that kicked it all off. (Top) Diagram of my PCR to detect excised Aux3 with primers shown as blue and orange arrows. (Bottom) My first gel image detecting excision of Aux3 in all samples but at different quantities. 

Luckily, a former PhD student, Daniel Unterweger, left me with the observation that while most strains with Aux3 carry a six-gene version, one non-pandemic V. cholerae strain (AM-19226) has ~45 phage-like genes in the Aux3 island. Stefan and I decided to take a step back and look at the greater V. cholerae population. I downloaded 749 cholera genomes and got to searching. I spent hours on my girlfriend’s (now fiancé’s) couch wrestling with R as her roommate binged Dawson’s Creek (each of us suffering emotionally in our own way). From this grind arose a crucial finding; while all pandemic V. cholerae isolates had the six-gene Aux3, nine environmental strains had a 40-plus gene version that looked a lot like an intact prophage. This realization gave me the idea that we were looking at a snapshot of T6SS development in that I had a prophage-like element bouncing around in the environment that was degraded to just the components that conferred a competitive edge in pandemic strains (Fig. 2). 

Figure 2. The Big Picture. (Left) Theoretical tree of V. cholerae evolution with Aux3(+) strains in green and Aux3(-) strains in blue. Green dashed arrows represent Aux3 lateral transfer. (Right) Schematics of the readily excised environmental Aux3 (bottom) and the domesticated pandemic Aux3 (top).

This finding still didn’t explain the differential excision in pandemic and environmental strains, until I looked more closely at the recombination machinery. I found that somewhere along the evolutionary path of pandemic V. cholerae, a piece of the Aux3 integrase was lost, reducing the enzyme’s functionality and Aux3’s ability to exit the chromosome. Further, the pandemic Aux3 lost a recombination directionality factor (this finding brought to you by three constructive reviewers and three months of Covid-19 lockdown in a small Denver apartment with nothing to do but stare at genomes and read papers). This small protein works with the integrase to drive excision. In short, Aux3 excision is blocked by two separate mechanisms in pandemic V. cholerae, indicating a selective pressure to maintain Aux3.

This story is the product of four years of confusion and enlightenment. In that time, I’ve learned to trust my gut, trust my reviewers, but never the first set of primers. In the end, we think these findings paint a picture of phage domestication in T6SS evolution and point to Aux3 as a potential pandemic factor in V. cholerae. I hope you enjoy the paper as much as I enjoyed the ride.

Frank Santoriello

PhD Candidate, University of Colorado Anschutz Medical Campus

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