What are the odds of ‘wrapping up’ a postdoctoral project ten years after its completion? Probably low when moving to a line of work that is unrelated to the previous one. And yet, this is exactly what happened with GIL01 thanks to a collaborative effort across continents.

GIL01 is a temperate bacteriophage (or phage for short) infecting the soil bacterium Bacillus thuringiensis in which it enjoys a double life of dormancy and occasional multiplication at the detriment of the host. With a single 15-kb DNA genome encapsulated in no more than a 50-nm-wide protein shell, GIL01 remains largely mysterious despite its small size. This is the case of most temperate phages, many are known, but very few are studied in detail. It does not come as a surprise then that I went on to make GIL01 the subject of both my doctoral and postgraduate work!

It is during the latter that GIL01 took me to the laboratory of Professor Margarita Salas, at the Centro de Biología Molecular Severo Ochoa in Madrid, between 2010 and 2014, to unravel its mechanisms. Margarita’s lab was the perfect environment to learn both biochemistry and Spanish. Equipped with GIL01 mutants that were unable to carry on latency, I decided to decipher each one of them. Mutations were found in non-coding sequences, such as promoter operators, as well as in coding sequences, such as ORF1 and ORF7. To study the mutants, I learned a repertoire of ‘old school’ techniques targeting protein-protein and protein-DNA interactions. In particular, I focused on identifying the roles of the proteins encoded by ORFs 1 and 7, that I named gp1 and gp7, respectively. Since GIL01’s cycle was in part regulated by the host SOS response to genomic stress, I hypothesized that gp1 and gp7 were either involved in the SOS response or worked independently and in parallel to control the phage cycle. It turns out that both scenarios were true: gp1 works on its own and in synch with gp7, which binds to and stabilizes LexA, the SOS response repressor.

At the same time, I turned to LexA experts in the Butala lab in Ljubljana to tap into their expertise with Surface Plasmon Resonance (SPR), a system that enables to precisely measure protein association to and dissociation from a substrate (such as DNA). This multi-site collaboration quickly turned into a fruitful endeavor that has since yielded many results and publications (PMIDs: 26138485; 26970840; 36434275). The latest chapter is fully dedicated to gp1, which we found binds to GIL01 promoters to regulate gene expression as well as GIL01’s vertical transmission. The exact mechanisms by which gp1 assumes these roles are not fully known but our latest study is one step further into deciphering GIL01’s switch from latency to the lytic cycle. A remarkable finding, which is the result of curiosity and a strong hunch, is that GIL01 will tend to evolve towards achieving latency. To cope with a mutation in gp1 that confines it to the lytic cycle, GIL01 will accrue mutations elsewhere to bypass the loss of a functional gp1. In other words, GIL01’s genome plasticity is in fact a consequence of a restricted lifestyle, one that aims to strive towards quiescence as much as possible.

Working with Margarita Salas was the privilege of a lifetime, an incredible opportunity to learn from one of the finest minds in phage biology and much more. By bringing the Butala lab on board, we complemented skills to investigate the biology of what is turning into its own textbook, a small phage with a grand lifestyle.