Most bacteria, including commonly used lab strains, carry one or several prophages – a fact that we (as many other bacteriologists) used to ignore while studying our favorite microbe Bacillus subtilis… Until we noted an intriguing pattern across series of laboratory evolution experiments: EVERY TIME we imposed prolonged sporulation/spore revival regime on B. subtilis, genetic rearrangements were occurring in its prophage regions. Moreover, millions of phages were being released to the medium, as a result of spontaneous induction of phage lytic cycle. 

De novo genome sequencing revealed something rather unexpected – indigenous prophage (SPβ) which was originally present in our B. subtilis strains, now contained DNA fragments of a foreign phage (phi3Ts)! The two phages (SPβ and phi3Ts) recombined with each other into various hybrids, some of which became prophages, while others multiplied solely via lytic cycle. We also identified extrachromosomal phage DNA, mostly being recombinant phi3Ts-SPβ fragments. Far from understanding what was really going on, I was certain of one thing: My future academic life will be committed to exploring prophage-host interactions.

After nearly 3 years of experiments, data analysis and discussions with collaborators and colleagues, we are slowly beginning to understand what happens with B. subtilis prophages upon prolonged sporulation/spore-revival selection regime. The phi3Ts is hitchhiking between Bacillus labs, hiding in stocks of commonly used B. subtilis lab strains. For years, phi3Ts can pass unnoticed, because only a combination of the right starting stock and the right selection regime allows this phage to multiply and recombine with indigenous B. subtilis prophage SPβ.  We see that phi3Ts and SPβ exchange certain fragments of their DNA more frequently (we call these, recombination hot spots). Finally, presence of phi3Ts or its recombinant form, may be beneficial under sporulation selection due to certain regulatory genes influencing sporulation and germination timing.  

Can a laboratory observation, caused by a contaminant phage teach us something about microbial ecology and evolution of a bacterial species? To answer this question, we screened over a 1000 prophages in over 300 Bacillus genomes. We soon realized than around 40% of natural B. subtilis isolates carry mosaic prophages which could originate from phi3Ts and SPβ. Genomes of these prophages are 4x the size of an average prophage, they encode for sophisticated communication systems, biological weapons and regulators, and they seem to pervasively recombine with each other. However coincidental at a start, the invasion of phi3Ts might have turn our gaze into the most ecologically relevant prophage group within B. subtilis species.  We are only at the beginning of a journey to understanding the role of these large mosaic prophages in Bacillus ecology and evolution. In the meantime, it is time to enjoy paper acceptance cake and sparkling wine.

Unfortunately, in 2020 the cake and wine cannot be shared with collaborators, who made this work possible. Virtual cheers to devoted students Priydarshini B. and Zahraa Hasan for conducting a controlled experiment which confirmed that sporulation selection regime promotes phi3Ts invasion and for molecular work confirming prophage rearrangements, respectively. As always, a credit goes to Gergo Maróti for bacteria and phage sequencing and genome analysis and to Paul Kempen for phage imaging. Thanks to Briana Burton and Baundana Bose for sharing sequencing data on phi3Ts-free american B. subtilis stocks, and thanks to Oskar Kuipers for sharing raw seq. data on phi3Ts-containing Groningen stocks. Big thank you to Charlotte Kaspar and Ilka Bischofs for help with germination kinetics and stimulating discussions. A huge credit to Mikael Lenz Strube, as the bioinformatics part would never be possible without him. And last but not least - thank you to Ákos T. Kovács for keeping the faith in the story, research advice and support along the way and for stimulating research environment where, despite the pandemics and series of  starting grants rejections, you still enjoy thriving in academia.

Cheers!

PS1. The work will continue at the Biotechnical Faculty, University of Ljubljana, supported by a grant ERC Complementary scheme from Slovenian Research Agency (ARRS) :-)

PS2. To all twitter colleagues –this is not a fungus but a Siphoviridae phage attached to bacterial cell.