Borreliella (syn. Borrelia) burgdorferi is a spirochete bacterium that causes tick-borne Lyme disease. Along its lifecycle B. burgdorferi develops several pleomorphic forms with unclear biological and medical relevance. Surprisingly, these morphotypes have never been compared at the global transcriptome level. To fill this void, we grew B. burgdorferi spirochete, round body, bleb, and biofilm-dominated cultures and recovered their transcriptomes by RNAseq profiling. We found that round bodies share similar expression profiles with spirochetes, despite their morphological differences. This sharply contrasts to blebs and biofilms that showed unique transcriptomes, profoundly distinct from spirochetes and round bodies. To better characterize differentially expressed genes in non-spirochete morphotypes, we performed functional, positional, and evolutionary enrichment analyses. Our results suggest that spirochete to round body transition relies on the delicate regulation of a relatively small number of highly conserved genes, which are located on the main chromosome and involved in translation. In contrast, spirochete to bleb or biofilm transition includes substantial reshaping of transcription profiles towards plasmids-residing and evolutionary young genes, which originated in the ancestor of Borreliaceae. Despite their abundance the function of these Borreliaceae-specific genes is largely unknown. However, many known Lyme disease virulence genes implicated in immune evasion and tissue adhesion originated in this evolutionary period. Taken together, these regularities point to the possibility that bleb and biofilm morphotypes might be important in the dissemination and persistence of B. burgdorferi inside the mammalian host. On the other hand, they prioritize the large pool of unstudied Borreliaceae-specific genes for functional characterization because this subset likely contains undiscovered Lyme disease pathogenesis genes.

Correspondence between evolution and development has been discussed for more than two centuries ¹ . Recent work reveals that phylogeny-ontogeny correlations are indeed present in developmental transcriptomes of eukaryotic clades with complex multicellularity ^2–10 . Nevertheless, it has been largely ignored that the pervasive presence of phylogeny-ontogeny correlations is a hallmark of development in eukaryotes ^{6, 10–12} . This perspective opens a possibility to look for similar parallelisms in biological settings where developmental logic and multicellular complexity are more obscure ^13–16 . For instance, it has been increasingly recognized that multicellular behaviour underlies biofilm formation in bacteria ^{13, 14, 17–19} . However, it remains unclear whether bacterial biofilm growth shares some basic principles with development in complex eukaryotes ^{14–16, 18, 20} . Here we show that the ontogeny of growing Bacillus subtilis biofilms recapitulates phylogeny at the expression level. Using time-resolved transcriptome and proteome profiles, we found that biofilm ontogeny correlates with the evolutionary measures, in a way that evolutionary younger and more diverged genes were increasingly expressed towards later timepoints of biofilm growth. Molecular and morphological signatures also revealed that biofilm growth is highly regulated and organized into discrete ontogenetic stages, analogous to those of eukaryotic embryos ^{11, 21} . Together, this suggests that biofilm formation in Bacillus is a bona fide developmental process comparable to organismal development in animals, plants and fungi. Given that most cells on Earth reside in the form of biofilms ²² and that biofilms represent the oldest known fossils ²³ , we anticipate that the widely-adopted vision of the first life as a single-cell and free-living organism needs rethinking.