Final Report Summary - BACTERIAL RESPONSE (New Concepts in Bacterial Response to their Surroundings) Bacteria in nature exhibit remarkable capacity to sense their surroundings and rapidly adapt to diverse conditions by gaining new beneficial traits. This extraordinary feature facilitates their survival when facing extreme environments. Utilizing Bacillus subtilis as our primary model organism, we studied two facets of this vital bacterial attribute: communication via extracellular nanotubes, a phenomenon we have previously discovered, and persistence as resilient spores while maintaining the potential to revive. In the past years we have made significant progress in understanding the bacterial response to their surroundings in two parallel aspects: I) the ability to communicate by producing extracellular nanotubes, and II) the capacity to reside as durable spores in response to environmental constraints and revive when possible. I will summarize our major achievements:I. We have previously identified a novel type of bacterial communication mediated by tubular extensions (nanotubes) that bridge neighboring cells. Using Bacillus subtilis as a model organism, we monitored the transfer of cytoplasmic fluorescent molecules, antibiotic resistance proteins and non-conjugative plasmids among adjacent cells (Dubey and Ben-Yehuda, 2011). Within the frame of our ERC grant, we have made significant progress in understanding these bacterial adaptive strategies according to our original specific aims: 1) By combining microscopy, biochemical and genetic studies, we characterized the molecular composition of nanotubes. We defined that nanotubes are composed of chains of membranous segments harboring a continuous lumen, directly emanating from cytoplasmic membrane (Dubey et al., 2016). We further identified the phosphodiesterase YmdB, as a major component required for nanotube formation and intercellular molecular trade (Dubey et al., 2016). Moreover, we elucidated that nanotubes emerge from widespread conserved components of the flagellar export apparatus, we termed CORE, and established the CORE-derived nanotube as a ubiquitous organelle, facilitating intercellular molecular trafficking across the bacterial kingdom (Bhattacharya et al., 2019). 2) We developed strategies for observing nanotube formation in living cells, by visualizing nanotube growth and dynamics, using newly developed fluorescence tools (Dubey et al., 2016). We subsequently observed the actual nanotube-delivered molecular cargo, at a single molecule resolution, using immuno-SEM (Stempler et al., 2017) and PALM (unpublished). 3) We studied whether nanotubes mediate phage infection, and revealed non-canonical modes of phage spreading in multispecies communities (Tzipilevich et al., 2017; Habusha et al., 2019). 4) We analyzed the formation and functionality of nanotubes connecting different bacterial species, and found nanotubes to impact colony formation, to be formed between the pioneering cells establishing the colony (Mamou et al., 2016) (Mamou et al., 2017), as well as to facilitate antagonistic interactions among species (Stempler et al., 2017; Bhattacharya et al., 2019). Yet, probably our most important and surprising discovery was that host-attached pathogenic bacteria extract nutrients directly from infected human cell cytoplasm via the conserved CORE-nanotube organelle (Pal et al., 2019). II. In parallel, we made important progress towards our understanding of spore robustness and their revival, by defining the entire spore revival proteome (Sinai et al., 2015), and by discovering that protein synthesis is carried out during germination, thereby fundamentally changing the current dogma of spore germination (Sinai et al., 2015). We further revealed the phosphoproteome of dormant and germinating spores for the first time (Rosenberg et al., 2015), and more recently, we found that dephosphorylation of arginine residues on key target proteins triggers germination by activating transcription and translational factors. Our results provide a whole new mechanism for spore awakening in bacteria (Zhou et al., in press). These achievements are in strong correlation with our original ERC goals.ReferencesBhattacharya, S., Baidya, A. K., Pal, R. R., Mamou, G., Gatt, Y. E., Margalit, H., Rosenshine, I., and Ben-Yehuda, S (2019). A ubiquitous platform for bacterial nanotube biogenesis. Cell Reports pii: S2211-1247(19)30234-7. doi: 10.1016/j.celrep.2019.02.055. [Epub ahead of print]. Dubey, G.P. and Ben-Yehuda, S. (2011). Intercellular nanotubes mediate bacterial communication. Cell 144, 590-600.Dubey, G.P. Malli Mohan, G.B. Dubrovsky, A., Amen, T., Tsipshtein, S., Rouvinski, A., Rosenberg, A., Kaganovich, D., Sherman, E., Medalia, O., et al. (2016). Architecture and Characteristics of Bacterial Nanotubes. Developmental cell 36, 453-461.Habusha, MS., Tzipilevich, ES., Fiyaksel, OS., and Ben-Yehuda, SPI. (2019). A mutant bacteriophage evolved to infect resistant bacteria gained a broader host range. Mol Microbiol. doi:10.1111/mmi.14231. [Epub ahead of print]. Mamou, G., Fiyaksel, O., Sinai, L., and Ben-Yehuda, S. (2017). Deficiency in Lipoteichoic Acid Synthesis Causes a Failure in Executing the Colony Developmental Program in Bacillus subtilis. Front Microbiol. 8:1991. Mamou, G., Malli Mohan, G.B. Rouvinski, A., Rosenberg, A., and Ben-Yehuda, S. (2016). Early Developmental Program Shapes Colony Morphology in Bacteria. Cell Rep 14, 1850-1857.Pal, R. R., Baidya, A.K. Mamou, G., Bhattacharya, S., Socol, Y., Kobi, S., Katsowich. N., Ben-Yehuda, S., and Rosenshine, I. (2019). Pathogenic E. coli extracts nutrients from infected host cells utilizing injectisome components. Cell pii:S0092-8674(19)30204-1. doi: 10.1016/j.cell.2019.02.022. [Epub ahead of print]. Rosenberg, A., Soufi, B., Ravikumar, V., Soares, N.C. Krug, K., Smith, Y., Macek, B., and Ben-Yehuda, S. (2015). Phosphoproteome dynamics mediate revival of bacterial spores. BMC biology 13, 76.Sinai, L., Rosenberg, A., Smith, Y., Segev, E., and Ben-Yehuda, S. (2015). The molecular timeline of a reviving bacterial spore. Molecular cell 57, 695-707.Stempler, O., Baidya, A., Bhattacharya, S., Malli Mohan, G.B. Tzipilevich, E., Sinai, L., Mamou, G., and Ben-Yehuda, S. (2017) Visualizing interspecies nutrient extraction and toxin delivery between bacteria. Nature Commun, 8(1), 315. Tzipilevich, E., Habusha, M., and Ben-Yehuda, S. (2017) Acquisition of phage sensitivity by bacteria through exchange of phage receptors. Cell, 168, 186-199.Zhou, B., Semanjski, M., Orlovetskie, N., Bhattacharya, S., Alon, S., Argaman, L., Jarrous, N., Zhang, Y., Macek, B., Sinai, L., and Ben-Yehuda, S. Arginine phosphorylation propels spore germination in bacteria. PNAS (direct submission), In press.
