Dynamical aspects of the regulation of bacterial proliferation

Bacterial proliferation has been studied for more than 100 years, but our knowledge of the mechanisms that control its dynamical aspects is still very limited. In this Thesis we have studied, at both singlecell and population levels, different aspects of the main regulators of bacterial proliferation, namely the cell cycle, biomass production and membrane stability. Our goal has been to shed light on how perturbation of these regulators affects their dynamical responses in a variety of situations. Specifically, we show that the periodic doubling of genes through the bacterial cell cycle partially entrains a genetic oscillator, but that significant coupling only arises when the oscillator is fed back to cell cycle. We have also studied perturbations in the ability of cells to produce biomass, for instance through antibiotics that affect ribosomal function. Our results suggest that survival in the presence of these antibiotics is determined by the ability of the cells to incorporate magnesium ions, which can be captured by membrane potential changes. Finally, we show that interplay between different bacterial species with diverse sensitivities to membrane-targeting antibiotics can have an unexpected outcome in co-culture, which can be explained in a simple manner by the sharing of the antibiotic between the two species.La proliferació bacteriana ha estat estudiada durant més de 100 anys, però el nostre coneixement dels mecanismes que controlen els aspectes dinàmics d’aquesta encara són molt limitats. En aquesta Tesi hem estudiat, tant en cèl.lules individuals com a nivell poblacional, diferents aspectes dels principals reguladors de la proliferació cel.lular, concretament el cicle cel.lular, la producció de biomassa i la estabilitat de la membrana. El nostre objectiu ha estat ajudar a explicar com pertorbacions en aquests reguladors afecten la dinàmica de les seves respostes en una varietat de situacions. Específicament, mostrem que el doblament periòdic de gens a través del cicle cel.lular bacterià entrena parcialment un oscil.lador genètic, però aquest acoblament només és significatiu quan l’oscil.lador retorna la resposta al cicle cel.lular. Tamée hem estudiat pertorbacions en l’habilitat de les cèl.lules de produir biomassa, per exemple a través d’antibiòtics que afecten la funció ribosomal. Els nostres resultats suggereixen que la supervivència sota l’efecte d’aquests antibiòtics ve determinada per l’habilitat de les cèl.lules d’incorporar ions de magnesi, la qual pot ser capturada a través de canvis en el potencial de membrana. Finalment, mostrem que les interaccions entre diferents espècies bacterianes amb diverses sensitivitats a antibiòtics que afecten la membrana cel.lular poden donar lloc a resultats inesperats quan es troben en co-cultiu, els quals poden ser explicats de forma senzilla a partir del compartiment de l’antibiòtic entre les dues espècies

Dynamical aspects of the regulation of bacterial proliferation

Bacterial proliferation has been studied for more than 100 years, but our knowledge of the mechanisms that control its dynamical aspects is still very limited. In this Thesis we have studied, at both singlecell and population levels, different aspects of the main regulators of bacterial proliferation, namely the cell cycle, biomass production and membrane stability. Our goal has been to shed light on how perturbation of these regulators affects their dynamical responses in a variety of situations. Specifically, we show that the periodic doubling of genes through the bacterial cell cycle partially entrains a genetic oscillator, but that significant coupling only arises when the oscillator is fed back to cell cycle. We have also studied perturbations in the ability of cells to produce biomass, for instance through antibiotics that affect ribosomal function. Our results suggest that survival in the presence of these antibiotics is determined by the ability of the cells to incorporate magnesium ions, which can be captured by membrane potential changes. Finally, we show that interplay between different bacterial species with diverse sensitivities to membrane-targeting antibiotics can have an unexpected outcome in co-culture, which can be explained in a simple manner by the sharing of the antibiotic between the two species.La proliferació bacteriana ha estat estudiada durant més de 100 anys, però el nostre coneixement dels mecanismes que controlen els aspectes dinàmics d’aquesta encara són molt limitats. En aquesta Tesi hem estudiat, tant en cèl.lules individuals com a nivell poblacional, diferents aspectes dels principals reguladors de la proliferació cel.lular, concretament el cicle cel.lular, la producció de biomassa i la estabilitat de la membrana. El nostre objectiu ha estat ajudar a explicar com pertorbacions en aquests reguladors afecten la dinàmica de les seves respostes en una varietat de situacions. Específicament, mostrem que el doblament periòdic de gens a través del cicle cel.lular bacterià entrena parcialment un oscil.lador genètic, però aquest acoblament només és significatiu quan l’oscil.lador retorna la resposta al cicle cel.lular. Tamée hem estudiat pertorbacions en l’habilitat de les cèl.lules de produir biomassa, per exemple a través d’antibiòtics que afecten la funció ribosomal. Els nostres resultats suggereixen que la supervivència sota l’efecte d’aquests antibiòtics ve determinada per l’habilitat de les cèl.lules d’incorporar ions de magnesi, la qual pot ser capturada a través de canvis en el potencial de membrana. Finalment, mostrem que les interaccions entre diferents espècies bacterianes amb diverses sensitivitats a antibiòtics que afecten la membrana cel.lular poden donar lloc a resultats inesperats quan es troben en co-cultiu, els quals poden ser explicats de forma senzilla a partir del compartiment de l’antibiòtic entre les dues espècies

Antithetic population response to antibiotics in a polybacterial community

Much is known about the effects of antibiotics on isolated bacterial species, but their influence on polybacterial communities is less understood. Here, we study the joint response of a mixed community of nonresistant Bacillus subtilis and Escherichia coli bacteria to moderate concentrations of the β-lactam antibiotic ampicillin. We show that when the two organisms coexist, their population response to the antibiotic is opposite to that in isolation: Whereas in monoculture B. subtilis is tolerant and E. coli is sensitive to ampicillin, in coculture it is E. coli who can proliferate in the presence of the antibiotic, while B. subtilis cannot. This antithetic behavior is predicted by a mathematical model constrained only by the responses of the two species in isolation. Our results thus show that the collective response of mixed bacterial ecosystems to antibiotics can run counter to what single-species potency studies tell us about their efficacy

IonoBiology: The functional dynamics of the intracellular metallome, with lessons from bacteria

Metal ions are essential for life and represent the second most abundant constituent (after water) of any living cell. While the biological importance of inorganic ions has been appreciated for over a century, we are far from a comprehensive understanding of the functional roles that ions play in cells and organisms. In particular, recent advances are challenging the traditional view that cells maintain constant levels of ion concentrations (ion homeostasis). In fact, the ionic composition (metallome) of cells appears to be purposefully dynamic. The scientific journey that started over 60 years ago with the seminal work by Hodgkin and Huxley on action potentials in neurons is far from reaching its end. New evidence is uncovering how changes in ionic composition regulate unexpected cellular functions and physiology, especially in bacteria, thereby hinting at the evolutionary origins of the dynamic metallome. It is an exciting time for this field of biology, which we discuss and refer to here as IonoBiology.We acknowledge Katherine Süel and Steve Lockless for helpful discussions. Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311. J.G.O. was supported by the Spanish Ministry of Science and Innovation and FEDER, under projects FIS2017-92551-EXP and PGC2018-101251-B-I00, by the “Maria de Maeztu” Programme for Units of Excellence in R\&D (grant CEX2018-000792-M), and by the Generalitat de Catalunya (ICREA Academia programme). G.M.S acknowledges funding from NIH/NIGMS R35 GM139645, NIH/NIGMS R01 GM121888, and Howard Hughes Medical Institute – Simons Foundation Faculty Scholar

A segmentation clock patterns cellular differentiation in a bacterial biofilm

Contrary to multicellular organisms that display segmentation during development, communities of unicellular organisms are believed to be devoid of such sophisticated patterning. Unexpectedly, we find that the gene expression underlying the nitrogen stress response of a developing Bacillus subtilis biofilm becomes organized into a ring-like pattern. Mathematical modeling and genetic probing of the underlying circuit indicate that this patterning is generated by a clock and wavefront mechanism, similar to that driving vertebrate somitogenesis. We experimentally validated this hypothesis by showing that predicted nutrient conditions can even lead to multiple concentric rings, resembling segments. We additionally confirmed that this patterning mechanism is driven by cell-autonomous oscillations. Importantly, we show that the clock and wavefront process also spatially patterns sporulation within the biofilm. Together, these findings reveal a biofilm segmentation clock that organizes cellular differentiation in space and time, thereby challenging the paradigm that such patterning mechanisms are exclusive to plant and animal development.We acknowledge Munehiro Asally, Tolga Çağatay, and Katherine Süel for helpful discussions. K.-T.C. acknowledges support from National Institutes of Health grant T32GM127235. J.G.-O. acknowledges support from the Spanish Ministry of Science, Innovation and Universities and FEDER (Project PGC2018-101251-B-I00 and CEX2018-000792-M), and from the Generalitat de Catalunya (ICREA Academia program). G.M.S. acknowledges support for this research from National Institute of General Medical Sciences grants R01 GM121888 and R35 GM139645. G.M.S. is a Howard Hughes Medical Institute - Simons Foundation Faculty Scholar

Magnesium flux modulates ribosomes to increase bacterial survival

Bacteria exhibit cell-to-cell variability in their resilience to stress, for example, following antibiotic exposure. Higher resilience is typically ascribed to "dormant" non-growing cellular states. Here, by measuring membrane potential dynamics of Bacillus subtilis cells, we show that actively growing bacteria can cope with ribosome-targeting antibiotics through an alternative mechanism based on ion flux modulation. Specifically, we observed two types of cellular behavior: growth-defective cells exhibited a mathematically predicted transient increase in membrane potential (hyperpolarization), followed by cell death, whereas growing cells lacked hyperpolarization events and showed elevated survival. Using structural perturbations of the ribosome and proteomic analysis, we uncovered that stress resilience arises from magnesium influx, which prevents hyperpolarization. Thus, ion flux modulation provides a distinct mechanism to cope with ribosomal stress. These results suggest new approaches to increase the effectiveness of ribosome-targeting antibiotics and reveal an intriguing connection between ribosomes and the membrane potential, two fundamental properties of cells.This work was supported by funding from The Spanish Ministry of Economy and Competitiveness and FEDER (project FIS2015-66503-C3-1-P) (to J.G.-O.), the ICREA Academia program (to J.G.-O.), and the Maria de Maeztu Program for Units of Excellence in Research and Development (Spanish Ministry of Economy and Competitiveness, MDM-2014-0370) (to J.G.-O.), the San Diego Center for Systems Biology (NIH P50 GM085764) (to G.M.S), National Institute of General Medical Sciences (R01 GM121888) (to G.M.S), and the Howard Hughes Medical Institute-Simons Foundation Faculty Scholars program (to G.M.S.)

Magnesium flux modulates ribosomes to increase bacterial survival

Bacteria exhibit cell-to-cell variability in their resilience to stress, for example, following antibiotic exposure. Higher resilience is typically ascribed to "dormant" non-growing cellular states. Here, by measuring membrane potential dynamics of Bacillus subtilis cells, we show that actively growing bacteria can cope with ribosome-targeting antibiotics through an alternative mechanism based on ion flux modulation. Specifically, we observed two types of cellular behavior: growth-defective cells exhibited a mathematically predicted transient increase in membrane potential (hyperpolarization), followed by cell death, whereas growing cells lacked hyperpolarization events and showed elevated survival. Using structural perturbations of the ribosome and proteomic analysis, we uncovered that stress resilience arises from magnesium influx, which prevents hyperpolarization. Thus, ion flux modulation provides a distinct mechanism to cope with ribosomal stress. These results suggest new approaches to increase the effectiveness of ribosome-targeting antibiotics and reveal an intriguing connection between ribosomes and the membrane potential, two fundamental properties of cells.This work was supported by funding from The Spanish Ministry of Economy and Competitiveness and FEDER (project FIS2015-66503-C3-1-P) (to J.G.-O.), the ICREA Academia program (to J.G.-O.), and the Maria de Maeztu Program for Units of Excellence in Research and Development (Spanish Ministry of Economy and Competitiveness, MDM-2014-0370) (to J.G.-O.), the San Diego Center for Systems Biology (NIH P50 GM085764) (to G.M.S), National Institute of General Medical Sciences (R01 GM121888) (to G.M.S), and the Howard Hughes Medical Institute-Simons Foundation Faculty Scholars program (to G.M.S.)