Iron acquisition is of fundamental importance for microorganisms, since this metal is generally poorly bioavailable under natural conditions. In the environment, most bacteria are found tightly packed within multicellular communities named biofilms. Here, using the soil Gram-positive bacterium Bacillus subtilis, we show that biofilm formation and the production of siderophores, i.e., small molecules specifically binding metals, are both essential to ensure Fe uptake from the medium and maintain cellular Fe homeostasis. The biofilm matrix appears to play an important role favoring the efficient usage of siderophores. Taken together, our results demonstrate a close link between biofilm formation and iron acquisition in B. subtilis, allowing a better comprehension of how bacteria can cope with metal limitation under environmental conditions.

Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer–ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.

Siderophores are soluble or membrane-embedded molecules that bind the oxidized form of iron, Fe(III), and play roles in iron acquisition by microorganisms. Fe(III)-bound siderophores bind to specific receptors that allow microbes to acquire iron. However, certain soil microbes release a compound (pulcherriminic acid, PA) that, upon binding to Fe(III), forms a precipitate (pulcherrimin) that apparently functions by reducing iron availability rather than contributing to iron acquisition. Here, we use Bacillus subtilis (PA producer) and Pseudomonas protegens as a competition model to show that PA is involved in a peculiar iron-managing system. The presence of the competitor induces PA production, leading to precipitation of Fe(III) as pulcherrimin, which prevents oxidative stress in B. subtilis by restricting the Fenton reaction and deleterious ROS formation. In addition, B. subtilis uses its known siderophore bacillibactin to retrieve Fe(III) from pulcherrimin. Our findings indicate that PA plays multiple roles by modulating iron availability and conferring protection against oxidative stress during inter-species competition.

Iron (Fe) is one of the most important micronutrients for most life forms on earth. While abundant in soil, Fe bioavailability in oxic soil is very low. In environmental conditions, bacteria need to acquire sufficient Fe to sustain growth while limiting the energy cost of siderophore synthesis. Biofilm formation might mitigate this Fe stress, since it was shown to accumulate Fe in certain Gram-negative bacteria and that this Fe could be mobilized for uptake. However, it is still unclear if and to what extent the amount of Fe accumulated in the biofilm can sustain growth, and if the mobilization of this local Fe pool is modulated by the availability of environmental Fe (i.e., Fe outside the biofilm matrix). Here we use a non-domesticated strain of the ubiquitous biofilm-forming soil bacterium, Bacillus subtilis, and stable Fe isotopes to precisely evaluate the origin of Fe during growth in the presence of tannic acid and hydroxides, used as proxies for different environmental conditions. We report that this B. subtilis strain can accumulate a large quantity of Fe in the biofilm, largely exceeding Fe associated with cells. We also report that only a fraction of biofilm-bound Fe is available for uptake in the absence of other sources of Fe in the vicinity of the biofilm. We observed that availability of environmental Fe modulates the usage of this pool of biofilm-bound Fe. Finally, our data suggest that consumption of biofilm-bound Fe relates to the efficacy of B. subtilis to transport Fe from the environment to the biofilm, possibly through siderophores. Importance Recent evidences suggest that Fe bound to the biofilm could assume at least two important functions; a local source of Fe for uptake and a support to extracellular metabolism such as extracellular electron transfer. Our results show that B. subtilis can use biofilm-bound Fe for uptake only if it does not compromise Fe homeostasis of the biofilm, i.e., maintaining a minimum Fe concentration in the biofilm for extracellular purposes. We propose a theoretical framework based on our results and recent literature to explain how B. subtilis manages biofilm-bound Fe and Fe uptake in response to environmental Fe availability. These results provide important insights into the management of biofilm-bound and environmental Fe by B. subtilis in response to Fe stress.

Siderophores are soluble or membrane-embedded molecules that play a major role in Fe acquisition by microorganisms. Pulcherriminic acid (PA) is a compound produced by different microbes that sequesters Fe in the precipitated pulcherrimin, but which role in Fe homeostasis remains elusive. Using Bacillus subtilis (PA producer) and Pseudomonas protegens as a competition model, we demonstrated that PA is involved in a yet undescribed Fe-managing system. When challenged by a competitor, PA production creates a local Fe(III) source, which can be retrieved via the bacillibactin siderophore produced by B. subtilis. Furthermore, precipitation of Fe(III) as pulcherrimin prevents oxidative stress in bacterial competition by restricting the Fenton reaction and deleterious ROS formation. Together, our findings uncover that PA is at the core of a counterintuitive Fe management strategy that capitalizes on controlled Fe precipitation when challenged by a competitor. This makes PA a unique and multifunction tool in the iron war.