Persisters are dormant phenotypic variants of bacterial cells that are tolerant to killing by antibiotics1. Persisters are associated with chronic infections and antibiotic treatment failure1–3. In Escherichia coli, toxin/antitoxin (TA) modules have been linked to persister formation4–6. The mechanism of persister formation in Gram-positive bacteria is unknown. Staphylococcus aureus is a major human pathogen, responsible for a variety of chronic and relapsing infections such as osteomyelitis, endocarditis and infections of implanted devices. Deleting TA modules in S. aureus did not affect the level of persisters. Here we show that S. aureus persisters are produced due to a stochastic entrance into stationary phase accompanied by a drop in intracellular ATP. Cells expressing stationary state markers are present throughout the growth phase, increasing in frequency with cell density. Cell sorting revealed that expression of stationary markers is associated with a 100–1000 fold increase in the likelihood of survival to antibiotic challenge. The ATP level of the cell is predictive of bactericidal antibiotic efficacy and explains bacterial tolerance to antibiotics.

Chronic bacterial infections are difficult to eradicate, though they are caused primarily by drug-susceptible pathogens. Antibiotic-tolerant persisters largely account for this paradox. In spite of their significance in the recalcitrance of chronic infections, the mechanism of persister formation is poorly understood. We previously reported that a decrease in ATP levels leads to drug tolerance in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. We reasoned that stochastic fluctuation in the expression of tricarboxylic acid (TCA) cycle enzymes can produce cells with low energy levels. S. aureus knockouts in glutamate dehydrogenase, 2-oxoketoglutarate dehydrogenase, succinyl coenzyme A (CoA) synthetase, and fumarase have low ATP levels and exhibit increased tolerance of fluoroquinolone, aminoglycoside, and β-lactam antibiotics. Fluorescence-activated cell sorter (FACS) analysis of TCA genes shows a broad Gaussian distribution in a population, with differences of over 3 orders of magnitude in the levels of expression between individual cells. Sorted cells with low levels of TCA enzyme expression have an increased tolerance of antibiotic treatment. These findings suggest that fluctuations in the levels of expression of energy-generating components serve as a mechanism of persister formation. IMPORTANCE Persister cells are rare phenotypic variants that are able to survive antibiotic treatment. Unlike resistant bacteria, which have specific mechanisms to prevent antibiotics from binding to their targets, persisters evade antibiotic killing by entering a tolerant nongrowing state. Persisters have been implicated in chronic infections in multiple species, and growing evidence suggests that persister cells are responsible for many cases of antibiotic treatment failure. New antibiotic treatment strategies aim to kill tolerant persister cells more effectively, but the mechanism of tolerance has remained unclear until now.

Persisters represent a small subpopulation of cells within a bacterial culture that are tolerant to killing by antibiotics. Persisters have been linked to recalcitrant infections caused by numerous bacterial pathogens, including A classic example is the incurable infection of the airways for patients with cystic fibrosis. The genetic mediators of persister formation for are poorly understood. We generated a high-density transposon insertion library of PAO1 and determined the relative frequency of each insertion following fluoroquinolone treatment using transposon sequencing (Tn-seq). Of the 4,411 disrupted genes included in the screen, 137 had a ≥10-fold impact on survival. The gene disruption that resulted in the lowest survival rate was disruption of, which codes for the large subunit of carbamoyl phosphate synthetase (CPSase). CPSase is a metabolic enzyme that is involved in pyrimidine and arginine synthesis. Disruption of resulted in survival rates that were reduced by up to 2,500-fold following antibiotic treatment, and this phenotype was abolished by the addition of uracil, highlighting the importance of pyrimidine biosynthesis for persister formation. Disruption of resulted in intracellular ATP accumulation, and lowering ATP levels using arsenate restored the antibiotic tolerance profile of the mutant to levels similar to those seen with the wild type. A decrease in ATP would lead to reduced antibiotic target activity and increased survival. Antibiotic treatment of residing in the lung of cystic fibrosis patients is ineffective. Treatment failure is attributed in part to antibiotic-tolerant phenotypic variants known as persister cells. Understanding how these cells emerge will likely inform future therapeutic strategies. In the current study, we identified, which codes for the large subunit of carbamoyl-phosphate synthetase, as a persister gene that contributes to multidrug tolerance in Disruption of resulted in a metabolic perturbation that increased cellular ATP and reduced persister formation. Conversely, lowering ATP in the mutant restored antibiotic tolerance. Our data support the hypothesis that a drop in intracellular ATP is a general mechanism of persister formation in bacteria.

Environmental strains of the soil bacterium Bacillus subtilis have valuable applications in agriculture, industry, and biotechnology; however, environmental strains are genetically less accessible. This reduced accessibility is in sharp contrast to laboratory strains, which are well known for their natural competence, and a limitation in their applications. In this study, we observed that robust biofilm formation by environmental strains of B. subtilis greatly reduced the frequency of competent cells in the biofilm. By using model strain 3610, we revealed a cross-pathway regulation that allows biofilm matrix producers and competence-developing cells to undergo mutually exclusive cell differentiation. We further demonstrated that the competence activator ComK represses the key biofilm regulatory gene sinI by directly binding to the sinI promoter, thus blocking competent cells from simultaneously becoming matrix producers. In parallel, the biofilm activator SlrR represses competence through three distinct mechanisms involving both genetic regulation and cell morphological changes. Finally, we discuss the potential implications of limiting competence in a bacterial biofilm. IMPORTANCE The soil bacterium Bacillus subtilis can form robust biofilms, which are important for its survival in the environment. B. subtilis also exhibits natural competence. By investigating competence development in B. subtilis in situ during biofilm formation, we reveal that robust biofilm formation often greatly reduces the frequency of competent cells within the biofilm. We then characterize a cross-pathway regulation that allows cells in these two developmental events to undergo mutually exclusive cell differentiation during biofilm formation. Finally, we discuss potential biological implications of limiting competence in a bacterial biofilm.

12 13 14 15a. These two authors contributed equally to this work. 16 (She contributed to about 50% of the results in the initial submission and the 17 revision; Hunter initiated the project and contributed to about 30% of the 18 results in the initial submission.) Abstract 31 Environmental strains of the soil bacterium Bacillus subtilis have valuable 32 applications in agriculture, industry, and biotechnology. They are capable of forming 33 robust biofilms and demonstrate excellent biological control activities in plant 34 protection. However, environmental strains are genetically less accessible, a sharp 35 contrast to the laboratory strains well known for their natural competence and a 36 limitation toward their application. In this study, we observed that robust biofilm 37 formation of the environmental strains greatly reduces the rate of competent cells 38 within the biofilm. By using the model strain 3610, we reveal a cross-pathway 39 regulation that allows biofilm matrix producers and competence-developing cells to 40 undergo mutually exclusive cell differentiation. We show that the competence 41 activator ComK represses the key biofilm regulatory gene sinI by directly binding to 42 the sinI promoter, thus blocking competent cells from simultaneously becoming 43 matrix producers. In parallel, the biofilm activator SlrR represses competence 44 through three distinct mechanisms, involving both genetic regulation and cell 45 morphological changes. We discuss potential implications of limiting competence in 46 a bacterial biofilm. 47 48 3 Importance 49The soil bacterium Bacillus subtilis is capable of forming robust biofilms, a 50 multicellular community important for its survival in the environment. B. subtilis also 51 exhibits natural competence, the ability of cells to acquire genetic materials directly 52 from the environment. By investigating competence development in situ during B. 53 subtilis biofilm formation, we reveal that robust biofilm formation, an important 54 feature of the environmental strains of B. subtilis, often greatly reduces the rate of 55 competent cells within the biofilm. We characterize a cross-pathway regulation that 56 allows cells associated with these two developmental events to undergo mutually 57 exclusive cell differentiation during biofilm formation. Finally, we discuss potential 58 biological implications of limiting competence in a bacterial biofilm. 59 60 Bacillus subtilis is a soil-dwelling, spore-forming bacterium widely present in 61 nature, and plays important roles in environment, agriculture, and industry. B. subtilis 62 is also a plant growth-promoting rhizobacterium (PGPR), and a biological control 63 agent that shows various beneficial activities in plant protection (1). Biological control 64 by B. subtilis is attributed to a number of important abilities of the bacterium, 65 including antibiotic production, inhibition of pathogenic fungi and parasites, induction 66 of plant systemic resistance, and formation of plant root-associated biofilms (2-4).

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