SinR is a mutational target for fine-tuning biofilm formation in laboratory-evolved strains of Bacillus subtilis

BMC Microbiology SinR is a mutational target for fine-tuning biofilm formation in laboratory-evolved strains of Bacillus subtilis Sara A Leiman 3 Laura C Arboleda 0 Joseph S Spina 0 2 Anna L McLoon 0 1 0 Biology Department, Colgate University , Hamilton, NY 13346 , USA 1 Current address: Department of Ecophysiology, MPI for Terrestrial Microbiology , D-35043 Marburg , Germany 2 Current address: Channing Division of Network Medicine, Brigham and Women's Hospital and Harvard Medical School , Boston, MA 02115 , USA 3 Department of Molecular and Cellular Biology, Harvard University , Cambridge, MA 02138 , USA Background: Bacteria often form multicellular, organized communities known as biofilms, which protect cells from a variety of environmental stresses. During biofilm formation, bacteria secrete a species-specific matrix; in Bacillus subtilis biofilms, the matrix consists of protein polymers and exopolysaccharide. Many domesticated strains of B. subtilis have a reduced ability to form biofilms, and we conducted a two-month evolution experiment to test whether laboratory culturing provides selective pressure against biofilm formation in B. subtilis. Results: Bacteria grown in two-month-long batch culture rapidly diversified their biofilm-forming characteristics, exhibiting highly diverse colony morphologies on LB plates in the initial ten days of culture. Generally, this diversity decreased over time; however, multiple types of colony morphology remained in our final two-month-old populations, both under shaking and static conditions. Notably, while our final populations featured cells that produce less biofilm matrix than did the ancestor, cells overproducing biofilm matrix were present as well. We took a candidate-gene approach to identify mutations in the strains that overproduced matrix and found point mutations in the biofilm-regulatory gene sinR. Introducing these mutations into the ancestral strain phenocopied or partially phenocopied the evolved biofilm phenotypes. Conclusions: Our data suggest that standard laboratory culturing conditions do not rapidly select against biofilm formation. Although biofilm matrix production is often reduced in domesticated bacterial strains, we found that matrix production may still have a fitness benefit in the laboratory. We suggest that adaptive specialization of biofilm-forming species can occur through mutations that modulate biofilm formation as in B. subtilis. Adaptation; Bacteria; Biofilms; Domestication; Laboratory; Selection - Background Many species of bacteria form multicellular communities called biofilms, in which aggregated bacterial cells are encased by an extracellular matrix that may comprise polysaccharides, proteins, and nucleic acids [1]. Despite the energetic cost of synthesizing extracellular matrix, cells in biofilms can have a fitness advantage over free-living cells [2]. Biofilms can help cells survive adverse conditions; for example, independent from genetic resistance mechanisms, cells within a biofilm are often more resistant to antibiotic treatment than are their planktonic counterparts [3,4]. Biofilms also allow bacteria to form robust communities on both biotic and abiotic surfaces, which can be ecologically beneficial in the environment but which often pose a threat in clinical and industrial settings [5,6]. Although biofilm regulatory pathways and the identities of matrix components are species-specific, the advantages of biofilm formation are widespread. Bacillus subtilis is an endospore-forming bacterium that is frequently found in the soil or associated with plants, and its biofilm-forming abilities have been studied in the laboratory for over a decade [7,8]. When B. subtilis approaches stationary phase in biofilm-promoting media, the bacteria initially a population of motile single cells and cell chains aggregate and become an ordered biofilm community [9]. Within the developing biofilm, a subset of cells secrete a matrix that contains complex polysaccharides, amyloid-like fibers of the protein TasA, and the hydrophobin BslA [10-13]. Notably, the operons responsible for matrix exopolysaccharide (epsA-O) and matrix protein (tapA-sipW-tasA) are under the control of the transcriptional repressor and biofilm master regulator SinR [12,14]. In the laboratory environment, B. subtilis is often cultured in conditions that do not induce robust biofilm formation, such as constant aeration and the standard laboratory medium LB. Notably, supplementing LB with glycerol and additional manganese can trigger high matrix production in stationary-phase B. subtilis [15]. Given the diversion of resources that occurs during matrix production, we hypothesized that typical culturing conditions might select for B. subtilis mutants that use all of their resources for growth rather than for producing biofilm matrix, even at low levels. Laboratory strains of B. subtilis and of other bacterial species often form less robust biofilms than do their wild ancestors, suggesting that biofilm attenuation is common during domestication [16,17]. Although we believe that historical contingencies (irradiation and repeated transfer from laboratory to laboratory) determined which mutations actually arose in commonly-used laboratory strains of B. subtilis such as 168, we set out to determine whether standard laboratory conditions alone (e.g., rich liquid media, constant aeration) could have selected for the loss of biofilm formation in domesticated B. subtilis. To examine biofilm formation during extended laboratory culture of B. subtilis, we grew multiple independent populations of a robust biofilm-forming strain of B. subtilis, NCIB3610 (referred to hereafter as either 3610 or the ancestor). We cultured cells for 60 days, using two different growth conditions, in LB. We regularly saved samples of each evolving population throughout the 60day period, allowing us to monitor colony morphologies over time, and to analyze strains isolated from our final, 60-day-old populations. To our surprise, we did not uniformly re-domesticate B. subtilis, but rather created populations whose members form biofilms with varying levels of robustness on rich medium. Our study suggests that laboratory conditions produce both bacteria with biofilmattenuating mutations and bacteria with biofilm-enhancing mutations. Neither class of mutation fixed in the population over 300 (shaking) or 150 (static) generations. Thus, it is unlikely that standard laboratory culturing alone led to the domestication of biofilm-forming B. subtilis. Results Extended culture produces coexisting cell-types with varied biofilm-forming abilities Ten independent populations of B. subtilis were founded from single colonies as either shaking or static cultures of the robust biofilm-forming strain 3610 and were serially transferred daily or every second day following vigorous vortexing. The cultures were maintained for 60 days, with a calculated average of 5.645 generations (...truncated)