You and I: The Effects of Intra- and Inter-Species Interactions on Microbial Biofilms, Growth, and Morphology by Christopher James Cody Davis Department of Biology Duke University Date:_______________________ Approved: ___________________________ Paul Magwene, Supervisor ___________________________ Rytas Vilgalys ___________________________ Fred Dietrich Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the Department of Biology in the Graduate School of Duke University 2016 i v ABSTRACT You and I: The Effects of Intra- and Inter-Species Interactions on Microbial Biofilms, Growth, and Morphology by Christopher James Cody Davis Department of Biology Duke University Date:_______________________ Approved: ___________________________ Paul Magwene, Supervisor ___________________________ Rytas Vilgalys ___________________________ Fred Dietrich An abstract of a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the Department of Biology in the Graduate School of Duke University 2016 i v Copyright by Christopher James Cody Davis 2016 Abstract Humanity is shaped by its relationships with microbes. From bacterial infections to the production of biofuels, industry and health often hinge on our control of microbial populations. Understanding the physiological and genetic basis of their behaviors is therefore of the highest importance. To this end I have investigated the genetic basis of plastic adhesion in Saccharomyces cerevisiae, the mechanistic and evolutionary dynamics of mixed species biofilms with Escherichia coli and S. cerevisiae, and the induction of filamentation in E. coli. Using a bulk segregant analysis on experimentally evolved populations, I detected 28 genes that are likely to mediate plastic adhesion in S. cerevisiae. With a variety of imaging and culture manipulation techniques, I found that particular strains of E. coli are capable of inducing flocculation and macroscopic biofilm formation via coaggregation with yeast. I also employed experimental evolution and microbial demography techniques to find that selection for mixed species biofilm association leads to lower fecundity in S. cerevisiae. Using culture manipulation and imaging techniques, I also found that E. coli are capable of inducing a filamentous phenotype with a secreted signal that has many of the qualities of a quorum sensing molecule. iv Contents Abstract ......................................................................................................................................... iv List of Tables .............................................................................................................................. viii List of Figures ............................................................................................................................... ix List of Abbreviations ................................................................................................................... xi 1. Plastic adhesion in S. cerevisiae ................................................................................................ 1 1.1 Introduction ....................................................................................................................... 1 1.2 Methods ............................................................................................................................. 3 1.2.1 Strains ............................................................................................................................ 3 1.2.2. Selection regime .......................................................................................................... 4 1.2.3 Detection of QTLs ........................................................................................................ 5 1.2.4 Quantification of plastic adhesion ............................................................................ 6 1.2.5 Measuring the effects of nutrient limitation on adhesion ..................................... 7 1.3 Results ................................................................................................................................ 8 1.3.1 Potential QTLs are abundant ..................................................................................... 8 1.3.2 Selection increased plastic adherence ..................................................................... 11 1.3.3 Nitrogen limitation increases adhesiveness .......................................................... 15 1.4 Discussion ........................................................................................................................ 16 2. Mixed species biofilms with S. cerevisiae and E. coli ........................................................... 21 2.2 Introduction ..................................................................................................................... 21 2.2 Methods ........................................................................................................................... 23 v 2.2.1. Flocculation measurements .................................................................................... 23 2.2.2 Biofilms ....................................................................................................................... 24 2.2.3 Long term co-culturing ............................................................................................. 26 2.2.4 TrackScar .................................................................................................................... 28 2.3 Results .............................................................................................................................. 28 2.3.1 DH5α and MACH1 induce flocculation via coaggregation with S. cerevisiae .. 28 2.3.2 DH5α and MACH1 form macroscopic biofilms when co-cultured with yeast 29 2.3.3 Biofilms are an intimate association of both species ............................................ 34 2.3.4 Biofilm formation requires live E. coli and is affected by deflocculation buffer ............................................................................................................................................... 37 2.3.5 Selection for biofilm association in S. cerevisiae leads to slower growth rates .. 38 2.4 Discussion ........................................................................................................................ 40 3. Filamentation in E. coli ........................................................................................................... 45 3.1 Introduction ..................................................................................................................... 45 3.2 Methods ........................................................................................................................... 47 3.2.1 Culturing .................................................................................................................... 47 3.2.2 Imaging and analysis ................................................................................................ 47 3.3 Results .............................................................................................................................. 48 3.3.1 E. coli can produce a signal that induces their own filamentation ..................... 48 3.3.2 There is variation in filamentation behavior among strains ............................... 50 3.3.3 Strain variation in filamentation is due to differences in reaction to inducing agent, not production of it ................................................................................................. 51 3.3.4 The PFIA is heat stable up to 80°C .......................................................................... 52 vi 3.3.5 Glucose is necessary for the production of the PFIA ........................................... 53 3.3.6 Low pH conditions and acclimatization are necessary for filamentation to occur ..................................................................................................................................... 54 3.4 Discussion ........................................................................................................................ 56 4. Conclusions .............................................................................................................................. 61 References .................................................................................................................................... 63 vii List of Tables Table 1: List of genes implicated in plastic adhesion by BSA/PROVEAN pipeline……...10 viii List of Figures Figure 1: G-statistic plots for PMY1683 and PMY1846. ........................................................... 9 Figure 2: Plastic adherence over the course of the selection regime in PMY1643, PMY1683, PMY1846, and PMY1847. ........................................................................................ 13 Figure 3: Colony counts over the course of the selection regime in PMY1643, PMY1683, PMY1846, and PMY1847 ............................................................................................................ 14 Figure 4: Distribution of all adherence assay measurements. .............................................. 15 Figure 5: Effects of nitrogen and glucose limitation on plastic adhesion ........................... 16 Figure 6: Effects of coculture with DH5α E. coli on floc size in a panel of S. cerevisiae strains ...........................................................................................................................................