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Investigating the Effect of Surfactin on Membrane Potential of Bacillus subtilis Biofilm

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Title:
Investigating the Effect of Surfactin on Membrane Potential of Bacillus subtilis Biofilm
Creator:
Guo, Hanling
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (58 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Biochemistry and Molecular Biology
Committee Chair:
BUNGERT,JORG
Committee Co-Chair:
HAGEN,STEPHEN JAMES
Committee Members:
PATRICK,ERIN E

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Subjects / Keywords:
biofilm -- membrane -- potential -- surfactin
Biochemistry and Molecular Biology -- Dissertations, Academic -- UF
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Biochemistry and Molecular Biology thesis, M.S.

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Abstract:
Forming biofilms is very common for most bacterial cells. A recent study first reported that membrane potential of Bacillus subtilis biofilm had oscillation behavior. This study also indicated biofilm formation is related to the membrane potential. Surfactin is a quorum sensing factor produced by Bacillus subtilis. It was reported to be very important in Bacillus subtilis biofilm formation and can cause potassium leakage across the lipid bilayer membrane. Surfactin has the potential to link the membrane potential with the biofilm formation. To further investigate the relationship between the membrane potential and the biofilm formation, surfactin was chosen to test the effect on the membrane potential of Bacillus subtilis biofilm. To detect the change of the membrane potential, we chose to use established voltage-sensitive fluorescent dyes, ThT and DiSC3(5) to represent the electrical potential of the inner membrane. In this project, we demonstrated that surfactin could make the membrane potential of Bacillus subtilis biofilm more negative by causing the leakage of potassium. Moreover, the effect of surfactin on the membrane potential was shown to be concentration related. Biofilm formation experiments of WT and the srfAA mutant further indicated that surfactin might have a dual function in Bacillus subtilis biofilm formation. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2017.
Local:
Adviser: BUNGERT,JORG.
Local:
Co-adviser: HAGEN,STEPHEN JAMES.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2018-06-30
Statement of Responsibility:
by Hanling Guo.

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Embargo Date:
6/30/2018
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LD1780 2017 ( lcc )

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INVESTIGATING THE EFFECT OF SURFACTIN ON MEMBRANE POTENTIAL OF BACILLUS SUBTILIS BIOFILM By HANLING GUO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCI ENCE UNIVERSITY OF FLORIDA 2017

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2017 Hanling Guo

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To my dear parents, my friends and everyone who helped me in my life

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4 ACKNOWLEDGMENTS First of all, I would like to thank my two mentors, Dr. Jorg Bungert and Dr. Stephen J. Hagen. They guided me in this study wi th great patience, sound advices, and continuous help They not only guided me through study and re search, but also helped me to overcome hard time s I want to thank Dr. Erin E. Patrick for be ing my committee member. Her knowledgeable comments and valuable suggestions assisted a lot in the project. I also thank all members of the Hagen Laboratory. They helped me in many ways in the project, and made it much easier for me Finally, I give my special thanks to Dr. Roberto Kolter from Department of Microbiology & Immunobiology, Harvard Medical School He generously sent us the srfAA mutant strain and th is strain have been an essential part of this project.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 1.1 Bacillus subtilis Biofilm ................................ ................................ ...................... 12 1.1.1 Definition of Biofilm & ECM. ................................ ................................ .... 12 1.1.2 Functions of Biofilm ................................ ................................ ................. 12 1.1.3 Benefits and Problems Associated with Biofilm. ................................ ...... 13 1.1.4 Components and Structure of Bacillus subtilis Biofilm ............................. 13 1.1.5 Metabolic Oscillation within Biofilm ................................ .......................... 14 1.2 Communication within Biofilm ................................ ................................ ........... 15 1.2.1 Quorum Sensing System ................................ ................................ ......... 15 1.2.2 Electrical Signaling in Bacteria ................................ ................................ 16 1.2.3 Membrane Potential Oscillation within Bacillus subtilis Biofilm ................ 16 1.3 Surfactin ................................ ................................ ................................ ............ 18 1.4 Motivation of This Project ................................ ................................ .................. 20 1.4.1 Goal of This Project ................................ ................................ ................. 20 1.4.2 Device and Reporter ................................ ................................ ................ 21 1.4.3 KCl Shock to Verify Dyes Can Report Membrane Potential .................... 21 1.4.4 Use Surfactin to See Its Influence on The Membrane Potential .............. 22 1.5 Significance of Studying Biofilm ................................ ................................ ........ 22 2 MATERIALS AND METHODS ................................ ................................ ................ 27 2.1 Materials ................................ ................................ ................................ ........... 27 2.2 Growth Con ditions ................................ ................................ ............................ 29 2.3 Data Collection ................................ ................................ ................................ 29 2.4 Data Analysis ................................ ................................ ................................ .... 30 3 RESULTS ................................ ................................ ................................ .............. 31 3.1 ThT and DiSC 3 (5) Were Verified to Report The Change of Membrane Potential Reliably. ................................ ................................ ................................ 31

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6 3.2 Surfactin Can Make The Membrane Potential of Bacillus subtilis Biofilm More Negative. ................................ ................................ ................................ .... 32 3.3 Surfactin Can Cause Potassium Leakage in Bacillus subtilis Biofilm ................ 34 3.4 The Membrane Potential Oscillation of Bacillus subtilis Biofilm Was Observed in Our Lab ................................ ................................ ........................... 35 3.5 High Concentration of Surfactin Can Eventually Make The Membrane Potential of Bacillus subtilis Biofilm More Positive ................................ ............... 36 3.6 Examination of The srfAA Mutant ................................ ................................ ..... 36 3.7 Low Concentration of Surfactin Can Hold The Membrane Pot ential of srfAA Mutant More Negative. ................................ ................................ ........................ 37 4 DISCUSSION ................................ ................................ ................................ ......... 47 4.1 Remarks on This Project ................................ ................................ ................... 47 4.2 Future Work ................................ ................................ ................................ ...... 49 LIST OF REFERENCES ................................ ................................ ............................... 52 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 58

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7 LIST OF TABLES Table page 2 1 List of strains used in this thesis ................................ ................................ ......... 30

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8 LIST OF FIGURES Figure page 1 1 Membrane Oscillation within Bacillus subtilis biofilm. ................................ ......... 23 1 2 Oscillations in ThT and growth rate. ................................ ................................ ... 24 1 3 Structure of surfactin. ................................ ................................ ......................... 25 1 4 Surfactin cause potassium leakage across the cell membrane .......................... 26 3 1 DiSC 3 (5) can report the change of biofilm membrane potential. ......................... 39 3 2 ThT can report the change of biofilm membrane potential.. ............................... 40 3 3 Surfactin can increase membrane potential of Bacillus subtilis biofilm.. ............. 41 3 4 Surfactin can cause potassium leakage across the cell membrane of Bacillus subtilis biofilm. ................................ ................................ ................................ .... 42 3 5 The membrane potential oscillation of Bacillus subtilis biofilm. ........................... 43 3 6 Long time surfactin shock on biofilm. ................................ ................................ .. 44 3 7 srfAA mutant examination. ................................ ................................ ................. 45 3 8 Low concentration surfactin can hold the membrane potential of srfAA mutant more negative. ................................ ................................ ................................ .... 46

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9 LIST OF ABBREVIATIONS ABC APG 4 ATCC CF DiSC 3 (5) ECM ATP binding cassette Asante Potassium Green 4 American Type Culture Collection Cystic fibrosis dipropylthiadicarbocyanine iodide Extracellular matrix EPS MEA Extracellular polysaccharides Microelectrode a rrays MRSA ThT WT Methicillin resistant Staphylococcus aureus Thioflavin T Wild type

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INVESTIGATING THE EFFECT OF SURFACTIN ON MEMBRANE POTENTIAL OF BACILLUS SUBTILIS BIOFILM By Hanling Guo December 2017 Chair: Jorg Bungert Cochair: Stephen J. Hagen Major: Biochemistry and Molecular Biology Forming biofilm s is very common for most bacterial cells. A recent study first reported that membrane potential of Bacillus subtilis biofil m had oscillation behavior. This study also indicated biofilm formation is related to the membrane potential. Surfactin is a quorum sensing factor p roduced by Bacillus subtilis It was reported to be very important in Bacillus subtilis biofilm formation and can cause potassium leakage across the lipid bila yer membrane. Surfactin has the potential to link the membrane potential with the biofilm formati on To further investigate the relationship between the membrane potential and the biofilm formation, surfactin was chosen to test the effect on the membrane potential of Bacillus subtilis biofilm To detect the change of the membrane potential, we chose to use established voltage sensitive fluorescent dyes, ThT and DiSC 3 (5) to represent the electrical potential of the inner membrane. In this project, we demonstrated that surfactin could make the membrane potential of Bacillus subtilis biofilm more negat ive by causing the leakage of potassium.

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11 Moreover, the effect of surfactin on the membrane potential was shown to be concentration related. B iofilm formation experiments of WT and the srfAA mutant furt her indicated that surfactin might have a dual function in Bacillus subtilis biofilm formation.

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12 CHAPTER 1 INTRODUCTION 1.1 Bacillus subtilis B iofilm 1.1.1 Definition of B iofilm & ECM A biofilm is a bacterial community that is enclosed by an extracellular matrix and can adhere to surfaces (Costerton et al., 1995). Forming biofilm, and associating to surface and interface is very common for the most bacterial cells in nature. Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus are good examples ( Vlamakis et al., 2013 ; Klausen et al., 2003 ; Singhal et al., 2011 ;). Biofilm requires the production of extracellular matrix (ECM) for formation and maintenance of structured multicellular communities (Lpez et al., 2010). The ECM, also called as extracellular poly saccharides(EPS), is produced by the cells, and it shows significant ability in regulating gene expression and coordinating cell behavior in bacterial biofilms (Steinberg et al., 2015). 1.1.2 Functions of B iofilm By forming biofilms, bacteria have the abi lity to adhere to almost every surface (Lpez et al., 2010). Moreover, forming biofilms offers other benefits for bacteria. One of the most significant biofilm benefits is that biofilm can provide protection for bacterial cells from the environment. For ex ample, the P.aeruginosa biofilm can dilute some antibiotics, and the ECM of P.aeruginosa biofilm has the ability to trap antimicrobial agents (Mah et al., 2001; Anderson et al., 2008). Biofilms can also benefit cells by protecting them from short term seve re environments, such as pH shifts, shear forces, and dehydration. Biofilms have even been found on radiation sources (Flemming et al., 1993; Lessel et al. 1975). Besides the protection benefits, biofilms can help bacterial

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13 cells in exchanging nutrients an d removing potentially toxic metabolites as well (Davey et al., 2000). Most bacterial cells are likely to form biofilms in natural settings based on these benefits. 1.1.3 Benefits and Problems Associated with B iofilm Antibiotics have been one of the m ost i mportant treatments for fight ing infectious disease caused by bact erial cells. The bacteria then, have two ways to deal with antibiotics. First, bacterial cells can contain antibiotic resistant genes and thus have antibiotic resistance. MRSA is a very goo d example of this situation. MRSA contains multiple antibiotic resistant genes and survives most known antibiotic treatment (Fitzgerald et al., 2001). Second, bacteria can survive from antibiotics by residing within biofilms. Bacteria growing within a biof ilm can have up to 1,000 fold resistance to antibiotics compared to the same species grown planktonically (Gilbert et al., 1997). For example, P.aeruginosa biofilm has much higher tolerance to antibiotics than planktonic P.aeruginosa (Mah et al., 2001). Th is antibiotic resistance makes it much harder for people to cure diseases associated with biofilms. It is important to note that biofilm s can also benefit human beings. Bacillus subtilis biofilm is a well known example. Bacillus subtilis biofilm can promot e plant growth and protect plants from an extensive collection of pathogens (Arkhipova et al., 200 5; Choudhary et al., 2009). Bacillus subtilis is widely used as a biofertilizer to promote the growth of crops (Lucy et al., 2004). 1.1. 4 Components and S tructure of Bacillus subtilis B iofilm Biofilm is composed of water, cells, extracellular matrix, and some DNA and RNA from lysed cells. Water is the main component, and it usually makes up over 90%, or up to 97% (Schmitt et al., 1999; Zhang et al., 1998). As for dry weight, the largest component is the cells. The extracellular matrix composes almost the rest of the biofilm.

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14 In the bacteria biofilm, exopolysaccharides, proteins and nucleic acids are the most extensively studied components of the extracellula r matrix (Branda et al., 2005). Exopolysaccharides in biofilm ECM were thought to impact bacterial virulence and promote capsule formation. The exopolysaccharides are made up of glucose, galactose, and N acetylgalactosamine. In the Bacillus subtilis biofil m, the eps A O operon is in charge of producing the exopolysaccharides (Chai et al., 2012). For proteinaceous components, amyloid fibers are mostly found to be the primary member in both Gram positive and Gram negative bacteria (Steinberg et al., 2015). In the Bacillus subtilis biofilm ECM, TasA amyloid fibers are the best characterized functional amyloid fibers, and these amyloid fibers are attached to the cell wall (Branda et al., 2006; Chai et al., 2013). TasA amyloid fibers can provide structural integri ty and work together with other ECM components to intermediate adhesion between cells (Romero et al., 2010). Another relevant biofilm ECM component is extracellular genomic DNA (eDNA). It has been found in many different bacterial biofilms and is very impo rtant for the young biofilm structure. eDNA shows important functions in regulating the cell surface performance, promoting cell adhesion and interaction with other biofilm ECM components (Okshevsky et al., 2015a; Okshevsky et al., 2015b; Das et al., 2013) eDNA, which is not correlated with cell lysis in biofilm, is also produced by Bacillus subtilis although it is only present about 0.1 g/mL (Lorenz et al., 1991; Zafra et al., 2012). 1.1.5 Metabolic Oscillation within B iofilm R esearchers in 2015 found a very interesting phenomenon called metabolic oscillation during Bacillus subtilis biofilm formation within a microfluidic device. The device is continuously flowing with media. The oscillation in biofilm growth happened

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15 spontaneously and had a relatively stable rhythm (Liu et al., 2015). The mechanism behind this behavior of Bacillus subtilis biofilm is still unknown. This novel discovery may lead us to understand the intracellular metabolic activity within the biofilms and find a new way to control the de velopment of biofilms. 1.2 Communication within B iofilm 1.2.1 Quorum Sensing S ystem Bacteria cells are not only living individually, but also in bacterial communities. They need to communicate within the single species community or even among different spe cies. Bacterial cells have many ways to communicate with each other, and quorum sensing is one of the most important ones. Quorum sensing has been studied less than 40 years. It was first discovered and reported in a luminous marine bacteria Vibrio fischer i in 1979 (Nealson et al., 1979). Quorum sensing is the chemical communication that requires small chemical molecules called autoinducers. Autoinducers are produced and released by cells and detected by corresponding proteins in cells. These proteins can respond to autoinducers, and activate or inactivate downstream events (Waters et al., 2005). Quorum sensing system widely exists in both Gram negative bacteria and Gram positive bacteria, although the mechanism s may have some difference s Many bacteria hav e more than one quorum sensing system. For the quorum sensing system in the Gram positive bacteria, peptides are chosen as the autoinducers for quorum sensing. The ATP binding cassette (ABC) transporters in the Gram positive bacteria secrete these peptides The two component adaptive response proteins of the Gram positive bacteria are used to detect these peptides and affect downstream activation or deactivation. A membrane sensor and a cognate response downstream regulator compose the two component adaptive

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16 response proteins. The membrane sensor, usually a kinase, can detect the extracellular concentration of the autoinducers. When the concentration reaches a certain level, the membrane sensor will phosphorylate the cognate response downstream regulator. The regulator can further control the downstream activities (Kleerebezem et al., 1997; Hoch et al., 1995; Lazazzera et al., 1998). 1.2.2 Electrical Signaling in B acteria Electrical signaling is very common in nature; the nervous syst em is the best known example. The nervous system can transport orders or stimulus fast and precisely via electrical signaling, and th is is very important for animals When it comes to bacteria, however, the electrical signaling has long been neglected comp ared to chemical signaling. Since electrical signaling in bacteria is not well studied, there is little research on the subject. Researchers have expressed and identified a voltage gated sodium channel in Bacillus halodurans called NaChBac. NaChBac is a tr ansmembrane protein which contains one six transmembrane segment NaChBac is activated by voltage, and it will be obstructed by calcium channel blockers (Ren et al., 2001). These characteristics enable it to serve in electrical signaling though further stu dy is required. Still, there are also some examples directly showing the signaling function of the ion channel. In 2015, researchers reported a bacterial potassium channel YugO enables electrical communication in Bacillus subtilis biofilm (Prindle et al., 2015). This will be discussed below. 1.2.3 Membrane Potential O scillation within Bacillus subtilis B iofilm In 2015, a group of researchers reported that when they measured the thioflavin T (ThT) fluorescence quantitatively, they surprisingly found the fluorescence within the Bacillus subtilis biofilm showed oscillation behavior. This oscillation happened

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17 spontaneously, and the whole biofilm edge showed oscillation at the same time a nd rate, as presented in Figure 1 1 (Prindle et al., 2015). They called this behavior a biofilm membrane potential oscillation. To further investigate the membrane potential oscillation, they used extracellular fluorescence dye APG 4 to report the potassium concentration in the media. This revealed potassium release. The potas sium release led them to investigate the potassium channel YugO. The YugO is the only experimentally described potassium channel in Bacillus subtilis. It contains an intracellular TrkA domain, which is in charge of gating the potassium flux. Moreover, the YugO potass ium channel is necessary for Bacillus subtilis biofilm formation (Lundberg et al., 2013; Cao et al. 2013). With several tests and comparison with yugO deletion mutant and trkA deletion mutant, the researchers successfully demonstrated the YugO c hannel gating could promote electrical communication within the Bacillus subtilis biofilm (Prindle et al., 2015). During Bacillus subtilis biofilm formation, the growth rate also showed oscillation. This is called metabolic oscillation. The metabolic oscil lation in Bacillus subtilis biofilm also happened spontaneously, and the whole biofilm edge showed oscillation at the same time and rate (Liu et al., 2015). This characteristic seems to be very similar to the membrane potential oscillation. Researchers com pared the oscillation in membrane potential and growth rate within the Bacillus subtilis biofilm and found that they are inversely correlated, as shown in Figure 1 2 (Prindle et al., 2015). Moreover, the YugO channel that can promote electrical communicati on within the Bacillus subtilis biofilm, is also necessary for biofilm formation. This indicates that there might be a close relationship between biofilm formation and membrane potential. There might also be

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18 some other factors besides the YugO potassium ch annel that can link membrane potential and biofilm formation together. 1.3 Surfactin Surfactin is a cyclic lipopeptide lactone secreted by Bacillus subtilis. It contains two acidic amino acids (glutamate and aspartate) besides five nonpolar residues and on e 3 hydroxy fatty acid (Kakinuma et al., 1969). The structure of surfactin is presented in Figure 1 3 (Kraas et al., 2010). Surfactin is produced by nonribosomal peptide synthetases SrfAA, SrfAB, SrfAC and a protein with high homology to external thioesterases of type II called SrfD (Kraas et al., 2010). These enzymes are encoded by corresponding genes that are organized in the srfA operon (Pratap et al. 2013). To link membrane potential and biofilm formation together, surfactin needs to participate in biofilm formation and have the ability to influence membrane potential. Surfactin is necessary for th e formation of Bacillus subtilis biofilm, despite the fact that its function can be partly replaced by molecules produced by other soil bacteria, such as nystatin from Streptomyces noursei (Lpez et al., 2009). Surfactin is well known as a quorum sensing f actor of Bacillus subtilis and plays a critical role in the biofilm formation of Bacillus subtilis by activating a protein kinase (Lpez et al., 2010). Researchers found that surfactin can be sensed by KinC, which is a membrane histidine kinase (Lpez et a l., 2009). KinC is a member of bacterial sensor histidine kinases family. In prediction, KinC has a PAS PAC sensor domain and two cross membrane segments containing seven extracellular residues (Mascher et al., 2006; Taylor et al. 1999). KinC can sense sur factin and phosphorylate Spo0A. Spo0A plays a dual signaling role, and the phosphorylation of Spo0A is critical for the biofilm formation. When the Spo0A~P level is low, phosphorylated Spo0A will activate the eps A O operon

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19 and tapA sipW tasA operon. The ac tivation of eps A O operon can promote exopolysaccharides production, and the activation of tapA sipW tasA operon can stimulate TasA amyloid fiber production. When the Spo0A~P level is high, phosphorylated Spo0A will inactivate these operons, stop ECM produ ction and trigger sporulation (Aguilar et al., 2010; Rubinstein et al., 2012; McLoon et al., 2011). When ECM reach a certain level, cells can grow fast in the ECM environment, and the bacterial biofilm can develop by repeating this cycle. In this way, surf actin can promote biofilm formation. Surfactin also has potential to influence membrane potential. The membrane potential is affected by the stable potassium flux rate through the cell membrane. If a factor has the ability to change the potassium flux rate through the cell membrane, it will probably in fluence membrane potential, like the YugO potassium channel. Surfactin can cause potassium leakage across the lipid bilayer membrane, which w ill increase potassium flux through the cell membrane and lead t o low intracellular potassium concentration (Sheppard et al., 1991) as shown in Figure 1 4 This provides the possibility for surfactin to influence the membrane potential within the Bacillus subtilis biofilm. Moreover, the kinase KinC can also respond to the lowered intracellular potassium concentration (McLoon et al., 2011). Taking all these results together, surfactin, intracellular potassium concentration, and KinC seem to have a functional linkage among them. The oscillation in fluorescence and growt h rate within the Bacillus subtilis biofilm both happened spontaneously and have a relatively stable rhythm (Liu et al., 2011; Prindle et al., 2011). Thus, a strong mechanism to synchronize them is required. A

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20 quorum sensing system seems to have good poten tial. The system mainly uses the extracellular concentration of autoinducer to regulate the transcription of target genes. The extracellular concentration of autoinducer can be considered uniform in a relatively long distance, so this might be the key reas on to explain how Bacillus subtilis is able to communicate over such long distance. Moreover, as an autoinducer in a quorum sensing system, surfactin can not only turn on or off the target gene, but also control the transcription level of it. Meanwhile, th is quorum sensing system is thought to have a negative feedback loop like most quorum systems, although it is not well understood in surfactin quorum sensing system. A negative feedback loop can control the production of surfactin dynamically. When extrace llular surfactin concentration is high, the loop should have a strong effect and inhibit surfactin production. When extracellular surfactin concentration is low, the loop should have a weak effect, and the surfactin production will increase. If surfactin c ould increase the membrane potential of Bacillus subtilis biofilm, the membrane potential would correlate with the extracellular surfactin concentration. These characteristics can provide strong support to the oscillation behavior. Surfactin seems to be a very promising candidate that can help us to decipher the correlation between membrane potential and the biofilm formation. 1.4 Motivation of This P roject 1.4.1 Goal of This P roject Investigating the relationship between membrane potential and biofilm form ation offers us a new way to study biofilm formation. Surfactin can cause potassium leakage across the lipid bilayer membrane, and play a critical role in the Bacillus subtilis biofilm formation. Thus surfactin has good

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21 potential to influence membrane pote ntial and link membrane potential and biofilm formation together, but it has not yet been directly reported to be able to influence membrane potential. The goal of this project is to study the effect of surfactin on membrane potential. 1.4.2 Device and R eporter To investigate the effect of surfactin on the membrane potential of Bacillus subtilis biofilm, the change of membrane potential needs to be measured in appropriate devices. The microfluidic devices were desi gned and made by our lab (Son et al., 2014). C ommercial micro fluidic slides (ibidi Inc.) were also used. To report the change of membrane poten tial, membrane potential was recorded time by time. Two established voltage dipropylth iadicarbocyanine iodide (DiSC 3 (5)) were used to detect membrane potential (Kralj et al., 2011; Strahl et al., 20 10). T he dye s are positively charged and can enter cell membrane. The inner membrane of bacteria has negative electrical potential (Hosoi et al. 1980) and thus will retain the dye s When the membrane has more negative electrical potential, it will contain a highe r concentration of these fluorescent dyes, and so have brighter fluorescence. 1.4.3 KCl Shock to Verify D ye s Can Report Membrane P otenti al Although ThT and DiSC 3 (5) were used to detect membrane potential in previous studies, we conduct ed a KCl shock experiment to verify that these two dyes can reliably report the membrane potential within the Bacillus subtilis biofilm. The idea o f this exp eriment is to use high concentration of potassium (300mM) to interrupt the normal potassium flux, thereby lowering the membrane potential. Then we can see whether the dye reports this change.

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2 2 1.4.4 Use Surfactin to S ee Its Influence on The Membrane P otenti al Extra surfactin was added, and we studied the effect on membrane potential and the change of membrane potential oscillation Meanwhile, APG 4 was used to measure the extracellular potassium concentration to verify that surfactin can cause potassium leak age. The srfAA gene deletion mutant was test ed to see if biofilm formation and membrane potential could be controlled by supplying it with surfactin. 1.5 Significance of S tudyin g B iofilm Studying biofilm formation is very important. On the one hand, there are still many unknown mechanisms and characteristics about biofilm s so studying biofilm can help us to understand more about biofilm itself. On the other hand, studying biofilm can al so benefit humanity. If we understand how cells form biofilm s and find a way to stop the formation of pathogenic biofilm s, we will largely improve the treatment of many diseases associated with biofilm s such as cystic fibrosis (CF) lung disease caused by P. aeruginosa (Collins 1992). Moreover, we could also help developing biofilms that can benefit humanity. For instance, Bacillus subtilis biofilm can promote plant growth and protect plants from an extensive collection of pathogens. If we can control Bacillus subtilis b iofilm formation and maintain it in the best concentration range, agriculture will be benefited. For this project, we studied Bacillus subtilis biofilm Bacillus subtilis is the best studied Gram positive bacteria. It is observed in many different environm ents and has l ong been used as a model Gram positive bacterium (Earl et al., 2008; Kunst et al., 1997).

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23 Figure 1 1. Membrane Oscillation within Bacillus subtilis biofilm a) Metabolic oscillation within the Bacillus subtilis biofilm b) Left is the dia gram of the microfluidic device. Right is the phase contrast image o f a biofilm. Scale bar is 100 m. c) Membrane potential oscillation, within the Bacillus subtilis biofilm reported by ThT Scale bar is 150 m d) Left is the edge region of the biofilm i n c. Right is the plot of fluorescence within edge region over time. e) Time traces plot of the data of the heat map shown in d. (Reproduced with permission from Reference [Prindle et al., 2015]; License Number: 4213850812388)

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24 Figure 1 2 Oscillations in ThT and growth rate The blue line is the time trace of membrane potential within the biofilm. The membrane potential is represented by the fluorescence of ThT. The black line is the time trace of the growth rate of the biofilm. (Reproduced with permiss ion from Reference [Prindle et al., 2015]; License Number: 4213850812388)

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25 Figure 1 3 Structure of surfactin The figure shows the structure of surfactin and lists the main variants of surfactin. The light blue box shows the lactone bond (Repr oduced with permission from Reference [Kraas et al., 2010]; License Number: 4213851138555)

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26 Figure 1 4. S urfactin cause potassium leakage across the cell membrane The cell membrane is s emipermeable and intracellular potassium concentration is higher than extracellular potassium concentration. When surfactin exist in the environment, it will attach to the lipid membrane and open channel s for potassium leakage.

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27 CHAPTER 2 MATERIALS AND METHODS 2.1 Materials Strain Strains are listed in Table 2 1. In most experiments, the wild type strain NCBI 3610 was used. This strain was brought from the American Type Culture Collection (ATCC), and the part number is ATCC 6051. The srfAA::erm mutant ZK3858 (Branda et al., 2001) wa s used in mutant experiments and compared with WT. ZK3858 is an isogenic srfA mutant of NCBI 3610. This strain was a gift from the Kolter l ab oratory Department o f Microbiology & Immunobiology, Harvard Medical School. Medium. For long time planktonic grow th, cells were cultured in LB medium. LB medium contained 5 g/L yeast extract, 10 g/L Tryptone and 10 g /L NaCl. LB medium was sterilized using an autoclave For the biofilm formation in experiments, MSgg medium (Prindle et al., 2011) was used. E very 100 mL MSgg medium contained 0.5 mmol potassium phosphate buffer (pH 7.0), 10 mmol MOPS buffer (pH 7.0), 0.2 mmol MgCl 2 70 mol CaCl 2 10 mol FeCl 3 5 mol MnCl 2 0.2 mol thiamine HCl, 0.1 mol ZnCl 2 0.5 mL glycerol and 0.5 g monosodium glutamate. In KCl sho ck experiment, MSgg (KCl shock) medium contained extra 300 mM KCl. In NaCl shock experiment, M Sgg (NaCl shock) medium contained extra 300 mM NaCl. MSgg medium was filter sterilized. Important stocks. dipropylthiadicarbocyanine i odide (DiSC 3 (5)) were used to report the change of biofilm membrane potential. ThT (ICN biomed Inc.) was solved in DMSO, and the stock concentration was 10 mM. DiSC 3 (5) (Thermo Fisher Inc.) was dis solved in deionized water, and the stock concentration was

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28 10 mM. Unless specifically described, ThT was used at 40 M, and DiSC 3 (5) was used at 1 0 M. Asante Potassium Green 4 (APG 4) was used to report the change of extracellular potassium concentration. APG 4 (TEFLabs Inc.; salt form) was dis solved in deionized water and the stock concentration was 2 mM. APG 4 was used at 2 M. Surfactin (Sigma Inc.) was dis solved in DMSO, and the stock concentration was 20 mM. Erythromycin ( ACROS Organics Inc.) was dis solved in EtOH, and the stock concentration was 20 mg/mL The working concentration of erythromycin was 20 g/mL Microfluidic device. The microfluidic device used for this project was designed and made in the Hagen lab oratory (Son et al., 2014). The device has three inlet channels and one outlet channel. The outlet channel was also used for loading cells. A 3 mm x 3 mm chamber, 4 mm x 4 mm chamber, 5 mm x 5 mm chamber were located on the corresponding channel. The depth of the chambers is 20 m. The microfluidic device was used to observe membrane potentia l oscillation of Bacillus subtilis biofilm. ibidi slides. A commercial microfluidic channel, ibidi Slide III 3in1 (catalog number: 80311) was used in KCl shock and surfactin shock experiments. It has three inlets, one large main chamber, and one outlet. The depth of the chamber is 0.4 m m. The inlets can connect to syringes with different medium. Pushing different syringe allows switching me dium in the main chamber 24 well plate s Falcon 24 Well Pol ystyrene Clear Flat Bottom Unt reated Cell Culture Plate s were used to test the effect of surfactin on Bacillus subtilis biofilm formation.

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29 2.2 Growth C ondition s To get the cell culture for experiments, 1 L cells from 80 C glycero l stock was inoculated into 3 mL LB medium and incubated in a shaker (37 C, 180 rpm) for 15 h. For microfluidic experiments, overnight cell culture was diluted 15 folds, and then centrifuged at 3000 rpm (LWS 815, LW Scien tific Inc.) for 2 min. T he supernatant was removed and the cell pellet was re suspend in the MSgg medium. The MSgg medium containing cells was loaded into the microfluidic device. The cells in the microfluidic device were incubated at 37 C for 1.5 h, no flow, and then in flowing medium at 37 C for another 2.5 h, with flow a rate at 0.02 mL /h. Before imaging, the temp e rature was set to 30 C until the experiment finished. For ibidi slides experiments, overnight cell culture was diluted 6 folds, and then centrifuged at 3000rpm (LWS 815, LW Scientific Inc.) for 2 min. The supernatant was removed and the cell pellet was r e suspend in the MSgg medium and then loaded into an ibidi slide. The MSgg medium containing cells were loaded into the ibidi slide. The cells in the slide were incu bated at 37 C for 1 h, no flow, and then in flowing medium at 37 C for another 1.5 h, wit h a flowing rate at 0.12 mL /h Before imaging, the temperature was set to 30 C and kept until the experiment finished. For 24 well plate experim ents, each well had 1.5 mL MSgg medium containing the corresponding concentration of surfactin (0 20 M ) and 1 L corresponding cell culture. The plate was incubated at 30 C for the whole experiment. 2.3 Data C ollection To collect phase contrast and fluorescence images, a Nikon eclipse TE2000U microscope and Photometrics CoolSNAP HQ2 camera were used. In most of the experiments, 20 X objectives were used to image the biofilms. The image size is 1040 x

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30 1392 pixels. Every 1 0 min in biofilm experiments and every 5 min in background tests, one phase contrast image, and one corresponding fluorescence image were taken. The exposure time for phase contrast image was 200 ms; t he exposure time for fluorescence image was 5000 ms. 2.4 Data A nalysis Custom scripts of MATLAB (MathWorks) were used for data analysis. To ensure the objective can match in all images of a timeline, images were registered. Unless specifically described, the mean fluorescence value of a 5 x 5 pixels region o n a biofilm was measured to represent membrane potential of this region at one time point. All the values of a timeline were collected together to generate a membrane potential curve. Table 2 1. List of strains used in this thesis S train Genotype Source Wild Type Bacillus subtilis NCBI 3610 ATCC ZK3858 srfAA::erm Branda et al., 2001

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31 CHAPTER 3 RESULTS 3.1 ThT and DiSC 3 (5) Were V erifie d to Report The Change of Membrane Potential R eliably This project focused on the membrane potential of Bacillus subtilis biofilm. Directly measuring the membrane potential of Bacillus subtilis biofilm is difficult. Indirectly but reliably reporting the membrane potential of Bacillus subtilis biofilm is more realistic. K + The positively charged dyes ThT and D iSC 3 (5) had already been used to report membrane potential in previous studies (Kralj et al., 2011; Strahl et al., 2010); however, it is preferable first to demonstrate their ability to report the change of biofilm membrane potential in our lab. To reach this goal, KCl shock was used to change the membrane potential of Bacillus subtilis biofilm manually. T he Goldman equation for the membrane potential is (3 1) ( Jackson, M. B. 2006 ) where V is the equilibrium potential; R is the universal gas constant (8.314 JK 1 mol 1 ); T is the temperature in Kelvin; F is Faraday's constant (96485 C/mol); P K is the permeability (ions/sec) for the potassium ion, P Na and P Cl are similar; [ K + ] out is the extracellular potassium concentration [ Na + ] out and [ Cl ] out are similar; [ K + ] in is the in tracellular potassium concentration [ Na + ] in and [ Cl ] in are similar From the Goldman equation, when a high concentration of potassium exist s in the extracellular environment, the membrane potential should be more positive. When the inner membrane is more positive, the positive ly charged fluorescent dyes will

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32 accumulate less within the inner membrane and so the level of fluorescence will be low er. When MSgg (KCl shock) medium (contain ing extra 300mM KCl) flows, biofilm should have more positive membrane potential compared to the potential when normal MSgg medium flows. Moreover, when MSgg medium flows again, the biofilm membrane potential should recover to the previous level. A reliable reporter should report these changes. For DiSC 3 (5), the background fluorescence of MSgg medium and MSgg (KCl shock) medium was relatively stable, as shown in Figure 3 1A. The fluorescence of biofilm decreased when MSgg (KCl shock) medium flowed and recovered when MSgg medium flowed again, as shown in Figure 3 1B. DiSC 3 (5) successfully reported the change of membrane potential. For ThT, the background fluorescence of MSgg medium and MSgg (KCl shock) medium was rela tively stable, as shown in Figure 3 2A. The fluorescence of biofilm decreased when MSgg (KCl shock) medium flowed and recovered when MSgg medium flowed again, as shown in Figure 3 2B. ThT successfully reported the change of membrane potential. In KCl shoc k experiments, ThT and DiSC 3 (5) both were demonstrated to be able to report the change of membrane potential. The experiments using these dyes to report membrane potential should give us reliable membrane potential data. 3.2 Surf actin Can M ake The Membrane P otential of Bacillus subtilis B iofilm More N egative Surfactin was not previously reported to influence the membrane potential of Bacillus subtilis biofilm. However surfactin was reported to cause potassium leakage

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33 across the lipid bilayer membrane, which will increase potassium flux rate through the cell membrane (Sheppard et al., 1991). The charged ions moving across the cell membrane can change the membrane potential (Prindle et al., 2015). We hypothesize that a higher potassium flux rate would lead to more negative membrane potential, so that surfactin would decrease the membrane potential of Bacillus subtilis The cell membrane is s emipermeable and the intracellular potassium concentration is higher than the extracellular potassium concentration. Surfactin can cause potassium leakage across the membrane, increasing the P K that appears in the Goldman equation given earlier. This will cause the membrane potential to become more negative. When the inner membrane is more negative, there will be more p ositive charged fluorescent dyes concentrated within the inner membrane and so have a higher level of corresponding fluorescence. Therefore if an area within the biofilm shows brighter fluorescence, the membrane potential of this area is more negative. To test this hypothesis, a surfactin shock experiment that was similar to a KCl shock experiment was designed. By this hypothesis, when MSgg (surfactin shock) medium (containing extra surfactin) flows, the membrane potential of Bacillus subtilis biofilm shoul d be more negative compared to the potential when MSgg medium flows. When MSgg medium flows again, the biofilm membrane potential should recover to the normal level. When ThT dye was chosen as a reporter, the background fluorescence of MSgg medium and MSg g (surfactin shock) medium was relatively stable, as shown in Figure 3 3A. As expected, the fluorescence of biofilm increased when MSgg (surfactin shock) medium flowed and recovered when MSgg medium flowed again, as shown in Figure 3

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34 3B and Figure 3 3C. T hus, surfactin was demonstrated to be able to make the membrane potential of Bacillus subtilis biofilm more negative. During the surfactin shock, the fluorescence increased at the beginning and then dropped back. This activity fits the fact that surfactin can cause potassium leakage. Initially surfactin caused higher P K and so from the Goldman equation, caused a more negative membrane potential. After several minutes, higher potassium flux caused lower intracellular potassium concentration ( [ K + ] in ) and higher extracellular potassium concentration ( [ K + ] out ) Thus the membrane potential later became more positive. Moreover, it seems that the membrane potential decreased more when high concentration surfactin shock (20 M surfactin ) medium flowed (Figure 3 3 B) compared to that when low concentration surfactin shock (2 M surfactin ) medium flowed (Figure 3 3C). This indicated that the effect of surfactin might be concentration related. This can be a good point for modeling and future work. 3.3 Surfactin Can Cau se Potassium L eakage in Bacillus subtilis B iofilm Asante Potassium Green 4 (APG 4) is an established reporter to measure the concentration of potassium, and the salt form of APG 4 cannot permeate the cell membrane ( Prindle et al., 2015 ). To test the speci ficity of APG 4, NaCl shock and KCl shock were used. As shown in Figure 3 4A, the fluorescence of APG 4 decreased slightly when MSgg (NaCl shock) medium flowed; and the fluorescence of APG 4 increased when MSgg (KCl shock) medium fl owed. This showed that A PG 4 could select potassium specifically. To verify that surfactin can cause potassium leakage in biofilm, APG 4 was used as the reporter in surfactin shock experiments. The background fluorescence of MSgg medium and MSgg (surfactin shock) medium was relat ively stable, as shown in Figure

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35 3 4B. As expected, the fluorescence of APG 4 increased when MSgg (surfactin shock) medium flowed and recovered when MSgg medium flowed again, as shown in Figure 3 4C. Thus, surfactin was demonstrated to be able to cause pot assium leakage in Bacillus subtilis biofilm. Moreover, the fluorescence of APG 4 increased more when high concentration surfactin shock (20 M surfactin ) medium flowed compared to that when low concentration surfactin shock (2 M surfactin ) medium flowed (Figure 3 4C). This again indicated that the effect of surfactin might be concentration related. 3.4 T he Membrane P otential O scillation of Bacillus subtilis B iofilm Was Observed in Our L ab We attempted to duplicate the observation of the membrane potential oscillation reported in 2015 (Prindle et al., 2015). In our microfluidic device, the biofilm had expanded much dur ing long time culture. The movement of the cells that were chosen to show the membrane potential cannot be ignored. To collect reliable data, a custom functio n of MATLAB was created to track specific area s locate d on the edge of biofilm in registered images, as shown in Figure 3 5A. Here, 3 areas located on the different positions of biofilm were cho sen. Each area equals to 25 x 20 pixels. The mean value of the fluorescence in this area represents the membrane potential of this area. DiSC 3 (5) was used as a reporter. Each line in Figure 3 5B represent the mean value of the fluorescence of corresponding area. The fluorescence showed oscil lation behavior ( Figure 3 5B ) Thus, the membrane potential oscillation of Bacillus subtilis biofilm was successfully repeated in our microfluidic device.

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36 3.5 High C oncentration of Surfactin Can Eventually Make T he Membrane P otential of Bacillus subtilis B iofilm More P ositive To further investigate the effect of surfactin on the membrane potential oscillation of Bacillus subtilis biofilm a long time surfactin shock experiment was designed. ThT was chosen as a report er in this experiment. As shown in Figure 3 6, in the first 420 minutes, the fluorescence showed oscillation behavior when MSgg medium flowed. Then, MSgg (surfactin shock) medium (containing 20 M surfactin ) flowed until the end of the experiment. At the be ginning of surfactin shock the fluorescence increa sed and then dropped back but he ld an overall higher level than the overall fluorescence when MSgg medium flowed. This result fits previous data that surfactin can make the membrane potential of Bacillus s ubtilis biofilm more negative However, an interesting phenomenon happened after the 560 minute time point. The fluorescence decreased and then he ld a relatively lower level than the overall fluorescence when MSgg medium flowed. This result showed that in continuous high concentration surfactin shock, the membrane potential of Bacillus subtilis biofilm will eventually become more positive This activity also could be explain ed by the fact that surfactin can cause potassium leakage. As mentioned above, after shock, the intracellular potassium concentration became lower. Eventually, a new balance with a lower potassium flux rate between inside and outside cell membrane established and made the more positive membrane potential. 3.6 Examination of T he srfAA M uta nt The Kolter lab oratory provided a sample of the ZK3858 mutant, which is an isogenic srfA mutant of Bacillus subtilis NCBI 3610. This mutant cannot produce surfactin.

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37 To compare ZK3858 with WT, and observe the influence of surfactin on biofilm formation, ZK3858 and WT were grown in MSgg medium containing the different concentration of surfactin on 24 well plate. As shown in Figure 3 7, WT formed biofilm in the normal MSgg medium (contain ing no extra surfactin). ZK3858 could not form biofilm in the MSgg medium (contain ing no extra surfactin) while it formed biofilm in MSgg medium containing 2 M surfactin. This result was in agreement with previous studies and demonstrated that surfactin is very important in Bacillus subtilis biofilm formation. Moreover, in MSgg medium containing high concentration surfactin, ZK3858 did not form a visible biofilm, while WT formed smaller biofilm than the biofilm in MSgg medium containing no extra surfactin and MSgg medium containing 2 M surfactin within 24h. This result ma y indicate that when surfactin concentration is too high, surfactin may prohibit Bacillus subtilis biofilm formation. Comparing WT grown in MSgg medium containing no extra surfactin and MSgg medium containing 2 M surfactin, Bacillus subtilis formed biofil m earlier or faster in MSgg medium containing 2 M surfactin. This result may indicate that low extra surfactin concentration may promote the formation of Bacillus subtilis biofilm. This suggests that surfactin pos sibly has a dual function in Bacillus subti lis biofilm formation. 3.7 Low C oncentration of Surfactin Can Hold T he Membrane P otential of srfAA Mutant More N egative A normal MSgg medium shock experiment was designed to investigate the effect of surfactin on the srfAA mutant. ThT was chosen as a repor ter. As shown above, the srfAA mutant ZK3858 could not form biofilm in the normal MSgg medium while it formed biofilm in MSgg medium containing 2 M surfactin. So the

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38 cells were cultured with MSgg (low surfactin concentration) medium (containing 2 M surfact in) in the device. As shown in Figure 3 8, the overall fluorescence when MSgg (low surfactin concentration) medium (containing 2 M surfactin) flowed is higher than that when normal MSgg medium flowed. This data showed that the low concentration could help the srfAA mutant to hold a more negative membrane potential. This might be because the srfAA mutant cannot produce surfactin, and so lack s an important way to export potassium In this way, the membrane potential of the srfAA mutant was more positive when normal MSgg medium flowed.

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39 Figure 3 1 DiSC 3 (5) can report the change of biofilm membrane potential. A) The background fluorescence of DiSC 3 (5) is relatively stable in the absence of biofilm B) The fluorescence of biofilm decreased when MSgg (KCl shock) medium flowed and recovered when MSgg medium flowed again. +300mM KCl A +300mM KCl B

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40 Figure 3 2. ThT can report the change of biofilm membrane potential. A) The background fluorescence of ThT is relatively stable i n the absence of biofilm. B) The fluorescence of biofilm decreased when MSgg (KCl shock) medium flowed and recovered when MSgg medium flowed again. +300mM KCl A + 300m M KCl B

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41 Figure 3 3 Surfactin can increase membrane potential of Bacillus subtilis biofilm. A) The background fluorescence of ThT is relatively stable in the absence of biofilm. B) The fluorescence of biofilm increased when MSgg (20 M surfactin shock) medium flowed and recovered when MSgg medium flowed again. C) The fluorescence of biofilm increased when MSgg (2 M surfactin shock) medium flowed and recovered when MSgg medium flowed again. A B C

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42 Figure 3 4 Surfactin can cause potassium leakage across the cell membrane of Bacillus subtilis biofilm. A) APG 4 can select potassium specifically B) The background fluorescence of APG 4 is relatively stable. The change of ThT fluorescence was not likely caused b y itself nor experiment al conditions, C) The fluorescence of biofilm increased when MSgg (2 M surfactin shock) medium and MSgg (20 M surfactin shock) medium flowed and recovered when MSgg medium flowed again. A B C +300mM KCl + 300mM Na Cl

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43 Figure 3 5 The membrane potential oscillation of Bacillus subtilis biofilm A) The white area s on the biofilm were tracked in all time points; the scale ba r is 20 M B) Each line showed the fluorescence of corresponding area on the biofilm. The fluorescence of the white area s showed oscillation behavior. A B 2 1 3

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44 Figure 3 6. Long time surfactin shock on biofilm. The fluo rescence of biofilm increased in the beginning when MSgg (surfactin shock) medium (containing 20 M surfactin ) flowed. Then the fluorescence dropped back but held a higher level until 560 min. After 560 min, the fluorescence decreased and kept a lower level.

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45 Figure 3 7 srfAA mutant examination A) WT and srfAA mutant were grown in MSgg medium containing different concentrations of surfactin in 24 we ll plate. The number s at left give the concentration of surfactin in micromolar units B) The WT and mutant growth conditions of different time points. A WT ZK3858 0 2 6 20 Control 48h WT ZK3858 WT WT WT ZK3858 ZK3858 ZK3858 0 2 6 20 14h 18h 24h B

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46 Figure 3 8 Low concentration surfactin can hold the membrane potential of srfAA mutant more negative The fluorescen ce of srfAA mutant decreased when normal MSgg medium flowed. This sh owed the low concentration surfactin he ld the membrane potential of srfAA mutant more negative

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47 CHAPTER 4 DISCUSSION 4.1 Remarks on This P roject In this project, fluorescent dyes were used to report the membrane potential of Bacillus subtilis biofilm. The dyes are positively charged and can enter cell membranes. The inner membrane of Bacillus subtili s biofilm is at a negative electrical potential with respect to the extracellular space and therefore will retain dyes. When the membrane has more negative electrical potential, it will contain a higher concentration of dyes and so have brighter fluorescen ce. Here, we successfully verified that ThT and DiSC 3 (5) could report the membrane potential of Bacillus subtilis biofilm in our lab oratory To test the influence of surfactin on the membrane potential of Bacillus subtilis biofilm, extra surfactin was adde d to the MSgg medium. The result demonstrated that surfactin could decrease the membrane potential of Bacillus subtilis biofilm. Moreover, APG 4 was used to verify that surfactin can cause potassium l eakage across the cell membrane This result demonstrat ed that surfactin renders the membrane potential of Bacillus subtilis biofilm more negative by causing potassium leakage. The data of different surfactin concentrations in these two experiments indicated that the effect of surfactin is concentration rela ted. This can be a good point for future work. The membrane potential oscillation of Bacillus subtilis biofilm was first reported in 2015 (Prindle et al., 2015). We successfully repeated it in the microfluidic device designed and made by our lab oratory (So n et al., 2014). Meanwhile, the phenomenon

PAGE 48

48 was also repeated in the commercial microfluidic channel, ibidi Slide III 3in1 which has much larger space than our microfluidic device. To test the influence of surfactin on Bacillus subtilis biofilm formatio n, WT and srfAA mutant ZK3858 were cultur ed in MSgg medium containing different concentration s of surfactin. In agreement with the previous study, the result successfully demonstrated that surfactin is very important in Bacillus subtilis biofilm formation. Surprisingly we found that low extra surfactin concentration may promote the formation of Bacillus subtilis biofilm while high extra surfactin concentration may prohibit Bacillus subtilis biofilm formation. This indicated that surfactin might play a dual role in Bacillus subtilis biofilm formation. The effect of surfactin on the membrane potential of Bacillus subtilis biofilm at long time s was tested by flowing with MSgg (surfactin shock) medium (containing 20 M surfactin ). The data surpr isingly showed that the high concentration surfactin eventually made the membrane potential of Bacillus subtilis biofilm more positive. The data on the effect of low concentration surfactin on the membrane potential of the srfAA mutant at long time s showed that low concentration of surfactin can help the mutant to hold a more negative potential. Taking into account the growth condition s with different surfactin concentration s these results indicate that biofilm formation is related to the overall membrane potential. The more negative membrane potential can promote the biofilm formation of Bacillus subtilis ; while the more positive membrane potential can prohibit it. If this indication can be verified, then we could further investigate the mechanism behind i t, e.g. what genetic events related to biofilm formation are involved

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49 in different membrane potential s or whether the different membrane potential will affect protein activities that can affect biofilm formation 4.2 Future W ork To further understand the m embrane potential in biofilm, there are many things to do. First, a method to report biofilm membrane potential directly is still needed. Using fluorescent dye s to report membrane potential ha s many limitations. Fluorescent dye is only a reporter, and it c annot directly indicate the va lue of the membrane potential Fluorescent dye s also have frequency limitation s When it comes to high fre quency changes, the fluorescent may not be considered to be reliable. Fluorescent dye s are also subject to photo bleachi ng which may make the result s less precise. Moreover, fluorescent dye s are extra component s which may have an unknown influence on the biofilm. Thus, finding a way to detect membrane potential of biofilm directly seems to be very important. Microelectrode a rrays (MEA) have good potential to do this. An MEA is a platform that contains an array of small size electrodes. The MEA method is widely used in stimulating and recording bioelectricity in cell and tissue cultures (Stett et al., 2003). Us ing MEA could directly detect the voltage of biofilm and record the change of membrane potential. More importantly, MEA method will not have t he disadvantages of fluorescent dye and can record reliable real time data of membrane potential. Second, further investigating the effect of surfactin is very important, especially the relationship between surfactin effect and surfactin concentration. In this project, the results indicate that low extra surfactin concentration may promote the formation of Bacillus su btilis biofilm while high extra surfactin concentration may prohibit the formation of Bacillus subtilis biofilm. Meanwhile, high extra surfactin concentration

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50 seems to increase membrane potential of Bacillus subtilis biofilm more than low surfactin concent ration. These results indicated that the effect of surfactin might be correlated with its concentration. Further investigation of the relationship between surfactin effect and surfactin concentration can help us to understand more about the role that surfa ctin plays in formation and membrane potential of Bacillus subtilis biofilm. With more data, a model for the relationship between surfactin and the membrane potential could be generated. Considering that surfactin is a quorum sensing factor in Bacillus sub tilis, the model might help us to find a feedback loop in the surfactin quorum sensing system. Third, understanding possible genetic mechanisms corresponding to these p rocesses is another direction for understand ing the relationship between biofilm format ion and biofilm membrane potential. There are many genetic activities related to the biofilm formation, such as eps A O operon activities in Bacillus subtilis biofilm formation. Regulation of these genes is an essential part of biofilm formation. Further st udies about how membrane potential can influence gene regulation could lead us to understand more about the relationship between biofilm formation and biofilm membrane potential. With these understandings, all the related genes could be linked together to generate a model for the genetic network. Moreover, further understanding about the surfactin effect could be a key to deciphering the secret of communication among different bacteria species. The previous study showed that nystatin from Streptomyces nours ei could partly replace the function of surfactin and induced the formation of Bacillus subtilis biofilm (Lpez et al., 2009). Investigating how nystatin induce the formation of the formation of Bacillus

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51 subtilis biofilm could help us to learn more about c ommunication between these two bacteria and provide insight into chemical communication among the Gram positive bacteria.

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58 BIOGRAPHICAL SKETCH Hanling Guo was born in Chengdu, Sichuan, China in 1993 He earned a Ba chelor of Science degree in biological s cience in Sichuan University (SCU) China in June 2015. His undergraduate research focused on construction of multi signal transduction c ell using Escherichia coli He then attended the University of Florida and got trained by Dr. Jorg Bungert and Dr. Stephen J. Hagen. After graduation, he plans to keep studying in biology and pursuit a doctoral degree.


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