Biofilm Formation by Escherichia coli csga and fima mutants

Transcription

1 Journl of Undergrdute Reserch t Minnesot Stte University, Mnkto Volume 14 Article Biofilm Formtion by Escherichi coli csga nd fima mutnts Nicole Snyder Minnesot Stte University, Mnkto Sen Willert Minnesot Stte University, Mnkto Follow this nd dditionl works t: Prt of the Genetics Commons Recommended Cittion Snyder, Nicole nd Willert, Sen (2014) "Biofilm Formtion by Escherichi coli csga nd fima mutnts," Journl of Undergrdute Reserch t Minnesot Stte University, Mnkto: Vol. 14, Article 9. Avilble t: This Article is brought to you for free nd open ccess by the Undergrdute Reserch Center t Cornerstone: A Collection of Scholrly nd Cretive Works for Minnesot Stte University, Mnkto. It hs been ccepted for inclusion in Journl of Undergrdute Reserch t Minnesot Stte University, Mnkto by n uthorized dministrtor of Cornerstone: A Collection of Scholrly nd Cretive Works for Minnesot Stte University, Mnkto.

2 Student Agreement: I m submitting my reserch rticle to be published in the JUR (The Journl of Undergrdute Reserch t Minnesot Stte University, Mnkto), n electronic journl of the Minnesot Stte University Undergrdute Reserch Center. I/We certify hve followed the ccepted stndrds of scientific, cretive, nd cdemic honesty nd ethics. I understnd tht my rticle submission will be blind-reviewed by fculty reviewers who will recommend cceptnce for publiction; cceptnce with revisions; or reject for publiction. I understnd tht s uthor, I retin the right to present ny prt of the reserch in ny form in other publictions. The JUR hs the right to reproduce nd reprint published submissions for instructionl or promotionl purposes. For complete detils, see Journl of Undergrdute Reserch t Minnesot Stte University, Mnkto policies pge. Mentor Agreement: I hve reviewed the submission, nd I support its inclusion in the JUR (The Journl of Undergrdute Reserch t Minnesot Stte University, Mnkto). I understnd tht I will be cknowledged s the fculty mentor for the student uthor(s). To the best of my knowledge, the student hs followed the ccepted stndrds of scientific, cretive, nd cdemic honesty nd ethics.

3 Snyder nd Willert: Biofilm Formtion by Escherichi coli csga nd fima mutnts Nicole Snyder nd Sen Willert nd Dr. Timothy Secott (Fculty Mentor) Deprtment of Biologicl Sciences Minnesot Stte University, Mnkto Biofilm Formtion by Escherichi coli csga nd fima mutnts Published by Cornerstone: A Collection of Scholrly nd Cretive Works for Minnesot Stte University, Mnkto,

4 Journl of Undergrdute Reserch t Minnesot Stte University, Mnkto, Vol. 14 [2014], Art. 9 Abstrct Biofilms re structured community of bcteril cells enclosed in self-produced polymeric mtrix nd dherent to n inert or living surfce. These structures nd the orgnisms tht cuse them cn pose very serious problem if they colonize on medicl devices. This is becuse biofilms hve the bility to communicte within the colony nd with other orgnisms tht might ttch to the surfce, cting like community working together. Biofilms llow the orgnism to be resistnt to hrsh nd unfvorble conditions llowing them to survive longer nd spred. Severl genes in Escherichi coli (E. coli) hve been ssocited with biofilm formtion by tht orgnism. Mny of those genes encode surfce ppendges such s flgell, fimbrie, nd pili. We creted muttions in genes encoding curli (csga) nd fimbrie (fima) with the im of compring their bility to form biofilms. The respective genes were selected on knmycincontining gr nd disrupted with knmycin resistnce gene. Biofilm formtion in nutrientrich medium nd nutrient-poor medium is currently in progress, nd the bility of the mutnt E. coli strins to form biofilms will be compred with tht of the prent wild type strin using crystl violet microplte ssy. 2

5 Snyder nd Willert: Biofilm Formtion by Escherichi coli csga nd fima mutnts Introduction Biofilms re structured communities of bcteri tht live in self-produced polymeric mtrix tht dhere to living or nonliving surfce. By doing so, they cn llow the orgnisms to be more resistnt to hrsh conditions, enbling them to survive longer nd spred. These structures nd the orgnisms tht re ble to form them cn cuse serious problems if they colonize on certin objects, such s medicl devices. Escherichi coli (E. coli) is one of these orgnisms. Severl genes in E. coli hve been ssocited with biofilm formtion, including the genes for curli (csga) nd fimbrie (fima). Muttions in these two genes were creted nd the orgnism grown in two different types of medi to: 1) compre the genes effect on biofilm formtion, nd 2) determine the effect on nutrient vilbility hs on biofilm formtion. The hypotheses of the experiment were tht E. coli crrying muttions in csga nd fima will not be ble to form biofilm s effectively s the wild type E. coli nd tht orgnisms grown in nutrient rich environments will produce more robust biofilms thn orgnisms grown in nutrient poor environments. Methods Formtion of Mutnts The orgnisms used in the experiment were isogenic, mening tht the only difference in the strins were the muttions we introduced. To obtin the mutnts, single-step inctivtion procedure ws followed (1). A strin of Escherichi coli BW crrying the plsmid PKD- 4, which contined knmycin resistnce gene, ws obtined nd the plsmid isolted. Gene disruption cssettes were formed by Polymerse Chin Rection (PCR) using primers designed to mplify the knmycin resistnce cssette nd introduce sequences from csga (primers CsgA- Published by Cornerstone: A Collection of Scholrly nd Cretive Works for Minnesot Stte University, Mnkto,

6 Journl of Undergrdute Reserch t Minnesot Stte University, Mnkto, Vol. 14 [2014], Art. 9 1 nd -2) nd fima (primers fima-1 nd -2). The PCR product ws purified nd electroported into wild E. coli contining the helper plsmid pkd64, which encoded mpicillin resistnce nd bcteriophge recombinse complex. Trnsformnts were obtined by plting the smple onto nutrient gr contining knmycin nd mpicillin to select for the strins contining both the PCR product nd the helper plsmid. Confirmtion of the trget genes ws verified by PCR to confirm tht the muttions were in the desired loction using specific primers. Biofilm Assy To do the biofilm ssy, the procedure suggested by George O Toole (2) ws followed. Once the mutnts were confirmed, overnight cultures of the orgnisms (both mutnt csga nd fima strins s well s the wild type) were prepred in either LB (nutrient-rich broth) or SMB (nutrient-poor) broth. The opticl densities of the cultures were djusted to OD550 nm= 1.0 nd the cultures were seeded onto microtiter pltes to test for their bility to form biofilms. The biofilms formed were stined with 0.1% crystl violet nd then the biofilms were quntified by dissolving in 30% cetic cid nd mesuring the bsorbnce t 550 nm with microplte spectrophotometer. Results Bsed on the dt collected in the ssy, it ws shown tht when compring the strins of mutnt E. coli with the wild type, the mount of biofilm formed by the mutnt types ws less thn the mount formed by the wild strin. For ech environment (csga in LB broth or fima in SMB for exmple) there were 192 replictes tken. These vlues were then nlyzed using vrious sttisticl tests nd then put into grphs for more visul representtion. In Figures 1 nd 2, it is shown tht the wild type strins, which did not hve the resistnce gene formed greter 4

7 Snyder nd Willert: Biofilm Formtion by Escherichi coli csga nd fima mutnts mounts of biofilm thn the mutnt types, which did hve the resistnce gene. Mening the wild type strin produced more biofilm formtion in the individul wells of the microtiter pltes leding to greter sttisticl dt. This supports one of the hypotheses for the experiment, tht E. coli with the muttions in either csga or fima re not ble to form s robust biofilms s the wild type E. coli. A nm b c 0.4 c c b 0.0 b n 0.0 A m Fig. 1 Fig. 2 Another vrible in biofilm formtion tested in this experiment ws to determine how the mount of nutrients present s the orgnism is growing hs n effect on the mount of biofilm formed. It ws hypothesized tht the orgnisms grown in nutrient rich environments (where LB medi ws used) would produce greter mount of biofilm thn orgnisms grown in nutrient poor environments (where SMB medi ws used). Figures 3 nd 4 show the dt collected displyed in the form of br grphs. The ptterned columns represent the wild strins while the non-ptterned columns represent the mutnt strins. Figure 3 is the dt from the ssys with the csga mutnt grown in either LB or SMB medi. When simply compring the medi, the orgnisms grown in the SMB medi formed Published by Cornerstone: A Collection of Scholrly nd Cretive Works for Minnesot Stte University, Mnkto,

8 0.0 Journl of Undergrdute Reserch t Minnesot Stte University, Mnkto, Vol. 14 [2014], Art. 9 greter mounts of biofilm. This did not support the hypothesis tht orgnisms grown in LB would form more biofilm thn the orgnisms grown in SMB. When looking t the difference between the wild strin nd mutnt strin on this grph, support of the first hypothesis ws reinforced. The wild strin of E. coli formed over twice the mount of biofilm s ws seen in the mutnt strin. The sme observtions were seen in the ssys run with the fima mutnt (dt shown in Figure 4). 0.8 A550 nm b A550 nm 0.6 b c b Fig. 3 Fig. 4 Discussion Our hypotheses were tht mutnt E. coli would not be ble to form biofilms s well s wild type E. coli nd tht orgnisms grown in nutrient-rich environments will produce more biofilm growth thn orgnisms frown in nutrient poor environments. More robust biofilms were produced by the wild type E. coli in both LB nd SMB, which supported our first hypothesis. However, our second hypothesis ws not supported becuse the mutnt strins produced more biofilm in SMB thn in LB in the csga nd the opposite for the fima strin. One reson for the discrepncy is simply there could hve been smll mount of contmintion. One of the 6

9 Snyder nd Willert: Biofilm Formtion by Escherichi coli csga nd fima mutnts resons n orgnism forms biofilm is to help it survive difficult conditions, nd it cn be rgued tht in LB, conditions become stressful only s the orgnisms begin to rech sttionry phse. In contrst, nutrient limittions present in cultures in SMB would be expected to be comprtively stressful throughout the orgnisms time in culture. Consequently, one might expect n increse in biofilm production for cells grown in miniml medi due to the unfvorble conditions. Further reserch should be ttempted to test this hypothesis, s well s the bility of csga-fima double mutnts to form biofilms. Our work shows tht csga nd fima re both resposible for the formtion of biofilms. With this informtion we mybe ble to produce products such s ctheters which re resistnt to either of these genes. Doing so would help reduce the formtion of biofilms nd directly reducing the occurrence of infections. With further reserch into double gene knock out we could possible discover yet nother gene tht my be responsible for the production of biofilms. Agin knowing which genes re responsible for the formtion of biofilms would llow us to produce products tht re resistnt to biofilms or even slow the formtion. Published by Cornerstone: A Collection of Scholrly nd Cretive Works for Minnesot Stte University, Mnkto,

10 Journl of Undergrdute Reserch t Minnesot Stte University, Mnkto, Vol. 14 [2014], Art. 9 References 1. Dtsenko, K.A., nd B.L. Wnner One-step inctivtion of chromosoml genes in Escherichi coli K-12 using PCR products. Proc Nt Acd Sci USA 12: O Toole, G.A Microtiter dish biofilm formtion ssy. J. Vis. Exp. 47: e

11 Snyder nd Willert: Biofilm Formtion by Escherichi coli csga nd fima mutnts Professionl Biogrphies Nicole Snyder I m from Trcy, Minnesot. I m currently n undergrdute student t Minnesot Stte University, Mnkto mjoring in Microbiology. I will be grduting in My 2014 with BS in Microbiology. I presented this reserch with my prtner t the 2014 Minnesot Undergrdute Scholrs Conference nd the 2014 Undergrdute Reserch Symposium. It is my gol to cquire job in qulity ssurnce or reserch. Sen Willert I m from Mnkto, Minnesot. I m n undergrdute student t Minnesot Stte University, Mnkto. I will grdute in the spring 2016 with degree in Humn Biology. I presented this reserch with my prtner t the 2014 Minnesot Undergrdute Scholrs Conference nd the 2014 Undergrdute Reserch Symposium. I would like to ttend medicl school nd pursue creer in the medicl field. Dr. Timothy Secott Dr. Secott received his BS in Biology from Millersville University in Pennsylvni. After working s reserch technicin in the Deprtment of Microbiology t Penn Stte University s Hershey Medicl Center, he supervised the ctivities of the Bcteriology nd Moleculr Dignostic Sections of the Pennsylvni Stte Veterinry Lbortory for more thn 10 yers before strting PhD studies t Purdue University. He joined the Deprtment of Biologicl Sciences fculty t Minnesot Stte University, Mnkto in Published by Cornerstone: A Collection of Scholrly nd Cretive Works for Minnesot Stte University, Mnkto,