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Biofilm Formation and Dispersal under the

Influence of the Global Regulator CsrA of
Escherichia coli
Debra W. Jackson, Kazushi Suzuki, Lawrence Oakford,
Jerry W. Simecka, Mark E. Hart and Tony Romeo
J. Bacteriol. 2002, 184(1):290. DOI:

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10.1128/JB.184.1.290-301.2002.

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decreasing antibiotic efficacy. Jackson. Intracellular to form microcolonies which eventually produce a mature bio- polyphosphate has pleiotropic effects on a variety of extracel- film (32. the expression of a chromosomally encoded csrAⴕ-ⴕlacZ translational fusion was dynamically regulated during biofilm formation in a pattern consistent with its role as a repressor. Finally. 3500 Camp Bowie Blvd. 2002. It is interesting that although factor needed for the transcription of stationary phase genes) motility is involved in the early stages of biofilm formation. The communities known as biofilms (13). OmpR and adherence (35. In E. al- vidual cells to a surface followed by migration and replication though the basis of this effect is unknown (34). 2014 by NATIONAL CHUNG HSING LIBRARY Department of Molecular Biology and Immunology1 and Department of Pathology and Anatomy.1 Mark E. 184. the regulatory mechanisms of biofilm development remain poorly defined and those of dispersal are unknown. 35). p. coli. University of North Texas Health Science The dispersal or shedding of planktonic cells from a biofilm Center at Fort Worth. TX 76107-2699. the biology of bacterial biofilms has become a major known as quorum sensing. 290–301 Vol. The impact of microbial biofilms is perva. No. flagella and type IV (outer membrane protein regulator) and RpoS or ␴s (a sigma pili have been implicated (32). 46). In E. to population density. and iron availability in Pseudomonas fluorescens and P. and chemical proper. Ectopic expression of the E. A two-component removal (13). Biofilm Formation and Dispersal under the Influence of the Global Regulator CsrA of Escherichia coli Debra W. aerugi- nosa. Biofilms often complicate chronic and difficult-to-treat infections by protecting bacteria from the immune system. While the biology of bacterial biofilms has become a major focus of microbial research. Fort Worth. whereby the concentration of a focus of microbial research. or regulatory factors previously implicated in biofilm formation. the typical three-dimensional structure of E. matrix-enclosed commu- nities known as biofilms.asm. diffusible autoinducer provides a regulatory signal in response Studies of the biophysical. including prostatitis. biliary tract infections.unt.g. coli K-12 csrA gene repressed biofilm formation by related bacterial pathogens. the biofilm in order to colonize new locations (14).1 and Tony Romeo1* Downloaded from http://jb. Hart. In contrast. thereby establishing microbial and Pseudomonas aeruginosa (discussed in reference 47). fla- gella. Bacteria have evolved elaborate mechanisms for adhering to lar gene expression is decreased in the biofilms of both E. Texas 76107-2699 Received 10 August 2001/Accepted 3 October 2001 The predominant mode of growth of bacteria in the environment is within sessile. Phone: (817) 735-2121. 46). Direct 290 . In medicine. coli biofilm. terial infections. Complemen- tation studies with glg genes provided further genetic evidence that the effects of CsrA on biofilm formation are mediated largely through the regulation of intracellular glycogen biosynthesis and catabolism. Fax: (817) 735-2118. is essential for the construction of a ties of biofilms have culminated in our present concept of the mature biofilm of P.JOURNAL OF BACTERIOLOGY. Nutritional cues.2 University of North Texas Health Science Center at Fort Worth.edu. in part because it and pillars that may facilitate nutrient exchange and waste activates the synthesis of type IV pili (33). mation of a mature biofilm in P. Mailing address: Department of Molecular complex and remains poorly understood in any species. coli. 1 0021-9193/02/$04. e. Biofilms represent a dis. Fort Worth. 35). it are apparently involved (1. The regulatory mechanisms that guide biofilm development and urinary catheter cystitis caused by Escherichia coli (14). also significantly affect biofilm formation (9. structural. have also come under recent scrutiny. and dispersing planktonic cells to distant body sites.1. aeruginosa (35). GacA. the regulation of biofilm development is highly * Corresponding author. respectively. E-mail: may be essential to permit bacteria to escape the confines of tromeo@hsc.org/ on April 11. though a biofilm is still formed in its absence (17). 38). In Pseudomonas. A biofilm is initiated by the attachment of indi- response regulator. coli and colonizing solid surfaces. extracellular polysaccharide colanic acid is needed to generate tinct lifestyle for bacteria which provides protection from del. American Society for Microbiology. The social behavior Thus. A variety of extracellular molecules and surface lular components and regulatory factors in P. and curli fimbriae are involved in attachment participate in biofilm formation (37. type I pili.290–301. aeruginosa which organelles participate in biofilm development. carbon is unfavorable for maintenance of a mature biofilm and flagel. Here we establish that the RNA binding global regulatory protein CsrA (carbon storage regulator) of Escherichia coli K-12 serves as both a repressor of biofilm formation and an activator of biofilm dispersal under a variety of culture conditions. A csrA knockout mutation enhanced biofilm formation in E.1 Lawrence Oakford. In contrast..2002 Copyright © 2002. Biology and Immunology. We propose that global regulation of central carbon flux by CsrA is an extremely important feature of E.. The Crc protein (catab- mature biofilm as a complex community exhibiting channels olite repression control) is also required.1128/JB. this csrA mutation did not affect biofilm formation by a glgA (glycogen synthase) knockout mutant. coli biofilm development.2 Jerry W. was also recently implicated. surface. All Rights Reserved. Clearly. al- eterious conditions. biofilms are known the extracellular polyuronide alginate is essential for the for- to complicate the majority of chronic and difficult-to-treat bac. aeruginosa (19).1 Kazushi Suzuki. sive throughout the biosphere. coli strains that were defective for extracellular.00⫹0 DOI: 10.184. Jan. Simecka.

2014 by NATIONAL CHUNG HSING LIBRARY MC4100 F⫺ ⌬(argF-lac)U169 rpsL relA flhD deoC ptsF rbsR 23 W3110 F⫺ mcrA mcrB IN(rrnD-rrnE)1 ␭⫺ 44 RG1-B MG1655 csrB::cam (a precise deletion of csrB) 22 MM5057 ⌬ motB urvC-279::Tn10 Mike Manson MHR 204 MC4100 csgA2::Tn105 23 RH106 MC4100 rpoS::Tn10 R. 276860.org/ on April 11. coli P18 24 E. Ampr Continued on following page . coli K-12 strains MG1655 F⫺ ␭⫺ Michael Cashel CF7789 MG1655 ⌬ lacI-Z(MluI) Michael Cashel KSA712 CF7789 ⌬ (␭ att-lom)::bla ␾(csrA⬘-⬘lacZ)1 (hyb) Ampr Kans This study AAECO72 MG1655 ⌬ fimB-H Ian Blomfield DHB6521 F⫺ ␭Inch1(Kanr)⌬lac(MS265) mel Nalr supF58 (⫽suIII⫹) 11 JM101 supE thi⌬ (lac-proAB)[F⬘ traD36 proAB lacIqZ⌬M115] 44 Downloaded from http://jb. freundii P5 24 E. enterica serovar Typhimurium 4 ATCC 14028 Bacteriophages P1vir Strictly lytic P1. Hengge-Aronis SG20043 MC4100 cpsE::Tn10 Valerie Stout TK821 MC4100 ompR331::Tn10 44 TR1-5 or TRa csrA::kanR 40 glgAb glgA::kanR This study DJ1 MG1655 csgA2::Tn105 This study DJ2 TRMG1655 csgA2::Tn105 This study DJ3 MG1655 cpsE::Tn10 This study DJ4 TRMG cpsE::Tn10 This study DJ6 TRAAECO72 This study DJ7 AAECO72 csgA2::Tn105 This study DJ8 TRAAECO72 csgA2::Tn105 This study DJ9 AAECO72 cpsE::Tn10 This study DJ10 TRAAECO72 cpsE::Tn10 This study DJ11 MG1655 csgA2::Tn105 cpsE::Tn10 This study DJ12 TR MG1655 csgA2::Tn105 cpsE::Tn10 This study DJ13 AAECO72 csgA2::Tn105 cpsE::Tn10 This study DJ14 TRAAECO72 csgA2::Tn105 cpsE::Tn10 This study DJ21 MG1655 ⌬motB uvrC-279::Tn10 This study DJ22 AAECO72 ⌬motB uvrC-279::Tn10 This study DJ24 TR ⌬motB uvrC-279::Tn10 This study DJ25 TRAAECO72 ⌬motB uvrC 279::Tn10 This study DJ30 MG1655 rpoS::Tn10 This study DJ31 TR MG1655 rpoS::Tn10 This study DJ32 MG1655 rpoS::Tn10 csgA2::Tn105 This study DJ33 TR MG1655 rpoS::Tn10 csgA2::Tn105 This study DJ34 AAECO72 rpoS::Tn10 This study DJ35 TR AAECO72 rpoS::Tn10 This study DJ36 AAECO72 csgA2::Tn105 rpoS::Tn10 This study DJ37 TRAAECO72 csgA2::Tn105 rpoS::Tn10 This study DJ40 MG1655 ompR333::Tn10 This study DJ41 TR MG1655 ompR333::Tn10 This study DJ42 MG1655 ompR333::Tn10 csgA2::Tn105 This study DJ43 TR MG1655 ompR333::Tn10 csgA2::Tn105 This study DJ44 AAECO72 ompR333::Tn10 This study DJ45 TR AAECO72 ompR333::Tn10 This study DJ46 AAECO72 ompR333::Tn10 csgA2::Tn105 This study DJ47 TR AAECO72 ompR333::Tn10 csgA2::Tn105 This study Clinical strains C. or plasmid Relevant genotype or characteristics Reference or source E. 184.asm. Ampr 44 pBR322 Cloning vector.VOL. Ampr 40 pSTCSR5 S. coli O157:H7 EF302 26 S. phage. 2002 REGULATION OF BIOFILM FORMATION AND DISPERSAL 291 TABLE 1. Bacterial strains. forms clear plaques Carol Gross ␭InCh1 For chromosomal integration of genes 11 Plasmids pUC19 Cloning vector. enterica serovar Typhimurium csrA in pUC19. Ampr Tetr 44 pCSR10 E. phage. and plasmids used in this study Strain. coli csrA gene in pUC19. GenBank This study accession no.

0. acetate metabolism. acetic acid (EM Science.. Growth of planktonic cells was determined biofilm dispersal. and the csrA homolog.65 g of sodium environmental signals influence the dispersal process (5). 44). Cultures for the ␤-galactosi- presses genes that are involved in the invasion of mammalian dase assays were grown in borosilicate test tubes at 26°C.6 g of NaCl2. 43. 200 ␮g/ml. The csrA gene determined using a microtiter plate reader (DynaTech. aeruginosa and P.). Ampicillin and kanamycin were used at 50 and 40 ␮g/ml. vapor (27). in-frame csrA⬘-⬘lacZ fusion.org/ on April 11. bound cells with crystal violet (BBL) (32). The coverslips were MATERIALS AND METHODS removed at various times. which was confirmed by PCR analysis. 20 ␮g/ml. including glycogen confirm that observed effects on biofilm formation in microtiter wells were not biosynthesis and catabolism and gluconeogenesis (40. as previously de- man pathogens. rinsing the wells with water (three times). and most of the ␭ DNA was eliminated to generate a stable widely distributed among eubacteria. Ampr 51 pMLB1034 For construction of ⬘lacZ translational fusions. under a single growth condition. chloramphenicol. 48). N.).C. rsmA. CsrA activates glycolysis. pH 7. expression studies. 1. and 5 mg of MnCl2. and plasmids used in these ␮g of acridine orange/ml (K&K Laboratories. The scribed (11). stained with 30 Bacterial strains and media. background staining was corrected by subtracting the crystal violet bound to uninoculated controls. coli K-12 strain CF7789 for csrA gene of Salmonella enterica serovar Typhimurium re. coli to adhere to culture tubes. .4-kb blunt-ended EcoRI- BglII restriction fragment from pTR151P1 into the SmaI site of pMLB1034. 10 ␮g/ml. protein. cultures were grown and tested simultaneously in new borosili- Conversely. Glycogen phenotypes were determined by staining colonies with iodine The purpose of the present study was to investigate the regu.3 g of Na2SO4. TABLE 1—Continued Strain. Biofilm was measured by dis- carding the medium. A highly repeated se. 2. 100 ␮g/ml. M63 salts solution contained (per liter) 13. csrA::kanR allele in pUC19. 12 g of urea. and molecular genetic techniques were performed by decreasing their decay rates (28. 50 mg of MgSO4. ␤-Galactosidase.) were carried out in artificial urine medium containing (per liter) 0. 0. including numerous hu. it has been suggested that dispersal of cells from a (NH4)2SO4. Sterile microscope coverslips were aseptically placed into sterile petri dishes along with 15 ml of a freshly inoculated culture.asm.6 g of KH2PO4. CsrA (carbon storage experiment. inoculated cultures were grown in a regulatory pathway has been shown to be capable of inducing 96-well polystyrene microtiter plate.). Fur. film dispersal in P. Tetr 25 pJF02 glgP in pUC19. The fusion was transduced into E.0 g of NH4Cl. 1. were used as required for biofilm studies at the following concentrations: ampi- tial expression of chitinase genes among a subpopulation of cillin. while cultures for biofilm assays were generally burgh. The construction of a chromosomal csrA⬘-⬘lacZ units of CsrA bind to an untranslated RNA molecule. or Roper Sensys cold-slow scan CCD camera (Roper Scientific Co. Phoenix. microscopic observation has revealed that physiological and H2O. In the microtiter plate assay. phage. standard procedures (30. the processes involved in Quantitative biofilm assay. Inc. Furthermore. Ampr 41 pTR151P1 PstI subclone from pTR151. by subcloning a 0. 11 g of K2HPO4 and 8.). 49).5 g of KH2PO4. ⬃18 sub. in 10-min assays. Ampr 40 Downloaded from http://jb. 50). Ariz. Va. latory role of CsrA in biofilm development. The differen. Bacteria were routinely cultured at 37°C in again. In summary. Ampr 27 pOP12 Contains asd-glgCAP⬘ in pBR322 Tetr 25 pOP245 glgA in pBR322. 2.1 g of creatinine. subcloning. and glycogen assays.J. All comparative analyses were conducted by regulator). regulates gene expression posttranscriptionally by binding to Molecular and genetic techniques.5 g used for Nomarski interference microscopy. 1. Gibbstown. citrate.Y. adjusted to pH 7. (29). P1 transduction or cotransduction of resis- mRNA transcripts of regulated genes and either increasing or tance markers. there is evidence that CsrA homologues of eubac. and no fresh medium. Pa.. gently rinsed with phosphate buffer (per liter. 2. 0. cells from biofilm (7).8 g of KH2PO4. Cary. Homologues of csrA are chromosome. This quence element found in the loop segments of CsrB hairpins fusion was transferred to ␭InCh1.292 JACKSON ET AL. precipitation with ice-cold 10% trichloroacetic acid and was quantified by using the bicinchoninic acid method with bovine serum albumin as the standard pro- teria are important in regulating host-microbe interactions. Thus.0.6 g of KCl. J.65 g of CaCl2 and the imaging program Image-Pro Lab software for MacIntosh (Scanalytics. N. and 1% tryptic soy broth (BBL. Chantilly.. during the construction of the csrA⬘-⬘lacZ fusion.4 with NaOH). 39). translational fusion first involved the preparation of a plasmid containing an which antagonizes CsrA activity (27).65 g of MgCl2 6H20. Microscopy. tein (45). The strains. grown at 26°C. phage. and the data were analyzed by Tukey Multigroup Analysis (StatView- SAS Institute Inc. kanamycin. Glycogen biosynthesis was assessed using Kornberg agar medium postulated to involve the degradation of alginate (2. To induced in the stationary phase of growth. Pitts- Luria-Bertani (LB) medium (30). Overnight cultures were inoculated 1:100 into biofilm dispersal are not clearly defined in any species. 2 g of thermore. BACTERIOL. and 1 ml of 1 M MgSO4. Antibiotics biofilm may be cell cycle mediated in E. pCAZ1. Each experiment was performed at least in motility (43. surface specific. Amp 27 pCSRB-SF Minimal csrB in pUC18. Cell protein was obtained by 16). coli (3).5 mg of FeSO4 7H20. Bio. For each encodes a global regulatory protein. b Strains names with the suffix glgA contained a glgA::kanR insertion. rinsed experiments are listed in Table 1. N. as previously described (41).4 (20). 12). Cockeysville. and staining It was previously observed. fluorescens has been Md. marine bacteria has been postulated to promote detachment of respectively.20 g of sodium oxalate. or plasmid Relevant genotype or characteristics Reference or source r pCSRH6-19 Produces recombinant CsrA under IPTG induction. which represses several metabolic pathways that are incubating strains within the same microtiter plate to minimize variability. the recombinant phage was moved into the may mediate this binding (27. and tetra- cycline. but are not apparent in eucaryotes (39). and cate glass test tubes (18 mm). 2014 by NATIONAL CHUNG HSING LIBRARY a Indicates that the wild-type csrA allele has been replaced by csrA::kanR.) (31). pH 7. All images were collected with a of yeast extract (Difco). CsrB. 4. by absorbance at 600 nm or total protein assay. The dye was solubilized with 33% that a csrA disruption caused E. 48. and mounted to the microscope slide with Immu-Mount (Shandon. A Nikon Microphot-FXA microscope with a 40⫻ Plan Apo objective lens was gen (CFA) medium containing (per liter) 10 g of Casamino Acids (Difco). CsrA is an RNA binding protein that triplicate. and absorbance at 630 nm was forming a coating suggestive of a biofilm (40). and planktonic cells gastrointestinal mucosa (4). chromosomal fusion.). ␤-Galactosidase activity was determined proteins involved in both plant disease and host response (15. Plainville. The biofilm was suspended in CFA medium plant pathogenic Erwinia species represses the production of with pipetting to disrupt cell aggregates. 0. Biofilm assays were typically carried out in colony-forming anti. of were separated from adherent cells.

10. 2014 by NATIONAL CHUNG HSING LIBRARY formed by MG1655 in the left panels (10. (B) Nomarski interference of biofilm Downloaded from http://jb. optical density at 600 nm.org/ on April 11. 2002 REGULATION OF BIOFILM FORMATION AND DISPERSAL 293 FIG. 1.asm. (A) Growth in polystyrene microtiter wells of planktonic cells of the wild-type strain MG1655 (filled squares) or its csrA mutant (filled triangles). and 24 h). 16. coli.VOL. 184. OD600. Biofilm formation in the same wells is shown as open squares or triangles. respectively. . top to bottom) or its csrA mutant in the right panels (3. and 24 h. Biofilm formation by wild-type and csrA mutant strains of E.

It was Biofilm formation in csrA mutant and wild-type strains. or M63 salts containing 0.asm. 10 ␮m) and a cross-section of a sagital view tilted (Q ⫽ 45°. along with a 2-␮m-thick cross-section at a depth of 6 ␮m in the right panel (scale bar. biofilm C-Apochromat objective lens was used to generate a topographical image and formation. In each strain. 14 ␮m. the Citrobacter freundii P5. Optical sections were collected at 2-␮m steps through a sample depth of 20 ␮m using 488 nm and 510 to 525 nm formation was observed in every growth medium that we have excitation and emission wave lengths. do not appear to be restricted to specific growth tative experiment (Fig. Thus. 2014 by NATIONAL CHUNG HSING LIBRARY FIG. BACTERIOL. 3A). 1A). 20 ␮m). every E. Va. Overexpression of csrA in E. was examined. Further ex. biofilm accumulated much Since a disruption in csrA dramatically increased biofilm for- more rapidly and extensively in the csrA mutant. BW3414. Two strains growth (Fig. crystal violet staining. 2). virtual color code depicts biofilm height above the microscope slide.A. The wild-type E. These include morpholinepropanesulfonic acid me- dium (40).2 N. examined.2% glucose (data not shown). 1B). ary phase of growth. ment was somewhat delayed until cultures were in the station. Fairfax. scanning Because biofilms are known to complicate a variety of in- confocal laser microscopy was utilized. the consequences of CsrA cultures was monitored by absorbance. J. tion appears to reflect normal. A Zeiss LSM 410 Confocal Microscope with a 40⫻ 1. Examples of apparent pillars (p) and channels (ch) are indicated. LB with or without 0. mutant exhibited features of a typical mature biofilm. coli P18 and and channels. unlike those of certain other fac- K-12 strain MG1655 and its isogenic csrA mutant exhibited similar growth in liquid CFA medium. the effects of csrA overexpression on biofilm formation amination of biofilms formed on microscope coverslips by No. To also observed under anaerobiosis (40). we examined whether csrA overexpression would in- of the mutant revealed a biofilm ⬃20 ␮m thick at 24 h of hibit biofilm formation in several clinical isolates. coli quantitatively assess the role of CsrA in biofilm development. Thus.2% glucose or glycerol. though accelerated. 2. from 0 to 20 ␮m. A topographical image fections. coli effects on biofilm formation. A topographical image of the biofilm formed by TR1-5MG1655 is shown in the left panel (white scale bar. A cross-section of this biofilm exhibited pillars isolated from colonized urinary catheters. F ⫽ 30°) in the bottom panel (scale bar. as shown in a represen. in 2-␮m increments). medium for cultivation to mimic their host environment (Fig. tors (18). Thereafter. W3110. including MC4100. CFA with or without 0.). coli K-12 and clinical isolates. . K-12 parental strain that has been tested exhibited this effect of biofilm growth was monitored by using an assay based on csrA. This stimulatory effect of a csrA mutation on biofilm cross-sections through the csrA mutant biofilm.2% glucose.org/ on April 11. Likewise.5% glucose. characteristic of a mature biofilm. Laser confocal microscopy of biofilm produced by a csrA mutant. MG1655. JM101. as visualized by confocal microscopy. were tested by using artificial urine increased adherence to abiotic surfaces caused by csrA disrup. conditions or strains. RESULTS Kornberg with 0. while planktonic growth in the same and others (data not shown). coli K-12 and its csrA mutant relative to the plasmid order to determine whether the biofilm formed by the csrA vector controls (Fig. Ectopic expression of csrA from a multicopy marski interference microscopy also confirmed that biofilm plasmid completely inhibited biofilm formation in both wild- formation was more prolific in the csrA mutant (Fig. E. Downloaded from http://jb. respectively. In type E. mation. biofilm develop.294 JACKSON ET AL.

3C). 27). biofilm was decreased by the motB mutation but was not strains (P ⬍ 0. Thus. and asterisks denote significant differences between tant. the effect (bars 1 to 3) and S. coli O157:H7 although chemotaxis is not required (35). coli. Biofilm formation was modestly inhibited factors. (B) defects (data not shown). ability to sequester ⬃18 subunits of this protein (27). overexpression. 3. colanic acid. 2002 REGULATION OF BIOFILM FORMATION AND DISPERSAL 295 TABLE 2. or the pUC19 control plasmid. ducted on these strains indicated that the differences in biofilm (A) MG1655 (bars 1 to 3) or its csrA mutant (bars 4 to 6) containing formation exhibited by these strains were not due to growth plasmids pCSR10 (csrA) (bars 2 and 5) or pUC19 (bars 3 and 6).016ⴱ 0. virtually eliminated biofilm formation (Fig. 4B). wild-type strain. formation (1. overexpressed its own csrA homolog. while colanic acid does not quantita- tively affect biofilm formation.VOL. pCSRB-SF.0001).003 a Biofilm formation in CFA medium was measured at 24 h by crystal violet staining in microtiter wells (A630). tested. a diarrheal pathogen (Fig.010 ⫾ 0. these two mutations had mini- CsrB RNA is an antagonist of CsrA. pUC19 (bars 3 and 6). ectopic expression While the addition of a curli (csgA) knockout mutation in the of csrA inhibited biofilm formation in these pathogenic rela. in the food-borne pathogens E. 4A. 4A). (C) Food-borne pathogens E. rpoS background yielded no additional effect.012 1. and csrB overexpression simulates a csrA dis- ruption (22. S. biofilm formation by this strain was sever- . pSTCSR5 (S. Results using strains lacking curli fimbriae.0001) were noted (ⴱ) between MG1655 and its csrB mutant.org/ on April 11. enterica serovar Typhimurium ATCC 14028 (bars of a ⌬motB mutation.170 ⫾ 0. and between strains that contained either a multi- copy csrB plasmid. an RpoS and OmpR have been reported to regulate biofilm enterohemorrhagic isolate. enterica serovar Typhi. Both flagella and motility are needed Urinary tract pathogens E. Therefore. biofilm formation was deficient in a csrB null mutant of E. relative to its isogenic parent.asm. In these strains.029 ⫾ 0. a csrB null mutation yields a phenotype similar to csrA in the csrA mutant. in the csrA mutant background only the loss of type I pili significantly decreased biofilm formation. coli K-12. 4B). freundii P5 (bars 4 to 6) containing plasmids pCSR10 (csrA) (bars 2 and 5) or for initial cell attachment during biofilm formation in E. These results confirm that curli fimbriae and type I pili play significant roles in biofilm forma- tion in the parent strain. was 4 to 6) containing plasmids pCSR10 (csrA) (bar 2). Statistical differences (P ⬍ 0. pCSRB-SF. 46). These studies re- vealed that a csrA mutant forms biofilm in the absence of each 3B). CsrA effects on biofilm formation by strains defective for extracellular and/or surface molecules or regulatory factors. 4C). and was stimulated by a multicopy plasmid clone of csrB.172ⴱ 0. eliminated (Fig. As a type I pili.21 ⫾ 0. and/or type I pili are shown in Fig. Effects of csrA on 24-h biofilms grown in microtiter wells.067 ⫾ 0. Nevertheless. 184. Biofilm formation by all of the isogenic csrA mutants was substantially greater than that of the wild-type parent.003ⴱ 0. Growth curves con- FIG. and rpoS only modestly decreased biofilm formation result. Interestingly. a disruption in motility enterica serovar Typhimurium csrA) (bar 5). Each result represents the mean values ⫾ standard errors of three independent experiments. the loss of curli fimbriae in the csrA mutant lacking colanic acid and type I pili reproducibly resulted in increased biofilm (Fig. enterica serovar Typhimurium and exhibited no significant effect in the csrA mutant (Fig. In the csrA motB mutant there was no additional effect of a type I pilus deletion.50 ⫾ 0. We observed that an rpoS disruption had murium ATCC 14028. RG1-B. coli. most significantly in the clinical isolates that type I pili and rpoS eliminated biofilm formation in the csrA apparently utilize biofilm formation as a virulence factor. coli P18 (bars 1 to 3) and C. MG1655. relative to a plasmid vector control (Table 2). coli. the loss of both tives of E. each conducted with triplicate Downloaded from http://jb. Effects of csrB on biofilm formationa Strains Results with plasmid (csrB genotype) None pCSRB-SF pUC19 MG1655 (csrB⫹) 0.011 RG1-B (csrB::cam) 0. or pUC19 (bars 3 and 6). apparently due to its mal effects in the csrA mutant. As predicted. its effects were examined in strains lacking one or more of the extracellular and/or surface factors that have been reported to participate in biofilm formation. which renders cells nonmotile. In the csrA wild-type strains. and S. To address the mechanism by which CsrA represses biofilm formation in E. In a csrA mu- Each bar shows the averages and standard errors of three separate experiments. 2014 by NATIONAL CHUNG HSING LIBRARY samples. Furthermore. coli O157:H7 strain EF302. Even the loss of curli fimbriae. overexpression of csrA was also inhibitory of these four previously reported extracellular and/or surface to biofilm formation. Note only a modest effect on biofilm formation in the parental strain that in the latter experiment.

nitrogen (36.g. Furthermore. the disruption of ompR and type I under nutrient limitation. This result again suggests that under certain situations. The csrA gene was first recognized as a olism might also be required for biofilm formation and the fact repressor of glycogen biosynthesis (40). and/or type I pili (D).296 JACKSON ET AL. lane 5). genes glgC (ADP-glucose synthetase) and glgA (glycogen syn- OmpR did have significant effects on biofilm formation in the thase) as well as the gene encoding the catabolic enzyme gly- parent strain but only modestly affected the csrA mutant (Fig. curli fimbriae. This occurs because CsrA posttranscriptionally and glgP resulted in even more biofilm production than that in represses the glgCAP operon. curli fimbriae. type I pili and/or motility (B). coli strain MG1655 and its isogenic csrA mutant. lane 3). MG1655. the glgA-carrying plasmid. Interestingly. 5A. csrA wild-type and mutant strains to become severely defective 4A). Each bar shows the averages and standard errors of three separate experiments (P ⬍ 0. 5A. for biofilm formation (Fig. Subsequent studies that the glgA insertion had a polar effect on glgP expression revealed that CsrA coordinately regulates glycogen biosynthe. 5A. or OmpR. which includes the biosynthetic the prototrophic parent (Fig. The possibility that glycogen catab- thesis and catabolism.asm. lane 4). and/or type I pili (A). we tested fimbriae almost eliminated biofilm formation in the csrA mu. Crystal violet staining of 24-h biofilms formed in microtiter wells by mutants disrupted in curli fimbriae. In contrast. 4. 42). com- titatively more significant than those of OmpR or RpoS under plemented the glycogen biosynthesis defect of this strain (data these in vitro growth conditions. the role of intra- 4D). mation (Fig. 4D). and/or type I pili (C). 5A. Weak accumulation by MG1655 was observed creased biofilm in the latter strain (Fig. While complementation by glgP alone catabolism. However. Therefore. MG1655 and its glgA mutant for glycogen accumulation on tant.0001). lanes 1 and 2 and lanes 6 and curli fimbriae may interfere with the function of some other 7). A glgA transposon insertion caused both in the type I pili and colanic acid-deficient csrA mutant (Fig. RpoS. biofilm formation did not differ quantitatively factor(s) involved in biofilm formation. colanic acid. CFA medium. pOP245 failed to restore biofilm for- Effects of CsrA are largely mediated through glycogen syn. ruption. both glgA mutant (50). the disruption of both ompR and curli fimbriae cellular glycogen in biofilm formation was examined. 4D). Likewise. alfold greater than that in the prototrophic parent. 2014 by NATIONAL CHUNG HSING LIBRARY FIG. Effects of extracellular and/or surface factors and global regulators on biofilm formation in E. We conclude that the for csrA wild-type or mutant strains containing the glgA dis- effects of CsrA are mediated independently of and are quan. J. BACTERIOL. Downloaded from http://jb. similar to its effect (data not shown). cogen phosphorylase (glgP) (50). were examined by complementation experiments with the glgP- sis in the early stationary phase of growth and its subsequent carrying plasmid pJF02. both processes are highly accelerated in a csrA did not restore biofilm formation (Fig. not shown). Because caused only an ⬃50% decrease in biofilm formation in the csrA glycogen synthesis is favored by the presence of excess carbon mutant (Fig. e. indicating that gly- .org/ on April 11.. the further disruption of curli fimbriae in. As expected. pOP245.

respectively. the relative significance of each induction were serially diluted and plated onto Kornberg me- of these metabolic pathways was examined.2% of three separate experiments. 2002 REGULATION OF BIOFILM FORMATION AND DISPERSAL 297 IPTG to the growth medium (data not shown). TR1-5JM101[pCSRH6-19]. Control experiments showed that IPTG did not affect glgA. Using the conditions given in the thetic genes as well as the gene encoding the catabolic enzyme legend to Fig. were compared. that glycogen accumulation is inhibited by the addition of In the biofilm a sharp decline in activity was observed during . This strain was allowed to grow for 24 h in CFA medium in borosilicate tubes in the absence of IPTG. pOP12. Each bar shows the averages and standard errors signal for biofilm dispersal in E. coli biofilm formation. was conducted in M63 salts. and the csrA glgA sence of induction (data not shown). 2014 by NATIONAL CHUNG HSING LIBRARY spent medium might have affected the results. induction releases viable planktonic cells from the biofilm we propose that intracellular glycogen. glucose and ampicillin with or without IPTG were added di- ences with respect to the parent strain (P ⬍ 0. when 0. 6A thetic or catabolic genes. The above olized. cells released from the biofilm following glycogen phosphorylase (50). pJF02. Microscopic examination of cells released from the biofilms revealed that a small proportion of cells had become motile in the fresh CFA medium but that cells in spent FIG. coli. To examine the potential role of CsrA in in planktonic and sessile cells. 6B). the preformed biofilm increased substantially in both the induced and uninduced cultures (Fig. 6C). the early stationary phase of growth and subsequently metab. Next a 24-h biofilm was rinsed and the spent medium was replaced with M63 salts solution containing am- picillin with or without IPTG. metabolite accumulation. pression is modulated during biofilm development. Based on these findings. con- double mutant. 184. csrA induction caused the biofilm to disperse (Fig. 5. or pOP12 carry glgA. or other conditions in the Downloaded from http://jb. or asd-glgBXCAP⬘ (deleted for most of glgP). little or no cell division should thetic genes or glgP in a csrA wild-type strain significantly have occurred during the experiment.0001). rectly to the spent medium. Plasmids pOP245.org/ on April 11. dium (Fig. the csrA mutant. conducted to monitor the planktonic cells that were released in Because a csrA mutant overexpresses the glycogen biosyn. 6C. Plating efficiency at 4 to enhanced biofilm formation. effect of CsrA on biofilm dispersal. Figure 5B demon. a chromo- Biofilm dispersal. suggesting that this nutrient can override the regulatory cogen biosynthesis and its subsequent turnover are both re. biofilm formation or dispersal in strains lacking the inducible respectively. (A) Effects of a polar glgA mutation. pBR322 (vector control). csrA. or both plasmids. 6D). ately prior to the appearance of the biofilm and for the fol- ible phenotype of this strain was confirmed by the observation lowing few hours. experiments revealed that CsrA plays an important regulatory tion of one or more adhesins or other factors in the stationary role in E. that of the control. indicated that csrA expression can serve as a general control). ␤-Galactosidase activity in planktonic cells declined immedi- lactopyranoside).asm. To assess whether csrA ex- phase of growth which are required for biofilm formation. These experiments. defective for shown). serves as a carbon and/or energy source for the forma. Again. This medium lacked a carbon and/or energy source for growth. The results depict data from biofilm dispersal.VOL. pression could be induced with IPTG (isopropyl-␤-D-thioga. 6A). along with ampicillin to maintain plasmid selection. glgA mutant. Effects of glycogen synthesis and catabolism on biofilm medium or M63 salts were uniformly nonmotile (data not formation at 24 h. respectively. Then IPTG (1 mM final concentra- tion) or sterile water was added directly to the culture medium. whereafter it remained relatively constant. ducted under three different physiological conditions (Fig. pJF02. each assayed in duplicate reactions (Fig. The csrA-induc. in relatively good agreement with data from sequent catabolism improves biofilm formation and that both the crystal violet assay. Isogenic derivatives of strain MG1655. the growth curves of for bars 1 to 7 were MG1655. 6E). and bio- film was monitored in quadruplicate cultures by the crystal violet assay. triplicate cultures. a strain was constructed in which csrA ex. The asterisks denote significant differ. Furthermore. glgA mutant containing the csrA-inducible strain were similar in the presence or ab- pOP245. This experiment established that csrA of these effects of CsrA are relevant. the dispersal experiment was repeated by removing the spent medium from a 24-h biofilm and gently rinsing it with and incubating it further in fresh sterile CFA medium containing ampicillin with or without IPTG. 7). or pUC19 (vector to C). glgP. Lanes 1 to 5 show MG1655 containing either no plasmid. which is synthesized in rather than dispersing the biofilm by causing cell death or lysis. or both. (B) Overexpression of glycogen biosyn. Because nutrient limitation. In this experiment csrA in- duction also caused biofilm to disperse over the following few hours (Fig. Induction of csrA resulted in the release of the biofilm over the following 4 to 6 h (Fig. Because the induction step of this experiment strates that ectopic expression of either the glycogen biosyn. Biofilm can serve as a nidus of infection somal csrA⬘-⬘lacZ translational fusion was constructed and from which planktonic cells can spread or disperse to other ␤-galactosidase activity encoded by this fusion was monitored sites in the body (14). A final experiment was quired for optimal biofilm formation. Conversely. The strain identities csrA gene (data not shown). CsrA expression during biofilm development. This experiment provided genetic 6 h was ⬃1 log10 greater from the IPTG-induced culture versus evidence that an increase in glycogen biosynthesis or its sub. pJF02. response to csrA induction.

coli increase to prebiofilm levels. the studies of its regulation have begun to reveal a variety of experiments with the csrA knockout mutants of E. Nevertheless. 27). the medium was discarded and fresh CFA containing ampicillin with or without IPTG was added (B). and planktonic cells that were released were recovered without disturbing the biofilm. the medium was replaced with a M63 salts solution containing ampicillin with or without IPTG (C). and after Ectopic expression studies provided evidence that CsrA also 1. Interestingly. ceased ␤-galactosidase activity increased moderately. This tatively greater than those of either of the regulatory factors finding allows that CsrA may regulate. was allowed to form a 24-h biofilm. overexpres- Biofilm formation is a complex developmental process. RpoS. csrA expression is dynam. K-12. the first few hours of growth such that activity was ultimately findings. biofilm formation is but one of the many clude a role for CsrA in the production of these same struc- physiological properties that are regulated by CsrA or its ho. which is essential for motility under various growth also see references 4. Dispersal of bacteria from preformed biofilm by csrA induction. 8. (E) The medium at 24 h was replaced with M63 salts solution containing ampicillin with or without IPTG (as for panel C). asterisks denote significant differences between the induced and uninduced cultures (P ⬍ 0. Thus. and were plated onto Kornberg medium. On the contrary. A strain containing an IPTG-inducible csrA gene. or OmpR the production of another factor(s). The present investigation demonstrates that the clearly establish a role for CsrA in biofilm formation in this RNA binding protein CsrA has a dramatic effect on E. CsrA stabilizes the flhDC mologs in gram-negative bacteria (reviewed in reference 38.5 to 2 days of incubation it was consistently observed to represses biofilm formation in pathogenic relatives of E. freundii ically regulated during the course of biofilm development. DISCUSSION we acknowledge that in the absence of other data. coli. Each value represents an average of at least two independent experiments. into a complex multicellular structure. notably in uropathogenic strains of E. coli and C. and asterisks denote significant differences between induced and uninduced cultures (P ⬍ 0. BACTERIOL. factors that are known to participate in this process.asm. Thus. directly or indirectly.298 JACKSON ET AL. Downloaded from http://jb. J. Each of the data points depicts the averages and standard errors of the two experiments. or 0. TRJM101[pCSRH6- 19]. CsrB was found to activate biofilm formation.org/ on April 11.0001). each including 3 countable plates (⬎30 but ⬍300 colonies) for each time point. coli K-12 influences. Biofilm remaining at the indicated times following csrA induction is shown. However. the absence of one or more of the extracellular or surface ported thus far. whereafter IPTG (1 mM final concentration) or sterile water (containing ampicillin in each case) was added directly to the medium (A). tural elements. As predicted by previous conditions (48). coli nonpathogenic bacterium and increase our confidence in the biofilm formation under a variety of growth conditions. To our knowledge CsrA Biofilm formation was stimulated by csrA disruption even in is the only repressor of biofilm formation to have been re. with quadruplicate samples. that have been previously studied in E. Plating efficiency (CFU/milliliter) was determined from two separate experiments. 6. a csrA mutant is motile in CFA . However. that were isolated from colonized urinary catheters.2% glucose and ampicillin with or without IPTG was added directly to the spent media (D). A csrA results of csrA overexpression analyses in the related patho- mutant of E. coli K-12 produces a biofilm that differentiates gens. 46). It is con- ceivable that elevated intracellular levels of CsrA could permit this protein to interact with nontarget RNA molecules. As biofilm growth activity (22. CsrA effects on biofilm formation are quanti.0001). and sion studies should be interpreted with caution. and 16). 2014 by NATIONAL CHUNG HSING LIBRARY FIG. message. which revealed that CsrB RNA antagonizes CsrA lower than that in the unattached cells. were serially diluted. it does not ex- (1.

It was interesting to established the requirement for glycogen phosphorylase (GlgP). CsrA also influences the expres.org/ on April 11. This would help to determine whether the redirection of central carbon flux. Closed squares and open tri. Average values (⫾ standard errors). Thus. indicating levels may provide a switch to direct biofilm formation and/or that the loss of motility does not account for the effect of dispersal. CsrA may affect a variety of properties that influence distinct sets of genes. the possibility that it provides an endogenous carbon and energy source for the synthesis of the extracellular glucans. as determined from three cultures assayed with duplicate samples. since the synthesis and the degradation or inactivation of a sion of genes involved in the synthesis of both curli and type I structure(s) that stabilize the biofilm should be accomplished pili under certain conditions (manuscript in preparation). Thus. aeruginosa biofilm formation rests Downloaded from http://jb. Previous studies have established that CsrA tional cues may play a critical role in biofilm dispersal. This has been assumed to result from the prolonged secretion of organic acids generated from the metabolism of this polymer. Because both synthe. Expression of a csrA⬘-⬘lacZ translational fusion in biofilm considered. Based on our results. Interestingly. should now be FIG. thereby offering potential therapeutic applications. Thus. by distinct mechanisms and thus require the expression of Thus. Perhaps the requirement for Crc in P. suggests that the intracellular availability of the CsrA protein thesis of one or more essential adhesins or other factors. A CsrA mimic essentially identical amounts of biofilm being formed in csrA should not be expected to affect the metabolism of eucaryotic wild-type and mutant strains. the participation of additional regula- tors of carbon metabolism in biofilm formation should be ex- amined in other eubacteria. not escaped our attention that the ability to induce biofilm dispersal by csrA induction offers a useful approach for inves- tigating the genetic and biochemical mechanisms of dispersal. Perhaps IPTG induction of csrA expression (Fig. . they did not provide insight into the means by phosphorylase. blocked biofilm dispersal by CsrA. The mechanism by which glycogen affects bio. reported that glycogen levels are positively cess that signals the release of cells from a biofilm. Because CsrA affects both of these pro- biofilm formation. Gly. 2014 by NATIONAL CHUNG HSING LIBRARY not only on its role in activating type IV pilus formation (33) but also on its influence on central carbon flux. cesses. 184. which are required for surface attachment and oral biofilm formation. We emphasize that there was no reason to suppose a priori due to the relatively high level of csrA-independent expression that CsrA should affect both biofilm formation and dispersal. The csrA mutant.VOL. of flhDC in this medium (48). and planktonic cells of strain KSA712. The induction of csrA within a preformed biofilm caused its angles represent determinations conducted on biofilm and planktonic dispersal under a variety of conditions. in order to attenuate bacterial virulence mechanisms has been ies.5 to 2 days of growth. To our knowledge this cells. The finding film formation remains to be elucidated. the glycogen-like intracellular polymer of Streptococcus mutans has long been recognized as a virulence factor for the forma- tion of dental caries (21). respectively. medium. a primary role of CsrA in biofilm formation is to Although csrA disruption and overexpression studies dem- influence the flux of carbon into glycogen and the subsequent onstrated that CsrA has a powerful influence on biofilm de- conversion of glycogen into glucose-1-phosphate by glycogen velopment. we propose that glycogen serves dynamically regulated during the course of biofilm formation as a carbon and energy source for the stationary-phase biosyn. which the cell modulates this potential of CsrA. itself may be a key feature of these processes. it is likely that intracellular glycogen elevation in csrA⬘-⬘lacZ expression at ⬃1. coli is perse antibiotic-sensitive planktonic cells from preexisting bio- through its regulatory role in the metabolism of glycogen. Error is the first such demonstration of extensive biofilm dispersal in bars are not visible where the standard error was less than the area response to a regulatory gene or signal in any species. We did not correlated with biofilm formation in Salmonella enterica sero. which was primarily utilized in the present studies. Complementation studies further hosts. This indicates that nutri- film formation. this was the only mutation that resulted in considered previously (reviewed in reference 6). In a film. 7. However. as they coordinately regulates both glycogen synthesis and catabolism do in biofilm formation (9. are shown. 35). (50). 6) glycogen on biofilm formation. It has occupied by a given symbol. a glgA (glycogen synthase) disruption was the concept of identifying compounds that modify gene expression most drastic down mutation that was examined in these stud. represents a general principle of bacterial biofilm formation. 2002 REGULATION OF BIOFILM FORMATION AND DISPERSAL 299 plays an important role in biofilm formation in the Enterobac- teriaceae. through glyco- gen or perhaps other energy storage compounds. find that the addition of glucose during induction apparently indicating that glycogen must be catabolized to promote bio. In addition.asm. CsrA cogen mutants are fully motile (data not shown). compounds that mimic CsrA activity or increase csrA This study provides compelling genetic evidence that the expression might both inhibit biofilm formation and also dis- primary effect of CsrA on biofilm formation in E. which lack apparent csrA homologs. that the expression of a csrA⬘-⬘lacZ translational fusion was sis and turnover are required. observe a statistically significant release of biofilm during the var Enteritidis (10). During the course of this study represents an extreme example of a typically more subtle pro- Bonafonte et al.

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