Biofilms, the preferred bacterial mode of living and survival, are employed
by most microorganisms—which tend to attach to surfaces—to gain physical support,
increase nutrient utilization and availability, and augment their resistance against
anti-bacterial agents. Rhodococcus ruber (C208) has been shown to form a
dense biofilm on polyethylene surfaces while degrading them. Bacterial biofilms
comprise bacterial cells embedded in self-secreted extracellular polymeric substances
(EPS) whose main components are polysaccharides, proteins and nucleic acids. Revealing
the roles of these components will enable further insight into biofilm development
and, therefore, the EPS structure-function relationship. The current study focuses
on contribution of extracellular DNA to biofilm formation and stability. This was
approached by investigating the influence of nucleases on biofilm formation via
degradation of their corresponding substrates within the biofilm of C208. RNase
application to cultures of C208 decreased biofilm formation. Degradation of biofilm
DNA by DNase reduced early-stage biofilm formation by 20% -25% but
had no significant effect on established, mature biofilm. Likewise, the addition
of DNA to cultures significantly enhanced early-stage biofilm formation by 50% -100%. RAPD-PCR
analysis revealed different band patterns from intra-cellular DNA and extra-cellular
DNA and also between the supernatant and biofilm fractions of extra-cellular DNA,
indicating that perhaps only certain DNA molecules are utilized as part of the biofilm.
References
[1]
A. Sivan, “New Perspectives in Plastic Biodegradation,” Current Opinion in Biotechnology, Vol. 22, No. 3, 2011, pp. 422-426.
[2]
R. Kolter and P. E. Greenberg, “Microbial Sciences: The Superficial Life of Microbes,” Nature, Vol. 441, No. 7091, 2006, pp. 300-302. http://dx.doi.org/10.1038/441300a
[3]
J. W. Costerton and P. S. Stewart, E. P. Greenberg, “Bacterial Biofilms: A Common Cause of Persistent Infections,” Science, Vol. 284, No. 5418, 1999, pp. 1318-1322. http://dx.doi.org/10.1126/science.284.5418.1318
[4]
T. C. Mah and G. A. O’Toole, “Mechanisms of Biofilm Resistance to Antimicrobial Agents,” Trends in Microbiology, Vol. 9, No. 1, 2001, pp. 34-39.
[5]
L. Hall-Stoodley and P. Stoodley, “Evolving Concepts in Biofilm Infections,” Cellular Microbiology, Vol. 11, No. 7, 2009, pp. 1034-1043. http://dx.doi.org/10.1111/j.1462-5822.2009.01323.x
[6]
S. Kjelleberg and S. Molin, “Is There a Role for Quorum Sensing Signals in Bacterial Biofilms?” Current Opinion in Microbiology, Vol. 5, No. 3, 2002, pp. 254-258.
[7]
M. R. Parsek and E. P. Greenberg, “Acyl-Homoserine Lactone Quorum Sensing in Gram-Negative Bacteria: A Signaling Mechanism Involved in Associations with Higher Organisms,” Proceedings of the National Academy of Sciences, Vol. 97, No. 16, 2000, pp. 8789-8793. http://dx.doi.org/10.1073/pnas.97.16.8789
[8]
N. A, Whitehead, A. M. L. Barnard, H. Slater, N. J. L. Simpson and G. P. C. Salmond, “Quorum-Sensing in Gram-Negative Bacteria,” FEMS Microbiology Reviews, Vol. 25, No. 4, 2001, pp. 365-404. http://dx.doi.org/10.1111/j.1574-6976.2001.tb00583.x
[9]
H. Withers, S. Swift and P. Williams, “Quorum Sensing as an Integral Component of Gene Regulatory Networks in Gram-Negative Bacteria,” Current Opinion in Microbiology, Vol. 4, No. 2, 2001, pp. 186-193.
[10]
B. A. Annous, P. M. Fratamico and J. L. Smith, “Quorum Sensing in Biofilms: Why Bacteria Behave the Way They Do,” Journal of Food Science, Vol. 74, No. 1, 2009, pp. R24-R37. http://dx.doi.org/10.1111/j.1750-3841.2008.01022.x
[11]
G. M. Dunny and B. A. B. Leonard, “Cell-Cell Communication in Gram-Positive Bacteria,” Annual Review of Microbiology, Vol. 51, No. 1, 1997, pp. 527-564. http://dx.doi.org/10.1146/annurev.micro.51.1.527
[12]
M. B. Miller and B. L. Bassler, “Quorum Sensing in Bacteria,” Annual Review of Microbiology, Vol. 55, No. 1, 2001, pp. 165-199. http://dx.doi.org/10.1146/annurev.micro.55.1.165
[13]
A. Sivan, M. Szanto and V. Pavlov, “Biofilm Development of the Polyethylene-Degrading Bacterium Rhodococcus Ruber,” Applied Microbiology and Biotechnology, Vol. 72, No. 2, 2006, pp. 346-352. http://dx.doi.org/10.1007/s00253-005-0259-4
[14]
V. V. Sakharovskii, D. I. Nikitin and V. G. Sakharovskii, “Physiological Characterization of the Survival of Some Gram-Negative Bacteria under Conditions of Carbon Deficiency,” Applied Biochemistry and Microbiology, Vol. 35, No. 4, 1999, p. 380.
[15]
S. L. Sanin, F. D. Sanin and J. D. Bryers, “Effect of Starvation on the Adhesive Properties of Xenobiotic Degrading Bacteria,” Process Biochemistry, Vol. 38, No. 6, 2003, pp. 909-914.
[16]
I. G. Orr, Y. Hadar and A. Sivan, “Colonization, Biofilm Formation and Biodegradation of Polyethylene by a Strain of Rhodococcus ruber,” Applied Microbiology and Biotechnology, Vol. 65, No. 1, 2004, pp. 97-104.
[17]
H. Flemming, T. R. Neu and D. J. Wozniak, “The EPS matrix: The ‘House of Biofilm Cells’,” Journal of Bacteriology, Vol. 189, No. 22, 2007, pp. 7945-7947. http://dx.doi.org/10.1128/JB.00858-07
[18]
S. Molin and T. Tolker-Nielsen, “Gene Transfer Occurs with Enhanced Efficiency in Biofilms and Induces Enhanced Stabilisation of the Biofilm Structure,” Current Opinion in Biotechnology, Vol. 14, No. 3, 2003, pp. 255-261.
[19]
I. W. Sutherland, “The Biofilm Matrix—An Immobilized but Dynamic Microbial Environment,” Trends in Microbiology, Vol. 9, No. 5, 2001, pp. 222-227.
[20]
R. M. Donlan, “Biofilms: Microbial Life on Surfaces,” Emerging Infectious Diseases journal, Vol. 8, No. 9, 2002, pp. 881-890. http://dx.doi.org/10.3201/eid0809.020063
[21]
B. R. Boles and A. R. Horswill, “Agr-Mediated Dispersal of Staphylococcus Aureus Biofilms,” PLOS Pathogens, Vol. 4, No. 4, 2008, Article ID: E1000052. http://dx.doi.org/10.1371/journal.ppat.1000052
[22]
P. Chaignon, I. Sadovskaya, C. Ragunah, N. Ramasubbu, J. Kaplan and S. Jabbouri, “Susceptibility of Staphylococcal Biofilms to Enzymatic Treatments Depends on Their Chemical Composition,” Applied Microbiology and Biotechnology, Vol. 75, No. 1, 2007, pp. 125-132. http://dx.doi.org/10.1007/s00253-006-0790-y
[23]
Z. Qin, Y. Ou, L. Yang, Y. Zhu, T. Tolker-Nielsen, S. Molin, et al., “Role of Autolysin-Mediated DNA Release in Biofilm Formation of Staphylococcus Epidermidis,” Microbiology, Vol. 153, No. 7, 2007, pp. 2083-2092. http://dx.doi.org/10.1099/mic.0.2007/006031-0
[24]
C. B. Whitchurch, T. Tolker-Nielsen, P. C. Ragas and J. S. Mattick, “Extracellular DNA Required for Bacterial Biofilm Formation,” Science, Vol. 295, No. 5559, 2002, p. 1487.http://dx.doi.org/10.1126/science.295.5559.1487
[25]
S. Vilain, J. M. Pretorius, J. Theron and V. S. Brözel, “DNA as an Adhesin: Bacillus Cereus Requires Extracellular DNA to Form Biofilms,” Applied and Environmental Microbiology, Vol. 75, No. 9, 2009, pp. 2861-2868.http://dx.doi.org/10.1128/AEM.01317-08
[26]
H. Mulcahy, L. Charron-Mazenod and S. Lewenza, “Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas Aeruginosa Biofilms,” PLoS Pathogens, Vol. 4, No. 11, 2008, Article ID: E1000213. http://dx.doi.org/10.1371/journal.ppat.1000213
[27]
P. S. Guiton, C. S. Hung, K. A. Kline, R. Roth, A. L. Kau, E. Hayes, et al., “Contribution of Autolysin and Sortase A during Enterococcus Faecalis DNA-Dependent Biofilm Development,” Infection and Immunity, Vol. 77, No. 9, 2009, pp. 3626-3638. http://dx.doi.org/10.1128/IAI.00219-09
[28]
E. S. Gloag, L. Turnbull, A. Huang, P. Vallotton, H. Wang, L. M. Nolan, et al., “Self-Organization of Bacterial Biofilms Is Facilitated by Extracellular DNA,” Proceedings of the National Academy of Sciences, Vol. 110, No. 28, 2013, pp. 11541-11546 http://dx.doi.org/10.1073/pnas.1218898110
[29]
V. C. Thomas, L. R. Thurlow, D. Boyle and L. E. Hancock, “Regulation of Autolysis-Dependent Extracellular DNA Release by Enterococcus Faecalis Extracellular Proteases Influences Biofilm Development,” Journal of Bacteriology, Vol. 190, No. 16, 2008, pp. 5690-5698. http://dx.doi.org/10.1128/JB.00314-08
[30]
V. C. Thomas, Y. Hiromasa, N. Harms, L. Thurlow, J. Tomich and L. E. Hancock, “A Fratricidal Mechanism Is Responsible for eDNA Release and Contributes to Biofilm Development of Enterococcus Faecalis,” Molecular Microbiology, Vol. 72, No. 4, 2009, pp. 1022-12036. http://dx.doi.org/10.1111/j.1365-2958.2009.06703.x
[31]
G. G. Rodriguez, D. Phipps, K. Ishiguro and H. F. Ridgway, “Use of a Fluorescent Redox Probe for Direct Visualization of Actively Respiring Bacteria,” Applied and Environmental Microbiology, Vol. 58, No. 6, 1992, pp. 1801-1808.
[32]
S. Yaqub, J. G. Anderson, S. J. MacGregor and N. J. Rowan, “Use of a Fluorescent Viability Stain to Assess Lethal and Sublethal Injury in Food-Borne Bacteria Exposed to High-Intensity Pulsed Electric Fields,” Letters in Applied Microbiology, Vol. 39, No. 6, 2004, pp. 246-251. http://dx.doi.org/10.1111/j.1472-765X.2004.01571.x
[33]
G. A. O’Toole and R. Kolter, “Initiation of Biofilm Formation in Pseudomonas Fluorescens WCS365 Proceeds via Multiple, Convergent Signalling Pathways: A Genetic Analysis,” Molecular Microbiology, Vol. 28, No. 3, 1998, pp. 449-461. http://dx.doi.org/10.1046/j.1365-2958.1998.00797.x
[34]
M. Carrolo, M. Jo?o Frias, F. R. Pinto, J. Melo-Cristino and M. Ramirez, “Prophage Spontaneous Activation Promotes DNA Release Enhancing Biofilm Formation in Streptococcus Pneumoniae,” PLoS One, Vol. 5, No. 12, 2010, Article ID: E15678. http://dx.doi.org/10.1371/journal.pone.0015678
[35]
E. Mahenthiralingam, M. Campbell, J. Foster, J. Lam and D. Speert, “Random Amplified Polymorphic DNA Typing of Pseudomonas Aeruginosa Isolates Recovered from Patients with Cystic Fibrosis,” Journal of Clinical Microbiology, Vol. 34, No. 5, 1996, pp. 1129-1135.
[36]
U. B?ckelmann, A. Janke, R. Kuhn, T. R. Neu, J. Wecke, J. R. Lawrence and U. Szewzyk, “Bacterial Extracellular DNA Forming a Defined Network-Like Structure,” FEMS Microbiology Letters, Vol. 262, No. 1, 2006, pp. 31-38. http://dx.doi.org/10.1111/j.1574-6968.2006.00361.x
[37]
M. Allesen-Holm, K. B. Barken, L. Yang, M. Klausen, J. S. Webb, S. Kjelleberg, S. Molin, M, Givskov and T. Tolker-Nielsen, “A Characterization of DNA Release in Pseudomonas Aeruginosa Cultures and Biofilms,” Molecular Microbiology, Vol. 59, No. 4, 2006, pp. 1114-1128. http://dx.doi.org/10.1111/j.1365-2958.2005.05008.x
[38]
M. Martí, M. P. Trotonda, M. á. Tormo-Más, M. Vergara-Irigaray, A. L. Cheung, I. Lasa and J. R. Penadés, “Extracellular Proteases Inhibit Protein-Dependent Biofilm Formation in Staphylococcus Aureus,” Microbes and Infection, Vol. 12, No. 1, 2010, pp. 55-64.
[39]
C. T. Steichen, C. Cho, J. Q. Shao and M. A. Apicella, “The Neisseria gonorrhoeae Biofilm Matrix Contains DNA, and an Endogenous Nuclease Controls Its Incorporation,” Infection and Immunity, Vol. 79, No. 4, 2011, pp. 1504-1511. http://dx.doi.org/10.1128/IAI.01162-10
[40]
R. Nakao, M. Ramstedt, S. N. Wai and B. E. Uhlin, “Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis,” PLoS ONE, Vol. 7, No. 12, 2012, Article ID: e51241. http://dx.doi.org/10.1371/journal.pone.0051241
[41]
F. C. Petersen, L. Tao and A. A. Scheie. “DNA BindingUptake System: A Link between Cell-to-Cell Communication and Biofilm Formation,” Journal of Bacteriology, Vol. 187, No. 13, 2005, pp. 4392-400. http://dx.doi.org/10.1128/JB.187.13.4392-4400.2005
[42]
K. Czaczyk and K. Myszka, “Biosynthesis of Extracellular Polymeric Substances (EPS) and Its Role in Microbial Biofilm Formation,” Polish Journal of Environmental Studies, Vol. 16, No. 6, 2007, pp. 799-806.
[43]
S. Nishimura, T. Tanaka, K. Fujita, M. Itaya, A. Hiraishi and Y. Kikuchi, “Extracellular DNA and RNA Produced by a Marine Photosynthetic Bacterium Rhodovulum Sulfidophilum,” Nucleic Acids Symposium Series, Vol. 3, No. 1, 2003, pp. 279-280. http://dx.doi.org/10.1093/nass/3.1.279
[44]
E. A. Izano, M. A. Amarante, W. B. Kher and J. B. Kaplan, “Differential Roles of Poly-N-Acetylglucosamine Surface Polysaccharide and Extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis Biofilms,” Applied and Environmental Microbiology, Vol. 74, No. 2, 2008, pp. 470-476. http://dx.doi.org/10.1128/AEM.02073-07
[45]
M. Harmsen, M. Lappann, S. Kn?chel and S. Molin, “Role of Extracellular DNA during Biofilm Formation by Listeria monocytogenes,” Applied and Environmental Microbiology, Vol. 76, No. 7, 2010, pp. 2271-2279. http://dx.doi.org/10.1128/AEM.02361-09
[46]
K. C. Rice, E. E. Mann, J. L. Endres, E. C. Weiss, J. E. Cassat, M. S. Smeltzer and K. W. Bayles, “The Cida Murein Hydrolase Regulator Contributes to DNA Release and Biofilm Development in Staphylococcus aureus,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, No. 19, 2007, pp. 8113-8118. http://dx.doi.org/10.1073/pnas.0610226104
[47]
W. Vollmer and U. Bertsche, “Murein (Peptidoglycan) Structure, Architecture and Biosynthesis in Escherichia coli,” Biochimica et Biophysica Acta (BBA)-Biomembranes, Vol. 1778, No. 9, 2008, pp. 1714-1734.
[48]
C. Heilmann, M. Hussain, G. Peters and F. G?tz, “Evidence for Autolysin-Mediated Primary Attachment of Staphylococcus epidermidis to a Polystyrene Surface,” Molecular Microbiology, Vol. 24, No. 5, 1997, pp. 1013-1024. http://dx.doi.org/10.1046/j.1365-2958.1997.4101774.x
[49]
C. Heilmann, G. Thumm, G. S. Chhatwal, J. Hartleib, A. Uek?tter and G. Peters, “Identification and Characterization of a Novel Autolysin (Aae) with Adhesive Properties from Staphylococcus epidermidis,” Microbiology, Vol. 149, No. 10, 2003, pp. 2769-2778. http://dx.doi.org/10.1099/mic.0.26527-0
[50]
R. E. Steinberger and P. A. Holden, “Extracellular DNA in Singleand Multiple-Species Unsaturated Biofilms,” Applied and Environmental Microbiology, Vol. 71, No. 9, 2005, pp. 5404-5410. http://dx.doi.org/10.1128/AEM.71.9.5404-5410.2005
[51]
M. J. Larkin, L. A. Kulakov and C. C. Allen, “Biodegradation and Rhodococcus—Masters of Catabolic Versatility,” Current Opinion in Biotechnology, Vol. 16, No. 3, 2005, pp. 282-290. http://dx.doi.org/10.1016/j.copbio.2005.04.007
[52]
S. Shimizu, H. Kobayashi, E. Masai and M. Fukuda, “Characterization of the 450-kb Linear Plasmid in a Polychlorinated Biphenyl Degrader, Rhodococcus sp. Strain RHA1,” Applied and Environmental Microbiology, Vol. 67, No. 5, 2001, pp. 2021-2028. http://dx.doi.org/10.1128/AEM.67.5.2021-2028.2001
[53]
J. de Vries and W. Wackernagel, “Integration of Foreign DNA during Natural Transformation of Acinetobacter sp. by Homology-Facilitated Illegitimate Recombination,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, No. 4, 2002, pp. 2094-2099. http://dx.doi.org/10.1073/pnas.042263399
