Resident Parking Only: Rhamnolipids Maintain Fluid Channels in Biofilms
TEXTczo, http://www.100md.com
When environmental conditions are favorable, bacteria abandon their planktonic lifestyle to form surface-attached communities known as biofilms. In environments where there is a constant flow of nutrients, biofilms of the opportunistic human pathogen Pseudomonas aeruginosa show common, defined structural patterns. Cells, embedded in the extracellular matrix, form mushroom- and pillar-like colonies interspersed with fluid-filled channels (5, 17). How does such elaborate architecture develop and subsist? In this issue of the Journal of Bacteriology, M. E. Davey et al. (6) present a series of elegant experiments demonstrating how these fluid channels are kept open by the action of rhamnolipid biosurfactants. Rhamnolipids alter cell-cell and cell-surface interactions. In this way the biofilm architecture is maintained, ensuring the flow of nutrients and oxygen into the community and the efflux of waste products. However, rhamnolipid production, which is controlled at the population level, not only guarantees the structure, and therefore the nutritional balance of the biofilm, but these surfactants also prevent outside invading bacteria from colonizing the open spaces in the resident biofilm. Thus, we may be looking both at a finely tuned self-control system for biofilm structure and a mechanism for the sessile community to preserve its territory.
Rhamnolipids are tensoactive glycolipids, secreted by P. aeruginosa, that contain one or two rhamnose molecules. They have been primarily studied in terms of their effect on biodegradation of organic compounds (12), but they are also considered virulence factors due to their deleterious effects on eukaryotic cells and their biocidal properties (2, 13). They have also been shown to alter cell surface properties (1). Synthesis of rhamnolipids occurs in liquid cultures in stationary phase and under certain environmental conditions (12, 15); it is partially under the control of RpoS (15) and is regulated via quorum sensing by the RhlI-RhlR and the LasI-LasR systems (12, 16). All of these data are pieces of a puzzle now put together, from the perspective of biofilm integrity, by Davey et al. (6).ml6\m, 百拇医药
Pseudomonas aeruginosa can efficiently colonize the surface of medical implants, catheters, and also live tissue and thus be responsible for a number of serious nosocomial infections (3). The fact that bacteria often live as communities associated with solid surfaces rather than as planktonic individuals is not a recent discovery (14). However, it has been only in the past few years that research on the biology of microbial biofilms has geared from a descriptive discipline toward the study of the molecular mechanisms underlying this form of bacterial life (18, 19, 20, 22). Due to its implications in health-related issues, surface colonization by P. aeruginosa is the subject of intensive research. The process can be monitored in vivo by nondisruptive techniques such as confocal-laser-scanning microscopy. From these studies, and the phenotypic changes observed throughout, a general sketch of how biofilm formation proceeds in this bacterium can be drawn (5, 22). Certain environmental conditions promote attachment of the bacterial cells to the solid surface. Initial adhesion and movement of the bacteria along the plane give rise to a monolayer of cells covering the surface. This monolayer will evolve toward a mature biofilm through cell growth and aggregation, leading to the formation of microcolonies first and then of large macrocolonies embedded in an extracellular matrix composed of exopolysaccharides and other polymers. Whereas a number of genes and gene products involved in initial attachment of different bacteria to abiotic and biotic surfaces have been characterized (7-9, 18, 19), relatively less is known about the later stages leading to a mature biofilm, in terms of molecular mechanisms. One of the open questions is what determines the typical architecture of a mature biofilm, with "mushroom" and "pillar" macrocolonies separated by the so-called water channels? It has been proposed that such a structure is key to the maintenance of the biofilm, since it would allow nutrients and oxygen to flow in and waste products to flow out (17) and, therefore, its formation and stability should be regulated. On the other hand, mathematical models predict these structures would appear under certain conditions, simply as the result of chance and of the changes in the flow and distribution of nutrients and oxygen that take place during biofilm growth (11). However, some mutants of Escherichia coli and P. aeruginosa can attach to solid surfaces and form thick cell layers but are unable to form structured biofilms. That is the case of E. coli mutants deficient in the exopolysaccharide colanic acid (4). Furthermore, overexpression of algC, a gene involved in the biosynthesis of alginate, gives rise to highly structured biofilms in P. aeruginosa (10). All of this suggests that biofilm architecture is not only a consequence of chance and flow conditions but also part of a genetic program.
In this context, the data obtained by Davey et al. strongly support the idea of a genetic program determining biofilm formation, development, and stability. The significance of these results has multiple facets. They imply that biofilm architecture is kept by means of an active process, coordinately regulated at a specific time point and at a general population level. The fact that quorum sensing plays a key role in the maturation of biofilms was known (20, 21), since mutants affected in lasI, a gene involved in the synthesis of a quorum-sensing signal, form unstructured biofilms (20). However, the mechanisms through which quorum sensing exerts its role were not precisely defined.}i1, http://www.100md.com
The implications of the present study are not limited to basic biology. Based on their properties and potential production levels, rhamnolipids have been proposed as an alternative to synthetic surfactants for a wide number of industrial and biotechnological applications, from cosmetics to bioremediation (12). The results obtained by the O'Toole lab prompt investigating the use of rhamnolipids as surface-coating agents to prevent biofilms from forming on catheters and medical implants.
However, perhaps the most enticing hypothesis that could be drawn from these results is that rhamnolipid production in pure-culture biofilms does more than keep the shape, it may also be a defensive mechanism for the surface-attached community. In fact, it prevents outside bacteria from colonizing the open spaces in the biofilm and thus clogging the channels, with the subsequent detrimental effects for the established community. One can envision a system invaded by a bacterial population metabolically more efficient in that particular niche than the preestablished biofilm. Even as a minority, if the newcomers were able to attach instead of being carried away by the flow, they might end up taking over and displacing the previous population, as happens in planktonic batch cultures (see, for example, the studies by Kolter and coworkers [23, 24] on the GASP [growth advantage in stationary phase] phenotype). However, by preventing the intruder from settling down, a biofilm population would persist in its toilsomely built estate. The obvious question that remains is: how does the biofilm prevent its own surfactant-induced destruction?
ACKNOWLEDGMENTS/{;, http://www.100md.com
Work in the M.E-U. lab on bacterial attachment to biotic surfaces is supported by a grant from the Ramón y Cajal Programme and by grant BMC2001-0576 from the Spanish Ministry for Science and Technology./{;, http://www.100md.com
The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM. /{;, http://www.100md.com
REFERENCES/{;, http://www.100md.com
Al-Tahhan, R. A., T. R. Sandrin, A. A. Bodour, and R. M. Maier. 2000. Rhamnolipid-induced removal of lipopolysaccharide from Pseudomonas aeruginosa: effect on cell surface properties and interaction with hydrophobic substrates. Appl. Environ. Microbiol. 66:3262-3268./{;, http://www.100md.com
Cosson, P., L. Zulianello, O. Join-Lambert, F. Faurisson, L. Gebbie, M. Benghezal, C. Van Delden, L. K. Curty, and T. Kohler. 2002. Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system. J. Bacteriol. 184:3027-3033./{;, http://www.100md.com
Costerton, J. W. 2001. Cystic fibrosis pathogenesis and the role of biofilms in persistent infection. Trends Microbiol. 9:50-52.
Danese, P. N., L. A. Pratt, and R. Kolter. 2000. Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J. Bacteriol. 182:3593-3596.:;3f, http://www.100md.com
Davey, M. E., and G. A. O'Toole. 2000. Microbial biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64:847-867.:;3f, http://www.100md.com
Davey, M. E., N. C. Caiazza, and G. A. O'Toole. 2003. Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J. Bacteriol. 185:1027-1036.:;3f, http://www.100md.com
Dörr, J., T. Hurek, and B. Reinhold-Hurek. 1998. Type IV pili are involved in plant-microbe and fungus-microbe interactions. Mol. Microbiol. 30:7-17.:;3f, http://www.100md.com
Espinosa-Urgel, M., A. Salido, and J. L. Ramos. 2000. Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds. J. Bacteriol. 182:2363-2369.:;3f, http://www.100md.com
Girón, J. A., A. G. Torres, E. Freer, and J. B. Kaper. 2002. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol. Microbiol. 44:361-379.
Hentzer, M., G. M. Teitzel, G. J. Balzer, A. Heydorn, S. Molin, M. Givskov, and M. R. Parsek. 2001. Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. J. Bacteriol. 183:5395-5401.n, 百拇医药
Kreft, J. U., C. Picioreanu, J. W. Wimpenny, and M. C. van Loosdrecht. 2001. Individual-based modeling of biofilms. Microbiology 147:2897-2912.n, 百拇医药
Maier, R. M., and G. Soberón-Chávez. 2000. Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications. Appl. Microbiol. Biotechnol. 54:625-633.n, 百拇医药
McClure, C. D., and N. L. Schiller. 1996. Inhibition of macrophage phagocytosis by Pseudomonas aeruginosa rhamnolipids in vitro and in vivo. Curr. Microbiol. 33:109-117.n, 百拇医药
Meadows, P. S. 1971. The attachment of bacteria to solid surfaces. Arch. Mikrobiol. 75:374-381.n, 百拇医药
Medina, G., K. Juárez, and G. Soberón-Chávez. 2003. The Pseudomonas aeruginosa rhlAB operon is not expressed during the logarithmic phase of growth even in the presence of its activator RhlR and the autoinducer N-butyryl-homoserine lactone. J. Bacteriol. 185:377-380.
Ochsner, U. A., and J. Reiser. 1995. Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 92:6424-6428./, 百拇医药
O'Toole, G., H. B. Kaplan, and R. Kolter. 2000. Biofilm formation as microbial development. Annu. Rev. Microbiol. 54:49-79./, 百拇医药
O'Toole, G. A., and R. Kolter. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol. Microbiol. 28:449-461./, 百拇医药
O'Toole, G. A., and R. Kolter. 1998. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30:295-304./, 百拇医药
Parsek, M. R., and E. P. Greenberg. 2000. Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in associations with higher organisms. Proc. Natl. Acad. Sci. USA 97:8789-8793./, 百拇医药
Riedel, K., M. Hentzer, O. Geisenberger, B. Huber, A. Steidle, H. Wu, N. Hoiby, M. Givskov, S. Molin, and L. Eberl. 2001. N-acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms. Microbiology 147:3249-3262./, 百拇医药
Sauer, K., A. K. Camper, G. D. Ehrlich, J. W. Costerton, and D. G. Davies. 2002. Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J. Bacteriol. 184:1140-1154./, 百拇医药
Zambrano, M. M., D. A. Siegele, M. Almirón, A. Tormo, and R. Kolter. 1993. Microbial competition: Escherichia coli mutants that take over stationary phase cultures. Science 259:1757-1760./, 百拇医药
Zinser, E. R., and R. Kolter. 1999. Mutations enhancing amino acid catabolism confer a growth advantage in stationary phase. J. Bacteriol. 181:5800-5807.(Manuel Espinosa-Urgel)
When environmental conditions are favorable, bacteria abandon their planktonic lifestyle to form surface-attached communities known as biofilms. In environments where there is a constant flow of nutrients, biofilms of the opportunistic human pathogen Pseudomonas aeruginosa show common, defined structural patterns. Cells, embedded in the extracellular matrix, form mushroom- and pillar-like colonies interspersed with fluid-filled channels (5, 17). How does such elaborate architecture develop and subsist? In this issue of the Journal of Bacteriology, M. E. Davey et al. (6) present a series of elegant experiments demonstrating how these fluid channels are kept open by the action of rhamnolipid biosurfactants. Rhamnolipids alter cell-cell and cell-surface interactions. In this way the biofilm architecture is maintained, ensuring the flow of nutrients and oxygen into the community and the efflux of waste products. However, rhamnolipid production, which is controlled at the population level, not only guarantees the structure, and therefore the nutritional balance of the biofilm, but these surfactants also prevent outside invading bacteria from colonizing the open spaces in the resident biofilm. Thus, we may be looking both at a finely tuned self-control system for biofilm structure and a mechanism for the sessile community to preserve its territory.
Rhamnolipids are tensoactive glycolipids, secreted by P. aeruginosa, that contain one or two rhamnose molecules. They have been primarily studied in terms of their effect on biodegradation of organic compounds (12), but they are also considered virulence factors due to their deleterious effects on eukaryotic cells and their biocidal properties (2, 13). They have also been shown to alter cell surface properties (1). Synthesis of rhamnolipids occurs in liquid cultures in stationary phase and under certain environmental conditions (12, 15); it is partially under the control of RpoS (15) and is regulated via quorum sensing by the RhlI-RhlR and the LasI-LasR systems (12, 16). All of these data are pieces of a puzzle now put together, from the perspective of biofilm integrity, by Davey et al. (6).ml6\m, 百拇医药
Pseudomonas aeruginosa can efficiently colonize the surface of medical implants, catheters, and also live tissue and thus be responsible for a number of serious nosocomial infections (3). The fact that bacteria often live as communities associated with solid surfaces rather than as planktonic individuals is not a recent discovery (14). However, it has been only in the past few years that research on the biology of microbial biofilms has geared from a descriptive discipline toward the study of the molecular mechanisms underlying this form of bacterial life (18, 19, 20, 22). Due to its implications in health-related issues, surface colonization by P. aeruginosa is the subject of intensive research. The process can be monitored in vivo by nondisruptive techniques such as confocal-laser-scanning microscopy. From these studies, and the phenotypic changes observed throughout, a general sketch of how biofilm formation proceeds in this bacterium can be drawn (5, 22). Certain environmental conditions promote attachment of the bacterial cells to the solid surface. Initial adhesion and movement of the bacteria along the plane give rise to a monolayer of cells covering the surface. This monolayer will evolve toward a mature biofilm through cell growth and aggregation, leading to the formation of microcolonies first and then of large macrocolonies embedded in an extracellular matrix composed of exopolysaccharides and other polymers. Whereas a number of genes and gene products involved in initial attachment of different bacteria to abiotic and biotic surfaces have been characterized (7-9, 18, 19), relatively less is known about the later stages leading to a mature biofilm, in terms of molecular mechanisms. One of the open questions is what determines the typical architecture of a mature biofilm, with "mushroom" and "pillar" macrocolonies separated by the so-called water channels? It has been proposed that such a structure is key to the maintenance of the biofilm, since it would allow nutrients and oxygen to flow in and waste products to flow out (17) and, therefore, its formation and stability should be regulated. On the other hand, mathematical models predict these structures would appear under certain conditions, simply as the result of chance and of the changes in the flow and distribution of nutrients and oxygen that take place during biofilm growth (11). However, some mutants of Escherichia coli and P. aeruginosa can attach to solid surfaces and form thick cell layers but are unable to form structured biofilms. That is the case of E. coli mutants deficient in the exopolysaccharide colanic acid (4). Furthermore, overexpression of algC, a gene involved in the biosynthesis of alginate, gives rise to highly structured biofilms in P. aeruginosa (10). All of this suggests that biofilm architecture is not only a consequence of chance and flow conditions but also part of a genetic program.
In this context, the data obtained by Davey et al. strongly support the idea of a genetic program determining biofilm formation, development, and stability. The significance of these results has multiple facets. They imply that biofilm architecture is kept by means of an active process, coordinately regulated at a specific time point and at a general population level. The fact that quorum sensing plays a key role in the maturation of biofilms was known (20, 21), since mutants affected in lasI, a gene involved in the synthesis of a quorum-sensing signal, form unstructured biofilms (20). However, the mechanisms through which quorum sensing exerts its role were not precisely defined.}i1, http://www.100md.com
The implications of the present study are not limited to basic biology. Based on their properties and potential production levels, rhamnolipids have been proposed as an alternative to synthetic surfactants for a wide number of industrial and biotechnological applications, from cosmetics to bioremediation (12). The results obtained by the O'Toole lab prompt investigating the use of rhamnolipids as surface-coating agents to prevent biofilms from forming on catheters and medical implants.
However, perhaps the most enticing hypothesis that could be drawn from these results is that rhamnolipid production in pure-culture biofilms does more than keep the shape, it may also be a defensive mechanism for the surface-attached community. In fact, it prevents outside bacteria from colonizing the open spaces in the biofilm and thus clogging the channels, with the subsequent detrimental effects for the established community. One can envision a system invaded by a bacterial population metabolically more efficient in that particular niche than the preestablished biofilm. Even as a minority, if the newcomers were able to attach instead of being carried away by the flow, they might end up taking over and displacing the previous population, as happens in planktonic batch cultures (see, for example, the studies by Kolter and coworkers [23, 24] on the GASP [growth advantage in stationary phase] phenotype). However, by preventing the intruder from settling down, a biofilm population would persist in its toilsomely built estate. The obvious question that remains is: how does the biofilm prevent its own surfactant-induced destruction?
ACKNOWLEDGMENTS/{;, http://www.100md.com
Work in the M.E-U. lab on bacterial attachment to biotic surfaces is supported by a grant from the Ramón y Cajal Programme and by grant BMC2001-0576 from the Spanish Ministry for Science and Technology./{;, http://www.100md.com
The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM. /{;, http://www.100md.com
REFERENCES/{;, http://www.100md.com
Al-Tahhan, R. A., T. R. Sandrin, A. A. Bodour, and R. M. Maier. 2000. Rhamnolipid-induced removal of lipopolysaccharide from Pseudomonas aeruginosa: effect on cell surface properties and interaction with hydrophobic substrates. Appl. Environ. Microbiol. 66:3262-3268./{;, http://www.100md.com
Cosson, P., L. Zulianello, O. Join-Lambert, F. Faurisson, L. Gebbie, M. Benghezal, C. Van Delden, L. K. Curty, and T. Kohler. 2002. Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system. J. Bacteriol. 184:3027-3033./{;, http://www.100md.com
Costerton, J. W. 2001. Cystic fibrosis pathogenesis and the role of biofilms in persistent infection. Trends Microbiol. 9:50-52.
Danese, P. N., L. A. Pratt, and R. Kolter. 2000. Exopolysaccharide production is required for development of Escherichia coli K-12 biofilm architecture. J. Bacteriol. 182:3593-3596.:;3f, http://www.100md.com
Davey, M. E., and G. A. O'Toole. 2000. Microbial biofilms: from ecology to molecular genetics. Microbiol. Mol. Biol. Rev. 64:847-867.:;3f, http://www.100md.com
Davey, M. E., N. C. Caiazza, and G. A. O'Toole. 2003. Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1. J. Bacteriol. 185:1027-1036.:;3f, http://www.100md.com
Dörr, J., T. Hurek, and B. Reinhold-Hurek. 1998. Type IV pili are involved in plant-microbe and fungus-microbe interactions. Mol. Microbiol. 30:7-17.:;3f, http://www.100md.com
Espinosa-Urgel, M., A. Salido, and J. L. Ramos. 2000. Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds. J. Bacteriol. 182:2363-2369.:;3f, http://www.100md.com
Girón, J. A., A. G. Torres, E. Freer, and J. B. Kaper. 2002. The flagella of enteropathogenic Escherichia coli mediate adherence to epithelial cells. Mol. Microbiol. 44:361-379.
Hentzer, M., G. M. Teitzel, G. J. Balzer, A. Heydorn, S. Molin, M. Givskov, and M. R. Parsek. 2001. Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. J. Bacteriol. 183:5395-5401.n, 百拇医药
Kreft, J. U., C. Picioreanu, J. W. Wimpenny, and M. C. van Loosdrecht. 2001. Individual-based modeling of biofilms. Microbiology 147:2897-2912.n, 百拇医药
Maier, R. M., and G. Soberón-Chávez. 2000. Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications. Appl. Microbiol. Biotechnol. 54:625-633.n, 百拇医药
McClure, C. D., and N. L. Schiller. 1996. Inhibition of macrophage phagocytosis by Pseudomonas aeruginosa rhamnolipids in vitro and in vivo. Curr. Microbiol. 33:109-117.n, 百拇医药
Meadows, P. S. 1971. The attachment of bacteria to solid surfaces. Arch. Mikrobiol. 75:374-381.n, 百拇医药
Medina, G., K. Juárez, and G. Soberón-Chávez. 2003. The Pseudomonas aeruginosa rhlAB operon is not expressed during the logarithmic phase of growth even in the presence of its activator RhlR and the autoinducer N-butyryl-homoserine lactone. J. Bacteriol. 185:377-380.
Ochsner, U. A., and J. Reiser. 1995. Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 92:6424-6428./, 百拇医药
O'Toole, G., H. B. Kaplan, and R. Kolter. 2000. Biofilm formation as microbial development. Annu. Rev. Microbiol. 54:49-79./, 百拇医药
O'Toole, G. A., and R. Kolter. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis. Mol. Microbiol. 28:449-461./, 百拇医药
O'Toole, G. A., and R. Kolter. 1998. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30:295-304./, 百拇医药
Parsek, M. R., and E. P. Greenberg. 2000. Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in associations with higher organisms. Proc. Natl. Acad. Sci. USA 97:8789-8793./, 百拇医药
Riedel, K., M. Hentzer, O. Geisenberger, B. Huber, A. Steidle, H. Wu, N. Hoiby, M. Givskov, S. Molin, and L. Eberl. 2001. N-acylhomoserine-lactone-mediated communication between Pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms. Microbiology 147:3249-3262./, 百拇医药
Sauer, K., A. K. Camper, G. D. Ehrlich, J. W. Costerton, and D. G. Davies. 2002. Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J. Bacteriol. 184:1140-1154./, 百拇医药
Zambrano, M. M., D. A. Siegele, M. Almirón, A. Tormo, and R. Kolter. 1993. Microbial competition: Escherichia coli mutants that take over stationary phase cultures. Science 259:1757-1760./, 百拇医药
Zinser, E. R., and R. Kolter. 1999. Mutations enhancing amino acid catabolism confer a growth advantage in stationary phase. J. Bacteriol. 181:5800-5807.(Manuel Espinosa-Urgel)