J Bacteriol 186(14): 4449-4456

Putative Exopolysaccharide Synthesis Genes Influence <em>Pseudomonas aeruginosa</em> Biofilm Development

Department of Microbiology and the W. M. Keck Microbial Communities and Cell Signaling Program, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242

^{Corresponding author. Mailing address: Department of Microbiology, 540 F Eckstein Medical Research Building, Carver College of Medicine, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-7775. Fax: (319) 335-7949. E-mail:}ude.awoiu@grebneerg-ttereve.

Received 2003 Dec 16; Accepted 2004 Feb 20.

Abstract

An analysis of the Pseudomonas aeruginosa genomic sequence revealed three gene clusters, PA1381-1393, PA2231-2240, and PA3552-3558, in addition to the alginate biosynthesis gene cluster, which appeared to encode functions for exopolysaccharide (EPS) biosynthesis. Recent evidence indicates that alginate is not a significant component of the extracellular matrix in biofilms of the sequenced P. aeruginosa strain PAO1. We hypothesized that at least one of the three potential EPS gene clusters revealed by genomic sequencing is an important component of P. aeruginosa PAO1 biofilms. Thus, we constructed mutants with chromosomal insertions in PA1383, PA2231, and PA3552. The mutant with a PA2231 defect formed thin unstructured abnormal biofilms. The PA3552 mutant formed structured biofilms that appeared different from those formed by the parent, and the PA1383 mutant formed structured biofilms that were indistinguishable from those formed by the parent. Consistent with a previous report, we found that polysaccharides were one component of the extracellular matrix, which also contained DNA. We suggest that the genes that were inactivated in our PA2231 mutant are required for the production of an EPS, which, although it may be a minor constituent of the matrix, is critical for the formation of P. aeruginosa PAO1 biofilms.

Abstract

Pseudomonas aeruginosa is an important opportunistic pathogen that is capable of causing several different types of infections, including chronic biofilm infections. For example, P. aeruginosa infects the lungs of people with the genetic disease cystic fibrosis (CF), and chronic CF lung infections are considered biofilm infections (5, 12, 30). Mature P. aeruginosa biofilms consist of groups of cells growing together in clusters embedded in an extracellular polymeric matrix (4, 5). Quite often, P. aeruginosa isolated from the lungs of CF patients exhibits a mucoid phenotype resulting from the overproduction of the exopolysaccharide (EPS) alginate (12, 34). However, strains that do not produce much alginate are capable of forming mature biofilms in vitro (18). In fact, the commonly studied laboratory strain PAO1 makes little or no alginate (17, 36).

If alginate is not the extracellular matrix for biofilms of strain PAO1, what is? A recent report showed that a major polymer in the extracellular matrix of P. aeruginosa PAO1 biofilms is DNA (35). However, this does not rule out the possibility that P. aeruginosa PAO1 makes an EPS other than alginate and that this polysaccharide is an important constituent of the extracellular polymeric matrix.

A clue that polysaccharides other than alginate might be involved in P. aeruginosa biofilms came from the P. aeruginosa genome sequencing project. The annotation of the genome suggested that there are gene clusters in addition to the alginate biosynthesis gene cluster that may be involved in the synthesis of EPSs. One of these clusters is PA1381-1393, a second is PA2231-2245, and a third is PA3552-3558 (31). The PA1381-1393 cluster has an anomalously low G+C content, and the encoded polypeptides show sequence similarity to polypeptides encoded by a gene cluster which is required for polymyxin resistance and for the aminoarabinose modification of lipid A in Salmonella enterica serovar Typhimurium (9). The PA3552-3558 cluster is adjacent to a cluster of alginate biosynthetic genes. Many of the polypeptides encoded by genes in the PA2231-2245 cluster show similarity to glycosyltransferases, export proteins, or polysaccharide polymerases. The first three genes in this cluster have been studied by Rocchetta et al. (23), who suggested that PA2232 is involved in the biosynthesis of the lipopolysaccharide (LPS) A-band O antigen.

We generated mutations in one gene selected from each cluster (PA1383, PA2231, and PA3552) and studied the influence of the mutations on biofilm development. Our PA2231 mutant showed a severe defect in biofilm formation. Thus, we studied this mutant in more detail. Our results, taken together with those reported in accompanying papers (8, 13), indicate that the PA2231 gene cluster is an important determinant of P. aeruginosa biofilm development. We have adopted the nomenclature of Jackson et al. (13), and we will refer to the genes of the PA2231-2245 cluster as psl (polysaccharide synthesis locus) genes throughout the remainder of this article.

Acknowledgments

Support for this study was provided by a grant from the National Institute of General Medicine (GM59026) and from the W. M. Keck Foundation.

The contents of this study do not necessarily represent the official views of the National Institute of General Medicine.

Acknowledgments

REFERENCES

References

1. Ausubel, F., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1997. Short protocols in molecular biology, 3rd ed. John Wiley & Sons, Inc., New York, N.Y.
2. Becher, A., and H. P. Schweizer. 2000. Integration-proficient Pseudomonas aeruginosa vectors for isolation of single-copy chromosomal lacZ and lux gene fusions. BioTechniques29:948-950, 952. [[PubMed]
3. Chugani, S. A., M. Whiteley, K. M. Lee, D. D'Argenio, C. Manoil, and E. P. Greenberg. 2001. QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA98:2752-2757.
4. Costerton, J. W., Z. Lewandowski, D. E. Caldwell, D. R. Korber, and H. M. Lappin-Scott. 1995. Microbial biofilms. Annu. Rev. Microbiol.49:711-745. [[PubMed]
5. Costerton, J. W., P. S. Stewart, and E. P. Greenberg. 1999. Bacterial biofilms: a common cause of persistent infections. Science284:1318-1322. [[PubMed]
6. Davies, D. G., M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, and E. P. Greenberg. 1998. The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science280:295-298. [[PubMed]
7. Fox, J. D., and J. F. Robyt. 1991. Miniaturization of three carbohydrate analyses using a microsample plate reader. Anal. Biochem.195:93-96. [[PubMed]
8. Friedman, L., and RKolter. 2004. Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix. J. Bacteriol.186:4457-4465. [Google Scholar]
9. Gunn, J. S., S. S. Ryan, J. C. Van Velkinburgh, R. K. Ernst, and S. I. Miller. 2000. Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar Typhimurium. Infect. Immun.68:6139-6146.
10. Hitchcock, P. J., and T. M. Brown. 1983. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol.154:269-277.
11. Hoang, T. T., R. R. Karkhoff-Schweizer, A. J. Kutchma, and H. P. Schweizer. 1998. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene212:77-86. [[PubMed]
12. Hoiby, N. 1993. Antibiotic therapy for chronic infection of Pseudomonas in the lung. Annu. Rev. Med.44:1-10. [[PubMed]
13. Jackson, K. D., M. Starkey, S. Kremer, M. R. Parsek, and D. J. Wozniak. 2004. Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation. J. Bacteriol.186:4466-4475.
14. Klausen, M., A. Aaes-Jorgensen, S. Molin, and T. Tolker-Nielsen. 2003. Involvement of bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms. Mol. Microbiol.50:61-68. [[PubMed]
15. Lesse, A. J., A. A. Campagnari, W. E. Bittner, and M. A. Apicella. 1990. Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Immunol. Methods126:109-117. [[PubMed]
16. Martin, P. R., A. A. Watson, T. F. McCaul, and J. S. Mattick. 1995. Characterization of a five-gene cluster required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa. Mol. Microbiol.16:497-508. [[PubMed]
17. Mathee, K., O. Ciofu, C. Sternberg, P. W. Lindum, J. I. Campbell, P. Jensen, A. H. Johnsen, M. Givskov, D. E. Ohman, S. Molin, N. Hoiby, and A. Kharazmi. 1999. Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology145:1349-1357. [[PubMed]
18. Nivens, D. E., D. E. Ohman, J. Williams, and M. J. Franklin. 2001. Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J. Bacteriol.183:1047-1057.
19. O'Toole, G. A., and R. Kolter. 1998. Flagellar and twitching motilities are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol.30:295-304. [[PubMed]
20. Parales, R. E., and C. S. Harwood. 1993. Construction and use of a new broad-host-range lacZ transcriptional fusion vector, pHRP309, for gram-negative bacteria. Gene133:23-30. [[PubMed]
21. Pearson, J. P., C. Van Delden, and B. H. Iglewski. 1999. Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J. Bacteriol.181:1203-1210.
22. Rashid, M. H., and A. Kornberg. 2000. Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA97:4885-4890.
23. Rocchetta, H. L., J. C. Pacan, and J. S. Lam. 1998. Synthesis of the A-band polysaccharide sugar d-rhamnose requires Rmd and WbpW: identification of multiple AlgA homologues, WbpW and ORF488, in Pseudomonas aeruginosa. Mol. Microbiol.29:1419-1434. [[PubMed]
24. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
25. Schuster, M., A. Hawkins, C. S. Harwood, and E. P. Greenberg. 2004. The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing. Mol. Microbiol.51:973-985. [[PubMed]
26. Schuster, M., C. P. Lostroh, T. Ogi, and E. P. Greenberg. 2003. Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J. Bacteriol.185:2066-2079.
27. Schweizer, HP. 1993. Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. BioTechniques15:831-833. [[PubMed][Google Scholar]
28. Simon, R., U. Priefer, and A. Puhler. 1983. A broad-host-range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Bio/Technology1:37-45. [PubMed]
29. Singh, P. K., M. R. Parsek, E. P. Greenberg, and M. J. Welsh. 2002. A component of innate immunity prevents bacterial biofilm development. Nature417:552-555. [[PubMed]
30. Singh, P. K., A. L. Schaefer, M. R. Parsek, T. O. Moninger, M. J. Welsh, and E. P. Greenberg. 2000. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature407:762-764. [[PubMed]
31. Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. L. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K.-S. Wong, Z. Wu, I. T. Paulsen, J. Reizer, M. H. Saier, R. E. W. Hancock, S. Lory, and M. V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature406:959-964. [[PubMed]
32. Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem.119:115-119. [[PubMed]
33. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem.218:97-106. [[PubMed]
34. Welsh, M. J., B. W. Ramsey, F. Accurso, and G. R. Cutting. 2001. Cystic fibrosis, p. 5121-5189. In C. L. Scriver, A. L. Beaudet, W. S. Sly, D. Valle, B. Childs, and B. Vogelstein (ed.), The metabolic and molecular basis of inherited disease, 8th ed. McGraw-Hill, New York, N.Y.
35. Whitchurch, C. B., T. Tolker-Nielsen, P. C. Ragas, and J. S. Mattick. 2002. Extracellular DNA required for bacterial biofilm formation. Science295:1487. [[PubMed]
36. Wozniak, D. J., T. J. Wyckoff, M. Starkey, R. Keyser, P. Azadi, G. A. O'Toole, and M. R. Parsek. 2003. Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms. Proc. Natl. Acad. Sci. USA100:7907-7912.
37. Yildiz, F. H., and G. K. Schoolnik. 1999. Vibrio cholerae O1 E1 Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation. Proc. Natl. Acad. Sci. USA96:4028-4033.