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Journal of Bacteriology, February 2001, p. 1047-1057, Vol. 183, No. 3
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.3.1047-1057.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Role of Alginate and Its O Acetylation in Formation of Pseudomonas aeruginosa Microcolonies and Biofilms

David E. Nivens,1 Dennis E. Ohman,2,3 Jessica Williams,4,5 and Michael J. Franklin4,5,*

Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee, 379961; Department of Microbiology and Immunology, Medical College of Virginia at Virginia Commonwealth University, Richmond, Virginia 232982; McGuire Veterans Affairs Medical Center, Richmond, Virginia 232493; and Department of Microbiology4 and Center for Biofilm Engineering,5 Montana State University, Bozeman, Montana 59717

Received 16 June 2000/Accepted 26 October 2000

Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT-IR) and scanning confocal laser microscopy (SCLM) were used to study the role of alginate and alginate structure in the attachment and growth of Pseudomonas aeruginosa on surfaces. Developing biofilms of the mucoid (alginate-producing) cystic fibrosis pulmonary isolate FRD1, as well as mucoid and nonmucoid mutant strains, were monitored by ATR/FT-IR for 44 and 88 h as IR absorbance bands in the region of 2,000 to 1,000 cm-1. All strains produced biofilms that absorbed IR radiation near 1,650 cm-1 (amide I), 1,550 cm-1 (amide II), 1,240 cm-1 (P==O stretching, C---O---C stretching, and/or amide III vibrations), 1,100 to 1,000 cm-1 (C---OH and P---O stretching) 1,450 cm-1, and 1,400 cm-1. The FRD1 biofilms produced spectra with an increase in relative absorbance at 1,060 cm-1 (C---OH stretching of alginate) and 1,250 cm-1 (C---O stretching of the O-acetyl group in alginate), as compared to biofilms of nonmucoid mutant strains. Dehydration of an 88-h FRD1 biofilm revealed other IR bands that were also found in the spectrum of purified FRD1 alginate. These results provide evidence that alginate was present within the FRD1 biofilms and at greater relative concentrations at depths exceeding 1 µm, the analysis range for the ATR/FT-IR technique. After 88 h, biofilms of the nonmucoid strains produced amide II absorbances that were six to eight times as intense as those of the mucoid FRD1 parent strain. However, the cell densities in biofilms were similar, suggesting that FRD1 formed biofilms with most cells at depths that exceeded the analysis range of the ATR/FT-IR technique. SCLM analysis confirmed this result, demonstrating that nonmucoid strains formed densely packed biofilms that were generally less than 6 µm in depth. In contrast, FRD1 produced microcolonies that were approximately 40 µm in depth. An algJ mutant strain that produced alginate lacking O-acetyl groups gave an amide II signal approximately fivefold weaker than that of FRD1 and produced small microcolonies. After 44 h, the algJ mutant switched to the nonmucoid phenotype and formed uniform biofilms, similar to biofilms produced by the nonmucoid strains. These results demonstrate that alginate, although not required for P. aeruginosa biofilm development, plays a role in the biofilm structure and may act as intercellular material, required for formation of thicker three-dimensional biofilms. The results also demonstrate the importance of alginate O acetylation in P. aeruginosa biofilm architecture.

* Corresponding author. Mailing address: Department of Microbiology, Montana State University, Bozeman, MT 59717. Phone: (406) 994-2420. Fax: (406) 994-4926. E-mail: umbfm{at}montana.edu.

Journal of Bacteriology, February 2001, p. 1047-1057, Vol. 183, No. 3
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.3.1047-1057.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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