Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms

Attenuated total reflection/Fourier transform-infrared spectrometry (ATR/FT -IR) and scanning confocal laser microscopy (SCLM) were used to study the r ole of alginate and alginate structure in the attachment and growth of Pseu domonas aeruginosa on surfaces. Developing biofilms of the mucoid (alginate -producing) cystic fibrosis pulmonary isolate FRD1, as well as mucoid and n onmucoid 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 produc ed biofilms that absorbed IR radiation near 1,650 cm(-1) (amide I), 1,550 c m(-1) (amide II), 1,230 cm(-1) (P=O stretching, C-O-C stretching, and/or am ide 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 increa se 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 compar ed to biofilms of nonmucoid mutant strains. Dehydration of an 88-h FRD1 bio film revealed other IR bands that were also found in the spectrum of purifi ed FRD1 alginate. These results provide evidence that alginate was present within the FRD1 biofilms and at greater relative concentrations at depths e xceeding 1 mum, 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. Ho wever, the cell densities in biofilms were similar, suggesting that FRD1 fo rmed biofilms with most cells at depths that exceeded the analysis range of the ATR/FT-IR technique. SCLM analysis confirmed this result, demonstratin g that nonmucoid strains formed densely packed biofilms that were generally less than 6 mum in depth. In contrast, FRD1 produced microcolonies that we re approximately 40 mum in depth. An algJ mutant strain that produced algin ate 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 snitched to the nonmucoid phenotype and formed uniform biofilms , similar to biofilms produced by the nonmucoid strains. These results demo nstrate that alginate, although not required for P. aeruginosa biofilm deve lopment, plays a role in the biofilm structure and may act as intercellular material, required for formation of thicker three-dimensional biofilms. Th e results also demonstrate the importance of alginate O acetylation in P. a eruginosa biofilm architecture.