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

Abstract

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 (PAO stretching, COOOC stretching, and/or amide III vibrations), 1,100 to 1,000 cm ؊1 (COOH and POO 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 (COOH stretching of alginate) and 1,250 cm ؊1 (COO 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 m, 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 m in depth. In contrast, FRD1 produced microcolonies that were approximately 40 m 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.

Calcium-Induced Virulence Factors Associated with the Extracellular Matrix of MucoidPseudomonas aeruginosaBiofilms

“…P. aeruginosa FRD875 algDϾxylE (69) was generously provided by Daniel Wozniak (Wake Forest University). For scanning confocal laser microscopy (SCLM) the strains expressed the gfpmut2 from a constitutive promoter from plasmid pMF230 (48).…”

“…The flow cell system for the SCLM experiments was similar to that described previously (48). Briefly, the system consisted of a onceflowthrough flow cell with a medium reservoir, pump, silicone tubing, and a waste container.…”

“…The biofilms were dried onto the cards in a laminar flow hood and stored under desiccation prior to analysis. The spectra were collected and processed as described previously (48).…”

“…These reports may reflect the dif-ferences in strains used in different studies. With mucoid P. aeruginosa FRD1 strains, alginate appears to be the primary constituent of the biofilm extracellular matrix material (48). Studies with this CF isolate and its mutant derivatives showed that alginate affects the three-dimensional architecture of P. aeruginosa FRD1 biofilms.…”

“…P. aeruginosa pulmonary infections are considered biofilm-associated diseases, where the bacteria colonize the surface of pulmonary tissue (36), are often encapsulated in extracellular polysaccharide matrix material (25,36,48), secrete quorumsensing signaling compounds (58), and have increased resistance to antibiotics (reviewed in reference 59) and to host defenses (2,30,45). Mutations in the cystic fibrosis transmembrane regulator of CF patients lead to physiological alteration of pulmonary fluid, with the most characteristic manifestation being the increased concentrations of thick pulmonary mucus that includes DNA and actin from lysed neutrophils (38).…”