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) (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 micrometer, 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 micrometer in depth. In contrast, FRD1 produced microcolonies that were approximately 40 micrometer 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.

FIG. 1

Use of ATR/FT-IR for biofilm analysis. An IR beam reflects within an IR-transparent substance (germanium) termed an IRE. The reflection generates a field of radiation (evanescent field) in the medium outside of the IRE. The intensity of the evanescent field decays exponentially to zero within approximately 1 μm of the IRE. Molecules of cellular biomass or extracellular polymers within the evanescent field absorb the IR radiation, thereby producing IR absorption spectra. Molecules outside of the evanescent field are not detected. Experimentally, opposing sides of the IRE are sealed with two stainless steel plates with O rings to create flow channels.

FIG. 2

Interfacial IR absorption spectra of attachment/growth of the mucoid CF isolate P. aeruginosa FRD1 and alg mutant strains plotted at 4-h intervals. Note scale changes to enhance the spectral features (panel B is fivefold greater than panels A and C). In all experiments, an inoculum of 10⁶ cells/ml was introduced into the flow system at time zero. After 20 min, the sterile medium was pumped though the flow cell to stimulate biofilm development. The fluctuations near 1,640 cm⁻¹ were attributed to the subtraction of a water band. (A) These spectra show the IR bands indicative of P. aeruginosa FRD1 biomass that absorbed near 1,650 cm⁻¹ (amide I), 1,550 cm⁻¹ (amide II), 1,250 cm⁻¹ (P⩵O stretching, C—O—C stretching, and/or amide III vibration), 1,090 cm⁻¹ (P—O and C—OH stretching), 1,060 cm⁻¹ (C—OH stretching of alginate), 1,450 cm⁻¹, and 1,400 cm⁻¹ (due in part to C—H deformations and symmetric stretching of carboxylates, respectively). The bands associated with FRD1 biomass were not detected until 24 h. (B) Interfacial IR absorption spectra of biofilms formed by nonmucoid strain P. aeruginosa FRD440. The IR bands indicative of biomass were detected at 8 h. The bands in the FRD440 spectra were similar to bands from the spectra of the mucoid P. aeruginosa FRD1, except for the absence of the band at 1,060 cm⁻¹. (C) Interfacial attachment/growth profile of mucoid P. aeruginosa FRD1153 algJ, a strain that lacked alginate O acetylation. At approximately 32 h, all of the bands indicative of biomass were detected, including the band at 1,060 cm⁻¹.

FIG. 3

Spectra of biofilms formed by mucoid and nonmucoid strains of P. aeruginosa following dehydration. Each biofilm was cultured for 88 h and then dried in a desiccator and analyzed. The drying process collapsed the biofilm onto the surface of the IRE. (A) Dehydrated FRD1 biofilm showing IR bands associated with the cells: 1,650 cm⁻¹ (amide I), 1,545 cm⁻¹ (amide II), 1,450 cm⁻¹ (C—H deformations), 1,405 cm⁻¹ (in part due to symmetric stretching of the carboxylate ions), 1,250 cm⁻¹ (P ⩵O stretching, C—O—C stretching, and/or amide III), and the 1,100- to 1,000-cm⁻¹ region (P—O and C—OH stretching). IR bands associated with the alginate were also detected: 1,735 cm⁻¹ (C⩵O stretching of esters), 1,615 cm⁻¹ (asymmetric stretching of the carboxylate ion), 1,405 cm⁻¹ and 1,375 cm⁻¹ (symmetric stretching of the carboxylate ion), 1,250 cm⁻¹ (C—O—C for the ester), and 1,060 cm⁻¹ (C—OH stretching of alcohols). (B) Spectrum of dried mucoid FRD1153 showing the lack of prominent alginate bands. The final three spectra show the IR bands associated with the dried nonmucoid strains FRD2 (C), FRD1131 (D), and FRD440 (E).

FIG. 4

(A) Spectral subtraction of dehydrated FRD1 biofilm and FRD2 biofilm. The band observed in the spectrum of the hydrated FRD1 biofilm at 1,060 cm⁻¹ (C—OH stretching) was present in the subtraction spectra. Other IR bands observed in the subtraction spectrum were 1,615cm⁻¹ (asymmetric carboxylate stretching), 1,410 cm⁻¹ (symmetric carboxylate stretching), 1,730 cm⁻¹ (C⩵O stretching), and 1,250 cm⁻¹ (C—O stretching). The IR bands at 1,730 and 1,250 were associated with the O-acetyl groups on the alginate that are linked to the mannuronate backbone by ester bonds. (B) The FT-IR spectrum of purified alginate from P. aeruginosa FRD1, showing bands similar to that observed from the FRD1-FRD2 subtraction spectrum. (C) The FT-IR spectrum of purified alginate from P. aeruginosa FRD1153 algJ. This spectrum was similar to the spectrum of purified FRD1 alginate but lacked IR bands at 1,730 and 1250 cm⁻¹ that resulted from ester bonds of O-acetylated alginate.

FIG. 5

Plot of amide II band absorbance (1,550 cm⁻¹) over time for mucoid and nonmucoid strains of P. aeruginosa. Open circles, FRD1; open diamonds, FRD1153 algJ; closed circles, FRD2 algT18; boxes, FRD440 algT:: Tn501–33; open triangles, FRD1131 algD::Tn501–31.

FIG. 6

SCLM analysis of 48-h biofilms of P. aeruginosa, as viewed from the top and at an angle. All strains constitutively expressed the mut2 GFP. (A) P. aeruginosa FRD1 viewed at ×100 magnification. (B) P. aeruginosa FRD1 viewed at ×400 magnification. (C) P. aeruginosa FRD2 algT18 viewed at ×400 magnification. (D) P. aeruginosa FRD1153 algJ viewed at ×400 magnification.