Monitoring Metal Ion Binding in Single-Layer Pseudomonas aeruginosa Biofilms Using ATR−IR Spectroscopy

Abstract

The binding of metal ions to Pseudomonas aeruginosa PAO1 cells attached to a ZnSe surface has been observed in this research through cation exchange experiments using ATR-IR spectroscopy. A biofilm consisting of a single layer of Pseudomonas aeruginosa PAO1 cells was formed on a ZnSe prism by flowing a bacterial suspension in a 0.03 mol L(-)(1) NaNO(3) solution at pH 5.0 across its surface. Exposure of the biofilm to chromium(III) nitrate solution resulted in increases in all band absorbances. This absorbance increase has been attributed to the binding of chromium(III) to the bacterial exopolymers associated with the prism surface. The chromium(III) binding causes the exopolymers to contract and move the bacterial cell closer to the ZnSe surface. Further study of chromium(III) ion exchange using a mutant P. aeruginosa with a truncated lipopolysaccharide (LPS) chain resulted in much smaller absorbance changes. This observation supports the view that the extension of bacterial exopolymers and hence the distance of the bacterial cell from the surface is strongly influenced by environmental factors such as the presence of metal cations. Following chromium(III) cation exchange, the bacterial band absorbances remained constant even when the bacteria were washed with a 0.03 mol L(-)(1) NaNO(3) solution, indicating that the chromium(III) was irreversibly bound. Ion exchange with nickel(II) and cobalt(II) nitrate solutions within identical biofilms showed that these cations caused relatively small increases in absorbances that were reversible, indicating that nickel(II) and cobalt(II) are less strongly bound than chromium(III) within P. aeruginosa biofilms. The absence of discernible IR spectral changes with metal binding appears to indicate a predominantly electrostatic mechanism for binding of Cr(III), Ni(II), and Co(II) ions by bacteria in the early stages of biofilm formation.

In Situ Monitoring of the Nascent Pseudomonas fluorescens Biofilm Response to Variations in the Dissolved Organic Carbon Level in Low-Nutrient Water by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy

“…These results are in agreement with a previous study of Korber et al (26) which reported that P. fluorescens bacteria obeyed monolayer attachment kinetics and the saturation of surfaces by a monolayer of colonizing bacteria does not equal the total available area during attachment of P. fluorescens. As observed previously in similar experiments with Pseudomonas aeruginosa (25), the rate of increase was reduced as the surface coverage increased. For this attachment period, the plot of the amide II band area (A) versus time can be fitted using the following equation: A ϭ A max (1 Ϫ e Ϫkt ), where A max is the amide II band area corresponding to a complete bacteria single layer, k is an absorption rate constant, and t is time (Fig.…”

Microscopic and infrared spectroscopic comparison of the underwater adhesives produced by germlings of the brown seaweed speciesDurvillaea antarcticaandHormosira banksii

“…Accordingly, any spectral contribution from sperm suspension is removed in the subtraction and the spectral evolution considered is representative of germling development only. The spectra are very similar to spectra of bacterial species, with distinct bands at 1651 (amide I), 1544 (amide II), 1454 (C-H deformation), 1400 (symmetric carboxylate stretch) and 1245 cm 21 (amide III, P¼O and C-O-C stretch) [85]. Some spectral changes are also visible in the 1100-1000 cm 21 region characteristic of polysaccharides (C-O stretch, C-C stretch, C-O-H stretch and bend, and ring vibrations).…”

Mechanisms of Cation Exchange by Pseudomonas aeruginosa PAO1 and PAO1 wbpL , a Strain with a Truncated Lipopolysaccharide

“…During the attachment process, the bacterial absorption intensity increased with time (data not shown), indicating that the number of cells attached to the ZnSe surface was increasing. Microscopic studies in our laboratory have previously shown that P. aeruginosa PAO1 readily forms a bacterial monolayer on ZnSe under similar flow conditions (10). The ATR-IR spectra exhibit distinct bands, around 1,646 cm Ϫ1 (amide I, mainly the CAO stretch), 1,548 cm Ϫ1 (amide II, mainly the NOH bend), 1,454 cm Ϫ1 (partly COH deformation), 1,402 cm Ϫ1 (partly symmetric carboxylate stretch), and 1,235 cm Ϫ1 (amide III, mainly the PAO, COOOC antisymmetric stretch of ester groups) and in the 1,100-to 1,000-cm Ϫ1 region (POO and COOH stretch) (31).…”

“…Pseudomonas aeruginosa is a ubiquitous, well-characterized microorganism used in many metal-binding investigations (10). Suggested metal ion binding sites in P. aeruginosa include the phosphate and carboxyl groups in the peptidoglycan (29) and charged groups in both the core oligosaccharide region and the O-antigenic side chains that make up its LPS (14,28).…”