Elucidation of Functional Groups on Gram-Positive and Gram-Negative Bacterial Surfaces Using Infrared Spectroscopy

Abstract

Surface functional group chemistry of intact Gram-positive and Gram-negative bacterial cells and their isolated cell walls was examined as a function of pH, growth phase, and growth media (for intact cells only) using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Infrared spectra of aqueous model organic molecules, representatives of the common functional groups found in bacterial cell walls (i.e., hydroxyl, carboxyl, phosphoryl, and amide groups), were also examined in order to assist the interpretation of the infrared spectra of bacterial samples. The surface sensitivity of the ATR-FTIR spectroscopic technique was evaluated using diatom cells, which possess a several-nanometers-thick layer of glycoprotein on their silica shells. The ATR-FTIR spectra of bacterial surfaces exhibit carboxyl, amide, phosphate, and carbohydrate related features, and these are identical for both Gram-positive and Gram-negative cells. These results provide direct evidence to the previously held conviction that the negative charge of bacterial surfaces is derived from the deprotonation of both carboxylates and phosphates. Variation in solution pH has only a minor effect on the secondary structure of the cell wall proteins. The cell surface functional group chemistry is altered neither by the growth phase nor by the growth medium of bacteria. This study reveals the universality of the functional group chemistry of bacterial cell surfaces.

Cell surface acid-base properties of the cyanobacterium Synechococcus : Influences of nitrogen source, growth phase and N:P ratios

“…Irrespective of the incubation time and the contents of the growth media, the FT-IR spectra collected for Synechococccus cells were relatively similar to each other and consistent with those obtained by other studies examining bacterial cells and proteins (after Zeroual et al, 1994;Kansiz et al, 1999;Benning et al, 2004;Jiang et al, 2004;Omoike and Chorover, 2004;Yee et al, 2004;Dittrich and Sibler, 2005;Kong and Yu, 2007;Leone et al, 2007;Meade et al, 2007;Movasaghi et al, 2008;Castro et al, 2010;Quilès et al, 2010;Alessi et al, 2014). This either means that there is a change in the type of phospholipids produced under these growth conditions, or the very sharp nature of this peak may indicate that the material may be more crystalline, and therefore, may be a relic of the sample preparation and relate to some residual media in the sample which crystallized upon drying.…”

“…Figure 1 and 2, in conjunction with work done by Jiang et al (2004), demonstrate that structural transformations and differences occurred in the Synechococcus cells under tested experimental growth conditions. With increasing growth stage, there are differences in the protein (amine) and carbohydrate (1155, 1080, and 1048 cm -1 ) regions which indicate the production of carboxylic acids and phosphorylated polysaccharides for cells grown on nitrate and ammonium.…”

“…FT-IR spectra of bacterial samples can provide insight into the biochemical composition of the cells (e.g., Jiang et al, 2004), and when combined with pKa values from the modeling of titration data and previous spectroscopic studies (see above), allow for tentative identification of the functional groups responsible for cell surface charge.…”

“…(Table 2). Based on spectroscopic studies of cell walls and metal binding to cells (e.g., Sarret et al, 1998;Hennig et al, 2001;Boyanov et al, 2003;Benning et al, 2004;Dittrich and Sibler, 2005), and analogy in acidity constants with respect to known compounds, the first group of acidic proton binding sites with pKa values in the range of 4.96-5.51 can be attributed to carboxylic groups (pKa = 2 to 6); the second group having pKa values of 6.67-7.42 to phosphoryl groups (pKa = 5.65 to 7.5); and the most basic pKa range of 8.13-9.95 to amine groups (pKa = 8.0 to 10.6) (Jiang et al, 2004;Konhauser, 2007;Castro et al, 2010). The total site concentration (N tot ) of Synechococcus cells, normalized to the dry weight of bacteria present, was in the range of 17.61 -34.18 mmol/g (Table 2) under the experimental conditions tested.…”

“…In contrast, other research indicated that bacterial surface compositions remain unchanged in response to growth phase. For example, Haas (2004) reported that growth phase had negligible influence on the ligand distributions of the Gram-negative Shewanella putrefaciens, and Jiang et al (2004) showed that the dominant functional groups on Pseudomonas stutzeri cell walls did not change over time with growth. Cumulatively these results suggest the effects of growth phase are species-specific.…”