In situ ATR-IR spectroscopic and electron microscopic analyses of settlement secretions of Undaria pinnatifida kelp spores.

Knowledge about the settlement of marine organisms on substrates is important for the development of environmentally benign new methods for control of marine biofouling. The adhesion to substrates by spores of Undaria pinnatifida, a kelp species that is invasive to several countries, was studied by scanning electron and transmission electron microscopies (SEM/TEM) as well as by in situ attenuated total reflection infrared (ATR-IR) spectroscopy. The IR spectra showed that adhesive secretion began approximately 15 min after initial settlement and that the adhesive bulk material contained protein and anionic polysaccharides. Energy dispersive X-ray microanalysis of the adhesive identified sulphur and phosphorus as well as calcium and magnesium ions, which facilitate the gelation of the anionic polysaccharides in the sea water. The adhesive may be secreted from Golgi bodies in the spore, which were imaged by TEM of spore thin sections. Additionally, an in situ settlement study on TiO(2) particle film by ATR-IR spectroscopy revealed the presence of phosphorylated moieties directly binding the substrate. The presence of anionic groups dominating the adhesive suggests that inhibition of spore adhesion will be favoured by negatively charged surfaces.

Characterization of spore surfaces from a Geobacillus sp. isolate by pH dependence of surface charge and infrared spectra.

AIMS: The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials. MATERIALS AND RESULTS: The surfaces of spores were characterized using transmission electron microscopy and infrared spectroscopy. Thin sections of spores stained with ruthenium red revealed an exosporium with a hair-like nap around the spores. Attenuated total reflection infrared spectra of the spores exposed to different pH solutions on a ZnSe prism revealed that pH-sensitive carboxyl and phosphodiester groups associated with proteins and polysaccharides contributed to the spore's negative charge which was revealed by our previous zeta potential measurements on the spores. Lowering the pH to the isoelectric point of spores resulted in an increase in intensity of all spectral bands, indicating that the spores moved closer to the zinc selenide (ZnSe) surface as the charged surface groups were neutralized and the spore surface polymers compressed. The attachment of spores to stainless steel was threefold higher at pH 3 compared with pH 7. CONCLUSIONS: This research showed that spore attachment to surfaces is influenced by electrostatic interactions, surface polymer conformation and associated steric interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesion of thermophilic spores is largely controlled by functional groups of surface polymers and polymer conformation.

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

The ability of bacterial cells to sequester cations is well recognized, despite the fact that the specific binding sites and mechanistic details of the process are not well understood. To address these questions, the cation-exchange behavior of Pseudomonas aeruginosa PAO1 cells with a truncated lipopolysaccharide (LPS) (PAO1 wbpL) and cells further modified by growth in a magnesium-deficient medium (PAO1 wbpL - Mg(2+)) were compared with that of wild-type P. aeruginosa PAO1 cells. P. aeruginosa PAO1 cells had a negative surface charge (zeta potential) between pH 11 and 2.2, due to carboxylate groups present in the B-band LPS. The net charge on PAO1 wbpL cells was increasingly positive below pH 3.5, due to the influence of NH(3)(+) groups in the core LPS. The zeta potentials of these cells were also measured in Na(+), Ca(2+), and La(3+) electrolytes. Cells in the La(3+) electrolyte had a positive zeta potential at all pH values tested. Growing P. aeruginosa PAO1 wbpL in magnesium-deficient medium (PAO1 wbpL - Mg(2+)) resulted in an increase in its zeta potential in the pH range from 3.0 to 6.5. In cation-exchange experiments carried out at neutral pH with either P. aeruginosa PAO1 or PAO1 wbpL, the concentration of bound Ca(2+) was found to decrease as the pH was reduced from 7.0 to 3.5. At pH 3.5, the bound Mg(2+) concentration decreased sharply, revealing the activity of surface sites for cation exchange and their pH dependence. Infrared spectroscopy of attached biofilms suggested that carboxylate and phosphomonoester functional groups within the core LPS are involved in cation exchange.

A surface lipid may control the permeability slump associated with entry into anhydrobiosis in the plant parasitic nematode Ditylenchus dipsaci.

The anhydrobiotic plant-parasitic nematode Ditylenchus dipsaci undergoes a decrease in permeability (the permeability slump) during the early stages of desiccation and this produces the slow rate of water loss necessary for its survival. There were no changes in annulation spacing, followed in individual nematodes by confocal microscopy, that would account for the permeability slump. Nile Red staining reveals that the surface of the nematode is coated with an extracuticular layer of lipid. This material can be seen in unstained desiccated nematodes where it forms an oil that adheres to the coverslip and to adjacent nematodes. The oily material leaves impressions on the coverslip (cuticle prints) after the nematode has detached upon rehydration. The presence of the surface lipid was confirmed using attenuated total reflection infrared spectroscopy. This material was shown to be a triglyceride and the proportion of fatty acids determined, using thin layer and gas chromatography. The production of the surface lipid material may be responsible for the permeability slump observed during the early phases of desiccation and its removal upon immersion in water may explain the paradox that cuticular permeability decreases during the permeability slump and yet desiccated nematodes are more permeable than are fully hydrated nematodes.

Recovery of spores from thermophilic dairy bacilli and effects of their surface characteristics on attachment to different surfaces.

Spores from four Geobacillus spp. were isolated from a milk powder manufacturing line in New Zealand. Liquid sporulation media produced spore yields of approximately 10(7) spores ml(-1); spores were purified using a two-phase system created with polyethylene glycol 4000 and 3 M phosphate buffer. The zeta potentials of the spores from the four isolates ranged from -10 to -20 mV at neutral pH, with an isoelectric point between pH 3 and 4. Through contact angle measurements, spores were found to be hydrophilic and had relative hydrophobicity values of 10 to 40%, as measured by the microbial adhesion to hexadecane assay. The most hydrophilic spore isolate with the smallest negative charge attached in the highest numbers to Thermanox and stainless steel (1 x 10(4) spores cm(-2)), with fewer spores attaching to glass (3 x 10(3) spores cm(-2)). However, spores produced by the other three strains attached in similar numbers (P > 0.05) to all substrata (approximately 1 x 10(3) spores cm(-2)), indicating that there was no simple relationship between individual physicochemical interactions and spore adherence. Therefore, surface modifications which limit the attachment of one strain may not be effective for all stains, and control regimens need to be devised with reference to the characteristics of the particular strains of concern.

Adsorption of bovine serum albumin (BSA) onto lecithin studied by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy.

The adsorption of bovine serum albumin (BSA) to lecithin was investigated by ATR-FTIR spectroscopy. Lecithin films were prepared by casting aliquots of 3.2 microg lecithin in methanol onto ZnSe ATR prisms. Surface morphology and the thickness of the films were investigated by laser scanning confocal electron microscopy and scanning electron microscopy and the thickness of the films used for adsorption studies was estimated to be 40 A. The dependency of the CO peak area on the lecithin mass in the calibration curve confirms that the thickness of the film is below the penetration depth of the infrared evanescent wave. Size exclusion HPLC and fluorescence spectroscopy show that BSA conformation in up to 1M NaCl and CaCl(2) solutions is similar to that in water with no aggregation or changes in protein conformation seen over 4h. The kinetics of BSA adsorption on the lecithin film from water, NaCl and CaCl(2) solutions demonstrates that ions promote the protein adsorption. BSA bound more in the presence of NaCl compared to CaCl(2) at equivalent concentrations. The adsorption appeared greatest at a 0.1M concentration for both NaCl and CaCl(2). The results are explained in terms of absorptive reactivity of BSA and lecithin surfaces upon salt addition.

In situ infrared spectroscopic analysis of the adsorption of aromatic carboxylic acids to TiO2, ZrO2, Al2O3, and Ta2O5 from aqueous solutions.

In situ infrared spectroscopy has been used to investigate the adsorption of a range of simple aromatic carboxylic acids from aqueous solution to metal oxides. Thin films of TiO2, ZrO2, Al2O3 and Ta2O5 were prepared by evaporation of aqueous sols on single reflection ZnSe prisms. Benzoic acid adsorbed very strongly to ZrO2, in a bridging bidentate fashion, but showed only weak adsorption to TiO2 and Ta2O5. Substituted aromatic carboxylic acids; salicylic, phthalic and thiosalicylic, were found to adsorb to each metal oxide. Salicylic and phthalic acids adsorbed to the metal oxides via bidentate interactions, involving coordination through both carboxylate and substituent groups. Thiosalicylic acid adsorbed to the metal oxides as a bridging bidentate carboxylate with no coordination through the thiol substituent group.

Resonance Raman and infrared spectral studies on radical anions of model photosynthetic reaction center quinones (naphthoquinone derivatives).

Quinones play a vital role in the processes of electron transfer in bacterial photosynthetic reaction centers. It is of interest to investigate photochemical reactions involving quinones with a view to elucidate structure-function relationships in biological processes. Resonance Raman and FTIR spectra of electrochemically generated radical anions of 2-methyl-1,4-naphthoquinone, and 2-methyl-3-phytyl-1,4-naphthoquinone, also known as Vitamin K3 and Vitamin K1, respectively, (model compound for QA in Rhodopseudomonas viridis, a bacterial photosynthetic reaction center) have been reported. The same study has also been extended to 1,4-naphthoquinone for comparison. The vibrational assignments were carried out on the basis of comparison with our earlier time resolved resonance Raman studies on photochemically generated radical anions of 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (Balakrishnan et al., J. Phys. Chem., 100, (1996), 16472-16478). These in vitro results have been compared with the reported vibrational spectral data under in vivo conditions.