Since the advent of the use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) as a tool for microbial characterization, efforts to increase the taxonomic resolution of the approach have been made. The rapidity and efficacy of the approach have suggested applications in counter-bioterrorism, prevention of food contamination, and monitoring the spread of antibiotic-resistant bacteria. Strain-level resolution has been reported with diverse bacteria, using library-based and bioinformatics-enabled approaches. Three types of characterization at the strain level have been reported: strain categorization, strain differentiation, and strain identification. Efforts to enhance the library-based approach have involved sample pre-treatment and data reduction strategies. Bioinformatics approaches have leveraged the ever-increasing amount of publicly available genomic and proteomic data to attain strain-level characterization. Bioinformatics-enabled strategies have facilitated strain characterization via intact biomarker identification, bottom-up, and top-down approaches. Rigorous quantitative and advanced statistical analyses have fostered success at the strain level with both approaches. Library-based approaches can be limited by effects of sample preparation and culture conditions on reproducibility, whereas bioinformatics-enabled approaches are typically limited to bacteria, for which genetic and/or proteomic data are available. Biological molecules other than proteins produced in strain-specific manners, including lipids and lipopeptides, might represent other avenues by which strain-level resolution might be attained. Immunological and lectin-based chemistries have shown promise to enhance sensitivity and specificity. Whereas the limits of the taxonomic resolution of MALDI TOF MS profiling of bacteria appears bacterium-specific, recent data suggest that these limits might not yet have been reached.

Little is known about the interaction of biosurfactants with bacterial cells. Recent work in the area of biodegradation suggests that there are two mechanisms by which biosurfactants enhance the biodegradation of slightly soluble organic compounds. First, biosurfactants can solubilize hydrophobic compounds within micelle structures, effectively increasing the apparent aqueous solubility of the organic compound and its availability for uptake by a cell. Second, biosurfactants can cause the cell surface to become more hydrophobic, thereby increasing the association of the cell with the slightly soluble substrate. Since the second mechanism requires very low levels of added biosurfactant, it is the more intriguing of the two mechanisms from the perspective of enhancing the biodegradation process. This is because, in practical terms, addition of low levels of biosurfactants will be more cost-effective for bioremediation. To successfully optimize the use of biosurfactants in the bioremediation process, their effect on cell surfaces must be understood. We report here that rhamnolipid biosurfactant causes the cell surface of Pseudomonas spp. to become hydrophobic through release of lipopolysaccharide (LPS). In this study, two Pseudomonas aeruginosa strains were grown on glucose and hexadecane to investigate the chemical and structural changes that occur in the presence of a rhamnolipid biosurfactant. Results showed that rhamnolipids caused an overall loss in cellular fatty acid content. Loss of fatty acids was due to release of LPS from the outer membrane, as demonstrated by 2-keto-3-deoxyoctonic acid and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and further confirmed by scanning electron microscopy. The amount of LPS loss was found to be dependent on rhamnolipid concentration, but significant loss occurred even at concentrations less than the critical micelle concentration. We conclude that rhamnolipid-induced LPS release is the probable mechanism of enhanced cell surface hydrophobicity.Cell surface properties result from the unique chemical structure of the cell surface. In the case of a gram-negative bacterium such as Pseudomonas aeruginosa, this structure is the outer membrane, the outer leaflet of which is primarily composed of lipopolysaccharide (LPS). The outer leaflet contains LPS molecules which are composed of three components (25). The first is the lipid A tail which is anchored into the hydrophobic region of the outer membrane. The second is the core oligosaccharide, which contains a unique eight-carbon sugar called 2-keto-3-deoxyoctonic acid (KDO). The core oligosaccharide is connected to the lipid A tail through its reducing end and is positioned at the surface of the membrane in a manner analogous to the glycerol-phosphate head group of a phospholipid. The core oligosaccharide is negatively charged, and the association of adjacent LPS molecules is stabilized by Mg 2ϩ ions at the membrane surface. The third component of LPS is the O antigen which consists of 15 to 20 repeating mon...

Forty percent of hazardous waste sites in the United States are co-contaminated with organic and metal pollutants. Data from both aerobic and anaerobic systems demonstrate that biodegradation of the organic component can be reduced by metal toxicity. Metal bioavailability, determined primarily by medium composition/soil type and pH, governs the extent to which metals affect biodegradation. Failure to consider bioavailability rather than total metal likely accounts for much of the enormous variability among reports of inhibitory concentrations of metals. Metals appear to affect organic biodegradation through impacting both the physiology and ecology of organic degrading microorganisms. Recent approaches to increasing organic biodegradation in the presence of metals involve reduction of metal bioavailability and include the use of metal-resistant bacteria, treatment additives, and clay minerals. The addition of divalent cations and adjustment of pH are additional strategies currently under investigation.

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A model cocontaminated system was developed to determine whether a metal-complexing biosurfactant, rhamnolipid, could reduce metal toxicity to allow enhanced organic biodegradation by a Burkholderia sp. isolated from soil. Rhamnolipid eliminated cadmium toxicity when added at a 10-fold greater concentration than cadmium (890 M), reduced toxicity when added at an equimolar concentration (89 M), and had no effect at a 10-fold smaller concentration (8.9 M). The mechanism by which rhamnolipid reduces metal toxicity may involve a combination of rhamnolipid complexation of cadmium and rhamnolipid interaction with the cell surface to alter cadmium uptake.Forty percent of hazardous waste sites on the U.S. Environmental Protection Agency's National Priority List are cocontaminated with organic and metal pollutants. Previous studies have shown that biodegradation of organic contaminants is often severely inhibited by toxic metals, such as cadmium (19,20). Increasing interest in bioremediation warrants development of strategies that can be successfully implemented in cocontaminated sites, yet few efforts have been made to develop such strategies. Effective strategies to enhance organic biodegradation in the presence of toxic metals include reducing the bioavailable concentration of the toxic metal and reducing interactions of the toxic metal with the cell.Attempts to reduce bioavailable metal concentrations in cocontaminated soils have included amendment with kaolinite and montmorillonite clays (2, 3, 11), wherein reductions in metal toxicity were observed. Recently, modified clay complexes (metal-chelating ligands bound to clay particles via a cationic surfactant) and a chelating resin (Chelex) were found to reduce cadmium toxicity during biodegradation of naphthalene by Pseudomonas putida ppo200 (14). Reductions in toxicity were assumed to be related to the metal-complexing characteristics of both the modified clay and the resin, despite the fact that metal chelators, such as EDTA, can alter cell surface properties through the release of lipopolysaccharide (LPS) (5)(6)(7)12). Because LPS confers a considerable negative charge upon the cell surface (18) which favors electrostatic interactions with cations, removal of LPS may reduce the magnitude of the negativity of the cell surface charge, thus reducing interactions with cations, such as cadmium. The mechanism by which metal-chelating agents reduce toxicity clearly warrants further exploration.We have previously studied a rhamnolipid biosurfactant produced by various Pseudomonas aeruginosa strains capable of selectively complexing cationic metal species, such as cadmium (Cd 2ϩ ), lead (Pb 2ϩ ), and zinc (Zn 2ϩ ) (8,17,21,22), increasing the bioavailability of substrates with limited aqueous solubilities (9, 24-27), and increasing cell surface hydrophobicity (1, 26). Delivery of a biosurfactant into cocontaminated sites for in situ treatment may be more environmentally compatible and more economical than using modified clay complexes or metal chelators, such as EDTA. For ...