Proteomic profiling of cell envelope-associated proteins from Staphylococcus aureus

The emergence of highly virulent community acquired Staphylococcus aureus and continued progression of resistance to multiple antimicrobials, including methicillin and vancomycin, marks the reemergence of S. aureus as a serious health care threat. Investigation of proteins localized to the cell surface could help to elucidate mechanisms of virulence and antibiotic resistance in S. aureus. In this study, proteomic profiling methods were developed to solubilize, display, and evaluate abundance levels of proteins present in the supernatants of the lysostaphin-digested cell envelope from cultured vancomycin-intermediate S. aureus (VISA) cells. Combining approaches of 2-DE or chromatographic separation of proteins with MS analyses resulted in the identification of 144 proteins of particular interest. Of these proteins, 48 contained predicted cell wall localization or export signal motifs, including 14 with distinct covalent peptidoglycan-anchor sites, four of which are uncharacterized to date. One of the two most abundant cell envelope proteins, which showed remarkably high variations in MW and pI in the 2-DE gel display, was the S. aureus surface protein G. The display of numerous secreted proteins that are not covalently cell wall-anchored, suggests that, in the exponential growth phase, secreted proteins can be retained physiologically in the cell envelope and may interact with cell wall-anchored proteins and carbohydrate structures in a manner yet to be determined. The remaining 96 proteins, devoid of recognizable motifs, were repeatedly profiled in the VISA cell envelope fractions. We describe a novel semiquantitative method to determine abundance factors of such proteins in 2-DE gels of cell envelope fractions relative to whole cell lysates and discuss these data in the context of true cell envelope localization versus experimentally caused cell lysis.     

Generation of novel-substrate-accepting biphenyl dioxygenases through segmental random mutagenesis and identification of residues involved in enzyme specificity

Aryl-hydroxylating dioxygenases are of interest for the degradation of persistant aromatic pollutants, such as polychlorobiphenyls (PCBs), or as catalysts for the functionalization of aromatic scaffolds. In order to achieve dioxygenation of technical mixtures of PCBs, enzymes with broadened or altered substrate ranges are essential. To alter the substrate specificity of the biphenyl dioxygenase (BphA) of Burkholderia xenovorans LB400, we applied a directed evolution approach that used structure-function relationship data to target random mutageneses to specific segments of the enzyme. The limitation of random amino acid (AA) substitutions to regions that are critical for substrate binding and the exclusion of AA exchanges from positions that are essential for catalytic activity yielded enzyme variants of interest at comparatively high frequencies. After only a single mutagenic cycle, 10 beneficial variants were detected in a library of fewer than 1,000 active enzymes. Compared to the parental BphA, they showed between 5- and 200-fold increased turnover of chlorinated biphenyls, with substituent patterns that rendered them largely recalcitrant to attack by BphA-LB400. Determination of their sequences identified AAs that prevent the acceptance of specific PCBs by the wild-type enzyme, such as Pro334 and Phe384. The results suggest prime targets for subsequent cycles of BphA modification. Correlations with a three-dimensional model of the enzyme indicated that most of the exchanges with major influence on substrate turnover do not involve pocket-lining residues and had not been predictable through structural modeling.     

Distinct properties of the five UDP-D-glucose/UDP-D-galactose 4-epimerase isoforms of Arabidopsis thaliana

Plant genomes contain genetically encoded isoforms of most nucleotide sugar interconversion enzymes. Here we show that Arabidopsis thaliana has five genes encoding functional UDP-D-glucose/UDP-D-galactose 4-epimerase (named UGE1 to UGE5). All A. thaliana UDP-d-glucose 4-epimerase isoforms are dimeric in solution, maximally active in vitro at 30-40 degrees C, and show good activity between pH 7 and pH 9. In vitro, UGE1, -3, and -5 act independently of externally added NAD+, whereas cofactor addition stimulates the activity of UGE2 and is particularly important for UGE4 activity. UGE1 and UGE3 are most efficiently inhibited by UDP. The five isoforms display kcatUDP-Gal values between 23 and 128 s(-1) and KmUDP-Gal values between 0.1 and 0.3 mm. This results in enzymatic efficiencies ranging between 97 and 890 mm(-1) s(-1) for UGE4 = UGE1 < UGE3 < UGE5 < UGE2. The KmUDP-Glc values, derived from the Haldane relationship, were 0.76 mm for UGE1, 0.56 mm for UGE4, and between 0.13 and 0.23 mm for UGE2, -3, and -5. The expression of UGE isoforms is ubiquitous and displays developmental and cell type-dependent variations. UGE1 and -3 expression patterns globally resemble enzymes involved in carbohydrate catabolism, and UGE2, -4, and -5 expression is more related to carbohydrate biosynthesis. UGE1, -2, and -4 are present in the cytoplasm, whereasUGE4 is additionally enriched close to Golgi stacks. All UGE genes tested complement the UGE4rhd1 phenotype, confer increased galactose tolerance in planta, and complement the galactose metabolization deficiency in the Saccharomyces cerevisiae gal10 mutant. We suggest that plant UGE isoforms function in different metabolic situations and that enzymatic properties, gene expression pattern, and subcellular localization contribute to the differentiation of isoform function.     

Rapidly in situ-forming degradable hydrogels from dextran thiols through Michael addition

Thiol-functionalized dextrans (dex-SH) (M(n,dextran) = 14K or 31K) with degrees of substitution (DS) ranging from 12 to 25 were synthesized and investigated for in situ hydrogel formation via Michael type addition using poly(ethylene glycol) tetra-acrylate (PEG-4-Acr) or a dextran vinyl sulfone conjugate with DS 10 (dex-VS DS 10). Dex-SH was prepared by activation of the hydroxyl groups of dextran with 4-nitrophenyl chloroformate and subsequent reaction with cysteamine. Hydrogels were rapidly formed in situ under physiological conditions upon mixing aqueous solutions of dex-SH and either PEG-4-Acr or dex-VS DS 10 at polymer concentrations of 10 to 20 w/v%. Rheological studies showed that these hydrogels are highly elastic. By varying the DS, concentration, dextran molecular weight, and type of cross-linker, hydrogels with a broad range of storage moduli of 9 to 100 kPa could be obtained. Varying the ratio of thiol to vinyl sulfone groups from 0.9 to 1.1 did not alter the storage modulus of the hydrogels, whereas larger deviations from equimolarity (thiol to vinyl sulfone ratios of 0.75 and 1.5) considerably decreased the storage modulus. The plateau value of hydrogel storage modulus was reached much faster at pH 7.4 compared to pH 7, due to a higher concentration of the thiolate anion at higher pH. These hydrogels were degradable under physiological conditions. Degradation times were 3 to 7 weeks for dex-SH/dex-VS DS 10 hydrogels and 7 to over 21 weeks for dex-SH/PEG-4-Acr hydrogels, depending on the DS, concentration, and dextran molecular weight.     

Neuroendocrine and behavioral responses to weak electric fields in adult sea lampreys (Petromyzon marinus)

We characterized the behavioral and neuroendocrine responses of adult sea lampreys (Petromyzon marinus) to weak electric fields. Adult sea lampreys, captured during upstream spawning migration, exhibited limited active behaviors during exposure to weak electric fields and spent the most time attached to the wall of the testing arena near the cathode (−). For adult male sea lampreys, exposure to weak electric fields resulted in increased lamprey (l) GnRH-I mRNA expression but decreased lGnRH-I immunoreactivities in the forebrain, and decreased Jun (a neuronal activation marker) mRNA levels in the brain stem. Similar effects were not observed in the brains of female sea lampreys after weak electric field stimulation. The influence of electroreception on forebrain lGnRH suggests that electroreception may modulate the reproductive systems in adult male sea lampreys. The changes in Jun expression may be associated with swimming inhibition during weak electric field stimulation. The results for adult sea lampreys are the opposite of those obtained using parasitic-stage sea lampreys, which displayed increased activity during and after cathodal stimulation. Our results demonstrate that adult sea lampreys are sensitive to weak electric fields, which may play a role in reproduction. They also suggest that electrical stimuli mediate different behaviors in feeding-stage and spawning-stage sea lampreys.     

Examining the dynamic energy landscape of an antiporter upon inhibitor binding

Previously, we applied single-molecule force spectroscopy to detect and locate interactions within the functional Na⁺/H⁺ antiporter NhaA from Escherichia coli. It was observed that the binding of the inhibitor 2-aminoperimidine established interactions different from those introduced by the binding of the native ligand. To understand the inhibitory mechanism of the inhibitor, we applied single-molecule dynamic force spectroscopy to reconstruct the energy landscape of NhaA. Dynamic force spectroscopy revealed that the energy landscape of the antiporter remained mainly unchanged except for the energy barrier of the functionally important transmembrane α-helix IX. Inhibitor binding set this domain into a newly formed deep and narrow energy minimum that kinetically stabilized α-helix IX and reduced its conformational entropy. The entropy reduction of α-helix IX is thought to inhibit its functionally important structural flexibility, while the deeper energy barrier shifted the population of active antiporters towards inhibited antiporters.