Engineering polyphosphate metabolism in Escherichia coli: implications for bioremediation of inorganic contaminants

Polyphosphate metabolism plays an important role in the bioremediation of phosphate contamination in municipal wastewater, and may play a key role in heavy metal tolerance and bioremediation. However, little is known about the regulation of polyphosphate metabolism in microorganisms and its role in heavy metal toxicity. We have manipulated polyphosphate metabolism in Escherichia coli by overexpressing the genes for polyphosphate kinase (ppk) and for polyphosphatase (ppx) under control of their native promoters and inducible promoters. Overexpression of ppk results in high levels of intracellular polyphosphate, improved phosphate uptake, but no increase in tolerance to heavy metals. Overexpression of both ppk and ppx results in lower levels of intracellular polyphosphate, secretion of phosphate from the cell, and increased tolerance to heavy metals. Metabolic flux analysis indicates that the cell responds to increased flux through the PPK-PPX pathway by altering flux through the TCA cycle.     

Conversion of corn milling fibrous co-products into ethanol by recombinant Escherichia coli strains K011 and SL40

Corn hulls and corn germ meal were both evaluated as feedstocks for production of ethanol for biofuel. Currently, these fibrous co-products are combined with corn steep liquor and the fermentation bottoms (if available) and marketed as cattle feed. Samples were obtained from wet and dry corn mills. The corn hulls and germ meal were evaluated for starch and hemicellulose compositions. Starch contents were 12 to 32% w/w and hemicellulose (arabinoxylans) contents were 23 to 64% w/w. Corn fibrous samples were hydrolysed, using dilute sulphuric acid, into mixed sugar streams containing arabinose, glucose and xylose. Total sugar concentrations in the hydrolysate varied from 8.4 to 10.8% w/v. The hydrolysates were fermented to ethanol using recombinant E. coli strains K011 and SL40. Ethanol yields were 0.38 to 0.41g ethanol produced/g total sugars consumed and fermentations were completed in 60h or less. However, residual xylose was detected for each hydrolysate fermentation and was especially significant for fermentations using strain SL40. Strain K011 was a superior ethanologenic strain compared with strain SL40 in terms of both ethanol yield and maximum productivity.     

Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH(2)-Gly(3) substrates

Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. We find that purified recombinant sortase hydrolyzed peptides bearing an LPXTG motif at the peptide bond between threonine and glycine. In the presence of NH(2)-Gly(3), sortase catalyzed exclusively a transpeptidation reaction, linking the carboxyl group of threonine to the amino group of NH(2)-Gly(3). In the presence of amino group donors the rate of sortase mediated cleavage at the LPXTG motif was increased. Hydrolysis and transpeptidation required the sulfhydryl of cysteine 184, suggesting that sortase catalyzed the transpeptidation reaction of surface protein anchoring via the formation of a thioester acyl-enzyme intermediate.     

A survey of yeast from the Yarrowia clade for lipid production in dilute acid pretreated lignocellulosic biomass hydrolysate

Applied Microbiology and Biotechnology - Yarrowia lipolytica is an oleaginous yeast species that has attracted attention as a model organism for synthesis of single cell oil. Among over 50 isolates...     

Protective effect of wogonin on endotoxin-induced lethal shock in D-galactosamine-sensitized mice.

The effects of wogonin, a major flavonoid from Scutellaria baicalensis Georgi, on lipopolysaccharide (LPS)-induced lethal shock in mice was investigated. Wogonin pretreatment prevented the lethal shock in mice injected with D-galactosamine (D-GalN) and LPS, but not in mice injected with a high dose of LPS. Wogonin definitely inhibited the hepatic injury in mice injected with D-GalN, and LPS and reduced the level of circulating tumor necrosis factor (TNF)-alpha. The reduction was more marked in mice injected with D-GalN and LPS compared with that in mice injected with a high dose of LPS. Wogonin pretreatment did not inhibit the lipid peroxidation in mice receiving either D-GalN and LPS or a high dose of LPS. Wogonin inhibited the in vitro production of TNF-alpha and nitric oxide in LPS-stimulated RAW 264.7 cells. The mechanism of the protective effect of wogonin on the lethal shock in mice injected with D-GalN and LPS is discussed.     

Impact of Harvest Time and Switchgrass Cultivar on Sugar Release Through Enzymatic Hydrolysis

Switchgrass (Panicum virgatum L.) is a native North American prairie grass being developed for bioenergy production in the central and eastern USA. The objective of this study was to identify the impacts of harvest time and switchgrass cultivar had on sugar release variables determined through enzymatic hydrolysis. Previously, we reported that delaying harvest of switchgrass until after frost and until after winter resulted in decreased yields of switchgrass but it reduced the amount of ash and nutrients in the biomass. The current study used near-infrared reflectance spectroscopy (NIRS) to broaden an existing set of calibration equations designed to predict composition and sugar release variables of switchgrass. These updated calibrations were then applied to the full set of samples from a multi-year and multi-location switchgrass harvest-management study. Composition and processor sugar yields were significantly affected by location, year, cultivar, and harvest time, of which the time of harvest was the most important. Delaying the time of harvest until after frost or post-winter increased the concentration of structural carbohydrates from 500 to over 570 g kg^?1 in the biomass and lignin content from 160 to over 200 g kg^?1. Conversely, delaying harvest time lowered the amounts of ash and soluble sugars. The later harvest times also yielded more sugars following processing with yields increasing over 20% from the first harvest. Increased sugar yields are attributable to both increased concentration of sugars in the biomass upon harvest and reduced biomass recalcitrance. Based upon processed sugar yields, it is estimated that a biorefinery producing 76 million liters of ethanol per year would require 229–373 km² of land cultivated with switchgrass.     

Anchor structure of staphylococcal surface proteins. IV. Inhibitors of the cell wall sorting reaction.

Surface proteins of Staphylococcus aureus are covalently linked to the bacterial cell wall by a mechanism requiring a COOH-terminal sorting signal with a conserved LPXTG motif. Cleavage between the threonine and the glycine of the LPXTG motif liberates the carboxyl of threonine to form an amide bond with the amino of the pentaglycine cross-bridge in the staphylococcal peptidoglycan. We asked whether antibiotic cell wall synthesis inhibitors interfere with the anchoring of surface proteins. Penicillin G, a transpeptidation inhibitor, had no effect on surface protein anchoring, whereas vancomycin and moenomycin, inhibitors of cell wall polymerization into peptidoglycan strands, slowed the sorting reaction. Cleavage of surface protein precursors did not require a mature assembled cell wall and was observed in staphylococcal protoplasts. A search for chemical inhibitors of the sorting reaction identified methanethiosulfonates and p-hydroxymercuribenzoic acid. Thus, sortase, the enzyme proposed to cleave surface proteins at the LPXTG motif, appears to be a sulfhydryl-containing enzyme that utilizes peptidoglycan precursors but not an assembled cell wall as a substrate for the anchoring of surface protein.     

Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-alanyl-glycine endopeptidase activity.

Bacteriophage muralytic enzymes degrade the cell wall envelope of staphylococci to release phage particles from the bacterial cytoplasm. Murein hydrolases of staphylococcal phages phi11, 80alpha, 187, Twort, and phiPVL harbor a central domain that displays sequence homology to known N-acetylmuramyl-L-alanyl amidases; however, their precise cleavage sites on the staphylococcal peptidoglycan have thus far not been determined. Here we examined the properties of the phi11 enzyme to hydrolyze either the staphylococcal cell wall or purified cell wall anchor structures attached to surface protein. Our results show that the phi11 enzyme has D-alanyl-glycyl endopeptidase as well as N-acetylmuramyl-L-alanyl amidase activity. Analysis of a deletion mutant lacking the amidase-homologous sequence, phi11(Delta181-381), revealed that the D-alanyl-glycyl endopeptidase activity is contained within the N-terminal 180 amino acid residues of the polypeptide chain. Sequences similar to this N-terminal domain are found in the murein hydrolases of staphylococcal phages but not in those of phages that infect other Gram-positive bacteria such as Listeria or Bacillus.