452 research outputs found
The Method of Adaptive Constrained Descent
Coordinated Science Laboratory changed its name from Control Systems LaboratoryContract DA-36-039-SC-8512
A Constraint Mapping Technique for System Organization
Coordinated Science Laboratory changed its name from Control Systems LaboratoryContract DA-36-039-SC-8512
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A mutant Escherichia coli that attaches peptidoglycan to lipopolysaccharide and displays cell wall on its surface
The lipopolysaccharide (LPS) forms the surface-exposed leaflet of the outer membrane (OM) of Gram-negative bacteria, an organelle that shields the underlying peptidoglycan (PG) cell wall. Both LPS and PG are essential cell envelope components that are synthesized independently and assembled by dedicated transenvelope multiprotein complexes. We have identified a point-mutation in the gene for O-antigen ligase (WaaL) in Escherichia coli that causes LPS to be modified with PG subunits, intersecting these two pathways. Synthesis of the PG-modified LPS (LPS*) requires ready access to the small PG precursor pool but does not weaken cell wall integrity, challenging models of precursor sequestration at PG assembly machinery. LPS* is efficiently transported to the cell surface without impairing OM function. Because LPS* contains the canonical vancomycin binding site, these surface-exposed molecules confer increased vancomycin-resistance by functioning as molecular decoys that titrate the antibiotic away from its intracellular target. This unexpected LPS glycosylation fuses two potent pathogen-associated molecular patterns (PAMPs). DOI: http://dx.doi.org/10.7554/eLife.05334.00
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Non-Proteinogenic Amino Acids in Lacticin 481 Analogues Result in More Potent Inhibition of Peptidoglycan Transglycosylation
Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain the thioether structures lanthionine and methyllanthionine and exert potent antimicrobial activity against Gram-positive bacteria. At present, detailed modes-of-action are only known for a small subset of family members. Lacticin 481, a tricyclic lantibiotic, contains a lipid II binding motif present in related compounds such as mersacidin and nukacin ISK-1. Here, we show that lacticin 481 inhibits PBP1b-catalyzed peptidoglycan formation. Furthermore, we show that changes in potency of analogues of lacticin 481 containing non-proteinogenic amino acids correlate positively with the potency of inhibition of the transglycosylase activity of PBP1b. Thus, lipid II is the likely target of lacticin 481, and use of non-proteinogenic amino acids resulted in stronger inhibition of the target. Additionally, we demonstrate that lacticin 481 does not form pores in the membranes of susceptible bacteria, a common mode-of-action of other lantibiotics.Chemistry and Chemical Biolog
Global citizens: Who are they?
A growing desire to instigate global citizenship programmes in Higher Education (HE) has led to the development of optional structured opportunities for students to engage in prosocial activities. One of the challenges facing such programmes is to demonstrate and plan for the personal growth of those students. This paper reports the dispositional, prosocial and attitudinal characteristics; knowledge and skills; and perceptions of social justice that students who undertake these activities bring to their initial participation. The findings indicate, that in comparison to a control group, the students differ significantly in a number of important ways (e.g. conscientiousness, extraversion, openness; Machiavellianism, prosocial behaviour; self-esteem; skills relating to social action and tolerance and understanding and their concern regarding social problems). However, consideration should be given to the ways in which those students can be developed within a framework for social justice. Further, recruitment procedures for citizenship programmes in general should encourage the participation of a more diverse group of students than currently appears to be the case
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D-Amino Acids Indirectly Inhibit Biofilm Formation in Bacillus subtilis by Interfering with Protein Synthesis
The soil bacterium Bacillus subtilis forms biofilms on surfaces and at air-liquid interfaces. It was previously reported that these biofilms disassemble late in their life cycle and that conditioned medium from late-stage biofilms inhibits biofilm formation. Such medium contained a mixture of d-leucine, d-methionine, d-tryptophan, and d-tyrosine and was reported to inhibit biofilm formation via the incorporation of these d-amino acids into the cell wall. Here, we show that l-amino acids were able to specifically reverse the inhibitory effects of their cognate d-amino acids. We also show that d-amino acids inhibited growth and the expression of biofilm matrix genes at concentrations that inhibit biofilm formation. Finally, we report that the strain routinely used to study biofilm formation has a mutation in the gene (dtd) encoding d-tyrosyl-tRNA deacylase, an enzyme that prevents the misincorporation of d-amino acids into protein in B. subtilis. When we repaired the dtd gene, B. subtilis became resistant to the biofilm-inhibitory effects of d-amino acids without losing the ability to incorporate at least one noncanonical d-amino acid, d-tryptophan, into the peptidoglycan peptide side chain. We conclude that the susceptibility of B. subtilis to the biofilm-inhibitory effects of d-amino acids is largely, if not entirely, due to their toxic effects on protein synthesis.Chemistry and Chemical Biolog
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Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function
ABSTRACT The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction
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The Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains
Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components.Chemistry and Chemical Biolog
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Reconstitution of Peptidoglycan Cross-Linking Leads to Improved Fluorescent Probes of Cell Wall Synthesis
The peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available. Here we report the synthesis of B. subtilis Lipid II and its use by purified B. subtilis PBP1 and E. coli PBP1A. While enzymes from both organisms assembled B. subtilis Lipid II into glycan strands, only the B. subtilis enzyme cross-linked the strands. Furthermore, B. subtilis PBP1 catalyzed the exchange of both d-amino acids and d-amino carboxamides into nascent peptidoglycan, but the E. coli enzyme only exchanged d-amino acids. We exploited these observations to design a fluorescent d-amino carboxamide probe to label B. subtilis PG in vivo and found that this probe labels the cell wall dramatically better than existing reagents
Toward a Prostate Specific Antigen-Based Prostate Cancer Diagnostic Assay: Preparation of Keyhole Limpet Hemocyanin -Conjugated Normal and Transformed Prostate Specific Antigen Fragments
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