Timeline: Neurospora: a model of model microbes

In the 1940s, studies with Neurospora pioneered the use of microorganisms in genetic analysis and provided the foundations for biochemical genetics and molecular biology. What has happened since this orange mould was used to show that genes control metabolic reactions? How did it come to be the fungal counterpart of Drosophila? We describe its continued use during the heyday of research with Escherichia coli and yeast, and its emergence as a biological model for higher fungi.     

A thermodynamic model describing the nature of the crista junction: a structural motif in the mitochondrion

The use of electron tomography has allowed the three-dimensional membrane topography of the mitochondrion to be better understood. The most striking feature of this topology is the crista junction, a structure that may serve to divide functionally the inner membrane and intermembrane spaces. In situ these junctions seem to have a preferred size and shape independent of the source of the mitochondrion with few exceptions. When mitochondria are isolated and have a condensed matrix the crista junctions enlarge and become nondiscrete. Upon permeation of the inner membrane and subsequent swelling of the matrix space, the uniform circular nature of the crista junction reappears. We examine the distribution of shapes and sizes of crista junctions and suggest a thermodynamic model that explains the distribution based on current theories of bilayer membrane shapes. The theory of spontaneous curvature shows the circular junction to be a thermodynamically stable structure whose size and shape is influenced by the relative volume of the matrix. We conclude that the crista junction exists predominantly as a circular junction, with other shapes as exceptions made possible by specific characteristics of the lipid bilayer.     

Evaluation of biotracers to monitor effluent retention time in constructed wetlands

AIMS: With concern surrounding the environmental impact of chemical tracers on the aquatic environment, this paper presents the initial evaluation of biotracers used to determine the effluent retention time, an important performance indicator, in a Free Water Surface Constructed Wetland. METHODS AND RESULTS: Production of the biotracers, coliphage MS2, and the bacteriophage of Enterobacter cloacae and antibiotic resistant endospores of Bacillus globigii is described in detail. Their subsequent use in three separate tracer experiments - January, March and June (2000) - revealed the variability of retention time with respect to effluent flow. The biotracer MS2 showed the constructed wetland had a retention time of 8-9 h at a mean discharge of 0.9 l s-1, increasing to 10-12 h at a mean discharge 0.3 l s-1. A similar retention of 9-10 h at a mean discharge of 0.3 l s-1 was calculated for the Ent. cloacae phage. In contrast, use of endospores revealed considerably longer retention times at these mean discharge rates; 12-24 h and 36-48 h, respectively. CONCLUSION: Biotracers could provide a useful and environmentally friendly technique to monitor effluent retention in constructed wetlands. At this stage the phage tracers appear particularly promising due to ease of isolation and recovery. SIGNIFICANCE AND IMPACT OF THE STUDY: Initial results are encouraging and have highlighted the potential of biotracers as alternatives to chemical tracers, even in microbially-rich waters.     

Structure-activity relationships of the peptide deformylase inhibitor BB-3497: modification of the methylene spacer and the P1' side chain

Structural modifications to the peptide deformylase inhibitor BB-3497 are described. In this paper, we describe the initial SAR around this lead for modifications to the methylene spacer and the P1' side chain. Enzyme inhibition and antibacterial activity data revealed that the optimum distance between the N-formyl hydroxylamine metal binding group and the P1' side chain is one unsubstituted methylene unit. Additionally, lipophilic P1' side chains that closely mimic the methionine residue in the substrate provided compounds with the best microbiological profile.     

Human monoclonal antibodies that inhibit binding of hepatitis C virus E2 protein to CD81 and recognize conserved conformational epitopes

The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.     

Clumping factor B (ClfB), a new surface-located fibrinogen-binding adhesin of Staphylococcus aureus

The surface-located fibrinogen-binding protein (clumping factor; ClfA) of Staphylococcus aureus has an unusual dipeptide repeat linking the ligand binding domain to the wall-anchored region. Southern blotting experiments revealed several other loci in the S. aureus Newman genome that hybridized to a probe comprising DNA encoding the dipeptide repeat. One of these loci is analysed here. It also encodes a fibrinogen-binding protein, which we have called ClfB. The overall organization of ClfB is very similar to that of ClfA, and the proteins have considerable sequence identity in the signal sequence and wall attachment domains. However, the A regions are only 26% identical. Recombinant biotinylated ClfB protein bound to fibrinogen in Western ligand blots. ClfB reacted with the α- and β-chains of fibrinogen in the ligand blots in contrast to ClfA, which binds exclusively to the γ-chain. Analysis of proteins released from the cell wall of S. aureus Newman by Western immunoblotting using antibody raised against the recombinant A region of ClfB identified a 124 kDa protein as the clfB gene product. This protein was detectable only on cells that were grown to the early exponential phase. It was absent from cells from late exponential phase or stationary phase cultures. Using a clfB mutant isolated by allelic replacement alone and in combination with a clfA mutation, the ClfB protein was shown to promote (i) clumping of exponential-phase cells in a solution of fibrinogen, (ii) adherence of exponential-phase bacteria to immobilized fibrinogen in vitro, and (iii) bacterial adherence to ex vivo human haemodialysis tubing, suggesting that it could contribute to the pathogenicity of biomaterial-related infections. However, in wild-type exponential-phase S. aureus Newman cultures, ClfB activity was masked by the ClfA protein, and it did not contribute at all to interactions of cells from stationary-phase cultures with fibrinogen. ClfB-dependent bacterial adherence to immobilized fibrinogen was inhibited by millimolar concentrations of Ca²⁺ and Mn²⁺, which indicates that, like ClfA, ligand binding by ClfB is regulated by a low-affinity inhibitory cation binding site.