Structural and biophysical characterization of the secreted, β-helical adhesin EtpA of Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a diarrhoeal pathogen associated with high morbidity and mortality especially among young children in developing countries. At present, there is no vaccine for ETEC. One candidate vaccine antigen, EtpA, is a conserved secreted adhesin that binds to the tips of flagellae to bridge ETEC to host intestinal glycans.

Elucidation of repeat motifs R1- and R2-related TRIOBP variants in autosomal recessive nonsyndromic hearing loss DFNB28 among indigenous South African individuals.

Background: DFNB28, a recessively inherited nonsyndromic form of deafness in humans, is caused by mutations in the TRIOBP gene (MIM #609761) on chromosome 22q13. Its protein TRIOBP helps to tightly bundle F-actin filaments, forming a rootlet that penetrates through the cuticular plate into the cochlear hair cell body. Repeat motifs R1 and R2, located in exon 7 of the TRIOBP-5 isoform, are the actin-binding domains.

Structural and biophysical characterization of the multidomain xylanase Xyl.

The depletion of fossil fuels, associated pollution, and resulting health hazards are of concern worldwide. Woody biomass constitutes an alternative source of cleaner and renewable energy. The efficient use of woody biomass depends on xylan depolymerisation as the endo-β-1,4-xylopyranosyl homopolymer is the main component of hemicellulose, the second most abundant component of wood.

Spectrum of Mutations in an Indigenous South African Population Further Elucidates the Nonsyndromic Autosomal Recessive Phenotype of DFNB2 to Include Both Homozygous and Compound Heterozygous Mutations.

gene encodes unconventional myosin VIIA, which, when mutated, causes a phenotypic spectrum ranging from recessive hearing loss DFNB2 to deaf-blindness, Usher Type 1B (USH1B). mutations are reported in nine DFNB2 families to date, none from sub-Saharan Africa.In DNA, from a cohort of 94 individuals representing 92 families from the Limpopo province of South Africa, eight variations were detected among 10 individuals.

Characterization and homology modelling of a novel multi-modular and multi-functional Paenibacillus mucilaginosus glycoside hydrolase.

Glycoside hydrolases, particularly cellulases, xylanases and mannanases, are essential for the depolymerisation of lignocellulosic substrates in various industrial bio-processes. In the present study, a novel glycoside hydrolase from Paenibacillus mucilaginosus (PmGH) was expressed in E. coli, purified and characterised.

Structural Characterization and Directed Evolution of a Novel Acetyl Xylan Esterase Reveals Thermostability Determinants of the Carbohydrate Esterase 7 Family.

A hot desert hypolith metagenomic DNA sequence data set was screened for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded an ∼36-kDa protein (Axe1). The synthesized gene was cloned and expressed, and the resulting protein was purified.

Resistance related metabolic pathways for drug target identification in Mycobacterium tuberculosis.

Background: Increasing resistance to anti-tuberculosis drugs has driven the need for developing new drugs. Resources such as the tropical disease research (TDR) target database and AssessDrugTarget can help to prioritize putative drug targets. Hower, these resources do not necessarily map to metabolic pathways and the targets are not involved in dormancy.

Expression and production of soluble H37Rv mycosin-3.

Mycobacteria encode five type VII secretion system (T7SS) or ESX for nutrient acquisition and virulence. Mycosins are membrane-anchored components of ESX with serine protease activity but an unidentified substrate range. Establishing the substrate specificity of individual mycosins will help to elucidate individual ESX functions.

Assessing the progress of Mycobacterium tuberculosis H37Rv structural genomics.

Tuberculosis threatens human health nowhere more than in developing countries with large malnourished and/or immune-compromised (e.g. HIV infected) populations.

Structure and functional characterization of pyruvate decarboxylase from Gluconacetobacter diazotrophicus.

Background: Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC has been produced successfully in a range of heterologous hosts.

Molecular mechanism of protrusion formation during cell-to-cell spread of Listeria.

The bacterial pathogen Listeria monocytogenes spreads within human tissues using a motility process dependent on the host actin cytoskeleton. Cell-to-cell spread involves the ability of motile bacteria to remodel the host plasma membrane into protrusions, which are internalized by neighboring cells. Recent results indicate that formation of Listeria protrusions in polarized human cells involves bacterial antagonism of a host signaling pathway comprised of the scaffolding protein Tuba and its effectors N-WASP and Cdc42.

Structural details of human tuba recruitment by InlC of Listeria monocytogenes elucidate bacterial cell-cell spreading.

The human pathogen Listeria monocytogenes is able to directly spread to neighboring cells of host tissues, a process recently linked to the virulence factor InlC. InlC targets the sixth SH3 domain (SH3-6) of human Tuba, disrupting its physiological interaction with the cytoskeletal protein N-WASP. The resulting loss of cortical actin tension may slacken the junctional membrane, allowing protrusion formation by motile Listeria.

γδ T cells exhibit multifunctional and protective memory in intestinal tissues.

The study of T cell memory and the target of vaccine design have focused on memory subsumed by T cells bearing the αβ T cell receptor. Alternatively, γδ T cells are thought to provide rapid immunity, particularly at mucosal borders. Here, we have shown that a distinct subset of mucosal γδ T cells mounts an immune response to oral Listeria monocytogenes (Lm) infection and leads to the development of multifunctional memory T cells capable of simultaneously producing interferon-γ and interleukin-17A in the murine intestinal mucosa.

Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex.

Photosynthesis uses chlorophylls for the conversion of light into chemical energy, the driving force of life on Earth. During chlorophyll biosynthesis in photosynthetic bacteria, cyanobacteria, green algae and gymnosperms, dark-operative protochlorophyllide oxidoreductase (DPOR), a nitrogenase-like metalloenzyme, catalyzes the chemically challenging two-electron reduction of the fully conjugated ring system of protochlorophyllide a. The reduction of the C-17=C-18 double bond results in the characteristic ring architecture of all chlorophylls, thereby altering the absorption properties of the molecule and providing the basis for light-capturing and energy-transduction processes of photosynthesis.

Crystal structure of the nitrogenase-like dark operative protochlorophyllide oxidoreductase catalytic complex (ChlN/ChlB)2.

During (bacterio)chlorophyll biosynthesis of many photosynthetically active organisms, dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the two-electron reduction of ring D of protochlorophyllide to form chlorophyllide. DPOR is composed of the subunits ChlL, ChlN, and ChlB. Homodimeric ChlL(2) bearing an intersubunit [4Fe-4S] cluster is an ATP-dependent reductase transferring single electrons to the heterotetrameric (ChlN/ChlB)(2) complex.

Structure of the heme biosynthetic Pseudomonas aeruginosa porphobilinogen synthase in complex with the antibiotic alaremycin.

The recently discovered antibacterial compound alaremycin, produced by Streptomyces sp. A012304, structurally closely resembles 5-aminolevulinic acid, the substrate of porphobilinogen synthase. During the initial steps of heme biosynthesis, two molecules of 5-aminolevulinic acid are asymmetrically condensed to porphobilinogen.

Structural basis for autoinhibition and activation of Auto, a virulence-associated peptidoglycan hydrolase of Listeria monocytogenes.

During a bacterial infection, each successive step is orchestrated by a dedicated set of virulence factors. In Gram-positive bacteria, the presentation or release of such factors is crucially dependent on the continual remodelling of the cell wall. We have investigated the autolysin or peptidoglycan hydrolase Auto (Lmo1076) from the human pathogen Listeria monocytogenes to structurally and biochemically underpin its role in host cell invasion.

Crystal structure and standardized geometric analysis of InlJ, a listerial virulence factor and leucine-rich repeat protein with a novel cysteine ladder.

We report on the crystal structure of the internalin domain of InlJ, a virulence-associated surface protein of Listeria monocytogenes, at 2.7-A resolution. InlJ is a member of the internalin family of listerial cell surface proteins characterized by a common N-terminal domain.

ATP-driven reduction by dark-operative protochlorophyllide oxidoreductase from Chlorobium tepidum mechanistically resembles nitrogenase catalysis.

During chlorophyll and bacteriochlorophyll biosynthesis in gymnosperms, algae, and photosynthetic bacteria, dark-operative protochlorophyllide oxidoreductase (DPOR) reduces ring D of aromatic protochlorophyllide stereospecifically to produce chlorophyllide. We describe the heterologous overproduction of DPOR subunits BchN, BchB, and BchL from Chlorobium tepidum in Escherichia coli allowing their purification to apparent homogeneity. The catalytic activity was found to be 3.

Thermodynamically reengineering the listerial invasion complex InlA/E-cadherin.

Biological processes essentially all depend on the specific recognition between macromolecules and their interaction partners. Although many such interactions have been characterized both structurally and biophysically, the thermodynamic effects of small atomic changes remain poorly understood. Based on the crystal structure of the bacterial invasion protein internalin (InlA) of Listeria monocytogenes in complex with its human receptor E-cadherin (hEC1), we analyzed the interface to identify single amino acid substitutions in InlA that would potentially improve the overall quality of interaction and hence increase the weak binding affinity of the complex.

Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase.

The initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR.

Crystal structure of the electron transfer complex rubredoxin rubredoxin reductase of Pseudomonas aeruginosa.

Crude oil spills represent a major ecological threat because of the chemical inertness of the constituent n-alkanes. The Gram-negative bacterium Pseudomonas aeruginosa is one of the few bacterial species able to metabolize such compounds. Three chromosomal genes, rubB, rubA1, and rubA2 coding for an NAD(P)H:rubredoxin reductase (RdxR) and two rubredoxins (Rdxs) are indispensable for this ability.

Extending the host range of Listeria monocytogenes by rational protein design.

In causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts.

Crystal structure of a non-discriminating glutamyl-tRNA synthetase.

Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS).