Insertional mutagenesis of pathogenic fungi

Screening insertional mutants for loss of virulence is an effective method for investigating the molecular genetic basis of bacterial pathogenesis, but has only recently been applied to fungal pathogens. For many pathogenic fungi transformation with heterologous plasmid DNA results in complex integration events. This problem can now be circumvented for some species using restriction enzyme mediated integration. Insertional mutagenesis of Fusarium oxysporum using the naturally occurring fungal transposon impala has been described, but transposon tagging for other fungi has yet to be developed. Although insertional mutagenesis has recently identified important virulence determinants of fungal phytopathogens, the lack of suitable screening strategies has so far limited its applicability for fungal pathogens of humans.     

A linear laser scanner to measure cross-sectional shape and area of biological specimens during mechanical testing

Measure of the cross-sectional area (CSA) of biological specimens is a primary concern for many biomechanical tests. Different procedures are presented in literature but besides the fact that noncontact techniques are required during mechanical testing, most of these procedures lack accuracy or speed. Moreover, they often require a precise positioning of the specimen, which is not always feasible, and do not enable the measure of the same section during tension. The objective of this study was to design a noncontact, fast, and accurate device capable of acquiring CSA of specimens mounted on a testing machine. A system based on the horizontal linear displacement of two charge-coupled device reflectance laser devices next to the specimen, one for each side, was chosen. The whole measuring block is mounted on a vertical linear guide to allow following the measured zone during sample tension (or compression). The device was validated by measuring the CSA of metallic rods machined with geometrical shapes (circular, hexagonal, semicircular, and triangular) as well as an equine superficial digital flexor tendon (SDFT) in static condition. We also performed measurements during mechanical testing of three SDFTs, obtaining the CSA variations until tendon rupture. The system was revealed to be very fast with acquisition times in the order of 0.1 s and interacquisition time of about 1.5 s. Measurements of the geometrical shapes yielded mean errors lower than 1.4% (n=20 for each shape) while the tendon CSA at rest was 90.29 ± 1.69 mm(2) (n=20). As for the tendons that underwent tension, a mean of 60 measures were performed for each test, which lasted about 2 min until rupture (at 20 mm/min), finding CSA variations linear with stress (R(2)>0.85). The proposed device was revealed to be accurate and repeatable. It is easy to assemble and operate and capable of moving to follow a defined zone on the specimen during testing. The system does not need precise centering of the sample and can perform noncontact measures during mechanical testing; therefore, it can be used to measure variations of the specimen CSA during a tension (or compression) test in order to determine, for instance, the true stress and transverse deformations.     

The x-ray structure of dTDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase

The repeating unit of the glycan chain in the S-layer of the bacterium Aneurinibacillus thermoaerophilus L420-91(T) is composed of four alpha-d-rhamnose molecules and two 3-acetamido-3,6-dideoxy-alpha-d-galactose moieties (abbreviated as Fucp3NAc). Formation of the glycan layer requires nucleotide-activated sugars as the donor molecules. Whereas the enzymes involved in the synthesis of GDP-rhamnose have been well characterized, less is known regarding the structures and enzymatic mechanisms of the enzymes required for the production of dTDP-Fucp3NAc. One of the enzymes involved in the biosynthesis of dTDP-Fucp3NAc is a 3,4-ketoisomerase, hereafter referred to as FdtA. Here we describe the first three-dimensional structure of this sugar isomerase complexed with dTDP and solved to 1.5 A resolution. The FdtA dimer assumes an almost jellyfish-like appearance with the sole alpha-helices representing the tentacles. Formation of the FdtA dimer represents a classical example of domain swapping whereby beta-strands 2 and 3 from one subunit form part of a beta-sheet in the second subunit. The active site architecture of FdtA is characterized by a cluster of three histidine residues, two of which, His(49) and His(51), appear to be strictly conserved in the amino acid sequences deposited to date. Site-directed mutagenesis experiments, enzymatic assays, and x-ray crystallographic analyses suggest that His(49) functions as an active site base.     

Molecular architecture of DesI: a key enzyme in the biosynthesis of desosamine

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found, for example, in such macrolide antibiotics as erthyromycin, azithromycin, and clarithromycin. The efficacies of these macrolide antibiotics are markedly reduced in the absence of desosamine. In the bacterium Streptomyces venezuelae, six enzymes are required for the production of dTDP-desosamine. The focus of this X-ray crystallographic analysis is the third enzyme in the pathway, a PLP-dependent aminotransferase referred to as DesI. The structure of DesI was solved in complex with its product, dTDP-4-amino-4,6-dideoxyglucose, to a nominal resolution of 2.1 A. Each subunit of the dimeric enzyme contains 12 alpha-helices and 14 beta-strands. Three cis-peptides are observed in each subunit, Phe 330, Pro 332, and Pro 339. The two active sites of the enzyme are located in clefts at the subunit/subunit interface. Electron density corresponding to the bound product clearly demonstrates a covalent bond between the amino group of the product and C-4' of the PLP cofactor. Interestingly, there are no hydrogen-bonding interactions between the protein and the dideoxyglucosyl group of the product (within 3.2 A). The only other sugar-modifying aminotransferase whose structure is known in the presence of product is PseC from Helicobacter pylori. This enzyme, as opposed to DesI, catalyzes amino transfer to the axial position of the sugar. A superposition of the two active sites for these proteins reveals that the major differences in ligand binding occur in the orientations of the deoxyglucosyl and phosphoryl groups. Indeed, the nearly 180 degrees difference in hexose orientation explains the equatorial versus axial amino transfer exhibited by DesI and PseC, respectively.     

Molecular architecture of DesV from Streptomyces venezuelae: a PLP-dependent transaminase involved in the biosynthesis of the unusual sugar desosamine

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in certain macrolide antibiotics such as the commonly prescribed erythromycin. Six enzymes are required for its biosynthesis in Streptomyces venezuelae. The focus of this article is DesV, which catalyzes the PLP-dependent replacement of a 3-keto group with an amino functionality in the fifth step of the pathway. For this study the three-dimensional structures of both the internal aldimine and the ketimine intermediate with glutamate were determined to 2.05 A resolution. DesV is a homodimer with each subunit containing 12 alpha-helical regions and 12 beta-strands that together form three layers of sheet. The structure of the internal aldimine demonstrates that the PLP-cofactor is held in place by residues contributed from both subunits (Asp 164 and Gln 167 from Subunit I and Tyr 221 and Asn 235 from Subunit II). When the ketimine intermediate is present in the active site, the loop defined by Gln 225 to Ser 228 from Subunit II closes down upon the active site. The structure of DesV is similar to another sugar-modifying enzyme referred to as PseC. This enzyme is involved in the biosynthesis of pseudaminic acid, which is a sialic acid-like nonulosonate found in the flagellin of Helicobacter pylori. In the case of PseC, however, the amino group is transferred to the C-4 rather than the C-3 position. Details concerning the structural analysis of DesV and a comparison of its molecular architecture to that of PseC are presented.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

Molecular structure of an N-formyltransferase from Providencia alcalifaciens O30

The existence of N-formylated sugars in the O-antigens of Gram-negative bacteria has been known since the middle 1980s, but only recently have the biosynthetic pathways for their production been reported. In these pathways, glucose-1-phosphate is first activated by attachment to a dTMP moiety. This step is followed by a dehydration reaction and an amination. The last step in these pathways is catalyzed by N-formyltransferases that utilize N¹⁰-formyltetrahydrofolate as the carbon source. Here we describe the three-dimensional structure of one of these N-formyltransferases, namely VioF from Providencia alcalifaciens O30. Specifically, this enzyme catalyzes the conversion of dTDP-4-amino-4,6-dideoxyglucose (dTDP-Qui4N) to dTDP-4,6-dideoxy-4-formamido-d-glucose (dTDP-Qui4NFo). For this analysis, the structure of VioF was solved to 1.9 Å resolution in both its apoform and in complex with tetrahydrofolate and dTDP-Qui4N. The crystals used in the investigation belonged to the space group R32 and demonstrated reticular merohedral twinning. The overall catalytic core of the VioF subunit is characterized by a six stranded mixed β-sheet flanked on one side by three α-helices and on the other side by mostly random coil. This N-terminal domain is followed by an α-helix and a β-hairpin that form the subunit:subunit interface. The active site of the enzyme is shallow and solvent-exposed. Notably, the pyranosyl moiety of dTDP-Qui4N is positioned into the active site by only one hydrogen bond provided by Lys 77. Comparison of the VioF model to that of a previously determined N-formyltransferase suggests that substrate specificity is determined by interactions between the protein and the pyrophosphoryl group of the dTDP-sugar substrate.