Biochemical and Structural Study of the Atypical Acyltransferase Domain from the Mycobacterial Polyketide Synthase Pks13

Pks13 is a type I polyketide synthase involved in the final biosynthesis step of mycolic acids, virulence factors, and essential components of the Mycobacterium tuberculosis envelope. Here, we report the biochemical and structural characterization of a 52-kDa fragment containing the acyltransferase domain of Pks13. This fragment retains the ability to load atypical extender units, unusually long chain acyl-CoA with a predilection for carboxylated substrates. High resolution crystal structures were determined for the apo, palmitoylated, and carboxypalmitoylated forms. Structural conservation with type I polyketide synthases and related fatty-acid synthases also extends to the interdomain connections. Subtle changes could be identified both in the active site and in the upstream and downstream linkers in line with the organization displayed by this singular polyketide synthase. More importantly, the crystallographic analysis illustrated for the first time how a long saturated chain can fit in the core structure of an acyltransferase domain through a dedicated channel. The structures also revealed the unexpected binding of a 12-mer peptide that might provide insight into domain-domain interaction.     

Engineered covalent leucotoxin heterodimers form functional pores: insights into S-F interactions

The staphylococcal alpha-toxin and bipartite leucotoxins belong to a single family of pore-forming toxins that are rich in beta-strands, although the stoichiometry and electrophysiological characteristics of their pores are different. The different known structures show a common beta-sandwich domain that plays a key role in subunit-subunit interactions, which could be targeted to inhibit oligomerization of these toxins. We used several cysteine mutants of both HlgA (gamma-haemolysin A) and HlgB (gamma-haemolysin B) to challenge 20 heterodimers linked by disulphide bridges. A new strategy was developed in order to obtain a good yield for S-S bond formation and dimer stabilization. Functions of the pores formed by 14 purified dimers were investigated on model membranes, i.e. planar lipid bilayers and large unilamellar vesicles, and on target cells, i.e. rabbit and human red blood cells and polymorphonuclear neutrophils. We observed that dimers HlgA T28C-HlgB N156C and HlgA T21C-HlgB T157C form pores with similar characteristics as the wild-type toxin, thus suggesting that the mutated residues are facing one another, allowing pore formation. Our results also confirm the octameric stoichiometry of the leucotoxin pores, as well as the parity of the two monomers in the pore. Correctly assembled heterodimers thus constitute the minimal functional unit of leucotoxins. We propose amino acids involved in interactions at one of the two interfaces for an assembled leucotoxin.     

Species-wide distribution of highly polymorphic minisatellite markers suggests past and present genetic exchanges among house mouse subspecies

Background  

Four hypervariable minisatellite loci were scored on a panel of 116 individuals of various geographical origins representing a large part of the diversity present in house mouse subspecies. Internal structures of alleles were determined by minisatellite variant repeat mapping PCR to produce maps of intermingled patterns of variant repeats along the repeat array. To reconstruct the genealogy of these arrays of variable length, the specifically designed software MS_Align was used to estimate molecular divergences, graphically represented as neighbor-joining trees.     

Survival of Desulfotomaculum spores from estuarine sediments after serial autoclaving and high-temperature exposure

Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are thought to be from hot environments and are distributed by ocean currents. Their cells and spores upper temperature limit for survival is unknown, as is whether they can survive repeated high-temperature exposure that might occur in hydrothermal systems. This was investigated by incubating estuarine sediments significantly above (40–80 °C) maximum in situ temperatures (∼23 °C), and with and without prior triple autoclaving. Sulphate reduction occurred at 40–60 °C and at 60 °C was unaffected by autoclaving. Desulfotomaculum sp. C1A60 was isolated and was most closely related to the thermophilic D. kuznetsovii^T (∼96% 16S rRNA gene sequence identity). Cultures of Desulfotomaculum sp. C1A60, D. kuznetsovii^Tand D. geothermicum B2T survived triple autoclaving while other related Desulfotomaculum spp. did not, although they did survive pasteurisation. Desulfotomaculum sp. C1A60 and D. kuznetsovii cultures also survived more extreme autoclaving (C1A60, 130 °C for 15 min; D. kuznetsovii, 135 °C for 15 min, maximum of 154 °C reached) and high-temperature conditions in an oil bath (C1A60, 130° for 30 min, D. kuznetsovii 140 °C for 15 min). Desulfotomaculum sp. C1A60 with either spores or predominantly vegetative cells demonstrated that surviving triple autoclaving was due to spores. Spores also had very high culturability compared with vegetative cells (∼30 × higher). Combined extreme temperature survival and high culturability of some thermophilic Desulfotomaculum spp. make them very effective colonisers of hot environments, which is consistent with their presence in subsurface geothermal waters and petroleum reservoirs.     

Experimental mapping of soluble protein domains using a hierarchical approach

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.     

Does Observation of Postural Imbalance Induce a Postural Reaction?

Background

Several studies bring evidence that action observation elicits contagious responses during social interactions. However automatic imitative tendencies are generally inhibited and it remains unclear in which conditions mere action observation triggers motor behaviours. In this study, we addressed the question of contagious postural responses when observing human imbalance.

Methodology/Principal Findings

We recorded participants'' body sway while they observed a fixation cross (control condition), an upright point-light display of a gymnast balancing on a rope, and the same point-light display presented upside down. Our results showed that, when the upright stimulus was displayed prior to the inverted one, centre of pressure area and antero-posterior path length were significantly greater in the upright condition compared to the control and upside down conditions.

Conclusions/Significance

These results demonstrate a contagious postural reaction suggesting a partial inefficiency of inhibitory processes. Further, kinematic information was sufficient to trigger this reaction. The difference recorded between the upright and upside down conditions indicates that the contagion effect was dependent on the integration of gravity constraints by body kinematics. Interestingly, the postural response was sensitive to habituation, and seemed to disappear when the observer was previously shown an inverted display. The motor contagion recorded here is consistent with previous work showing vegetative output during observation of an effortful movement and could indicate that lower level control facilitates contagion effects.     

4′-Phosphopantetheinyl Transferase PptT,a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo

The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, contains lipids with unusual structures. These lipids play a key role in both virulence and resistance to the various hostile environments encountered by the bacteria during infection. They are synthesized by complex enzymatic systems, including type-I polyketide synthases and type-I and -II fatty acid synthases, which require a post-translational modification to become active. This modification consists of the covalent attachment of the 4′-phosphopantetheine moiety of Coenzyme A catalyzed by phosphopantetheinyl transferases (PPTases). PptT, one of the two PPTases produced by mycobacteria, is involved in post-translational modification of various type-I polyketide synthases required for the formation of both mycolic acids and lipid virulence factors in mycobacteria. Here we identify PptT as a new target for anti-tuberculosis drugs; we address all the critical issues of target validation to demonstrate that PptT can be used to search for new drugs. We confirm that PptT is essential for the growth of M. bovis BCG in vitro and show that it is required for persistence of M. bovis BCG in both infected macrophages and immunodeficient mice. We generated a conditional expression mutant of M. tuberculosis, in which the expression of the pptT gene is tightly regulated by tetracycline derivatives. We used this construct to demonstrate that PptT is required for the replication and survival of the tubercle bacillus during the acute and chronic phases of infection in mice. Finally, we developed a robust and miniaturized assay based on scintillation proximity assay technology to search for inhibitors of PPTases, and especially of PptT, by high-throughput screening. Our various findings indicate that PptT meets the key criteria for being a therapeutic target for the treatment of mycobacterial infections.