The synthesis and function of the Escherichia coli hemolysin and related RTX exotoxins

Abstract The RTX group of exotoxins represents a branch of a family of exoproteins produced by Gram-negative bacteria which share the properties of being secreted by a leader-independent pathway and a tandemly-repeated nine-amino-acid sequence that is responsible for calcium binding. The Escherichia coli hemolysin (HlyA) is the prototype for the RTX exotoxin family which includes the leukotoxins of Pasteurella haemolytica and Actinobacillus actinomycetemcomitans and hemolysins from four Gram-negative genera. A review of the genetics, synthesis, export and target cell reactivity of the E. coli hemolysin is given. An evolutionary tree of the RTX toxin family based on amino acid sequence similarity is presented.     

Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl₂, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over-expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.     

Unilateral lower limb muscle fatigue induces bilateral effects on undisturbed stance and muscle EMG activities

The study investigated the effects of an unilateral ankle muscle fatigue onto independent postural control parameters including the trajectories of the estimated resultant CoP (CoPres) and his components: the centre of gravity (CG) and CoP–CG trajectories.Nine healthy men realized series of 10 toe-lift immediately followed by 10 knee flexions until exhaustion with one (Ex) leg. Maximal isometric voluntary contractions, postural sway measures of each leg, and muscular activities of the ankle muscles were recorded before and immediately after the fatiguing exercise.As expected, the latter induced a decrease in maximal voluntary peak force associated with a greater variability of the relative contribution of each leg on the CoPres, enhanced all postural parameters of the non-exercised leg. A significant decreased of the tibialis anterior EMG activity for the Ex leg and an increased one for the NoEx leg. Finally, following unilateral fatigue, the body sway destabilisation seemed to occur only along the medio-lateral (ML) axis.The enhanced and greater variability of the variance along ML axis might be explained by the recourse at the loading–unloading strategy choice and suggests a central attempt to compensate for pain sensation.     

The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharides

The ability to form biofilm is seen as an increasingly important colonization strategy among both pathogenic and environmental Klebsiella pneumoniae strains. The aim of the present study was to identify abiotic surface colonization factors of K. pneumoniae using different models at different phases of biofilm development. A 2200 K. pneumoniae mutant library previously obtained by signature-tagged mutagenesis was screened in static and dynamic culture models to detect clones impaired at early and/or mature stages of biofilm formation. A total of 28 mutants were affected during late phases of biofilm formation, whereas 16 mutants displayed early adhesion defect. These mutants corresponded to genes involved in potential cellular and DNA metabolism pathways and to membrane transport functions. Eight mutants were deficient in capsule or LPS production. Gene disruption and microscopic analyses showed that LPS is involved in initial adhesion on both glass and polyvinyl-chloride and the capsule required for the appropriate initial coverage of substratum and the construction of mature biofilm architecture. These results give new insight into the bacterial factors sequentially associated with the ability to colonize an abiotic surface and reveal the dual roles played by surface exopolysaccharides during K. pneumoniae biofilm formation.     

<Emphasis Type="Italic">AtMRP6</Emphasis>/<Emphasis Type="Italic">AtABCC6</Emphasis>, an ATP-Binding Cassette transporter gene expressed during early steps of seedling development and up-regulated by cadmium in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

ABC proteins constitute one of the largest families of transporters found in all living organisms. In Arabidopsis thaliana, 120 genes encoding ABC transporters have been identified. Here, the characterization of one member of the MRP subclass, AtMRP6, is described.     

A highly multiplexed quantitative phosphosite assay for biology and preclinical studies

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho‐signaling in drug‐treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.     

Ethanol Tolerance in the Yeast Saccharomyces cerevisiae Is Dependent on Cellular Oleic Acid Content

In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Δ⁹Z-C_16:1) and oleic acid (Δ⁹Z-C_18:1), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C_16:0) and stearic acid (C_18:0), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Δ⁹ and Δ¹¹) and substrate chain-length preferences (i.e., C_16:0 and C_18:0); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Δ¹¹Z-C_18:1), whereas neither Δ¹¹Z-C_16:1 nor palmitoleic acid (Δ⁹Z-C_16:1) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.     

Biofilm dispersal: multiple elaborate strategies for dissemination of bacteria with unique properties

In most environments, microorganisms evolve in a sessile mode of growth, designated as biofilm, which is characterized by cells embedded in a self‐produced extracellular matrix. Although a biofilm is commonly described as a “cozy house” where resident bacteria are protected from aggression, bacteria are able to break their biofilm bonds and escape to colonize new environments. This regulated process is observed in a wide variety of species; it is referred to as biofilm dispersal, and is triggered in response to various environmental and biological signals. The first part of this review reports the main regulatory mechanisms and effectors involved in biofilm dispersal. There is some evidence that dispersal is a necessary step between the persistence of bacteria inside biofilm and their dissemination. In the second part, an overview of the main methods used so far to study the dispersal process and to harvest dispersed bacteria was provided. Then focus was on the properties of the biofilm‐dispersed bacteria and the fundamental role of the dispersal process in pathogen dissemination within a host organism. In light of the current body of knowledge, it was suggested that dispersal acts as a potent means of disseminating bacteria with enhanced colonization properties in the surrounding environment.     

The perforated stones of the Doi Pha Kan burials (Northern Thailand): A Mesolithic singularity?

Throughout continental Southeast Asia, the Hoabinhian techno-complex stands out in clear contrast with the universal chrono-cultural model essentially established on the basis of western prehistory. Following this model, early authors considered perforated stones and associated lithic artefacts as markers of what was then believed to pertain to a Southeast Asian Mesolithic. However, Southeast Asian Mesolitithic has progressively been abandoned in favour of a ubiquitous Hoabinhian spanning from 30,000 to 3000 BP. Here, we present and discuss the discovery of perforated stones at the Doi Pha Kan site in northern Thailand. Perforated stones have almost never been found in undisturbed stratigraphic conditions nor dated with any sufficient degree of certainty. At Doi Pha Kan site, such a kind of artefacts was found in burials intersecting sedimentary layers that could be ascertained as Hoabinhian. In contrast with similar perforated stones described in the literature, that found at Doi Pha Kan are well-dated (13,000 BP), thus providing a time-reference for a putative Southeast Asian Mesolithic. We therefore advocate that such non-Hoabinhian artefacts support the early authors’ hypothesis of the existence of a Southeast Asian Mesolithic. Finally, the funerary practices, the unusually high stature of individuals found at Doi Pha Kan in conjunction with the particular lithic assemblages further contributes to raise the question of the co-occurrence of several cultures or populations at the Pleistocene–Holocene interface in continental Southeast Asia.     

RNA hairpin invasion and ribosome elongation arrest by mixed base PNA oligomer

Recently, we have shown that peptide nucleic acid (PNA) tridecamers targeted to the codon 74, 128 and 149 regions of Ha-ras mRNA arrested translation elongation in vitro. Our data demonstrated for the first time that PNAs with mixed base sequence targeted to the coding region of a messenger RNA could arrest the translation machinery and polypeptide chain elongation. The peculiarity of the complexes formed with PNA tridecamers and Ha-ras mRNA rests upon the stability of PNA-mRNA hybrids, which are not dissociated by cellular proteins or multiple denaturing conditions. In the present study, we show that shorter PNAs such as a dodecamer or an undecamer targeted to the codon 74 region arrest translation elongation in vitro. The 13, 12, and 11-mer PNAs contain eight and the 10-mer PNA seven contiguous pyrimidine residues. Upon binding with parallel Hoogsteen base-pairing to the PNA-RNA duplex, six of the cytosine bases and one thymine base of a second PNA can form C.G*C(+) and T.A*T triplets. Melting experiments show two well-resolved transitions corresponding to the dissociation of the third strand from the core duplex and to melting of duplex at higher temperature. The enzymatic structure mapping of a target 27-mer RNA revealed a hairpin structure that is disrupted upon binding of tri-, dodeca-, undeca- and decamer PNAs. We show that the non-bonded nucleobase overhangs on the RNA stabilize the PNA-RNA hybrids and probably assist the PNA in overcoming the stable secondary structure of the RNA target. The great stability of PNA-RNA duplex and triplex structures allowed us to identify both 1:1 and 2:1 PNA-RNA complexes using matrix-assisted laser desorption/ionization time-of -flight mass spectrometry. Therefore, it is possible to successfully target mixed sequences in structured regions of messenger RNA with short PNA oligonucleotides that form duplex and triplex structures that can arrest elongating ribosomes.