Studies on pyruvate decarboxylase-catalysed acyloin formation and the effects of surfactants on lipase-catalysed hydrolysis of esters

The effect of surfactants on the hydrolysis of achiral and chiral substrates by crude and purified porcine pancreatic lipase (PPL; EC 3.1.1.3) has been studied. Rather than accelerating the reactions, surfactants slowed down ("inhibited") the reactions relative to the rate in the absence of surfactant, despite effective emulsification of the substrate. Surfactants varied in the extent to which the reaction was inhibited and inhibition occurred below the critical micelle concentration of surfactants. Inhibition was accompanied by a loss of enantioselectivity with the crude enzyme but not the purified enzyme, indicating the presence of more than one activity in the crude PPL preparation. In general, there would seem to be no advantage to be gained from the use of surfactants in the hydrolysis of compounds of low water solubility with lipolytic enzymes; the use of an immiscible cosolvent is more effective. The pyruvate decarboxylase (EC 4.1.1.1) from Zymomonas mobilis strain CP4 ATCC 31821 was purified from recombinant Escherichia coli harbouring the plasmid pLOI295, which contained the gene coding for the enzyme. The purified recombinant enzyme catalysed acyloin condensations with a number of aldehyde acceptors. The substrate specificity of the Zymomonas enzyme was very similar to that observed with the enzyme from Saccharomyces carlsbergensis. However, the Zymomonas enzyme was found to catalyse the formation of acyloins from acetaldehyde at a rate four orders of magnitute greater than that observed with yeast enzyme. By comparing the stereochemistry of acyloin condensations catalysed by the Zymomonas and yeast enzymes, differences in the architecture of the active sites of these closely related enzymes have emerged

Assembly of α-Glucan by GlgE and GlgB in Mycobacteria and Streptomycetes

Actinomycetes, such as mycobacteria and streptomycetes, synthesize α-glucan with α-1,4 linkages and α-1,6 branching to help evade immune responses and to store carbon. α-Glucan is thought to resemble glycogen except for having shorter constituent linear chains. However, the fine structure of α-glucan and how it can be defined by the maltosyl transferase GlgE and branching enzyme GlgB were not known. Using a combination of enzymolysis and mass spectrometry, we compared the properties of α-glucan isolated from actinomycetes with polymer synthesized in vitro by GlgE and GlgB. We now propose the following assembly mechanism. Polymer synthesis starts with GlgE and its donor substrate, α-maltose 1-phosphate, yielding a linear oligomer with a degree of polymerization (∼16) sufficient for GlgB to introduce a branch. Branching involves strictly intrachain transfer to generate a C chain (the only constituent chain to retain its reducing end), which now bears an A chain (a nonreducing end terminal branch that does not itself bear a branch). GlgE preferentially extends A chains allowing GlgB to act iteratively to generate new A chains emanating from B chains (nonterminal branches that themselves bear a branch). Although extension and branching occur primarily with A chains, the other chain types are sometimes extended and branched such that some B chains (and possibly C chains) bear more than one branch. This occurs less frequently in α-glucans than in classical glycogens. The very similar properties of cytosolic and capsular α-glucans from Mycobacterium tuberculosis imply GlgE and GlgB are sufficient to synthesize them both

Structure of the Mycobacterium smegmatis alpha-maltose-1-phosphate synthase GlgM

Mycobacterium tuberculosis produces glycogen (also known as alpha-glucan) to help evade human immunity. This pathogen uses the GlgE pathway to generate glycogen rather than the more well known glycogen synthase GlgA pathway, which is absent in this bacterium. Thus, the building block for this glucose polymer is alpha-maltose-1-phosphate rather than an NDP-glucose donor. One of the routes to alpha-maltose-1-phosphate is now known to involve the GlgA homologue GlgM, which uses ADP-glucose as a donor and alpha-glucose-1-phosphate as an acceptor. To help compare GlgA (a GT5 family member) with GlgM enzymes (GT4 family members), the X-ray crystal structure of GlgM from Mycobacterium smegmatis was solved to 1.9 angstrom resolution. While the enzymes shared a GT-B fold and several residues responsible for binding the donor substrate, they differed in some secondary-structural details, particularly in the N-terminal domain, which would be expected to be largely responsible for their different acceptor-substrate specificities

The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae

Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli. However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae. The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_ 3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate

A temperature-sensitive Mycobacterium smegmatis glgE mutation leads to a loss of GlgE enzyme activity and thermostability and the accumulation of α-maltose-1-phosphate

Background: The bacterial GlgE pathway is the third known route to glycogen and is the only one present in mycobacteria. It contributes to the virulence of Mycobacterium tuberculosis. The involvement of GlgE in glycogen biosynthesis was discovered twenty years ago when the phenotype of a temperature-sensitive Mycobacterium smegmatis mutation was rescued by the glgE gene. The evidence at the time suggested glgE coded for a glucanase responsible for the hydrolysis of glycogen, in stark contrast with recent evidence showing GlgE to be a polymerase responsible for its biosynthesis. Methods: We reconstructed and examined the temperature-sensitive mutant and characterised the mutated GlgE enzyme. Results: The mutant strain accumulated the substrate for GlgE, α-maltose-1-phosphate, at the non-permissive temperature. The glycogen assay used in the original study was shown to give a false positive result with α-maltose-1-phosphate. The accumulation of α-maltose-1-phosphate was due to the lowering of the kcat of GlgE as well as a loss of stability 42 °C. The reported rescue of the phenotype by GarA could potentially involve an interaction with GlgE, but none was detected. Conclusions: We have been able to reconcile apparently contradictory observations and shed light on the basis for the phenotype of the temperature-sensitive mutation. General significance: This study highlights how the lowering of flux through the GlgE pathway can slow the growth mycobacteria

Pseudomonas syringae addresses distinct environmental challenges during plant infection through the coordinated deployment of polysaccharides

Prior to infection, phytopathogenic bacteria face a challenging environment on the plant surface, where they are exposed to nutrient starvation and abiotic stresses. Pathways enabling surface adhesion, stress tolerance, and epi phytic survival are important for successful plant pathogenesis. Understanding the roles and regulation of these path ways is therefore crucial to fully understand bacterial plant infections. The phytopathogen Pseudomonas syringae pv. tomato (Pst) encodes multiple polysaccharides that are implicated in biofilm formation, stress survival, and virulence in other microbes. To examine how these polysaccharides impact Pst epiphytic survival and pathogenesis, we ana lysed mutants in multiple polysaccharide loci to determine their intersecting contributions to epiphytic survival and infection. In parallel, we used qRT–PCR to analyse the regulation of each pathway. Pst polysaccharides are tightly coordinated by multiple environmental signals. Nutrient availability, temperature, and surface association strongly af fect the expression of different polysaccharides under the control of the signalling protein genes ladS and cbrB and the second messenger cyclic-di-GMP. Furthermore, functionally redundant, combinatorial phenotypes were observed for several polysaccharides. Exopolysaccharides play a role in mediating leaf adhesion, while α-glucan and alginate together confer desiccation tolerance. Our results suggest that polysaccharides play important roles in overcoming environmental challenges to Pst during plant infection

Trehalose-6-phosphate-mediated toxicity determines essentiality of OtsB2 in Mycobacterium tuberculosis in vitro and in mice

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors

Developmental delay in a Streptomyces venezuelae glgE null mutant is associated with the accumulation of alpha-maltose 1-phosphate

The GlgE pathway is thought to be responsible for the conversion of trehalose into a glycogen-like alpha-glucan polymer in bacteria. Trehalose is first converted to maltose, which is phosphorylated by maltose kinase Pep2 to give alpha-maltose 1-phosphate. This is the donor substrate of the maltosyl transferase GlgE that is known to extend alpha-1,4-linked maltooligosaccharides, which are thought to be branched with alpha-1,6 linkages. The genome of Streptomyces venezuelae contains all the genes coding for the GlgE pathway enzymes but none of those of related pathways, including glgC and glgA of the glycogen pathway. This provides an opportunity to study the GlgE pathway in isolation. The genes of the GlgE pathway were upregulated at the onset of sporulation, consistent with the known timing of a-glucan deposition. A constructed Delta glgE null mutant strain was viable but showed a delayed developmental phenotype when grown on maltose, giving less cell mass and delayed sporulation. Pre-spore cells and spores of the mutant were frequently double the length of those of the wild-type, implying impaired cross-wall formation, and spores showed reduced tolerance to stress. The mutant accumulated alpha-maltose 1-phosphate and maltose but no alpha-glucan. Therefore, the GlgE pathway is necessary and sufficient for polymer biosynthesis. Growth of the Delta glgE mutant on galactose and that of a Delta pep2 mutant on maltose were analysed. In both cases, neither accumulation of alpha-maltose 1-phosphate/alpha-glucan nor a developmental delay was observed. Thus, high levels of alpha-maltose 1-phosphate are responsible for the developmental phenotype of the Delta glgE mutant, rather than the lack of a-glucan

Trehalose and α-glucan mediate distinct abiotic stress responses in Pseudomonas aeruginosa

An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PAO1 and define its role in abiotic stress protection. We show that trehalose metabolism in PAO1 is integrated with the biosynthesis of branched α-glucan (glycogen), with mutants in either biosynthetic pathway significantly compromised for survival on abiotic surfaces. While both trehalose and α-glucan are important for abiotic stress tolerance, we show they counter distinct stresses. Trehalose is important for the PAO1 osmotic stress response, with trehalose synthesis mutants displaying severely compromised growth in elevated salt conditions. However, trehalose does not contribute directly to the PAO1 desiccation response. Rather, desiccation tolerance is mediated directly by GlgE-derived α-glucan, with deletion of the glgE synthase gene compromising PAO1 survival in low humidity but having little effect on osmotic sensitivity. Desiccation tolerance is independent of trehalose concentration, marking a clear distinction between the roles of these two molecules in mediating responses to abiotic stress

International consensus conference recommendations on ultrasound education for undergraduate medical students

Objectives: The purpose of this study is to provide expert consensus recommendations to establish a global ultrasound curriculum for undergraduate medical students. Methods: 64 multi-disciplinary ultrasound experts from 16 countries, 50 multi-disciplinary ultrasound consultants, and 21 medical students and residents contributed to these recommendations. A modified Delphi consensus method was used that included a systematic literature search, evaluation of the quality of literature by the GRADE system, and the RAND appropriateness method for panel judgment and consensus decisions. The process included four in-person international discussion sessions and two rounds of online voting. Results: A total of 332 consensus conference statements in four curricular domains were considered: (1) curricular scope (4 statements), (2) curricular rationale (10 statements), (3) curricular characteristics (14 statements), and (4) curricular content (304 statements). Of these 332 statements, 145 were recommended, 126 were strongly recommended, and 61 were not recommended. Important aspects of an undergraduate ultrasound curriculum identified include curricular integration across the basic and clinical sciences and a competency and entrustable professional activity-based model. The curriculum should form the foundation of a life-long continuum of ultrasound education that prepares students for advanced training and patient care. In addition, the curriculum should complement and support the medical school curriculum as a whole with enhanced understanding of anatomy, physiology, pathophysiological processes and clinical practice without displacing other important undergraduate learning. The content of the curriculum should be appropriate for the medical student level of training, evidence and expert opinion based, and include ongoing collaborative research and development to ensure optimum educational value and patient care. Conclusions: The international consensus conference has provided the first comprehensive document of recommendations for a basic ultrasound curriculum. The document reflects the opinion of a diverse and representative group of international expert ultrasound practitioners, educators, and learners. These recommendations can standardize undergraduate medical student ultrasound education while serving as a basis for additional research in medical education and the application of ultrasound in clinical practice