Science & Insights

@article{Tropis2005,

title = {The Crucial Role of Trehalose and Structurally Related Oligosaccharides in the Biosynthesis and Transfer of Mycolic Acids in Corynebacterineae},

author = {Marielle Tropis and Xavier Meniche and Andreas Wolf and Henrike Gebhardt and Sergey Strelkov and Mohamed Chami and Dietmar Schomburg and Reinhard Krämer and Susanne Morbach and Mamadou Daffé},

url = {https://trenzyme.com/protein-production-services/custom-protein-expression-service/, ➥Custom Protein Expression Service},

doi = {10.1074/jbc.m502104200},

issn = {0021-9258},

year  = {2005},

date = {2005-07-00},

urldate = {2005-07-00},

journal = {Journal of Biological Chemistry},

volume = {280},

number = {28},

pages = {26573--26585},

publisher = {Elsevier BV},

abstract = {Trehalose (alpha-D-glucopyranosyl-alpha'-D-glucopyranoside) is essential for the growth of the human pathogen Mycobacterium tuberculosis but not for the viability of the phylogenetically related corynebacteria. To determine the role of trehalose in the physiology of these bacteria, the so-called Corynebacterineae, mutant strains of Corynebacterium glutamicum unable to synthesize trehalose due to the knock-out of the genes of the three pathways of trehalose biosynthesis, were biochemically analyzed. We demonstrated that the synthesis of trehalose under standard conditions is a prerequisite for the production of mycolates, major and structurally important constituents of the cell envelope of Corynebacterineae. Consistently, the trehalose-less cells also lack the cell wall fracture plane that typifies mycolate-containing bacteria. Importantly, however, the mutants were able to synthesize mycolates when grown on glucose, maltose, and maltotriose but not on other carbon sources known to be used for the production of internal glucose phosphate such as fructose, acetate, and pyruvate. The mycoloyl residues synthesized by the mutants grown on alpha-D-glucopyranosyl-containing oligosaccharides were transferred both onto the cell wall and free sugar acceptors. A combination of chemical analytical approaches showed that the newly synthesized glycolipids consisted of 1 mol of mycolate located on carbon 6 of the non reducing glucopyranosyl unit. Additionally, experiments with radioactively labeled trehalose showed that the transfer of mycoloyl residues onto sugars occurs outside the plasma membrane. Finally, and in contradiction to published data, we demonstrated that trehalose 6-phosphate has no impact on mycolate synthesis in vivo.},

keywords = {Corynebacterineae, trehalose},

pubstate = {published},

tppubtype = {article}

}

@article{Silva2005,

title = {The High-Affinity Maltose/Trehalose ABC Transporter in the Extremely Thermophilic Bacterium            \textit{Thermus thermophilus}            HB27 Also Recognizes Sucrose and Palatinose},

author = {Zélia Silva and Maria-Manuel Sampaio and Anke Henne and Alex Böhm and Ruben Gutzat and Winfried Boos and Milton S. da Costa and Helena Santos},

url = {https://trenzyme.com/protein-production-services/custom-protein-expression-service/, ➥Custom Protein Expression Service},

doi = {10.1128/jb.187.4.1210-1218.2005},

issn = {1098-5530},

year  = {2005},

date = {2005-02-15},

urldate = {2005-02-15},

journal = {J Bacteriol},

volume = {187},

number = {4},

pages = {1210--1218},

publisher = {American Society for Microbiology},

abstract = {We have studied the transport of trehalose and maltose in the thernophilic bacterium Thermus thermophilus HB27, which grows optimally in the range of 70 to 75 degrees C. The K(m) values at 70 degrees C were 109 nM for trehalose and 114 nM for maltose; also, a high K(m) (424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while d-galactose, d-fucose, l-rhamnose, l-arabinose, and d-mannose were not competitive inhibitors. In the recently published genome of T. thermophilus HB27, two gene clusters designated malEFG1 (TTC1627 to -1629) and malEFG2 (TTC1288 to -1286) and two monocistronic genes designated malK1 (TTC0211) and malK2 (TTC0611) are annotated as trehalose/maltose and maltose/maltodextrin transport systems, respectively. To find out whether any of these systems is responsible for the transport of trehalose, the malE1 and malE2 genes, lacking the sequence encoding the signal peptides, were expressed in Escherichia coli. The binding activity of pure recombinant proteins was analyzed by equilibrium dialysis. MalE1 was able to bind maltose, trehalose, and sucrose but not glucose or maltotetraose (K(d) values of 103, 67, and 401 nM, respectively). Mutants with disruptions in either malF1 or malK1 were unable to grow on maltose, trehalose, sucrose, or palatinose, whereas mutants with disruption in malK2 or malF2 showed no growth defect on any of these sugars. Therefore, malEFG1 encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose/palatinose ABC transporter, and malK1 encodes the ATP-binding subunit of this transporter. Despite the presence of an efficient transporter for trehalose, this compound was not used by HB27 for osmoprotection. MalE1 and MalE2 exhibited extremely high thermal stability: melting temperatures of 90 degrees C for MalE1 and 105 degrees C for MalE2 in the presence of 2.3 M guanidinium chloride. The latter protein did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose.},

keywords = {trehalose},

pubstate = {published},

tppubtype = {article}

}

@article{Qu2004,

title = {TreT, a Novel Trehalose Glycosyltransferring Synthase of the Hyperthermophilic Archaeon Thermococcus litoralis},

author = {Qiuhao Qu and Sung-Jae Lee and Winfried Boos},

url = {https://trenzyme.com/protein-production-services/custom-protein-expression-service/, ➥Custom Protein Expression Service},

doi = {10.1074/jbc.m404955200},

issn = {0021-9258},

year  = {2004},

date = {2004-11-00},

urldate = {2004-11-00},

journal = {Journal of Biological Chemistry},

volume = {279},

number = {46},

pages = {47890--47897},

publisher = {Elsevier BV},

abstract = {The gene cluster in Thermococcus litoralis encoding a multicomponent and binding protein-dependent ABC transporter for trehalose and maltose contains an open reading frame of unknown function. We cloned this gene (now called treT), expressed it in Escherichia coli, purified the encoded protein, and identified it as an enzyme forming trehalose and ADP from ADP-glucose and glucose. The enzyme can also use UDP- and GDP-glucose but with less efficiency. The reaction is reversible, and ADP-glucose plus glucose can also be formed from trehalose and ADP. The rate of reaction and the equilibrium favor the formation of trehalose. At 90 degrees C, the optimal temperature for the enzymatic reaction, the half-maximal concentration of ADP-glucose at saturating glucose concentrations is 1.14 mm and the V(max) is 160 units/mg protein. In the reverse reaction, the half-maximal concentration of trehalose at saturating ADP concentrations is 11.5 mm and the V(max) was estimated to be 17 units/mg protein. Under non-denaturating in vitro conditions the enzyme behaves as a dimer of identical subunits of 48 kDa. As the transporter encoded in the same gene cluster, TreT is induced by trehalose and maltose in the growth medium.},

keywords = {trehalose},

pubstate = {published},

tppubtype = {article}

}

@article{Molnos2003,

title = {A continuous coupled enzyme assay for bacterial malonyl–CoA:acyl carrier protein transacylase (FabD)},

author = {Juliette Molnos and Rana Gardiner and Glenn E Dale and Roland Lange},

url = {https://trenzyme.com/gene-synthesis-custom-cloning/, ➥Gene Synthesis and Custom Cloning},

doi = {10.1016/s0003-2697(03)00327-0},

issn = {0003-2697},

year  = {2003},

date = {2003-08-00},

urldate = {2003-08-00},

journal = {Analytical Biochemistry},

volume = {319},

number = {1},

pages = {171--176},

publisher = {Elsevier BV},

abstract = {Bacterial malonyl-CoA:acyl carrier protein transacylase catalyzes the transfer of a malonyl moiety from malonyl-CoA to the free thiol group of the phosphopantetheine arm of acyl carrier protein. Malonyl-ACP, the product of this enzymatic reaction, is the key building block for de novo fatty acid biosynthesis. Here, we describe a continuous enzyme assay based on the coupling of the malonyl-CoA:acyl carrier protein transacylase reaction to alpha-ketoglutarate dehydrogenase (KDH). KDH-dependent consumption of the coenzyme A generated by malonyl-CoA:acyl carrier protein transacylase is accompanied by a reduction of nicotinamide adenine dinucleotide, oxidized (NAD(+)) to nicotinamide adenine dinucleotide, reduced. The rate of NAD(+) reduction is continuously monitored as a change in fluorescence using a microtiter plate reader. We show that this coupled enzyme assay is amenable to routine chemical compound screening.},

keywords = {malonyl-CoA:acyl},

pubstate = {published},

tppubtype = {article}

}

@article{Eitel2002,

title = {The YadA Protein of\textit{Yersinia pseudotuberculosis}Mediates High-Efficiency Uptake into Human Cells under Environmental Conditions in Which Invasin Is Repressed},

author = {Julia Eitel and Petra Dersch},

url = {https://trenzyme.com/protein-production-services/custom-protein-expression-service/, ➥Custom Protein Expression Service},

doi = {10.1128/iai.70.9.4880-4891.2002},

issn = {1098-5522},

year  = {2002},

date = {2002-09-00},

urldate = {2002-09-00},

journal = {Infect Immun},

volume = {70},

number = {9},

pages = {4880--4891},

publisher = {American Society for Microbiology},

abstract = {The YadA protein is a major adhesin of Yersinia pseudotuberculosis that promotes tight adhesion to mammalian cells by binding to extracellular matrix proteins. In this study, we first addressed the possibility of competitive interference of YadA and the major invasive factor invasin and found that expression of YadA in the presence of invasin affected neither the export nor the function of invasin in the outer membrane. Furthermore, expression of YadA promoted both bacterial adhesion and high-efficiency invasion entirely independently of invasin. Antibodies against fibronectin and β1 integrins blocked invasion, indicating that invasion occurs via extracellular-matrix-dependent bridging between YadA and the host cell β1 integrin receptors. Inhibitor studies also demonstrated that tyrosine and Ser/Thr kinases, as well as phosphatidylinositol 3-kinase, are involved in the uptake process. Further expression studies revealed that yadA is regulated in response to several environmental parameters, including temperature, ion and nutrient concentrations, and the bacterial growth phase. In complex medium, YadA production was generally repressed but could be induced by addition of Mg2+. Maximal expression of yadA was obtained in exponential-phase cells grown in minimal medium at 37°C, conditions under which the invasin gene is repressed. These results suggest that YadA of Y. pseudotuberculosis constitutes another independent high-level uptake pathway that might complement other cell entry mechanisms (e.g., invasin) at certain sites or stages during the infection process.},

keywords = {YadA},

pubstate = {published},

tppubtype = {article}

}