On the 230-kilobase-pair (kb) virulence plasmid of Shigella flexneri 2a strain YSH6000, at least seven separate genetic determinants have been identified. One of them, an approximately 4-kb region, virG, that is required for the Sereny reaction, was extensively studied to examine the role of the virG region. The phenotype of a VirG-mutant (M94) of YSH6000 in the cytoplasm of cultured MK cels was characterized by a kinetic study of the invading shigellae. The observed phenotype of M94 in the cytoplasm indicated that the virG locus is not required for multiplication of the invading shigellae, but is essential for their spread to adjacent cells. The DNA region necessary for the VirG function was localized to a 3.6-kb DNA sequence on the 230-kb plasmid. A 130-kilodalton polypeptide was confirmed to be the virG product. External labeling of bacteria with 1251 indicated that the 130-kilodalton virG protein is exposed on the bacterial surface. The nucleotide sequence of 4,472 bp, which contains the functional virG gene and its own regulatory sequence, was determined, and a large open reading frame encoding 1,102 amino acid residues was identified.

BackgroundThe integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed.ResultsA chimeric Embden-Meyerhof (EM) pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinant E. coli strains overproducing either one of the seven glycolytic enzymes of Thermus thermophilus, the cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase of Thermococcus kodakarensis. By coupling this pathway with the Thermus malate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31.ConclusionsIn this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as “synthetic metabolic engineering”. Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be potentially applicable to the biocatalytic manufacture of any chemicals or materials on demand.

Abstract-Evidence indicates that the high density lipoprotein (HDL) subfraction containing apolipoprotein A-I without apolipoprotein AII (LP-AI) is more antiatherogenic than HDL particles containing apolipoprotein A-I and apolipoprotein A-II (LP-AIϩAII). This study examined the effect of extended-release niacin (niacin-ER) and gemfibrozil on LP-AI and LP-AIϩAII particles in patients with low levels of HDL cholesterol (HDL-C). Mechanisms by which these agents modulate HDL particles were investigated by in vitro studies using human hepatoblastoma (Hep G2) cells. A total of 139 patients with low HDL-C (Յ40 mg/dL) were randomized to niacin-ER or gemfibrozil in a multicenter double-blind trial. Patients were dose-escalated with once-nightly niacin-ER (1 to 2 g) or gemfibrozil (1.2 g) for 19 weeks. Niacin-ER had a greater effect in raising HDL-C and apolipoprotein A-I levels than did gemfibrozil. Niacin-ER at 1-and 2-g doses increased LP-AI levels by 8.7Ϯ4.0% (Pϭ0.033) and 24.0Ϯ4.4% (PϽ0.001), respectively. Gemfibrozil had no consistent effect on LP-AI levels. LP-AIϩAII levels increased 5% to 8% by both agents. In vitro studies showed that niacin, but not gemfibrozil, selectively decreased the uptake of 125 I-labeled LP-AI holoparticles by Hep G2 cells. The uptake of [ 3 H]cholesterol ester was Ϸ75% greater from LP-AI versus LP-AIϩAII particles, but neither niacin nor gemfibrozil affected cholesterol ester uptake. These data indicate that unlike gemfibrozil, niacin selectively increases LP-AI compared with LP-AIϩAII particle concentration in patients with low HDL-C levels. The mechanism of action of increased LP-AI concentration appears to be mediated by decreased hepatic removal of LP-AI particles, which are more efficient in reverse cholesterol transport, thus suggesting an additional mechanism by which niacin mediates its antiatherogenic properties.

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The diffusive behavior of the cation and anion species of the PEO-type polymer electrolyte has been investigated using pulsed gradient spin-echo NMR to elucidate the carrier conduction mechanism of the polymer electrolyte. Compared with the random diffusive behavior generally observed in electrolyte solutions, the carriers in the PEO-type polymer electrolyte showed a characteristic migration feature of a restricted condition especially after the application of stress on the membrane. This is attributed to carrier hopping along the polymer chains through Coulombic interaction between the carriers and the ethylene oxide sites on the chains. The restricted feature was in agreement with the simple boundary restriction model. The diffusion time dependence of the echo intensity change also supported the belief that the carrier migration in the polymer electrolyte followed the simple boundary model. Considering the actual situation of the polymer electrolyte, the polymer chains spread in all directions to create a random network structure, which consequently permits three-dimensional migration as random diffusion under the condition of long-time limit. The diffusion manner of the cation species along and across the stretched direction was different at 80 °C. This was due to the difference in the diffusion coefficient between the two directions from the fitted results according to the simple boundary model. This confirmed that the alignment of the sites in the polymer electrolyte by strain would be effective for creating a highly conductive pathway for lithium ion transport even if application of stress is disadvantageous for segmental mobility.