In vitro synthesis of heparosan using recombinant Pasteurella multocida heparosan synthase PmHS2

In vertebrates and bacteria, heparosan the precursor of heparin is synthesized by glycosyltransferases via the stepwise addition of UDP-N-acetylglucosamine and UDP-glucuronic acid. As heparin-like molecules represent a great interest in the pharmaceutical area, the cryptic Pasteurella multocida heparosan synthase PmHS2 found to catalyze heparosan synthesis using substrate analogs has been studied. In this paper, we report an efficient way to purify PmHS2 and to maintain its activity stable during 6 months storage at −80 °C using His-tag purification and a desalting step. In the presence of 1 mM of each nucleotide sugar, purified PmHS2 synthesized polymers up to an average molecular weight of 130 kDa. With 5 mM of UDP-GlcUA and 5 mM of UDP-GlcNAc, an optimal specific activity, from 3 to 6 h of incubation, was found to be about 0.145 nmol/μg/min, and polymers up to an average of 102 kDa were synthesized in 24 h. In this study, we show that the chain length distribution of heparosan polymers can be controlled by change of the initial nucleotide sugar concentration. It was observed that low substrate concentration favors the formation of high molecular weight heparosan polymer with a low polydispersity while high substrate concentration did the opposite. Similarities in the polymerization mechanism between PmHS2, PmHS1, and PmHAS are discussed

Population Diversity of Listeria monocytogenes LO28: Phenotypic and Genotypic Characterization of Variants Resistant to High Hydrostatic Pressure ▿

A comparative phenotype analysis of 24 Listeria monocytogenes LO28 stress-resistant variants obtained after high-pressure treatment was performed to assess their robustness and growth performance under a range of food-relevant conditions. In addition, genetic analysis was conducted to characterize the promoter regions and open reading frames of the class I and III transcriptional repressors CtsR and HrcA, which control production of specific sets of stress proteins. Analysis of stress survival capacity, motility, biofilm formation, and growth under various conditions showed all variants to be more resistant to pressure and heat than the wild type; however, differences among variants were observed in acid resistance, growth rate, motility, and biofilm-forming capacity. Genetic analysis revealed no variation in the genetic make-up of hrcA and its upstream region, but two variants had deletions in the upstream region of ctsR and seven variants had mutations in the ctsR gene itself. The results of the characterization were cluster analyzed to obtain insight into the diversity of variants. Ten unique variants and three clusters with specific features could be identified: one cluster consisting of seven variants having a mutation in the CtsR regulator gene, one cluster containing two variants with an aerobic biofilm formation capacity similar to that of the wild type, and a cluster composed of five immotile variants. The large population diversity of L. monocytogenes stress-resistant variants signifies the organism's genetic flexibility, which in turn may contribute to the survival and persistence of this human pathogen in food-processing environments