Commensal Bacteria: Not Just Innocent Bystanders.

Neisseria gonorrhoeae is quickly becoming untreatable due to its acquisition of resistance to multiple antimicrobials. It is vital that we begin to understand the mechanisms by which this is occurring. The paper by C. E. Rouquette-Loughlin, J. L. Reimche, J. T. Balthazar, V. Dhulipala, et al. (mBio 9:e02281-18, https://doi.org/10.1128/mBio.02281-18) has shown that horizontal transfer of DNA from a nasopharyngeal commensal, Neisseria polysaccharea, has resulted in multiple sequence changes in the mtr locus that affect both regulatory and structural regions of the MtrCDE pump, resulting in low-level azithromycin resistance. Studies such as this are increasingly important in our understanding of the movement of resistance between species and for devising strategies to overcome such events.

Synthesis of Aesculetin and Aesculin Glycosides Using Engineered Escherichia coli Expressing Neisseria polysaccharea Amylosucrase.

Because glycosylation of aesculetin and its 6-glucoside, aesculin, enhances their biological activities and physicochemical properties, whole-cell biotransformation and enzymatic synthesis methodologies using Neisseria polysaccharea amylosucrase were compared to determine the optimal production method for glycoside derivatives. High-performance liquid chromatography analysis of reaction products revealed two glycosylated products (AGG1 and AGG2) when aesculin was used as an acceptor, and three products (AG1, AG2, and AG3) when using aesculetin. The whole-cell biotransformation production yields of the major transfer products for each acceptor (AGG1 and AG1) were 85% and 25%, respectively, compared with 68% and 14% for enzymatic synthesis. These results indicate that whole-cell biotransformation is more efficient than enzymatic synthesis for the production of glycoside derivatives.

Synthesis of aesculetin and aesculin glycosides using engineered Escherichia coli expressing Neisseria polysaccharea amylosucrase.

Because glycosylation of aesculetin and its 6-glucoside, aesculin, enhances their biological activities and physicochemical properties, whole-cell biotransformation and enzymatic synthesis methodologies using Neisseria polysaccharea amylosucrase were compared to determine the optimal production method for glycoside derivatives. High performance liquid chromatography analysis of reaction products revealed two glycosylated products (AGG1 and AGG2) when aesculin was used as an acceptor and three products (AG1, AG2, and AG3) when using aesculetin. The whole-cell biotransformation production yields of the major transfer products for each acceptor (AGG1 and AG1) were 85% and 25%, respectively, compared to 68% and 14% for enzymatic synthesis. These results indicate that whole-cell biotransformation is more efficient than enzymatic synthesis for the production of glycoside derivatives.

Fluorescence detection of the transglycosylation activity of amylosucrase.

The purpose of this study was to investigate the novel fluorescence-based assay for the transglycosylation activity of amylosucrase (ASase). The transglycosylation activity of ASase from Deinococcus geothermalis (DGAS), ASase from Neisseria polysaccharea (NPAS), and DGAS-B (chimeric ASase wherein the B domain from DGAS was exchanged with the B domain of NPAS in a DGAS background) was applied to modify 4-methlylumberlliferone (MU) to 4-methylumberlliferone glucoside (MUG) using MU as an acceptor and sucrose as a glucoside donor. The result of HPLC (high performance liquid chromatography) show that the bioconversion of MUG with ASases was successfully accomplished using sucrose and MU. Kinetic studies of ASases were performed to determine kinetic parameter for sucrose and MU. The order of overall performance (kcat/Km) of transglycosylation activity for MU among DGAS, DGAS-B and NPAS was as follows: DGAS-B (8.1) > DGAS (5.0) > NPAS (0.4). The fluorescence-based transglycosylation assay using MU has a potential to be used as the detection of transglycosylation activity of ASase and to screen novel ASase variants, which may be improved in their transglycosylation activities.

Enzymatic Process for High-Yield Turanose Production and Its Potential Property as an Adipogenesis Regulator.

Turanose is a sucrose isomer naturally existing in honey and a promising functional sweetener due to its low glycemic response. In this study, the extrinsic fructose effect on turanose productivity was examined in Neisseria amylosucrase reaction. Turanose was produced, by increasing the amount of extrinsic fructose as a reaction modulator, with high concentration of sucrose substrate, which resulted in 73.7% of production yield. In physiological functionality test, lipid accumulation in 3T3-L1 preadipocytes in the presence of high amounts of pure glucose was attenuated by turanose substitution in a dose-dependent manner. Turanose treatments at concentrations representing 50%, 75%, and 100% of total glucose concentration in cell media significantly reduced lipid accumulation by 18%, 35%, and 72%, respectively, as compared to controls. This result suggested that turanose had a positive role in controlling adipogenesis, and enzymatic process of turanose production has a potential to develop a functional food ingredient for controlling obesity and related chronic diseases.

An unusual chimeric amylosucrase generated by domain-swapping mutagenesis.

Amylosucrase (ASase; EC 2.4.1.4) synthesizes α-1,4-glucans using sucrose as a sole substrate. The aim of this study was to compare the enzymatic properties of four recombinant ASase genes to determine the underlying mechanisms thereof. Following cloning and expression in Escherichia coli, we determined that the ASase enzyme from Deinococcus geothermalis (DGAS) had the highest thermostability whereas ASase from Neisseria polysaccharea (NPAS) showed the greatest polymerization activity. Chimeric ASases were constructed using dgas and npas genes by overlap extension polymerase chain reaction. Two of the six chimeric ASases generated, NPAS-B' and DGAS-B, showed ASase activity using sucrose as the sole substrate. However, DGAS-B was not able to produce longer α-1,4-glucans; the highest degree of polymerization was <12. In the kinetic study, not only the substrate binding affinity but also the production rate of DGAS-B was greater than those of DGAS. Molecular dynamic computational simulation suggested that DGAS-B could not synthesize longer glucan chains because of the change in flexibilities of loops 4, 7, and 8as compared to those of DGAS.