Bioelectrochemical sensor based on PQQ-dependent glucose dehydrogenase

Bioelectrochemical responses of pyrroloquinoline quinone (PQQ) dependent glucose dehydrogenase (PQQ-GDH) have been studied with the use of two principal electrode configurations: (i) glassy carbon electrode, coated with Nafion layer, containing sorbed N-methylphenazonium (NMP), and overcoated by an enzyme layer, crosslinked by glutaraldehyde, and (ii) glassy carbon electrode, containing an electropolymerized layer of either Toluidine blue, or o-phenylenediamine, or pyrrole, and overcoated by a crosslinked enzyme layer. When operated within the potential window of 0.0–0.3 V, Nafion-coated and NMP-soaked bioelectrode shows an anodic current response to glucose without the presence of electron transfer mediator in solution. The current–concentration profile obtained resemble to that, typical for enzyme catalyzed reaction. Other configurations studied showed bioelectrochemical response to glucose only in the presence of soluble mediator NMP. Without any mediator, no electrochemical responses have been registered. It indicates that direct electron transfer between PQQ-GDH and all types of electrodes modified during current work is undetectable.     

Pantoea Bacteriophage vB_PagS_MED16—A Siphovirus Containing a 2′-Deoxy-7-amido-7-deazaguanosine-Modified DNA

A novel siphovirus, vB_PagS_MED16 (MED16) was isolated in Lithuania using Pantoea agglomerans strain BSL for the phage propagation. The double-stranded DNA genome of MED16 (46,103 bp) contains 73 predicted open reading frames (ORFs) encoding proteins, but no tRNA. Our comparative sequence analysis revealed that 26 of these ORFs code for unique proteins that have no reliable identity when compared to database entries. Based on phylogenetic analysis, MED16 represents a new genus with siphovirus morphology. In total, 35 MED16 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phage–host interactions, and DNA metabolism. In addition, a gene encoding a preQ₀ DNA deoxyribosyltransferase (DpdA) is present in the genome of MED16 and the LC–MS/MS analysis indicates 2′-deoxy-7-amido-7-deazaguanosine (dADG)-modified phage DNA, which, to our knowledge, has never been experimentally validated in genomes of Pantoea phages. Thus, the data presented in this study provide new information on Pantoea-infecting viruses and offer novel insights into the diversity of DNA modifications in bacteriophages.     

The First Step of Biodegradation of 7-Hydroxycoumarin in Pseudomonas mandelii 7HK4 Depends on an Alcohol Dehydrogenase-Type Enzyme

Coumarins are well known secondary metabolites widely found in various plants. However, the degradation of these compounds in the environment has not been studied in detail, and, especially, the initial stages of the catabolic pathways of coumarins are not fully understood. A soil isolate Pseudomonas mandelii 7HK4 is able to degrade 7-hydroxycoumarin (umbelliferone) via the formation of 3-(2,4-dihydroxyphenyl)propionic acid, but the enzymes catalyzing the α-pyrone ring transformations have not been characterized. To elucidate an upper pathway of the catabolism of 7-hydroxycoumarin, 7-hydroxycoumarin-inducible genes hcdD, hcdE, hcdF, and hcdG were identified by RT-qPCR analysis. The DNA fragment encoding a putative alcohol dehydrogenase HcdE was cloned, and the recombinant protein catalyzed the NADPH-dependent reduction of 7-hydroxycoumarin both in vivo and in vitro. The reaction product was isolated and characterized as a 7-hydroxy-3,4-dihydrocoumarin based on HPLC-MS and NMR analyses. In addition, the HcdE was active towards 6,7-dihydroxycoumarin, 6-hydroxycoumarin, 6-methylcoumarin and coumarin. Thus, in contrast to the well-known fact that the ene-reductases usually participate in the reduction of the double bond, an alcohol dehydrogenase catalyzing such reaction has been identified, and, for P. mandelii 7HK4, 7-hydroxycoumarin degradation via a 7-hydroxy-3,4-dihydrocoumarin pathway has been proposed.     

Transient N4-Acyl-DNA Protection against Cleavage by Restriction Endonucleases

A set of five N⁴-acyl-modified 2′-deoxycytidine 5′-triphosphates were incorporated into modified DNA by using phi29 DNA polymerase, and cleavage by selected restriction endonucleases was studied. Modified DNA containing N⁴-acyl functional groups in either one or both strands of a DNA molecule was resistant to the majority of restriction enzymes tested, whereas modifications outside of the recognition sequences were well tolerated. The N⁴-acylated cytidine derivatives were subjected to competitive nucleotide incorporation by using phi29 DNA polymerase, showing that a high-fidelity phi29 DNA polymerase efficiently used the modified analogues in the presence of its natural counterpart. These N⁴ modifications were also demonstrated to be easily removed in an aqueous ethanolamine solution, in which all steps, including primer extension, demodification, and cleavage by restriction endonuclease, could be performed in a one-pot procedure that eliminated additional purification stages. It is suggested that N⁴-modified nucleotides are promising building blocks for a programmable; transient; and, most importantly, straightforward DNA protection against specific endonucleases.