An archaeal transcription factor EnfR with a novel 'eighth note' fold controls hydrogen production of a hyperthermophilic archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1, a hyperthermophilic carboxydotrophic archaeon, produces H2 through CO oxidation catalyzed by proteins encoded in a carbon monoxide dehydrogenase (CODH) gene cluster. TON_1525 with a DNA-binding helix-turn-helix (HTH) motif is a putative repressor regulating the transcriptional expression of the codh gene cluster. The T55I mutation in TON_1525 led to enhanced H2 production accompanied by the increased expression of genes in the codh cluster. Here, TON_1525 was demonstrated to be a dimer. Monomeric TON_1525 adopts a novel 'eighth note' symbol-like fold (referred to as 'eighth note' fold regulator, EnfR), and the dimerization mode of EnfR is unique in that it has no resemblance to structures in the Protein Data Bank. According to footprinting and gel shift assays, dimeric EnfR binds to a 36-bp pseudo-palindromic inverted repeat in the promoter region of the codh gene cluster, which is supported by an in silico EnfR/DNA complex model and mutational studies revealing the implication of N-terminal loops as well as HTH motifs in DNA recognition. The DNA-binding affinity of the T55I mutant was lowered by ∼15-fold, for which the conformational change of N-terminal loops is responsible. In addition, transcriptome analysis suggested that EnfR could regulate diverse metabolic processes besides H2 production.

Tackling Vibrio parahaemolyticus in ready-to-eat raw fish flesh slices using lytic phage VPT02 isolated from market oyster.

The opportunistic pathogen V. parahaemolyticus is a major causative agent for seafood-borne illness worldwide. It also causes severe vibriosis in aquaculture animals, affecting seafood production with huge economic loss. These issues are getting worse due to the current global warming in oceans, spread of antibiotic resistance, and changes in consumer preference toward ready-to-eat (RTE) food items including seafood. To answer the urgent need for sustainable biocontrol agents against V. parahaemolyticus, we isolated and characterized a novel lytic bacteriophage VPT02 from market oyster. VPT02 lysed antibiotic resistant V. parahaemolyticus strains including FORC_023. Moreover, it exhibited notable properties as a biocontrol agent suitable for seafood-related settings, like short eclipse/latent periods, high burst size, broad thermal and pH stability, and no toxin/antibiotic resistance genes in the genome. Further comparative genomic analysis with the previously reported homologue phage pVp-1 revealed that VPT02 additionally possesses genes related to the nucleotide scavenging pathway, presumably enabling the phage to propagate quickly. Consistent with its strong in vitro bacteriolytic activity, treatment of only a small quantity of VPT02 (multiplicity of infection of 10) significantly increased the survival rate of V. parahaemolyticus-infected brine shrimp (from 16.7% to 46.7%). When applied to RTE raw fish flesh slices, the same quantity of VPT02 achieved up to 3.9 log reduction of spiked V. parahaemolyticus compared with the phage untreated control. Taken together, these results suggest that VPT02 may be a sustainable anti-V. parahaemolyticus agent useful in seafood-related settings including for RTE items.