Agarose biodegradation by deep-sea bacterium Vibrio natriegens WPAGA4 with the agarases through horizontal gene transfer.

This study aimed to reveal the importance of horizontal gene transfer (HGT) for the agarose-degrading ability and the related degradation pathway of a deep-sea bacterium Vibrio natriegens WPAGA4, which was rarely reported in former works. A total of four agarases belonged to the GH50 family, including Aga3418, Aga3419, Aga3420, and Aga3472, were annotated and expressed in Escherichia coli cells. The agarose degradation products of Aga3418, Aga3420, and Aga3472 were neoagarobiose, while those of Aga3419 were neoagarobiose and neoagarotetraose. The RT-qPCR analysis showed that the expression level ratio of Aga3418, Aga3419, Aga3420, and Aga3472 was stable at about 1:1:1.5:2.5 during the degradation, which indicated the optimal expression level ratio of the agarases for agarose degradation by V. natriegens WPAGA4. Based on the genomic information, three of four agarases and other agarose-degrading related genes were in a genome island with a G + C content that was obviously lower than that of the whole genome of V. natriegens WPAGA4, indicating that these agarose-degrading genes were required through HGT. Our results demonstrated that the expression level ratio instead of the expression level itself of agarase genes was crucial for agarose degradation by V. natriegens WPAGA4, and HGT occurred in the deep-sea environment, thereby promoting the deep-sea carbon cycle and providing a reference for studying the evolution and transfer pathways of agar-related genes.

Polarity-Specific and Pyrimidine-over-Purine Adaptive Triplex DNA Recognition by a Near-Infrared Fluorogenic Molecular Rotor.

Triplex DNA structures have displayed a wide range of applications including nanosensing, molecule switching, and drug delivering. Therefore, it is of great importance to effectively recognize triplex DNA structures by a simple and highly selective manner. Herein, we found that a near-infrared fluorogenic probe of NIAD-4 with a molecular rotor (MR) merit can selectively recognize triplex DNA structures over G-quadruplex, i-motif, and duplex structures (Tri-over-QID selectivity), which is competent over the widely used MR probe of thioflavin T (ThT). Furthermore, NIAD-4 exhibits as well a high selectivity toward the 'pyrimidine-type' triplex structures (Y:R-Y type) with respect to the 'purine-type' triplex structures (R:R-Y type) (a Y-over-R selectivity). Interestingly, NIAD-4 recognizes the Y:R-Y triplex structures by a polarity-dependent manner. The 3' end triplet is the preferential binding field of NIAD-4 with respect to the 5' end one (a 3'-over-5' selectivity) as the 3' end triplet is more stable than the 5' end one in the Hoogsteen hydrogen bond. It is expected that the adaptive stacking interaction between NIAD-4 and the 3' end triplet favors the Tri-over-QID, Y-over-R, and 3'-over-5' selectivities since this MR probe has three rotating shafts matching well with the triplet in topology. Such a high selectivity of NIAD-4 opens a new route in designing sensors with DNA structures switching between triplex, i-motif, and G-quadruplex structures.

Agarose-Degrading Characteristics of a Deep-Sea Bacterium Vibrio Natriegens WPAGA4 and Its Cold-Adapted GH50 Agarase Aga3420.

Up until now, the characterizations of GH50 agarases from Vibrio species have rarely been reported compared to GH16 agarases. In this study, a deep-sea strain, WPAGA4, was isolated and identified as Vibrio natriegens due to the maximum similarity of its 16S rRNA gene sequence, the values of its average nucleotide identity, and through digital DNA-DNA hybridization. Two circular chromosomes in V. natriegens WPAGA4 were assembled. A total of 4561 coding genes, 37 rRNA, 131 tRNA, and 59 other non-coding RNA genes were predicted in the genome of V. natriegens WPAGA4. An agarase gene belonging to the GH50 family was annotated in the genome sequence and expressed in E. coli cells. The optimum temperature and pH of the recombinant Aga3420 (rAga3420) were 40 °C and 7.0, respectively. Neoagarobiose (NA2) was the only product during the degradation process of agarose by rAga3420. rAga3420 had a favorable stability following incubation at 10-30 °C for 50 min. The Km, Vmax, and kcat values of rAga3420 were 2.8 mg/mL, 78.1 U/mg, and 376.9 s-1, respectively. rAga3420 displayed cold-adapted properties as 59.7% and 41.2% of the relative activity remained at 10 3 °C and 0 °C, respectively. This property ensured V. natriegens WPAGA4 could degrade and metabolize the agarose in cold deep-sea environments and enables rAga3420 to be an appropriate industrial enzyme for NA2 production, with industrial potential in medical and cosmetic fields.