Aeromonas hydrophila associated with red spot disease in Macrobrachium nipponense and host immune-related gene expression profiles.

In September 2018, a serious disease causing high mortality with red spot syndrome occurred in a Macrobrachium nipponense aquaculture farm in Jintan County, Jiangsu Province, China. In this study, a pathogenic isolate 5-S3 was isolated from diseased M. nipponense and was identified as Aeromonas hydrophila by phenotypically and molecularly. The pathogenicity of the isolate 5-S3 to M. nipponense was determined by challenge experiments. Results of artificial challenge showed A. hydrophila was pathogenic to M. nipponense, the LD50 was 9.58 × 104 CFU/mL, and histopathological analysis revealed that the hepatopancreas of infected M. nipponense exhibited obvious inflammatory responses to A. hydrophila infection. The isolate showed significant phenotypical activities such as the lecithinase, esterase, caseinase and hemolysin which are indicative of their virulence potential. Besides, virulence genes such as aerA, act, fla, ahpß, alt, lip, eprCAI, hlyA, acg and gcaT were detected in the isolate 5-S3. Subsequently, the immune-related genes expression in M. nipponense were evaluated by quantitative real-time PCR (qRT-PCR), and the results showed that the expression levels of dorsal, relish, crustin1, crustin2, anti-lipopolysaccharide factors 1 (ALF1), anti-lipopolysaccharide factors 2 (ALF2), hemocyanin, i-lysozyme and prophenoloxidase were significantly up-regulated in hepatopancreas of M. nipponense after A. hydrophila infection, the stat, p38, crustin3, anti-lipopolysaccharide factors 3 (ALF3) genes had no significant change during the infection. The present results reveal that A. hydrophila was an etiological agent causing red spot syndrome and mass mortality of M. nipponense and the influence of A. hydrophila infection on host immune genes.

Biochemical characterization of a thermostable DNA ligase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5.

DNA ligases are essential enzymes for DNA replication, repair, and recombination processes by catalyzing a nick-joining reaction in double-stranded DNA. The genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes a putative ATP-dependent DNA ligase (Tba ligase). Herein, we characterized the biochemical properties of the recombinant Tba ligase. The enzyme displays an optimal nick-joining activity at 65-70 °C and retains its DNA ligation activity even after heated at 100 °C for 2 h, suggesting the enzyme is a thermostable DNA ligase. The enzyme joins DNA over a wide pH spectrum ranging from 5.0-10.0, and its optimal pH is 6.0-9.0. Tba ligase activity is dependent on a divalent metal ion: Mn2+, Mg2+, or Ca2+ is an optimal ion for the enzyme activity. The enzyme activity is inhibited by NaCl with high concentrations. Tba ligase is ATP-dependent and can also use UTP as a weak cofactor; however, the enzyme with high concentrations could function without an additional nucleotide cofactor. Mass spectrometric result shows that the residue K250 of Tba ligase is AMPylated, suggesting that the enzyme is bound to AMP. The substitution of K250 of Tba ligase with Ala abolishes the enzyme activity. In addition, the mismatches at the first position 3' to the nick suppress Tba ligase activity more than those at the first position 5' to the nick. The enzyme also discriminates more effectively mismatches at 3' to the nick than those at 5' to the nick in a ligation cycling reaction, suggesting that the enzyme might have potential application in single nucleotide polymorphism.