Cloning, heterologous expression, and molecular characterization of a highly active and stable non-specific endonuclease from Pseudomonas fluorescens.

Non-specific endonucleases can be used for the digestion of nucleic acids because they hydrolyze DNA/RNA into 3-5 base pairs (bp) length oligonucleotide fragments without strict selectivity. In this work, a novel non-specific endonuclease from Pseudomonas fluorescens (PfNuc) with high activities for both DNA and RNA was successfully cloned and expressed in Escherichia coli. The production of PfNuc in flask scale could be achieved to 1.73 × 106 U/L and 4.82 × 106 U/L for DNA and RNA by investigation of the culture and induction conditions. The characterization of PfNuc indicated that it was Mg2+-dependent and the catalytic activity was enhanced by 3.74 folds for DNA and 1.06 folds for RNA in the presence of 5 mM Mg2+. The specific activity of PfNuc for DNA was 1.44 × 105 U/mg at pH 8.0 and 40 °C, and 3.93 × 105 U/mg for RNA at pH 8.5 and 45 °C. The Km of the enzyme for both DNA and RNA was close to 43 µM. The Vmax was 6.40 × 105 U/mg and 1.11 × 106 U/mg for DNA and RNA, respectively. There was no observed activity loss when PfNuc was stored at 4 °C and - 20 °C after 28 days or 10 repeated freeze-thaw cycles at - 80 °C. Molecular docking revealed that PfNuc formed 17 and 19 hydrogen bonds with single-stranded RNA and double-stranded DNA, respectively. These results could explain the high activity and stability of PfNuc, suggesting its great potential applications in the industry and clinic.

A stomach-specific lectin plays a crucial role in the innate immunity of red swamp crayfish, Procambarus clarkii.

C-type lectins (CTLs) play important roles in innate immune system of crustaceans as pattern recognition receptors (PRRs). In this study, a novel CTL gene was obtained from the red swamp crayfish, Procambarus clarkii, designated as PcLec. PcLec encodes a peptide with 175 amino acids, with a signal peptide and a single carbohydrate recognition domain (CRD). The PcLec transcripts were specifically expressed in crayfish stomach and were induced by bacterial challenge. In vitro assays with recombinant PcLec protein revealed that it had bacterial binding activity, polysaccharide binding activity, bacterial agglutination activity, and antimicrobial activity. Most importantly, PcLec knockdown significantly impaired the survivability of crayfish upon oral infection with its pathogen A. hydrophila. According to these results, we infer that the PcLec plays a crucial role in antibacterial defense of crayfish.

Characterization of an immune deficiency (IMD) homolog from the oriental river prawn, Macrobrachium nipponense.

The immune deficiency (IMD) signal pathway mediates innate immunity against Gram-negative bacteria in crustaceans. In the present study, an IMD homolog (MnIMD) from the oriental river prawn, Macrobrachium nipponense was identified. The full-length cDNA of MnIMD was 782bp with an open reading frame of 549 bp that encodes a putative protein of 182 amino acids including a death domain at the C-terminus. Multiple alignment analysis showed that IMDs in prawn M. nipponense and other crustaceans shared high similarity. The recombinant protein of MnIMD was expressed and purified for further functional analyses. Western blot analysis indicated that MnIMD was present in many tissues, but with the highest level in the gills, which was consistent with the qRT-PCR results. After Vibrio parahaemolyticus challenge, MnIMD was significantly induced in gills. RNA interference analysis showed that the IMD pathway was involved in regulating the expression of different antimicrobial peptide (AMP) genes, including Cru4 and Cru6. These results are helpful in promoting research on the innate immunity in M. nipponense.