Characterization of Porcine Reproductive and Respiratory Syndrome Virus Deletion Mutant

The genome of the isolate of porcine reproductive and respiratory syndrome virus （PRRSV） from China, designated HPBEDV, was sequenced and analyzed. The size of the genome of HPBEDV was 15 334 nucleotides （nt）. Comparative analysis of HPBEDV with the genomic sequences of the domestic and other isolates （JXA 1, HUN4, CH-1 a, B J-4, VR2332, and LV） revealed that HPBEDV shared 98.4, 98.0, 89.0, 88.7, and 88.6% identity with the American strain JXA1, HUN4, CH- 1a, BJ-4, and VR2332, respectively, but only 54.7% identity with the European reference strain Lelystad virus. The NSP2 gene had 2 850 nt and encoded 950 amino acids （aa）, with two discontiguous deletions of 1 aa and 29 aa at positions 482 and 534-562, respectively, relative to VR-2332. Also, phylogenetic analysis with the published PRRSV genomic sequences indicated that the newly emerging isolate form a clade with the VR-2332 isolates. Therefore, HPBEDV was a novel strain with deletions in NSP2 gene.

Evaluation of the function of a luciferase-like monooxygenase homologue in 4,4´-dithiodibutyric acid catabolism in Rhodococcus erythropolis MI2

The bacterium Rhodococcus erythropolis MI2 uses 4,4´-dithiodibutyric acid(DTDB)as carbon source to synthesize polythioesters(PTE).The first step for the production of PTE using DTDB is catalyzed by an NADH:flavin oxidoreductase(nox)as it was previously shown in our laboratory,and the second step is catabolized by a putative luciferase-like monooxygenase(Llm).In the current study,experiments were carried out to identify the function of Llm.Hence,the llm gene,which encodes for the Llm protein,was amplified from the genomic DNA of MI2 using polymerase chain reaction and expressed in Escherichia coli BL21 cells.Protein purification was done using His Spin Trap affinity columns.Enzyme assay was carried out using the purified protein and p-coumaric acid as substrate giving a specific activity of 1.6 U/mg protein for the purified Llm.The responsible gene(llm)was deleted in the genome of MI2,and a single deletion mutant was subsequently generated.Finally,growth of the wild-type strain(MI2)and the mutant strain(MI2Δllm)were compared using DTDB or succinate as carbon sources.Whereas the wild type was successfully grown with DTDB or succinate,the llm-negative mutant exhibited low grow with DTDB although it grows very well with succinate.