Thermococcus sp. KS-1 PPIase as a fusion partner improving soluble production of aromatic amino acid decarboxylase.

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) catalyzes the racemization reaction of proline residues on a polypeptide chain. This enzyme is also known to function as a molecular chaperon to stabilize protein conformation during the folding process. In this study, we noted FK506 binding protein (FKBP)-type PPIase from a hyperthemophilic archaeon Thermococcus sp. strain KS-1 (PPIase KS-1) to improve the solubility of Pseudomonas putida aromatic amino acid decarboxylase (AADC) that is an indispensable enzyme for fermentative production of plant isoquinoline alkaloids. AADC fused N-terminally with the PPIase KS-1 (PPIase KS-1-AADC), which was synthesized utilizing Escherichia coli host, showed improved solubility and, consequently, the cell-free extract from the recombinant strain exhibited 2.6- to 3.4-fold elevated AADC activity than that from the control strain that expressed the AADC gene without PPIase KS-1. On the other hand, its thermostability was slightly decreased by fusing PPIase KS-1. The recombinant E. coli cells expressing the PPIase KS-1-AADC gene produced dopamine and phenylethylamine from L-dopa and phenylalanine by two- and threefold faster, respectively, as compared with the control strain. We further demonstrated that the efficacy of PPIase KS-1-AADC in solubility and activity enhancement was a little but obviously higher than that of AADC fused N-terminally with NusA protein, which has been assumed to be the most effective protein solubilizer. These results suggest that PPIase KS-1 can be used as one of the best choices for producing heterologous proteins as active forms in E. coli.

Nationwide survey of salmonella prevalence in environmental dust from layer farms in Japan.

A nationwide survey was conducted to determine Salmonella prevalence in airborne dust from layer farms. Of the 4,090 layer farms in Japan, 203 were surveyed and 48 (23.6%) of these were positive for Salmonella. Salmonella isolation rates were higher in the eastern (24.3%), central (25.6%), western (23.9%), and southern (27.5%) prefectures than they were in the northern (13.3%) prefecture. We recovered 380 Salmonella isolates and identified 34 different Salmonella serovars. Salmonella Infantis was the most prevalent serovar (42 [11.1%] of 380), followed by Salmonella Agona (39 [10.3%] of 380), Salmonella Mbandaka (37 [9.7%] of 380), Salmonella Cerro (32 [8.4%] of 380), Salmonella Thompson (29 [7.6%] of 380), and Salmonella Braenderup (27 [7.1%] of 380). Of the 380 isolates, 273 (71.8%) were resistant to more than one antibiotic. Salmonella Infantis (41 [97.6%] of 42), Salmonella Agona (38 [97.4%] of 39), and Salmonella Mbandaka (34 [91.9%] of 37) showed the highest resistance rates. We found 18 different resistance patterns and the most common (179 [47.1%] of 273) was resistant to dihydrostreptomycin. One of the 13 Salmonella Hadar isolates was resistant to eight antibiotics. To investigate characteristics of Salmonella Agona, Salmonella Infantis, and Salmonella Mbandaka isolates across different prefectures, we performed pulsed-field gel electrophoresis by using XbaI and BlnI. The Salmonella Agona and Salmonella Mbandaka dendrograms were grouped into seven clusters, with 80 and 70% similarity, respectively. Because the Salmonella Infantis dendrogram showed low similarity, there is a possibility of genetic diffusion of this serovar across Japan. This report is the first to describe Salmonella contamination in airborne dust from layer farms in Japan. Our findings should be useful for future Salmonella infection monitoring and control.

Identification and characterization of murine DNAM-1 (CD226) and its poliovirus receptor family ligands.

The leukocyte adhesion molecule DNAM-1 (CD226) is a member of the immunoglobulin superfamily and constitutively expressed on the majority of CD4+ and CD8+ T lymphocytes, natural killer (NK) cells, monocytes/macrophages, and a subset of B lymphocytes. The poliovirus receptor (PVR; CD155) and its family member nectin 2 (CD112) have recently been identified as the ligands for DNAM-1. Interaction of DNAM-1 with the ligands induces NK cell- and CD8+ T cell-mediated cytotoxicity and cytokine secretion. However, in vivo function of the receptor-ligand interaction has remained unclear. Here, we identified murine DNAM-1 and PVR homologues that physically and functionally bind each other. We demonstrated that ligand binding of murine DNAM-1 induced a costimulatory signal in antigen-specific CD8+ T cells. These results should provide a useful animal model to explore a role of DNAM-1 in immune responses in vivo.