No abstract

In this study, a β-agarase gene, agaB-4, was isolated for the first time from the agar-degrading bacterium Paenibacillus agarexedens BCRC 17346 by using next-generation sequencing. agaB-4 consists of 2652 bp and encodes an 883-amino acid protein with an 18-amino acid signal peptide. agaB-4 without the signal peptide DNA was cloned and expressed in Escherichia coli BL21(DE3). His-tagged recombinant AgaB-4 (rAgaB-4) was purified from the soluble fraction of E. coli cell lysate through immobilized metal ion affinity chromatography. The optimal temperature and pH of rAgaB-4 were 55 °C and 6.0, respectively. The results of a substrate specificity test showed that rAgaB-4 could degrade agar, high-melting point agarose, and low-melting point agarose. The Vmax and Km of rAgaB-4 for low-melting point agarose were 183.45 U/mg and 3.60 mg/mL versus 874.61 U/mg and 9.29 mg/mL for high-melting point agarose, respectively. The main products of agar and agarose hydrolysis by rAgaB-4 were confirmed to be neoagarotetraose. Purified rAgaB-4 can be used in the recovery of DNA from agarose gels and has potential application in agar degradation for the production of neoagarotetraose.Electronic supplementary materialThe online version of this article (10.1186/s13568-018-0581-8) contains supplementary material, which is available to authorized users.

From 2004 to 2010, pork carcass swabs from state-inspected slaughter plants in Taiwan were intermittently analyzed to determine the prevalence of selected pathogenic microorganisms associated with foodborne illness. The prevalences of Staphylococcus aureus each year from 2006 to 2010 were 6.6, 10.8, 5.1, 6.4, and 7.4%, respectively, while those of Listeria monocytogenes were 1.2% in 2004, 1.3% in 2005, and 3.5% in 2008. The prevalences of Clostridium perfringens were 0.9% in 2004, 3.2% in 2005, and 1.1% in 2008. Campylobacter jejuni and Campylobacter coli had a higher recovery rate than the other surveyed microorganisms, with prevalences during 2004, 2005, and 2008 of 21.1, 13.7, and 8.1%, respectively. Salmonella strains were analyzed each year, and their prevalences ranged between 3.0 and 6.9%. Derby, Typhimurium, Anatum, Choleraesuis, and Agona were the five serovars most frequently identified among the Salmonella isolates. Escherichia coli O157:H7 was not detected in 2004, 2005, or 2010. Routine baseline surveying of pork carcasses to determine the prevalence of selected pathogens of concern for food safety can provide valuable information regarding the effectiveness of the slaughtering procedures or the need for interventions.

ApxI exotoxin is an important virulence factor derived from Actinobacillus pleuropneumoniae that causes pleuropneumonia in swine. Here, we investigate the role of lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), a member of the β2 integrin family, and the involvement of the integrin signaling molecules focal adhesion kinase (FAK) and Akt in ApxI cytotoxicity. Using Western blot analysis, we found that ApxI downregulated the activity of FAK and Akt in porcine alveolar macrophages (AMs). Preincubation of porcine AMs with an antibody specific for porcine CD18 reduced ApxI-induced cytotoxicity as measured by a lactate dehydrogenase release assay and decreased ApxI-induced FAK and Akt attenuation, as shown by Western blot analysis. Pretreatment with the chemical compounds PMA and SC79, which activate FAK and Akt, respectively, failed to overcome the ApxI-induced attenuation of FAK and Akt and death of porcine AMs. Notably, the transfection experiments revealed that ectopic expression of porcine LFA-1 (pLFA-1) conferred susceptibility to ApxI in ApxI-insensitive cell lines, including human embryonic kidney 293T cells and FAK-deficient mouse embryonic fibroblasts (MEFs). Furthermore, ectopic expression of FAK significantly reduced ApxI cytotoxicity in pLFA-1-cotransfected FAK-deficient MEFs. These findings show for the first time that pLFA-1 renders cells susceptible to ApxI and ApxI-mediated attenuation of FAK activity via CD18, thereby contributing to subsequent cell death.