Heterologous expression and characterization of CpI, OcpA, and novel serine-type carboxypeptidase OcpB from Aspergillus oryzae

In the genome of Aspergillus oryzae, 12 genes have been predicted to encode serine-type carboxypeptidases. However, the carboxypeptidase activities of the proteins encoded by these genes have not yet been confirmed experimentally. In this study, we have constructed three of these 12 genes overexpressing strains using Aspergillus nidulans and characterized their overproduced recombinant proteins. Of these three genes, one was previously named cpI; the other two have not been reported yet, and hence, we named them ocpA and ocpB. The recombinant proteins released amino acid residues from the C terminus of peptides, and the activity of the enzymes was inhibited by phenylmethylsulfonyl fluoride, indicating the enzymes to be serine-type carboxypeptidases. Recombinant OcpA, OcpB, and CpI were stable at 45°C, 55°C, and 55°C, respectively, at a low pH. The enzymatic properties of recombinant OcpB were different from those of any reported serine-type carboxypeptidase. On the other hand, recombinant OcpA had similar enzymatic properties to A. oryzae carboxypeptidases O1 and O2. The DNA and N-terminal amino acid sequences of carboxypeptidases O1 and O2 from A. oryzae IAM2640 were similar to those of OcpA. Result of transcriptional analysis of ocpA, ocpB, and cpI suggest differences in transcriptional regulation between these genes

The Roles and Acting Mechanism of Caenorhabditis elegans DNase II Genes in Apoptotic DNA Degradation and Development

DNase II enzymes are acidic endonucleases that have been implicated in mediating apoptotic DNA degradation, a critical cell death execution event. C. elegans genome contains three DNase II homologues, NUC-1, CRN-6, and CRN-7, but their expression patterns, acting sites, and roles in apoptotic DNA degradation and development are unclear. We have conducted a comprehensive analysis of three C. elegans DNase II genes and found that nuc-1 plays a major role, crn-6 plays an auxiliary role, and crn-7 plays a negligible role in resolving 3′ OH DNA breaks generated in apoptotic cells. Promoter swapping experiments suggest that crn-6 but not crn-7 can partially substitute for nuc-1 in mediating apoptotic DNA degradation and both fail to replace nuc-1 in degrading bacterial DNA in intestine. Despite of their restricted and largely non-overlapping expression patterns, both CRN-6 and NUC-1 can mediate apoptotic DNA degradation in many cells, suggesting that they are likely secreted nucleases that are retaken up by other cells to exert DNA degradation functions. Removal or disruption of NUC-1 secretion signal eliminates NUC-1's ability to mediate DNA degradation across its expression border. Furthermore, blocking cell corpse engulfment does not affect apoptotic DNA degradation mediated by nuc-1, suggesting that NUC-1 acts in apoptotic cells rather than in phagocytes to resolve 3′ OH DNA breaks. Our study illustrates how multiple DNase II nucleases play differential roles in apoptotic DNA degradation and development and reveals an unexpected mode of DNase II action in mediating DNA degradation

Intracellular Phospholipase A1 and Acyltransferase, Which Are Involved in Caenorhabditis elegans Stem Cell Divisions, Determine the sn-1 Fatty Acyl Chain of Phosphatidylinositol

Phosphatidylinositol (PI) is unique in the abundance of stearic acid at the sn-1 position. This fatty acid is thought to be incorporated through fatty acid remodeling. Here, we identified a phospholipase and acyltransferases involved in the fatty acid remodeling at the sn-1 position of PI and provide a link between the sn-1 fatty acid of PI and asymmetric cell division

Free-Living Turtles Are a Reservoir for Salmonella but Not for Campylobacter

Different studies have reported the prevalence of Salmonella in turtles and its role in reptile-associated salmonellosis in humans, but there is a lack of scientific literature related with the epidemiology of Campylobacter in turtles. The aim of this study was to evaluate the prevalence of Campylobacter and Salmonella in free-living native (Emys orbicularis, n=83) and exotic (Trachemys scripta elegans, n=117) turtles from 11 natural ponds in Eastern Spain. In addition, different types of samples (cloacal swabs, intestinal content and water from Turtle containers) were compared. Regardless of the turtle species, natural ponds where individuals were captured and the type of sample taken, Campylobacter was not detected. Salmonella was isolated in similar proportions in native (8.0±3.1%) and exotic (15.0±3.3%) turtles (p=0.189). The prevalence of Salmonella positive turtles was associated with the natural ponds where animals were captured. Captured turtles from 8 of the 11 natural ponds were positive, ranged between 3.0±3.1% and 60.0±11.0%. Serotyping revealed 8 different serovars among four Salmonella enterica subspecies: S. enterica subsp. enterica (n = 21), S. enterica subsp. salamae (n = 2), S. enterica subsp. diarizonae (n = 3), and S. enterica subsp. houtenae (n = 1). Two serovars were predominant: S. Thompson (n=16) and S. typhimurium (n=3). In addition, there was an effect of sample type on Salmonella detection. The highest isolation of Salmonella was obtained from intestinal content samples (12.0±3.0%), while lower percentages were found for water from the containers and cloacal swabs (8.0±2.5% and 3.0±1.5%, respectively). Our results imply that free-living turtles are a risk factor for Salmonella transmission, but do not seem to be a reservoir for Campylobacter. We therefore rule out turtles as a risk factor for human campylobacteriosis. Nevertheless, further studies should be undertaken in other countries to confirm these results.This work was supported by the Conselleria de Infraestructura, Territorio y Medio Ambiente for their assistance and financial support (Life09-Trachemys, Resolution 28/02/12 CITMA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Marín, C.; Ingresa-Capaccioni, S.; González Bodí, S.; Marco Jiménez, F.; Vega Garcia, S. (2013). Free-Living Turtles Are a Reservoir for Salmonella but Not for Campylobacter. PLoS ONE. 8(8):1-6. https://doi.org/10.1371/journal.pone.0072350S1688(2012). The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food‐borne Outbreaks in 2010. EFSA Journal, 10(3). doi:10.2903/j.efsa.2012.2597Kapperud, G. (2003). Factors Associated with Increased and Decreased Risk of Campylobacter Infection: A Prospective Case-Control Study in Norway. American Journal of Epidemiology, 158(3), 234-242. doi:10.1093/aje/kwg139Mermin, J., Hutwagner, L., Vugia, D., Shallow, S., Daily, P., … Bender, J. (2004). Reptiles, Amphibians, and HumanSalmonellaInfection: A Population‐Based, Case‐Control Study. Clinical Infectious Diseases, 38(s3), S253-S261. doi:10.1086/381594De Jong, B., Andersson, Y., & Ekdahl, K. (2005). Effect of Regulation and Education on Reptile-associated Salmonellosis. Emerging Infectious Diseases, 11(3), 398-403. doi:10.3201/eid1103.040694NAKADAI, A., KUROKI, T., KATO, Y., SUZUKI, R., YAMAI, S., YAGINUMA, C., … HAYASHIDANI, H. (2005). Prevalence of Salmonella spp. in Pet Reptiles in Japan. Journal of Veterinary Medical Science, 67(1), 97-101. doi:10.1292/jvms.67.97Lafuente, S., Bellido, J. B., Moraga, F. A., Herrera, S., Yagüe, A., Montalvo, T., … Caylà, J. A. (2013). Salmonella paratyphi B and Salmonella litchfield outbreaks associated with pet turtle exposure in Spain. 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Applied and Environmental Microbiology, 70(1), 613-615. doi:10.1128/aem.70.1.613-615.2004Van Meervenne, E., Botteldoorn, N., Lokietek, S., Vatlet, M., Cupa, A., Naranjo, M., … Bertrand, S. (2009). Turtle-associated Salmonella septicaemia and meningitis in a 2-month-old baby. Journal of Medical Microbiology, 58(10), 1379-1381. doi:10.1099/jmm.0.012146-0Williams, L. P. (1965). Pet Turtles as a Cause of Human Salmonellosis. JAMA: The Journal of the American Medical Association, 192(5), 347. doi:10.1001/jama.1965.03080180005001Feeley, J. C., & Treger, M. D. (1969). Penetration of Turtle Eggs by Salmonella braenderup. Public Health Reports (1896-1970), 84(2), 156. doi:10.2307/4593527Mermin, J., Hoar, B., & Angulo, F. J. (1997). Iguanas and Salmonella Marina Infection in Children: A Reflection of the Increasing Incidence of Reptile-associated Salmonellosis in the United States. PEDIATRICS, 99(3), 399-402. doi:10.1542/peds.99.3.399Rodgers, G. L., Long, S. S., Smergel, E., & Dampier, C. (2002). Salmonella Infection Associated With a Pet Lizard in Siblings With Sickle Cell Anemia: An Avoidable Risk. Journal of Pediatric Hematology/Oncology, 24(1), 75-76. doi:10.1097/00043426-200201000-00020Tu, Z.-C., Zeitlin, G., Gagner, J.-P., Keo, T., Hanna, B. A., & Blaser, M. J. (2004). Campylobacter fetus of Reptile Origin as a Human Pathogen. Journal of Clinical Microbiology, 42(9), 4405-4407. doi:10.1128/jcm.42.9.4405-4407.2004Hidalgo-Vila, J., Díaz-Paniagua, C., Pérez-Santigosa, N., de Frutos-Escobar, C., & Herrero-Herrero, A. (2008). Salmonella in free-living exotic and native turtles and in pet exotic turtles from SW Spain. Research in Veterinary Science, 85(3), 449-452. doi:10.1016/j.rvsc.2008.01.011Harris, J. R., Neil, K. P., Behravesh, C. B., Sotir, M. J., & Angulo, F. J. (2010). Recent Multistate Outbreaks of HumanSalmonellaInfections Acquired from Turtles: A Continuing Public Health Challenge. Clinical Infectious Diseases, 50(4), 554-559. doi:10.1086/649932Geue, L., & Löschner, U. (2002). Salmonella enterica in reptiles of German and Austrian origin. Veterinary Microbiology, 84(1-2), 79-91. doi:10.1016/s0378-1135(01)00437-0Sánchez-Jiménez, M. M., Rincón-Ruiz, P. A., Duque, S., Giraldo, M. A., Ramírez-Monroy, D. M., Jaramillo, G., & Cardona-Castro, N. (2011). Salmonella enterica in semi-aquatic turtles in Colombia. The Journal of Infection in Developing Countries, 5(05), 361-364. doi:10.3855/jidc.1126HEALTH SURVEY OF WILD AND CAPTIVE BOG TURTLES (CLEMMYS MUHLENBERGII) IN NORTH CAROLINA AND VIRGINIA. (2002). Journal of Zoo and Wildlife Medicine, 33(4), 311-316. doi:10.1638/1042-7260(2002)033[0311:hsowac]2.0.co;2Richards, J. M., Brown, J. D., Kelly, T. R., Fountain, A. L., & Sleeman, J. M. (2004). ABSENCE OF DETECTABLE SALMONELLA CLOACAL SHEDDING IN FREE-LIVING REPTILES ON ADMISSION TO THE WILDLIFE CENTER OF VIRGINIA. 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Two very long chain fatty acid acyl-CoA synthetase genes, acs-20 and acs-22, have roles in the cuticle surface barrier in Caenorhabditis elegans

10.1371/journal.pone.0008857PLoS ONE51e885