title: Short communication: Homologous expression of recombinant and native thurincin H in an engineered natural producer.
abstract: The Bacillus bacteriocin thurincin H exhibits a wide inhibitory spectrum of activity against various foodborne pathogens, such as Listeria monocytogenes, and dairy spoilage bacteria, especially different Bacillus species commonly existing in dairy products. Previously, we constructed 3 plasmids to express native thurincin H homologously in an engineered natural producer, Bacillus thuringiensis SF361thnH(-). This host is deficient in thurincin H production because of an in-frame deletion of structural genes thnA1, thnA2, and thnA3 from the chromosome of the natural producer B. thuringiensis SF361. The previously constructed expression vectors were constructed by cloning the native thurincin H promoter, 3 (or 1) copies of structural genes, and the native (or Cry protein) terminator into an Escherichia coli-B. thuringiensis shuttle vector pHT315. In this study, 3 corresponding expression vectors (pGW134, pGW135, and pGW136) were constructed to express recombinant thurincin H-His6 in the same host, in which a 6-histidine tag was fused to the C terminus of each structural gene. The resulting low level of bacteriocin production indicated that the His tag might negatively interfere with subsequent posttranslational modification or exportation processes after the thurincin H-His6 prepeptide was translated. Additionally, in order to overexpress native thurincin H, 2 additional plasmids (pGW137 and pGW138) were constructed, consisting of the sporulation-dependent Cry protein dual promoter BtI and BtII, the thnA1 structural gene, and the thurincin H native or Cry protein terminator. However, production was low on Luria broth plates and absent on sporulation plates. It is possible that the resulting thurincin H prepeptide was not correctly modified or exported to the extracellular environment, due to the undesired biochemical and physiological changes during the sporulation phase.
authors.affiliation: Department of Food Science, New York State Agricultural Experiment Station, Cornell University, 630 W North Street, Geneva 14456.
authors.forename: G
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Food Science, New York State Agricultural Experiment Station, Cornell University, 630 W North Street, Geneva 14456.
authors.forename: D C
authors.initials: DC
authors.lastname: Manns
authors.affiliation: Department of Food Science, New York State Agricultural Experiment Station, Cornell University, 630 W North Street, Geneva 14456.
authors.forename: G K P
authors.initials: GK
authors.lastname: Guron
authors.affiliation: Department of Food Science, New York State Agricultural Experiment Station, Cornell University, 630 W North Street, Geneva 14456.
authors.forename: J J
authors.initials: JJ
authors.lastname: Churey
authors.affiliation: Department of Food Science, New York State Agricultural Experiment Station, Cornell University, 630 W North Street, Geneva 14456. Electronic address: rww8@cornell.edu.
authors.forename: R W
authors.initials: RW
authors.lastname: Worobo
mesh_terms: D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001430:Bacteriocins / Q000096:biosynthesis*; D053898:Biosynthetic Pathways; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D015202:Protein Engineering
chemical_list: D001426:Bacterial Proteins; D001430:Bacteriocins; C568940:thurincin H
keywords: Bacillus bacteriocin; Cry protein promoter; Cry protein terminator; heterologous expression; histidine tag