Short communication: Naturally sensitive Bacillus thuringiensis EG10368 produces thurincin H and acquires immunity after heterologous expression of the one-step-amplified thurincin H gene cluster.
Heterologous expression of bacteriocin genetic determinants (or operons) has long been a research interest for the functional analysis of genes involved in bacteriocin biosynthesis, regulation, modification, and immunity. Previously, construction of genomic libraries of the bacteriocin producer strains was usually required to identify new bacteriocin operons, a method that is tedious and time consuming. For the first time, we directly amplified an 8.14-kb bioinformatically identified thurincin H gene cluster using a one-step PCR method with 100% accuracy. This amplified gene cluster was cloned into plasmid pHT315, resulting in plasmid pGW139, and subsequently transformed to Bacillus thuringiensis EG10368, a strain naturally sensitive to thurincin H. Heterologous expression of the gene cluster makes the sensitive B. thuringiensis EG10368 produce thurincin H at a higher level compared with the wild-type producer, B. thuringiensis SF361. Moreover, B. thuringiensis EG10368pGW139 acquired complete immunity to thurincin H. The results indicated that one-step PCR is a promising tool to accurately amplify long bacteriocin gene clusters used in bacteriocin functional analysis studies and it is an effective way to produce bacteriocins at a higher level, without the need to clone large chromosomal fragments.
Department of Food Science, New York State Agricultural Experiment Station, Cornell University, Geneva 14456.
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G
Wang
Department of Food Science, New York State Agricultural Experiment Station, Cornell University, Geneva 14456.
D C
DC
Manns
Department of Food Science, New York State Agricultural Experiment Station, Cornell University, Geneva 14456.
J J
JJ
Churey
Department of Food Science, New York State Agricultural Experiment Station, Cornell University, Geneva 14456. Electronic address: rww8@cornell.edu.
R W
RW
Worobo
D001413:Bacillus thuringiensis / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001430:Bacteriocins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D003611:Dairy Products / Q000382:microbiology; D005520:Food Preservatives / Q000737:chemistry; D005784:Gene Amplification; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005810:Multigene Family; D010957:Plasmids / Q000235:genetics
D001430:Bacteriocins; D005520:Food Preservatives; C568940:thurincin H
bacteriocin; gene cluster amplification; heterologous expression; immunity