Molecular genetic analysis of curvaticin FS47, an antilisterial bacteriocin produced by Lactobacillus curvatus FS47
Abstract
Curvaticin FS47 is a novel antilisterial bacteriocin produced by Lactobacillus curvatus FS47. Based on the N-terminal amino acid sequence of curvaticin FS47, a biotinylated 28-mer-oligonucleotide probe was constructed for identifying the structural gene (cutA) of curvaticin FS47. Southern hybridization analysis of both genomic and plasmid DNA from the producer strain revealed the chromosomal-encoded nature of curvaticin FS47; whereas, Northern hybridization analysis of the total RNAs suggested that cutA may be transcribed as part of a polycistronic mRNA with a size about 2.0 kb. The structural gene cutA was cloned into Escherichia coli DH5α and the nucleotide sequence of the gene and its adjacent region (total 4376 base pairs long) were determined. Sequence analyses of this region revealed the presence of at least nine open reading frames (ORFs), apparently organized into three operon-like structures and transcribed in different directions. The cutA is located in the putative cutA operon that consists of six ORFs. Curvaticin FS47 appears to be synthesized as a 69-amino acid precursor with an 18-amino acid leader sequence containing a double-glycine type of processing site. The peptides potentially encoded by the second (cutB) and forth (cutC) ORFs in the cutA operon exhibited strong similarities to Class II LAB bacteriocins. Protein similarity searches revealed the resemblance of CutA/CutB with several two-component bacteriocins while the 61-amino acid CutC showed strong homology with Class IIa LAB bacteriocins including the YGNGXC N-terminal consensus motif. The ORFs downstream of cutB (orf3), cutC (orf5 ), and ORF8 (orf7) could be the immunity genes for curvaticin. The deduced 273-amino acid from region at the 5′ end of the truncated ORF (cutT) upstream of cutA showed significant homology to bacterial ABC transporters especially these dedicated for bacteriocins with double glycine type of leader peptides, suggesting it also may play a role in the secretion of curvaticin FS47. Expression of curvaticin FS47 using the cloned AcE fragment (without cutT) in Lb. curvatus ATCC 25601 and E. coli was unsuccessful, suggesting that the construct might have been detrimental to both host cells. Expression of curvaticin FS47 was achieved in Escherichia coli as a fusion protein with a maltose-binding protein (MBP). The fusion protein, MBP-curvaticin FS47, displayed bacteriocin activity in the SDS-PAGE overlay and the spot-on-lawn test of the cell lysate, suggesting that the N-terminal attachment of MBP did not inhibit the bacterioicin activity of curvaticin FS47.
Degree
Ph.D.
Advisors
Muriana, Purdue University.
Subject Area
Food science|Microbiology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.