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Abstract
The structural gene for the lactococcal lantibiotic lacticin 481 (lct) has been identified and cloned using a degenerated 20-mer DNA oligonucleotide based on the amino-terminal 7 amino acid residues of the purified protein. The transcription of the lct gene was analyzed, and its promoter was mapped. DNA sequence analysis of the lct gene revealed an open reading frame encoding a peptide of 51 amino acids. Comparison of its deduced amino acid sequence with the amino-terminal sequence and the amino acid composition of lacticin 481 indicates that the 51-residue peptide is prelacticin 481, containing a 27-residue carboxyl-terminal propeptide and a 24-residue amino-terminal leader peptide which lacks the properties of a typical signal sequence and which is significantly different from the leaders of other lantibiotics. The predicted amino acid sequence of prolacticin 481 contains 3 cysteines, 2 serines, and 2 threonines which were not detectable in amino acid analyses of mature lacticin 481. Based on these results and on characterization by two-dimensional NMR techniques, a structural model is proposed in which 2 cysteine residues are involved in lanthionine and one in beta-methyllanthionine formation, and a 4th threonine residue is dehydrated. This model predicts a molecular mass for lacticin 481 of 2,901, which is in excellent agreement with that obtained from mass spectrometry.
Highlights
To cite this version: Jean-Christophe Piard, Oscar P
Lacticin 481-containing samples were prepared in methanokacetic acid, 99:l (v/v), and 10 pl was introduced into theion source a t a flow rate of 3 pl rnin"
IL1441 (Lct-) were hybridized with the degenerated P25 icant similarity (26% identity within a stretch of 88 amino oligonucleotide based on the amino-terminal amino acid se- acids) with the transposase from the E. coli IS4 insertion quence of lacticin 481
Summary
No of residues/molecule in M~~~probableno. of Lacticin 481' Prolacticin 481' processed residues. Of Lacticin 481' Prolacticin 481' processed residues. This mixture was heated for 5 min a t 65 'C and subsequently allowed to cool at mom temperature. The pellet was washed with 70% ethanol and dissolved in 3 pl of water and 3 p1 of sequencing dye. The samples were heated for 3 min a t 98 "C and loaded on an acrylamide sequencing gel. Lacticin 481-containing samples were prepared in methanokacetic acid, 99:l (v/v), and 10 pl was introduced into theion source a t a flow rate of 3 pl rnin". 4. Electrospray mass spectrumof lacticin 481 representing therelative abundance as a functionof mass to charge ratio (m/z)of the multiply chargedions. The calculated molecular mass for prolacticin 481 is 2973 Da. A potentialShine-Dalgarno sequence (GGAG) is located 7 base pairs upstream of the ATG start codon of the kt gene Twentyseven base pairs downstream of the k t stop codon an inverted repeat was identified which could act as a rho-independent
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