Genetic and Biochemical Analysis of Mutacin 1140, a Lantibiotic from Streptococcus mutans

Abstract

Streptococcus mutans JH1000 and its derivatives were previously shown (J. D. Hillman, K. P. Johnson, and B. I. Yaphe, Infect. Immun. 44:141–144, 1984) to produce a low-molecular-weight, broad-spectrum bacteriocin-like inhibitory substance (BLIS). The thermosensitive vector pTV1-OK harboring Tn917 was used to isolate a BLIS-deficient mutant, DM25, and the mutated gene was recovered by shotgun cloning inEscherichia coli. Sequence analysis of insert DNA adjacent to Tn917 led to the identification of four open reading frames including two (lanA and lanB) which have substantial homology to the Staphylococcus epidermidisstructural gene (epiA) and a modifying enzyme gene (epiB) for biosynthesis of the lantibiotic epidermin, respectively. Although the BLIS activity could not be recovered from broth cultures, high yields were obtained from a solid medium consisting of Todd-Hewitt broth containing 0.5% agarose that was stab inoculated with JH1140 (a spontaneous mutant of JH1000 that produces threefold-elevated amounts of activity). Agar could not substitute for agarose. Chloroform extraction of the spent medium produced a fraction which yielded two major bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The faster-migrating band was absent in chloroform extracts of the mutant, DM25. The amino acid sequence of this band was determined by Edman sequencing and mass spectroscopy. The results showed that it is a lantibiotic, which we have named mutacin 1140, and that the sequence corresponded to that deduced from thelanA sequence. We observed a number of similarities of mutacin 1140 to epidermin and an S. mutans lantibiotic, B-Ny266, but it appears to have significant differences in the positions of its thioether bridges. It also has other unique features with regard to its leader sequence and posttranslational modification. A proposed structure for mutacin 1140 is presented.

Covalent structure of mutacin 1140 and a novel method for the rapid identification of lantibiotics

“…Purified mutacin 1140 was analyzed by ESI-MS and MS/MS. The calculated mass of 2263 Da was identical to that measured for mutacin 1140 in crude preparations [3] and indicated complete post-translational modifications. MS/MS of the doubly charged molecular ion yielded a complex spectrum of daughter ions ( Table 1).…”

“…Subsequent Edman sequencing yields an amino-acid sequence with uncertain identification of dehydrated and thioether residues (for example, see Fig. 1A) [3]. In order to discriminate between unsaturated amino acids and residues that form thioether rings, we developed a double-labeling protocol in which we first hydrogenated the double bonds and subsequently reduced the thioether linkages with ethanethiol.…”

“…Hillman's group also presented a tentative structure of mutacin 1140 [3]. This tentative structure was based primarily on Edman sequencing and mass spectrometry, but the authors noted that``(the thioether bridge) assignment [would] have to be resolved, most probably by nuclear magnetic resonance spectroscopy'' [3]. In the work presented here, we have determined the structure of mutacin 1140 using NMR spectroscopy.…”

“…In a previous study, Hillman and coworkers reported the genetic and biochemical analysis of mutacin 1140 [3]. In this work, they isolated the mutacin 1140 gene cluster, determined the spectrum of antibacterial activity, characterized a mutant strain of S. mutans (DM25) deficient in antibacterial activity, and reported the isolation and purification of mutacin 1140.…”

“…In this work, they isolated the mutacin 1140 gene cluster, determined the spectrum of antibacterial activity, characterized a mutant strain of S. mutans (DM25) deficient in antibacterial activity, and reported the isolation and purification of mutacin 1140. Hillman's group also presented a tentative structure of mutacin 1140 [3]. This tentative structure was based primarily on Edman sequencing and mass spectrometry, but the authors noted that``(the thioether bridge) assignment [would] have to be resolved, most probably by nuclear magnetic resonance spectroscopy'' [3].…”