GENETIC AND BIOCHEMICAL-ANALYSIS OF MUTACIN-1140, A LANTIBIOTIC FROM STREPTOCOCCUS-MUTANS

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 bacterioc in-like inhibitory substance (BLIS). The thermosensitive vector pTV1-O K harboring Tn917 was used to isolate a BLIS-deficient mutant, DM25, a nd the mutated gene was recovered by shotgun cloning in Escherichia co li. Sequence analysis of insert DNA adjacent to Tn917 led to the ident ification of four open reading frames including two (lanA and lanB) wh ich have substantial homology to the Staphylococcus epidermidis struct ural gene (epiA) and a modifying enzyme gene (epiB) for biosynthesis o f 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% ag arose that was stab inoculated with JH1140 (a spontaneous mutant of JH 1000 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 s ulfate-polyacrylamide gel electrophoresis. The faster-migrating band w as absent in chloroform extracts of the mutant, DM25. The amino acid s equence of this band was determined by Edman sequencing and mass spect roscopy. The results showed that it is a lantibiotic, which we have na med mutacin 1140, and that the sequence corresponded to that deduced f rom the lanA sequence. We observed a number of similarities of mutacin 1140 to epidermin and an S. mutans lantibiotic, B-Ny266, but it appea rs to have significant differences in the positions of its thioether b ridges. It also has other unique features with regard to its leader se quence and posttranslational modification. A proposed structure for mu tacin 1140 is presented.