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Infect Immun, June 1998, p. 2743-2749, Vol. 66, No. 6
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

J. D. Hillman,1,* Jan Novák,2 Edy Sagura,3 Juan A. Gutierrez,1,dagger T. A. Brooks,1 Paula J. Crowley,1 M. Hess,1 Abdul Azizi,1 K.-P. Leung,1 Dennis Cvitkovitch,1,Dagger and A. S. Bleiweis1

Department of Oral Biology, University of Florida College of Dentistry,1 and Interdisciplinary Center for Biological Research Protein Chemistry Core, University of Florida College of Medicine,3 Gainesville, Florida 32610, and Departments of Microbiology and Oral Biology, University of Alabama at Birmingham, Birmingham, Alabama 352942

Received 24 November 1997/Returned for modification 18 December 1997/Accepted 4 March 1998

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 in Escherichia 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 epidermidis structural 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 the lanA 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.

* Corresponding author. Mailing address: University of Florida School of Dentistry, Box 100424, Gainesville, FL 32610. Phone: (352) 846-0792. Fax: (352) 392-2361. E-mail: jhillman{at}dental.ufl.edu.

dagger Present address: Millennium Pharmaceuticals, Inc., Cambridge, MA 02189.

Dagger Present address: University of Toronto Dental Research Institute, Toronto, Canada M5G 1G6.

Infect Immun, June 1998, p. 2743-2749, Vol. 66, No. 6
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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