Structure-activity study of the lantibiotic mutacin II from Streptococcus mutans T8 by a gene replacement strategy

Abstract

Mutacin II, elaborated by group II Streptococcus mutans, is a ribosomally synthesized and posttranslationally modified polypeptide antibiotic containing unusual thioether and didehydro amino acids. To ascertain the role of specific amino acid residues in mutacin II antimicrobial activity, we developed a streptococcal expression system that facilitates the replacement of the mutA gene with a single copy of a mutated variant gene. As a result, variants of mutacin II can be designed and expressed. The system was tested by constructing the following mutant peptides: delta N1, V7A, P9A, T10A, T10S, C15A, C26A, and C27A. All of these mutacin II variants except delta N1 and T10A, which were not secreted, were isolated, and their identities were verified by mass spectrometry. Variants P9A, C15A, C26A, and C27A failed to exert antimicrobial activity. Because the P9A and T10A variants comprise the "hinge" region of mutacin II, these observations suggest that in addition to the thioether and didehydro amino acids, the hinge region is essential for biological activity and biosynthesis or export of the peptide. Tandem mass spectrometry of the N-terminal part of the wild-type molecule and its C15A variant confirmed that the threonine at position 10 is dehydrated and present as a didehydrobutyrine residue. This analysis of the active T10S variant further suggested that a didehydro amino acid at this position is specific for antimicrobial activity and that the biosynthetic machinery does not discriminate between threonine and serine. In contrast, the lack of production of mutacin variants with alanine substituted for threonine at position 10, as well as the deletion of asparagine at the N terminus (delta N1), indicates that specific residues in the propeptide may be crucial for certain steps in the biosynthetic pathway of this lantibiotic.

FIG. 1

Construction of strain CBM0 (ΔmutA). (a) Region of S. mutans T8 chromosome encoding mutA and start of mutM. The position of the mutacin promoter (P) is indicated by an arrow. (b) Up- and downstream portions of mutA were PCR amplified and cloned into the pCRII vector. (c) Two fragments were ligated into the pCRII vector. (d) tetM gene was inserted at the HpaI site. The final plasmid was treated with restriction enzyme XbaI and then used to transform wild-type S. mutans T8. A transformant which was tetracycline resistant and mutacin negative was designated S. mutans CBM0.

FIG. 2

Primary structure of the unmodified propeptide of mutacin II and mutations that were introduced. T in position 10 is shown as dehydrated residue.

FIG. 3

MS-MS of the C15A variant of mutacin II. The doubly charged ion was allowed to move into the second quadrupole, where it collided with argon atoms. The fragments were then analyzed with the third quadrupole. The spectrum shows part of the N-terminal sequence with a series of b ions corresponding to the N-terminal amino acid sequence (NRWWQGVVPdhBV−); the m/z values were 271, 457, 643, 771, 828, 928, 1,027, 1,124, 1,207, and 1,306 for b2 to b11, respectively. The signals with m/z values of 1,027, 1,124, 1,207, and 1,306 represent fragments of the N termini ending with Val, Pro, Dhb, and Val, respectively.

FIG. 4

EIS-MS analysis of purified P9A variant of mutacin II. The molecular mass calculated from multiply charged molecular ions was 3,218.4 ± 1.2 Da.