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 themutA 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: ΔN1, V7A, P9A, T10A, T10S, C15A, C26A, and C27A. All of these mutacin II variants except ΔN1 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 (ΔN1), indicates that specific residues in the propeptide may be crucial for certain steps in the biosynthetic pathway of this lantibiotic.

The biology of lantibiotics from the lacticin 481 group is coming of age

“…All three (Me)Lan residues are thus strongly involved in biological activity. Interestingly, the T9S mutation, which leads to the replacement of MeLan by Lan, did not impair lantibiotic activity (Chen et al, 1998a). This indicated that it is the presence of a (Me)Lan residue, rather than its nature, which is important.…”

“…Moreover, variacin, nukacin ISK-1 and mutacin II possess a proline (position 8 in Fig. 1) that creates a rigid hinge connecting their linear and globular parts (Chen et al, 1998a). This feature is not shared by the other members of the group (Fig.…”

“…The determination of structure-activity relationships of lacticin 481-related lantibiotics thus requires specific studies. A mutational analysis performed on mutacin II showed that MeLan9-14 and Lan11-25 (numbering according to Figs 1 and 2) are both essential to antimicrobial activity, as replacement of either Cys14 or Cys25 by an alanine led to the production of inactive mutants (Chen et al, 1998a). Disruption of Lan18-26 by the C26A mutation reduced, but did not abolish, the antimicrobial activity (Chen et al, 1998a).…”

Determination of the amino acid residues required for the activity of the anti-rhizobial peptide antibiotic trifolitoxin

“…The lanthionine ring (ring c) and ring d of the antibiotic epidermin are required for activity (Augustin 1992). In addition, the lanthionine rings of mutacin II and subtilin are required for activity, although the subtilin variant only shows this phenotype against Bacillus cereus T spores (Liu and Hansen 1993;Chen et al 1998). Many ring structures including lanthionine, b-methyllanthionine and oxazole-thiazole rings have been found to be important although not required for antibiotic activity (Augustin 1992;Dodd et al 1992;Bierbaum et al 1994;Dodd et al 1995;Kuipers et al 1996;Sinha Roy et al 1999).…”

“…Site-directed mutagenesis of ribosomally synthesized peptide antibiotics has been used for structure-activity analyses of these molecules. The antibiotic microcin B17 as well as the lantibiotics nisin, subtilin, pep5, gallidermin, epidermin, mersacidin and mutacin II have all been subjected to analysis by this method (Augustin 1992;Dodd et al 1992Dodd et al , 1995Dodd et al , 1996Bierbaum et al 1994;Kuipers et al 1996;Chen et al 1998;Szekat et al 2003). Like TFX, these antibiotics are gene-encoded and require posttranslational modifications provided by adjacently coded proteins.…”