# Nisin E, a New Nisin Variant Produced by _Streptococcus equinus_ MDC1
## 4 Discussion
These additional indicator bacteria may be useful for the detection of subtle differences in the inhibitory spectra of bacterios of interest, as evidenced by the testing results listed in Table 2. Comparison of the inhibitory spectra of nisin E and its homologo nisin U revealed that not only fewer species were sensitive to the antibacterial effects of nisin E but also that the potency of nisin E appeared reduced against some species, e.g., _Streptococcus mitis_ (Table 2). This could be explained by the amino acid differences between nisin E and nisin U (Figure 2). Although there are only two amino acid differences between the two peptides, isoleucine15\(\rightarrow\)alanine and leucine21\(\rightarrow\)isoleucine, of which the latter is a conserved substitution, nisin E has an additional asparagine residue at its C-terminus (Figure 2). As shown in Figure 2, these amino acid differences are not expected to affect the post-translational modification of the various residues essential in forming the modified amino acids didehydrodanarine, didehydrobutyrine, lanthionine and methyl-lanthionine, along with the associated internal thioether bridges. However, the isoleucine15\(\rightarrow\)alanine substitution and the presence of the additional asparagine might influence the overall structure of the nisin E propeptide, thus compromising its interaction with the cell envelopes of certain streptococcal species such as _Streptococcus gordonii_ and _Streptococcus anginos_. Unlike the unmodified class II bacterios which can be chemically synthesized and readily tested, nisin E requires extensive post-translational modification of the _nmdA_ gene product, hence the requirement for at least the _nmdBTC_ and _nmdP_ genes to be present. Future work to explore engineered variants of nisin E and nisin U that either target specific pathogens or kill a wider range of bacteria may involve expression systems already established for the expression of lantibiotic gene clusters [26; 27]. One of the unexpected findings during deferred antagonism testing was the partial inhibition of _S. equinus_ MDC1 by nisin U. As both strain MDC1 and _S. uberis_ ATCC 27958 produce nisin U-like bacterios, and their respective biosynthetic loci possessed the immunity genes (e.g., _nmdFEG_ and _nmdl_), it was expected that both strains would be cross-immune to the other's bacteriocin. This could be explained by the considerable differences in their putative immunity proteins (Table 3), which range from 47.1% identity (68.4% similarity) to 74.8% identity (85.2% similarity) for NmdI/Nsul and NmdF/NsuF, respectively. As producer self-immunity relies on the concerted interactions of the ABC transporter (comprising the NisFEG/NsuFEG/NmdFEG proteins) and the membrane-bound NisI/Nsul/NmdI proteins to pump out the bacteriocin [28], it is possible that the NmdFEGI complex is optimized for nisin E efflux and therefore unable to protect adequately against nisin U due to the inability to fully recognize nisin U. In contrast, either the NsuFEG and NsuI proteins are able to protect _S. uberis_ ATCC 27958 against a range of nisin-like peptides (including nisin E). An alternative explanation is that nisin E itself is intrinsically unable to effect pore formation in _S. uberis_. Although beyond the scope of the present study, future work could be conducted to answer this intriguing question, perhaps by expressing _nmdFEGI_ in _S. uberis_ and examining its effect on immunity to nisin E.  
During the course of this research project, the genome of _S. equinus_ MDC1 was sequenced, primarily motivated by the aim of locating the bacteriocin-encoding biosynthetic locus. Indeed, the MDC1 genome sequence has served this purpose. However, _S. equinus_ MDC1 has an unusual growth characteristic on mutans selective agar (a medium containing 20% sucrose and the antibiotic bactircain) routinely used to cultivate the cariogenic pathogen _Streptococcus mutans_ and related organisms [29].