# Nisin E, a New Nisin Variant Produced by _Streptococcus equinus_ MDC1
## 3 Results
### Genome Sequence Analysis and Identification of the Nisin E Biosynthetic Locus  
The genome of _Streptococcus equinus_ MDC1 comprises a single 1,936,555-bp chromosome containing seven copies of the ribosomal RNA (16S, 23S and 5S) operon, 69 tRNA genes and 1899 putative coding sequences (CDS). The overall G + C content of the genome is 37.4%, which is consistent with those obtained with other streptococcal genomes. When the MDC1 genome sequence was analyzed by BAGEL4 and antiSMASH, both algorithms detected the presence of a Antibiotic-encoding locus, specifically that of nisin U, thus confirming that strain MDC1 did indeed produce a nisin-like lantibiotic (now called nisin E).  
The nisin E biosynthetic locus, \(nmd\), in the same way as its nisin A and nisin U counterparts, comprises 11 genes that encode proteins in several functional modules (Figure 1): \(nmdA\) (nisin E precursor peptide), \(nmdBCT\) (modification and transport proteins), \(nmd\)-_PRK_ (peptide processing and regulation of synthesis), and \(nmdFEGI\) (producer immunity). Although the genetic loci of the various nisins are comparable in length (13,164 (\(nmd\)) to 13,540 bp (\(nsu\))), the organization of each is different with respect to the juxtaposition of the functional modules (Figure 1). Whereas the nisin A locus is organized in the order \(nisABTCIPRKFE\), i.e., with the structural gene being the first, followed by the modification/transport, regulation and immunity genes [21], the nisin U locus appears to be swapped such that the processing/regulation and immunity genes are at the 5\({}^{\prime}\)-end with the structural gene in the middle of the cluster (Figure 1) [10]. The organization of the \(nmd\) locus essentially follows that of the \(nsu\) locus, except that \(nmdA\) is located between the regulation and immunity modules rather than being immediately upstream of the modification/transport genes (Figure 1). However, this unusual arrangement of genes obviously does not affect the overall expression of the \(nmd\) locus as functional nisin E is produced.  
\begin{table}
\begin{tabular}{c c c c} \hline  & \multicolumn{3}{c}{**Producer Strain (Bacteriocin Produced)**} \\ \cline{2-4}  & _S. uberis_ **ATCC 27958 (Nisin U)** & _S. equinus_ **MDC1 (Nisin E)** \\ \hline
**Indicator Bacteria** & **No. of Strains** & **Potency of Inhibitory** & **No. of Strains** & **Potency of Inhibitory** \\
**(no. of Strains)** & **Inhibited** & **Activity 1** & **Inhibited** & **Activity 1** \\ \hline _Bacillus tropicus_ (1) & \(0/1\) & - & 0/1 & - \\ _Escherichia coli_ (1) & \(0/1\) & - & 0/1 & - \\ \hline \end{tabular}  
* The potency of inhibitory activity is semi-quantitative and based on the width (in mm) of growth inhibition beyond the producer growth streak: +++, >15 mm; ++, >10 and up to 15 mm; +, up to 10 mm; -, no growth inhibition detected. 2 Partial growth inhibition of _S. equinus_ MDC1 by nisin U was observed, i.e., there was some growth within the _S. uberis_ ATCC 27958 product streak. 3 For clarity, the indicator bacteria have been divided into three categories: (I) where both bacterios exhibit identical inhibitory activity spectra; (II) where both bacterios display different inhibitory activity spectra (including species that are not inhibited by nisin E); (III) strains not inhibited by both bacterios.  
\end{table}
Table 2: _Cont._  
Figure 1: Comparison of the nisin A (_L. lactis_) [21], nisin U (_S. uberis_) [10] and nisin E (_S. equinus_) biosynthetic loci.  
Whilst the NmdA and NsuA precursor peptides exhibit relatively high homology scores (83.9% identity, 92.9% similarity), mainly due to differences in their leader peptides, the mature nisin E and nisin U peptides differ by only two amino acids, with nisin E having one extra asparagine residue at its C-terminus (Figure 2). However, there is considerably less homology (47% to 76% identity, 68% to 89% similarity) between the other proteins of the biosynthetic machinery, e.g., the immunity protein NmdI displaying only 47.4% identity (68.4% similarity) to NsuI (Table 3).