Actual and expected frequencies of hinge amino acids from randomly selected representatives of the hinge mutant bank.

<p>Actual and expected frequencies of hinge amino acids from randomly selected representatives of the hinge mutant bank.</p

Deferred Antagonism and Specific Activity Results.

<p>The zone of inhibition is expressed as the area of the zone of inhibition minus the area of the ‘spot’ in mm². MIC: Minimum Inhibitory Concentration. ND: Not Determined.</p

Oligonucleotides used in this study.

<p>PHO – 5’- Phosphate modification. Emboldened – Degenerate codons, emboldened & underlined – locations for site-directed mutagenesis.</p

A - Nisin A mature peptide, B - AAA Producer (Deferred Antagonism Assay).

<p>The structure of the mature nisin A peptide and the zone of inhibition exhibited by the nisin A derivative, AAA. Panel A depicts the amino acid changes that produce the AAA derivative in the nisin ‘hinge’ region. The modified amino acids dehydroalanine and dehydrobutyrine are denoted as Dha and Dhb respectively with the five (β-methyl)lanthionine rings labelled A to E. Panel B depicts the zones of inhibition produced by <i>L. lactis</i> NZ9800 pDF05(left) and AAA ‘hinge’ variant (right) against <i>L. lactis</i> HP.</p

Nisin A ‘hinge’ variants with enhanced bioactivity identified through the initial screen.

<p>The three letter code corresponds to the amino acids located at each of the three ‘hinge’ sites.</p

Frequency with which amino acids are located at each hinge site among the derivatives presented in Table 3.

<p>Frequency with which amino acids are located at each hinge site among the derivatives presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079563#pone-0079563-t003" target="_blank">Table 3</a>.</p

Bioactivity of K12A, K12S and K12T producers against various Gram-positive targets.

<p>Values are the mean of triplicate deferred antagonism assays and represent zone of inhibition (diameter of zone minus diameter of bacterial growth) expressed as a percentage compared to that of the wild-type nisin producer at 100%. S.D.: Standard Deviation; Relative Standard Deviation<10% for each given value. All values in bold reached statistical significance compared to nisin control (K) (Student's t-test: P<0.05). Strains marked with an asterisk are drug resistant isolates.</p

Minimum inhibitory concentrations of purified nisin (WT) and nisin K12A against various Gram-positive targets.

<p>Results from minimum inhibitory concentration assays of purified nisin (WT) and nisin K12A against various Gram-positive targets. Values given are identical results from three independent determinations. Fold Difference represents the improvement of K12A compared to nisin against the relevant indicator.</p

Oligonucleotides used in this study.

<p>PHO indicates 5′ phosphate modification. Underlined sequences represent degenerate codon (N = A+C+G+T, K = G+T, M = A+C). Lower-case letters indicate site-directed mutation.</p

Structure of nisin A.

<p>The structure of nisin A is depicted, showing the location of its five (β-methyl)lanthionine rings (A–E) and modified residues dehydroalanine (yellow) and dehydrobutyrine (green). Position K12 is highlighted in red and the hinge region is boxed.</p