Journal: Journal of Bacteriology

Article Title: Biosynthesis and Transport of the Lantibiotic Mutacin 1140 Produced by Streptococcus mutans

doi: 10.1128/jb.02531-14

Figure Lengend Snippet: Figure 1. Lantibiotic structural elements. A) Covalent structures for nisin and mutacin 1140 with 547 the lanthionine rings labeled from N- to C-terminus. B) Leader sequence alignments of 548 structurally related Class I lantibiotics; i.e. nisin produced by Lactococcus lactis, subtilin 549 produced by Bacillus subtilis, epidermin produced by Staphylococcus epidermidis, gallidermin 550 produced by Staphylococcus gallinarium, Pep5 produced by Staphylococcus epidermidis, 551 epilancin K7 produced by Staphylococcus epidermidis, mutacin Ny266 produced by S. mutans 552 Ny266, mutacin III produced by S. mutans UA787, mutacin 1140 produced by S. mutans 553 JH1140, and for comparison a class II lantibiotic lacticin 481 produced by Lactococcus lactis 554 (16, 18, 47-53). The F(N/D)LD box and C-terminal proline in class I lantibiotics is underlined 555 and in bold print in the sequences. C) Secondary structure prediction using SOPMA for mutacin 556 1140 leader peptide; h (alpha helix), e (extended strand), c (random coil), t (beta turn). Alpha 557 helical regions are underlined, while random coils are in bold in the leader peptide sequence. 558 559 Figure 2. Identification of structural elements within the mutacin 1140 leader peptide that are 560 important for bioactivity. A) Covalent structure representation of the mutations made on the 561 leader peptide. Bioactivity for leader and core peptide mutants were measured as the percent 562 difference in the zone of inhibition between wild-type and the mutant strains. ∆lanA strain was 563 used as a negative control for bioactivity in all experiments. The change in activity was measured 564 for: B) mutations near the LanP cleavage site, C) mutations within the newly discovered 565 cleavage site, and D) mutations in the transporter lanT and core peptide regions responsible for 566 ring formation. For each mutation, the bioactivity has been compared to the activity of wild-type 567 S. mutans JH1140 strain. All the activities are determined in the absence of trypsin in the top 568 agar of the overlays. Statistical method used was Student t-test and the asterisk signifies 569 statistical significance (p<0.05). 570 571 Figure 3. HPLC chromatograms of crude extracts obtained from modified THyex media 572 inoculated with A) wild-type S. mutans JH1140 B) and S. mutans ∆lanP. There is more than a 573 two-fold increase in isolated product from the S. mutans ∆lanP strain. 574 575 Figure 4. Characterization of the leader peptide ∆(-7-2) mutant. A) Overlay assay using M. 576 luteus as an indicator strain. From top left to bottom right: 5 µL spotted of purified mutacin 1140 577 (10 µg/mL) in acetonitrile:water (1:1); (negative control) 5 µL spotted of acetonitrile:water (1:1); 578 deferred antagonism assay of S. mutans ∆(-7-2); deferred antagonism assay of S. mutans 579 ∆lanP∆(-7-2); 5 µL spotted of trypsin digestion of product from S. mutans ∆(-7-2); and 5 µL 580 spotted of trypsin digestion of product from S. mutans ∆lanP∆(-7-2). B) MALDI-MS data of 581 product from S. mutans ∆(-7-2). C) MALDI-MS data of product from S. mutans ∆lanP∆(-7-2). 582 D) MALDI-MS data of the 2-mercaptoethanol (BME) derivitization of the isolated products 583 from S. mutans ∆(-7-2) strain. 584 585 Figure 5. Transport efficiencies of mutacin 1140 core peptide variants. Alanine substitutions 586 were made to disrupt ring A (C(7)A), ring B (C(11)A), ring C (C(21)A), ring D (S(19)A), rings 587 AB (C(7)A:C(11)A), and rings CD (C(21)A:S(19)A). Three independent extractions of each 588 mutant strain were characterized by RP-HPLC at 220 nm. Peak volumes of the variants of 589 mutacin 1140 fractions were compared to wild-type strain JH1140 and are represented as percent 590 of product relative to wild-type strain. Statistical method used was Student t-test and the asterisk 591 signifies statistical significance (p<0.05). 592 593

Article Snippet: THyex broth (30 g/L Todd Hewitt Broth, 3 g/L yeast extract), THyex agar media 439 (30 g/L Todd Hewitt Broth, 3 g/L yeast extract, 15 g/L agar; Bacto, Sparks, MD) and Top agar 440 media (30 g/L Todd Hewitt Broth, 3 g/L yeast extract, 7.5 g/L agar; Bacto, Sparks, MD) was 441 used to culture S. mutans JH1140 ATCC 55676 and Micrococcus luteus ATCC 10240 at 37 °C.

Techniques: Labeling, Sequencing, Produced, Comparison, Inhibition, Mutagenesis, Negative Control, Activity Assay, Modification, Isolation, Overlay Assay, Purification