Primers used in this study.

<p>Primers used in this study.</p

Antibacterial activity of IVK45 wild type compared to nukacin deletion mutant and complemented mutant.

<p>A: IVK 45 wild type (1); nukacin-deficient mutant Δ<i>nukA</i> (2); and complemented mutant (3) on <i>M</i>. <i>luteus</i> lawns. B: <i>M</i>. <i>luteus</i> cultures supplemented with 20% and 40% supernatant (SN) of IVK 45 wild type and nukacin-deficient mutant; C: <i>M</i>. <i>catarrhalis</i> cultures in spent medium or supplemented with 4-fold concentrated activity of IVK 45 wild type and nukacin-deficient mutant; D: <i>C</i>. <i>accolens</i> cultures in spent medium or supplemented with 50% supernatant of IVK 45 wild type and nukacin-deficient mutant; E: Nukacin insensitive <i>S</i>. <i>aureus</i> Newman cultures supplemented with 40% and 80% supernatant of IVK 45 wild type and nukacin-deficient mutant as negative control.</p

Frequency and activity spectra of antimicrobial substances produced by nasal <i>Staphylococcus</i> isolates.

<p>Pattern and intensity of test strain inhibition by nasal <i>Staphylococcus</i> isolates is shown as a heat map. They are ordered hierarchically by activity patterns according to the number and identity of inhibited strains (indicated by numbers in the last column) against <i>Actinobacteria</i> (A-E); <i>Proteobacteria</i> (F-G); <i>Firmicutes</i> (H-J). (i) indicates inducible bacteriocin production, which was only visible under iron-limitation stress. MLST types are given for 19 <i>S</i>. <i>epidermidis</i> strains and <i>spa</i>-types are indicated for all <i>S</i>. <i>aureus</i> isolates. (n.t.; non-typeable).</p

Co-cultivation of <i>M</i>. <i>catarrhalis</i> and <i>S</i>. <i>epidermidis</i> IVK45 strains.

<p><i>S</i>. <i>epidermidis</i> IVK45 wild type and mutant IVK45 Δ<i>nukA</i> (black) were inoculated at ratios of 3:1 with <i>M</i>. <i>catarrhalis</i> (grey) on solid agar. <i>M</i>. <i>catarrhalis</i> is overgrown by the nukacin-producing IVK45 wild type after 48 hours. In contrast, the numbers of the nukacin-deficient mutant IVK45 Δ<i>nukA</i> and <i>M</i>. <i>catarrhalis</i> shift towards a ratio of 1:1 after 48 hours. Significant differences between the IVK45 wild type and mutant Δ<i>nukA</i> ratios after 48 hours were analyzed by two tailed paired t-test (** <i>P</i> < 0.005).</p

Frequency of antimicrobial activitiy in nasal <i>Staphylococcus</i> isolates.

<p>Frequency of antimicrobial activitiy in nasal <i>Staphylococcus</i> isolates.</p

Nukacin IVK 45 operon, predicted peptide structure and composition of plasmid pIVK45.

<p>A: comparison of the operon structures from <i>S</i>. <i>warneri</i> ISK-1 (top) and <i>S</i>. <i>epidermidis</i> IVK45 (bottom), Tn: insertion site of the transposon. B: Predicted structure of nukacin IVK45. Amino acid positions of nukacin IVK45, which are different in corresponding peptides from <i>S</i>. <i>warneri</i> ISK-1 and <i>S</i>. <i>hominis</i> KQU-131 are shown in grey. The additional different amino acid in <i>S</i>. <i>hominis</i> KQU-131 is shown in a grey pattern; A-S-A, lanthionine (thioether bridge between cysteine and serine); Abu-S-A, 3-methyllanthionine (thioether bridge between cysteine and threonine); Abu, aminobutyrate (threonine within the methyllanthionine ring); Dhb, dehydrobutyrine (dehydrated threonine). C: Intact genes or fragments for transposases, recombinases, IS- and IS-like elements indicate multiple recombination events in the genesis of pIVK45. Outer ring of plasmid: identified genes are indicated by arrows. Inner ring: The color of the various segments indicates their most likely species origin (analyzed by BLAST). Red: <i>S</i>. <i>warneri</i>, blue: <i>S</i>. <i>aureus</i>, light green: <i>S</i>. <i>epidermidis</i>, dark green: <i>S</i>. <i>aureus</i> and <i>S</i>. <i>epidermidis</i>, yellow: <i>S</i>. <i>lugdunensis</i>, lilac: many different <i>Staphylococcus</i> species, light blue: IS-like element. White segments show unique DNA fragments with no homologies in available databases.</p

Bacteriocin induction by iron limitation or H₂O₂.

<p>Intensity of inhibitory activities of IVK strains 1–96 against <i>M</i>. <i>luteus</i> or <i>S</i>. <i>aureus</i> without stressors (normal) or in the presence of 2,2’-bipyridine (iron limitation) or H₂O₂ (H₂O₂ stress).</p

AGOS: A Plug-and-Play Method for the Assembly of Artificial Gene Operons into Functional Biosynthetic Gene Clusters

The generation of novel secondary metabolites by reengineering or refactoring biochemical pathways is a rewarding but also challenging goal of synthetic biology. For this, the development of tools for the reconstruction of secondary metabolite gene clusters as well as the challenge of understanding the obstacles in this process is of great interest. The artificial gene operon assembly system (AGOS) is a plug-and-play method developed as a tool to consecutively assemble artificial gene operons into a destination vector and subsequently express them under the control of a de-repressed promoter in a <i>Streptomyces</i> host strain. AGOS was designed as a set of entry plasmids for the construction of artificial gene operons and a SuperCos1 based destination vector, into which the constructed operons can be assembled by Red/ET-mediated recombination. To provide a proof-of-concept of this method, we disassembled the well-known novobiocin biosynthetic gene cluster into four gene operons, encoding for the different moieties of novobiocin. We then genetically reorganized these gene operons with the help of AGOS to finally obtain the complete novobiocin gene cluster again. The production of novobiocin precursors and of novobiocin could successfully be detected by LC–MS and LC–MS/MS. Furthermore, we demonstrated that the omission of terminator sequences only had a minor impact on product formation in our system

Mixed-terpenoid secondary metabolites of <i>Streptomyces</i> sp. CNQ-509.

<p>Mixed-terpenoid secondary metabolites of <i>Streptomyces</i> sp. CNQ-509.</p

Phylogenetic tree of ABBA prenyltransferases of the phenol / phenazine family.

<p>Data include previously biochemically characterised ABBA prenyltransferases and those investigated in this study. The tree was constructed with MEGA6 using default parameter for multiple sequence alignment (CLUSTALW) and neighbour-joining method. Bootstrap values (in percent) calculated from 1000 replications are shown at the respective nodes. The fungal indole prenyltransferase DMATS (shares PT barrel) serves as a root.</p