SDS-PAGE and western immunoblot analysis of whole cell lysates and cell fractions of <i>M. marinum</i> M harbouring plasmids expressing LM-M8 and MlsA2_His.

<p>(A) SDS-PAGE separation and Coomassie-stained protein gel of 10 μg of whole cell lysate of <i>M. marinum</i> M with LM-M8 and <i>mlsA2</i> (TPS8313) and empty vector control (TPS8256); (B) Western immunoblot of (A) using an anti-AT domain antibody; (C) Western immunoblot of (A) using an anti-His antibody; (D) Western immunoblot of cell fractions from <i>M. marinum</i> M harbouring plasmids expressing LM-M8 and MlsA2_His using an anti-His antibody, showing MlsA2 is present only in the cell wall (P27) fraction. The reactivity of the anti-His antibody to a protein with a mass ∼65 kDa in panels (C) and (D) is the known cross-reactivity with the polyhistidines of mycobacterial GroEL (Hsp65) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070520#pone.0070520-Noens1" target="_blank">[37]</a>.</p

Gene, module and domain organisation in the bimodular MlsA-derived PKS.

<p>(A) Arrangement using the MlsA1 load module from <i>M. ulcerans</i> Agy99 for the biosynthesis of methyl-triketide lactone; (B) Arrangement using the MlsA1 load module from <i>M. ulcerans</i> Liflandii for the biosynthesis of ethyl-triketide lactone.</p

Schematic view of the mycobacterial expression vectors developed for this study.

<p>(A) pTPS333, pYUB412-based integrating vector with <i>mlsA1</i> LM-M8 under the control of the <i>mlsA1</i> promoter; (B) pTPS629, pMUM001-based low-copy number vector with <i>mlsA1</i> LM-M8 under the control of the <i>mlsA1</i> promoter; (C) pTPS334, pYUB412-based integrating vector with <i>mlsA2</i> under the control of the <i>mlsA1</i> promoter; (D) pTPS338 pMV261-based vector with mup038 and mup045 in an operon and under the control of the <i>ermE</i> promoter.</p

Bacterial strains used in the study.

<p>Bacterial strains used in the study.</p

Expression of mycolactone accessory enzymes encoded by mup045 and mup038.

<p>(A) RT-PCR of mup045, mup038 and <i>crtI</i> on RNA extracted from <i>M. marinum</i> M containing plasmids expressing LM-M8 and mup045-mup038 under control of the P<i>_ermE</i> promoter. Western immunoblots showing: (B) presence of Mup038 in the cell wall fraction of <i>M. marinum</i> TPS8334; (C) presence of Mup045 in whole cell lysates from <i>M. marinum</i> M expressing LM-M8 and mup045-mup038 (TPS8334) as well as <i>M. ulcerans</i> 06-3844 harbouring pTPS331 (TPS8164) or pTPS333 (TPS8162); (D) Localization of Mup045 to the cell wall in <i>M. ulcerans</i> 06-3844 wild type and <i>M. marinum</i> TPS8334 cell fractions, showing reactivity against Mup045 in all strains, and localized to the cell wall fraction in both strains. Positive controls are purified, recombinant Mup038 and Mup045. (D).</p

Stability of pTPS629 in <i>M. marinum</i> M cultured in the absence of apramycin antibiotic selection.

<p>A late log-phase culture of <i>M. marinum</i> harbouring pTPS629 (and <i>mlsA2</i> on pTPS334) grown in the presence of apramycin and hygromycin, was shifted to media without apramycin and then monitored at successive time points by determining the cfu/ml on media with the antibiotic (squares) and calculating the percentage of <i>M. marinum</i> cells retaining apramycin resistance (right hand Y-axis, triangles). These results depict the mean and standard deviation of at least biological triplicates.</p

Chromatogram of fragment ions at m/z 73.3 and 97.3 from MS/MS analysis of [M+H]+ ion at m/z 115.1.

<p>(A) control; (B) sample and (C) standard compound, 5-Hydroxyhexanoic acid lactone. Insets are the MS/MS spectrum for [M+H]+ ion at m/z 115.</p

Plasmids used in this study.

*<p>LM-M8 refers to the loading module and module 8 regions encoded within <i>mlsA1</i>; LM_P-M8 refers to the loading module from <i>M. ulcerans</i> Liflandii <i>mlsB</i> fused with <i>mlsA1</i> M8 from <i>M. ulcerans</i> Agy99.</p

Expression analysis of recombinant <i>M. marinum</i> TPS8334.

<p>(A) Coomassie stained SDS-PAGE and (B) Western immunoblot of cell wall fractions of <i>M. marinum</i> expressing LM-M8, MlsA2_His, Mup038 and Mup045 (TPS8334) using an anti-AT domain antibody demonstrating the presence of the heterologously expressed PKSs in the cell wall fraction of this strain. The positive control is purified, recombinant acyltransferase from MlsA2.</p

SDS-PAGE and western immunoblot analysis of <i>M. ulcerans</i> 06-3844 expressing TKL constructs.

<p>(A) SDS-PAGE separation and Coomassie-stained protein of 10 μg of <i>M. ulcerans</i> 06-3844 containing <i>mlsA1</i> LM-M8 (TPS8162), LM_P-M8 (TPS8307) or empty vector (TPS8164) cell fractions (B) Western immunoblot analysis of (A) with an anti-AT domain antibody showing the presence of a ∼400 kDa protein produced by <i>M. ulcerans</i> harbouring either LM-M8 or LM_P-M8 (lane 1 and 4). Positive control is purified, recombinant acyltransferase derived from MlsA2.</p