Table 1.
Growth stimulation of various M. ulcerans strains by starch.
Figure 1.
Effects of defined carbohydrates on M. ulcerans growth.
A- Growth curves of M. ulcerans 1615 strain grown in MGIT medium containing carbohydrate. The growth has been monitored with the BACTEC system. The dotted line corresponds to the Growth Index (GI) 500. B- Growth Index 500 corresponding to the growth curves from A. The control medium is indicated in black. The conditions in which M. ulcerans growth is significantly stimulated are indicated in red (p<0.001).
Figure 2.
Macroscopic phenotypes of M. ulcerans 1615 strain grown on carbohydrates-containing 7H10 media.
A- Phenotypes on 7H10 plates and picture of the corresponding pellets. B- Quantification of aggregative abilities of M. ulcerans 1615 strain grown on 7H10 medium containing 7.5% of glucose or starch by measuring the decrease in OD600 upon sedimentation of bacterial aggregates in static liquid cultures. Optical density has been measured and compared to initial optical density (t = 0). Relative OD600 is indicated. C- Electrophoretic mobility of M. ulcerans 1615 strain grown on 7H10 medium containing 7.5% of glucose or starch, measured according to NaCl concentration in the suspending medium at constant pH.
Figure 3.
Morphological features of M. ulcerans are not modified by carbohydrates added to the medium.
M. ulcerans strain 1615 was grown in MGIT medium alone or supplemented with 7.5% glucose, maltose, maltopentaose or starch, fixed in the absence (A) or presence (B–F) of 0.1% Malachite green and processed for transmission electron microscopy (TEM). A- Cell wall ultrastructure of bacilli grown in normal medium. Note the presence of the thin electron translucent layer (ETL) and the dense outer layer (OL). Inserts show enlarged view of cell wall (rectangle). B- Morphological appearance of a bacterial clump surrounded by OL (small arrows). Lipids (long arrows) fill the space between bacilli. Note the thick extracellular matrix (ECM). C- Morphological appearance of lipid-rich vesicles (long arrows) surrounded by OL (small arrow) in the ECM. D, E, F- Formation of lipid-rich blebs at the bacterial surface (arrows).
Figure 4.
Total lipids analysis of M. ulcerans.
A- Lipids from M. ulcerans were separated by TLC using CHCl3/CH3OH/H2O [20∶4∶0.5, by volume] as the solvent system. Lipids were revealed with alpha-naphthol (left panel) or cupric sulfate (right panel) followed by heating. B- Lipids from M. ulcerans were separated by TLC using CHCl3/CH3OH [98∶2, by volume] as the solvent system. TLC are shown before (left panel) and after revelation with cupric sulfate and heating (right panel). Arrows highlight new lipid forms in the bacteria grown in 7.5% glucose and maltose-enriched media compared to regular 7H10. Gluc, 7H10 glucose; Malto, 7H10 maltose; MP, 7H10 maltopentaose; starch, 7H10 starch. TDM, trehalose dimycolate; TMM, trehalose monomycolate; GMM, glucose monomycolate; MMM, maltose monomycolate. The compound migrating between TMM and TDM in glucose-grown bacteria does not stain with alpha-naphthol indicating that it is not a glycolipid. Its identity was not determined here.
Figure 5.
Mycolactone quantification by HPLC from M. ulcerans.
Relative quantities have been calculated after normalization to the proteins quantities in each sample. The percentages compared to the 7H10 medium are indicated in the chart. *** p<0.001. ** p<0.05.
Figure 6.
Effect of carbohydrates on mycolactone biosynthesis gene expression in M. ulcerans.
A- Schematic representation of mycolactone biosynthesis genes and representation of their function on the mycolactone structure [9], [10]. The six genes coding for proteins involved in mycolactone synthesis are located on the giant plasmid named pMUM001. The genes mlsA1 (50,973 bp) and mlsA2 (7,233 bp) encode modular type I polyketide synthase (PKS) required for the biosynthesis of the mycolactone core (highlighted in orange). The side chain enzyme is encoded by mlsB gene (42,393 bp) in pink. The mls genes encode 11 different functional domains that are repeated along the gene. Among these modules, the two load modules (LM, in black) are present at 5′end of mlsA1 and mlsB genes and 15 ketoreductase (KR, in grey) are present. There are also three genes coding for potential polyketide-modifying enzymes, including a P450 hydroxylase (mup053, in purple), probably responsible for hydroxylation at carbon 12 of the side chain, a potential FabH-like type III ketosynthases (KS) with an acyltransferase activity that might catalyze the C–O bond between the mycolactone core and side-chain (mup045, in green) and a putative type II thioesterase (mup038, in blue) that may be required for removal of short acyl chains from the PKS loading modules, arising by aberrant decarboxylation. B- Analysis of mycolactone-associated gene expression in M. ulcerans grown on various 7.5% sugar-enriched medium by qRT-PCR. Gene expression has been normalized to the constitutively expressed housekeeping gene ppk encoding the polyphosphate kinase. Relative genes expression is expressed as fold-changes relative to the 7H10 medium. Data are presented as the mean and SD of at least three biological repeats. LM, load module domain.
Table 2.
Effect of carbohydrates on the production of some enzymes involved in mycolactone biosynthesis.
Figure 7.
Identification of mycobactins.
LC/MS analysis (in positive mode) of the orange compound. Shown are the ion traces at m/z 909.45 (A) and at m/z 911.47 (B) which are diagnostic for mycobactins differing by one unsaturation.