Mass-Spectrometry-Based Quantification of Protein-Bound Fatty Acid Synthesis Intermediates from <i>Escherichia coli</i>

The production of fatty acids from simple nutrients occurs via a complex biosynthetic pathway with dozens of intermediate compounds and multiple branch points. Despite its importance for microbial physiology and biotechnology, critical aspects of fatty acid biosynthesis, especially dynamics of in vivo regulation, remain poorly characterized. We have developed a liquid chromatography/mass spectroscopy (LC–MS) method for relative quantification of fatty acid synthesis intermediates in <i>Escherichia coli</i>, a model organism for studies of fatty acid metabolism. The acyl carrier protein, a vehicle for the substrates and intermediates of fatty acid synthesis, is extracted from <i>E. coli</i>, proteolytically digested, resolved using reverse-phase LC, and detected using electrospray ionization coupled with a tandem MS. Our method reliably resolves 21 intermediates of fatty acid synthesis, with an average relative standard deviation in ratios of individual acyl-ACP species to total ACP concentrations of 20%. We demonstrate that fast sampling and quenching of cells is essential to accurately characterize intracellular concentrations of ACP species. We apply our method to examine the rapid response of fatty acid metabolism to the antibiotic cerulenin. We anticipate that our method will enable the characterization of in vivo regulation and kinetics of microbial fatty acid synthesis at unprecedented detail and will improve integration of fatty acid synthesis into models of microbial metabolism

Eradication of MRSA V0608892/1 from the explants after application of bacteriophage solution and mupirocin.

<p>Data are presented as the mean CFU ± standard deviation of three experiments; time is hours after adhesion.</p

Evaluation of porcine mucosa explants after cultivation by means of light microscopy.

<p>Sections of 4 µm thickness were stained by immunohistochemistry to evaluate the apoptosis of cells. TUNEL-positive cells in the epithelium are indicated with white arrows (panel A). Panel B shows the thickness of the epithelium after staining with haematoxylin-eosin (indicated with a white arrow).</p

MRSA recovery in the <i>in vivo</i> experiment.

<p>Culture results for nasal samples in the <i>in vivo</i> experiment. Numbers of MRSA are displayed in CFU/swab. The error bars represent standard deviation. Each group consisted of 4 piglets. Group A) piglets that were treated with bacteriophage solution and received mupirocin ointment; B) piglets that received a placebo without bacteriophage and were treated with mupirocin ointment; group C) received a bacteriophage treatment, but no mupirocin and group D) was administered a placebo without bacteriophage and was not treated with mupirocin. △) indicate bacteriophage or placebo treatment; ■) indicate ointment with mupirocin. If a sample was obtained on the same day as treatment took place the sample was taken before treatment was applied.</p

Effect of bacteriophage solution on the growth of MRSA V0608892/1.

<p>OD values reflect bacterial concentration in presence of the bacteriophage solution, placebo and no-treated control. The results are presented as the mean OD ± standard deviation of 3 different experiments in duplicate.</p

Scanning electron micrographs of porcine nasal epithelium.

<p>Epithelial cells at 0 h (A) and after 72 h (B) of <i>ex vivo</i> cultivation.</p

MRSA colonization of the porcine mucosa explants.

<p>Log scale presence of MRSA isolates S0462 (▾), S0385-1 (▴) and S0385-2 (▪) on the porcine nasal mucosa explants. Data are presented is the mean CFU ± standard deviation (error bars) of five different pig experiments.</p

Average epithelial thickness of the porcine mucosa explants at different time points.

<p>Data are presented as mean ± standard deviation (error bars) of five independent experiment.</p