Amidation of the cell wall precursor lipid II <i>in vitro</i>.

<p>(A) Amidation is catalyzed by the cooperative action of GatD and MurT (lane 1) and MurT cannot be replaced by MurE ligase (lane 2) or any other Mur-ligase (not shown). <b>Interdependency of GatD/MurT.</b> (B) Increasing concentrations of purified GatD (dots) or MurT (squares) proteins (0–2 µg) were incubated in the presence of glutamine, ATP and [¹⁴C]-lipid II in the presence of a fixed concentration of 2 µg MurT and GatD, respectively. A maximum conversion to amidated lipid II was observed only at equimolar ratio, considering the molecular masses of 49.2 kD (MurT) and 29.7 kD (GatD).</p

Primers used in this study.

a<p>- restriction sites are underlined.</p>b<p>- nucleotide exchange in bold.</p

Model for the GatD/MurT-catalyzed amidation during cell wall biosynthesis in <i>S. aureus</i>.

<p>The GatD/MurT bi-enzyme complex uses glutamine as the primary nitrogen donor and ammonia is shuttled from the GatD glutaminase active site to the MurT synthetase active-site. MurT finally catalyzes the amidation in an ATP-dependent fashion to the acceptor substrate which could be lipid II or, as depicted here, lipid II-Gly₅.</p

<i>In vitro</i> activity of MurT and GatD.

<p>(A) Purified recombinant GatD and MurT enzymes were incubated with purified lipid II. In the negative control (lane 1) the reaction was immediately stopped by the addition of BuOH/Pyr/Ac. <b>Time dependency of the GatD/MurT catalyzed reaction.</b> (B) Purified [¹⁴C]-lipid II was incubated in the presence of 2 µg MurT and GatD each, glutamine and ATP. The reaction was stopped by the addition of BuOH/PyrAc and the amount of amidated lipid II was quantified using phosphoimaging; mean values of three independent experiments are given.</p

Mass spectrometry of non-amidated and amidated Lipid II.

<p>ESI-MS spectra were obtained with a micrOTOF-Q instrument running in negative mode. Peaks at m/z 936.52 correspond to lipid II (A) and at m/z 936.02 to amidated lipid II (B) for the doubly charged molecules, corresponding to a neutral mass of 1875 and 1874, respectively.</p

Acceptor substrate specificity of the GatD/MurT catalyzed reaction.

<p>(A) Radiolabeled lipid I, lipid II and lipid II-Gly₅ were incubated together with glutamine and ATP in the presence (gray bars) and absence (white bars) of GatD/MurT. The addition of a 5- and 10-fold molar excess of UDP-MurNAc-pentapeptide (black bars) only resulted in minor reduction of amidated lipid II synthesized. <b>Amidation is not catalyzed in concert with MurA-F.</b> (B) Purified MurA-F enzymes were incubated in the presence and absence of GatD/MurT enzymes. After incubation the reaction product was added to a MraY-catalyzed lipid I synthesis assay using C₅₅-P. No change in migration behavior of the respective lipid I reaction products was observed.</p

Migration behavior of lipid II and amidated lipid II on thin layer chromatography (TLC).

<p>TLC of lipid II incubated in the absence (lane 1) and presence (lane 2) of <i>S. aureus</i> membrane preparations. Only when supplemented with ATP and glutamine (lane 2), a modified lipid II was separated. Omission of glutamine and ATP almost completely abolished synthesis of the lipid II variant (lane 3).</p

Discovery, Isolation, and Structure Elucidation of Dretamycin

The Candida albicans fitness test is a whole cell screening platform that utilizes a mixed-pool of C. albicans mutants, each of which carries a heterozygous deletion of a particular gene. In the presence of an antifungal inhibitor, a subset of these mutants exhibits a growth phenotype of hypersensitivity or hyposensitivity. Collectively these mutants reflect aspects of the mechanism of action of the compound in question. In the course of screening natural products a culture of Streptomyces sp. MS-1-4 was discovered to produce a compound, dretamycin, which yielded a fitness profile exhibiting significant hypersensitivity of the <i>DRE2</i> heterozygote and hyposensitivity of the <i>DIP5</i> heterozygote. Herein we report the production, isolation, and structure elucidation of dretamycin

Determination of Cidal or Static Terminal Phenotypes

<div><p>A representative example of cidal and static terminal phenotypes for <i>GFA1</i> and <i>TRR1</i> p<i>NiiA</i>-CPR mutants is shown.</p><p>(A) p<i>NiiA</i>-GFA1 displayed a cidal terminal phenotype as a dramatic (greater than 90%) reduction in CFU was observed after incubation in p<i>NiiA</i>-repressing conditions for 24 or 48 h.</p><p>(B) p<i>NiiA</i>-<i>TRR1</i> revealed a static terminal phenotype as no significant reduction in CFU counts was detected after 48-h incubation under repressing conditions. A summary of all additional cidal/static terminal phenotypes for A. fumigatus genes displaying 4+ qualitative growth phenotypes is provided (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030024#ppat-0030024-t002" target="_blank">Table 2</a>).</p></div

Analysis of p<i>NiiA</i>-CPR Associated Morphological Terminal Phenotypes

<p>Terminal growth phenotypes of p<i>NiiA</i>-CPR mutants were observed under a microscope (×160) with conidia grown for 36 to 40 h at 30 °C under standard repressing conditions. A continuum of conidia germination phenotypes of high penetrance was observed; ranging from those completely failing to undergo polarized growth <i>(SEC31, SLY1)</i> or swollen and highly disorganized condidia <i>(GFA1),</i> to those displaying stunted <i>(TUB1, ERG10)</i> or nonbranching germlings with swollen conidia <i>(HEM15)</i> with only rudimentary polarized growth. Micromycelial colonies were observed for a p<i>NiiA</i>-<i>FKS1</i> mutant and resembling the morphology of wild-type A. fumigatus when grown in the presence of minimum effective concentration (MEC) of the FKS1p inhibitor, caspofungin [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030024#ppat-0030024-b021" target="_blank">21</a>]. Growth phenotypes under inducing conditions are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030024#ppat-0030024-sg003" target="_blank">Figure S3</a>.</p