Disrupting the Acyl Carrier Protein/SpoT Interaction In Vivo: Identification of ACP Residues Involved in the Interaction and Consequence on Growth

In bacteria, Acyl Carrier Protein (ACP) is the central cofactor for fatty acid biosynthesis. It carries the acyl chain in elongation and must therefore interact successively with all the enzymes of this pathway. Yet, ACP also interacts with proteins of diverse unrelated function. Among them, the interaction with SpoT has been proposed to be involved in regulating ppGpp levels in the cell in response to fatty acid synthesis inhibition. In order to better understand this mechanism, we screened for ACP mutants unable to interact with SpoT in vivo by bacterial two-hybrid, but still functional for fatty acid synthesis. The position of the selected mutations indicated that the helix II of ACP is responsible for the interaction with SpoT. This suggested a mechanism of recognition similar to one used for the enzymes of fatty acid synthesis. Consistently, the interactions tested by bacterial two-hybrid of ACP with fatty acid synthesis enzymes were also affected by the mutations that prevented the interaction with SpoT. Yet, interestingly, the corresponding mutant strains were viable, and the phenotypes of one mutant suggested a defect in growth regulation

Protein–Protein Interactions: Oxidative Bacterial Two Hybrid

International audienceProtein-protein interaction studies are essential to understand how proteins organize themselves into interaction networks and thus influence cellular processes. Protein binding specificity depends on the correct three-dimensional folding of the polypeptide sequences. One of the forces involved in the structuring and stability of proteins is the formation of disulfide bonds. These covalent bonds are formed post-transcriptionally by the oxidation of a pair of cysteine residues and can serve structural, catalytic, or signaling roles. Here, we describe an engineered E. coli adenylate cyclase mutant strain with an oxidative cytoplasm that promotes correct folding of proteins with disulfide bonds. This genetic background expands the set of host strains suitable for studying protein-protein interactions in vivo by the adenylate-cyclase two-hybrid approach

Plasmids.

<p>ts: thermosensitive replication; Amp^R: carrying resistance to Ampicillin gene, Kana^R; carrying resistance to Kanamycin gene; Cam^R: carrying resistance to Chloramphenicol. ori: origin of replication; FRT: recombination site for Flipase. The plasmids containing the mutations in <i>acpP</i> are not listed. They correspond to mutagenesis of pEB379, pEB375, pEB1154, and pEB1334 plasmids.</p

<i>E. coli</i> K12 strains.

<p>Ts: thermosensitive. Kana^R; carrying resistance to Kanamycin gene; Cam^R: carrying resistance to Chloramphenicol. FRT: recombination site for Flipase.</p

Interactions of the ACP mutants with SpoT and MukB. A.

<p>Co-purification on calmodulin beads was performed as described in Experimental procedures on extracts prepared from cultures of W3110SpoT-SG transformed with the indicated pT18-ACP mutant plasmids. Purified samples were analyzed by SDS-PAGE (10% for MukB and SpoT-SG and 12% for T18-ACP) and Western blotting, with anti-T18 to detect the purified ACP mutants, an anti-MukB antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036111#pone.0036111-denBlaauwen1" target="_blank">[45]</a> to detect the wild type endogenous MukB, and finally PAP antibody to detect the endogenous tagged SpoT protein. <b>B.</b> BTH101 strain was transformed by pT18-SpoT (pEB596) and the pT25-ACP mutant plasmids. ß-galactosidase assay was performed on 3 independent clones for each pair, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036111#s4" target="_blank">Material and Methods</a>.</p

morphology of the ACP mutant strains.

<p>Strain MG1665Δ<i>acpP</i>::kana^R complemented by the pKO3-<i>acpP</i> mutant series was grown overnight at 30°C in LB containing Chloramphenicol. The cultures were then diluted 100X in 3 ml of LB containing Chloramphenicol. At OD_600nm = 0.5 (<b>A</b>) and after overnight growth (<b>B</b>), the cultures were observed by phase contrast microscopy with a 100X objective.</p

Growth of the ACP mutant strains.

<p>Strain MG1665Δ<i>acpP</i>::kana^R complemented by the pKO3-<i>acpP</i> mutant series was grown overnight at 30°C in LB containing Chloramphenicol. The cultures were then diluted 100X in LB containing Chloramphenicol and aliquots of 150 µl were grown in a TECAN microplate reader at 30°C with continuous shaking. Note that the indicated OD_600nm is 4 times lower than the usually OD measured with a standard spectrophotometer.</p

In vitro acylation assay of purified ACP mutants.

<p>The indicated purified ACP mutants were assayed for acylation with oleate using acyl-transferase Aas enzyme (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036111#s4" target="_blank">Material and Methods</a>). ACP species were analyzed on a 15% SDS-PAGE stained with Coomassie Blue. Molecular weight markers in kDa are indicated on the left-hand of the gel.</p

Interaction profiles of ACP mutants.

<p>The two-hybrid interaction profiles have been obtained by assaying the interaction between the T18-ACP mutants and the indicated T25- constructions (first 4 columns), and also by assaying the T25-ACP mutants against T18-SpoT (middle column; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036111#pone-0036111-g004" target="_blank">Figure 4B</a>). The co-purification results correspond to the experiment presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036111#pone-0036111-g004" target="_blank">figure 4A</a>. The results obtained for the ACP/SpoT interaction in the three different assays (2-hybrid against T18-ACP, 2–hybrid against T25-ACP, and co-purification of SpoT-SG with T18-ACP on Calmodulin beads) are indicated.</p

ACP mutants.

<p>ACP mutants number 3 to 149 were obtained by random mutagenesis. Mutants ACP745 and ACP749 were obtained by site-directed mutagenesis. Mutant ACP(S36T) was described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036111#pone.0036111-Gully2" target="_blank">[15]</a>. Functionality corresponds to the result of the complementation test by transduction of the Δ<i>acpP</i>::kana^R deletion in strain MG1655 containing the pKO3-<i>acpP</i> mutant construction (see text). + indicates growth on LB plate equivalent to the growth of the wild type; -indicates that no clone was obtained; +/− indicates that colonies were obtained, but growth was severely delayed; nd (not determined) indicates that the corresponding ACP mutants were not tested for functionality. The six mutants further characterized in the study are indicated in bold letters.</p