25 research outputs found
Identification of the SlmA Active Site Responsible for Blocking Bacterial Cytokinetic Ring Assembly over the Chromosome
Bacterial cells use chromosome-associated division inhibitors to help coordinate the processes of DNA replication and segregation with cytokinesis. SlmA from Escherichia coli, a member of the tetracycline repressor (TetR)–like protein family, is one example of this class of regulator. It blocks the assembly of the bacterial cytokinetic ring by interfering with the polymerization of the tubulin-like FtsZ protein in a manner that is dramatically stimulated upon specific DNA binding. Here we used a combination of molecular genetics and biochemistry to identify the active site of SlmA responsible for disrupting FtsZ polymerization. Interestingly, this site maps to a region of SlmA that in the published DNA–free structure is partially occluded by the DNA-binding domains. In this conformation, the SlmA structure resembles the drug/inducer-bound conformers of other TetR–like proteins, which in the absence of inducer require an inward rotation of their DNA-binding domains to bind successive major grooves on operator DNA. Our results are therefore consistent with a model in which DNA-binding activates SlmA by promoting a rotational movement of the DNA-binding domains that fully exposes the FtsZ-binding sites. SlmA may thus represent a special subclass of TetR–like proteins that have adapted conformational changes normally associated with inducer sensing in order to modulate an interaction with a partner protein. In this case, the adaptation ensures that SlmA only blocks cytokinesis in regions of the cell occupied by the origin-proximal portion of the chromosome where SlmA-binding sites are enriched
Expected colony phenotypes for <i>slmA</i> mutants in strain HC328/pHC534 [Δ<i>slmA</i> P<sub>sbs</sub>::<i>lacZ</i>/pUC-2xSBS].
a<p>For cells plated on LB X-gal supplemented with 1 mM IPTG.</p
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The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli
Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix
Location of amino acid substitutions on the SlmA structure.
<p>Shown are two views of the SlmA dimer structure <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003304#pgen.1003304-Tonthat1" target="_blank">[26]</a> with one subunit colored cyan and the other green. On the left view, the HTH motif is facing the bottom of the page and on the right it is facing into the page. N- and C- termini as well as helices 1–9 are labeled for reference. The location of SlmA residues identified in this study as being important for FtsZ regulation are highlighted in purple. Residues R73 and T33 for which the effect of substitutions has been previously studied are highlighted in red and orange, respectively. Note that F65 is occluded by the HTH domain.</p
Nucleoid occlusion activity of the SlmA variants.
<p>A. Overnight cultures of TB57 [P<sub>ara</sub>::<i>minCDE</i>], HC278 [P<sub>ara</sub>::<i>minCDE</i> Δ<i>slmA</i>], and HC278 containing integrated expression plasmids producing the indicated SlmA variant were diluted and plated on the indicated medium as described in the legend for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003304#pgen-1003304-g002" target="_blank">Figure 2</a>. B–C. The same strains were grown in LB broth supplemented with 1 mM IPTG to an OD<sub>600</sub> of 0.6. Protein extracts were prepared and proteins in 10 and 20 µg of total extract were separated by SDS-PAGE. SlmA was then detected by immunoblotting with affinity-purified anti-SlmA antibodies (B). The division phenotype of cells from the resulting cultures was also observed using DIC optics. Images for a representative set of strains are shown (C–H). Bar equals 5 microns.</p
Model for SlmA activation.
<p>The SlmA monomer is represented as a two domain structure formed by intersecting ovals. The vertical oval represents the dimerization domain and the angled oval with the small protrusion represents the DNA-binding domain. The small protrusion is the recognition helix, and the blue oval represents the identified FtsZ-interaction interface. The green circles represent FtsZ monomers within a protofilament. At least two conformations of SlmA dimers are envisioned to exist in solution: an open and a closed conformation. The closed conformation is the only one thought to have a fully exposed/functional FtsZ-interaction interface necessary for antagonizing FtsZ polymerization. We propose that in addition to promoting SlmA dimer formation at lower protein concentrations, DNA-binding also stabilizes the closed SlmA conformation thus stimulating its anti-FtsZ activity. See text for a detailed description of the model and the rationale behind it.</p
Amino acid substitutions in SlmA generated by site-directed mutagenesis.
<p>Amino acid substitutions in SlmA generated by site-directed mutagenesis.</p
Subcellular localization of GFP-SlmA fusions.
<p>Cells of HC259 [Δ<i>slmA</i>] with the integrated expression plasmids: pHC625 [P<sub>lac</sub>::<i>gfp-slmA</i>(WT)] (A), pHC505 [P<sub>lac</sub>::<i>gfp-slmA</i>(T33A)] (B), pHC482 [P<sub>lac</sub>::<i>gfp-slmA</i>(R73D)] (C), pHC628 [P<sub>lac</sub>::<i>gfp-slmA</i>(F65I)] (D), pHC684 [P<sub>lac</sub>::<i>gfp-slmA</i>(F65A)] (E), pHC631 [P<sub>lac</sub>::<i>gfp-slmA</i>(L94Q)] (F), pHC629 [P<sub>lac</sub>::<i>gfp-slmA</i>(G97D)] (G), pHC685 [P<sub>lac</sub>::<i>gfp-slmA</i>(R101D)] (H), pHC627 [P<sub>lac</sub>::<i>gfp-slmA</i>(N102S)] (I), or pHC630 [P<sub>lac</sub>::<i>gfp-slmA</i>(L105Q)] (J) were grown to an OD<sub>600</sub> of 0.8–1.0 in LB supplemented with 1 mM IPTG and imaged with DIC (panel 1) and GFP (panel 2) optics. Bar equals 3 microns. Note that as reported previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003304#pgen.1003304-Bernhardt1" target="_blank">[23]</a> the GFP-SlmA(WT) signal closely resembles that of nuceloids stained with the DNA-stain DAPI.</p
Effect of SlmA variants on Z-ring formation in the presence of multi-copy SBS.
<p>Cells of HC290/pHC534 [<i>zapA-gfp</i> Δ<i>slmA</i>/pUC-2xSBS] with the integrated expression plasmids: pHC531 [P<sub>lac-m3</sub>::<i>slmA</i>(WT)] (A), pHC544 [P<sub>lac-m3</sub>::<i>slmA</i>(T33A)] (B), pHC543 [P<sub>lac-m3</sub>::<i>slmA</i>(R73D)] (C), pHC678 [P<sub>lac-m3</sub>::<i>slmA</i>(F65A)] (D), and pHC610 [P<sub>lac-m3</sub>::<i>slmA</i>(N102S)] (E), were grown to an OD<sub>600</sub> of 0.6 in LB supplemented with ampicillin (50 µg/ml) and 1 mM IPTG and imaged with DIC (panel 1) and GFP (panel 2) optics. Bar equals 3 microns. Note that a GFP fusion to the FtsZ-binding protein ZapA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003304#pgen.1003304-GueirosFilho1" target="_blank">[42]</a> is used as a proxy for Z-ring formation and that the ZapA-GFP rings in panels B–E are identical to those observed in wild-type cells.</p
Selection for mutants producing SlmA variants defective in FtsZ regulation.
<p>A. Shown is a diagram of the P<sub>sbs</sub>::<i>lacZ</i> reporter construct indicating the relative positions of the SBS and promoter elements. The synthetic promoter replaces the <i>lac</i> promoter at the native <i>lac</i> locus in the chromosome. B. A frozen aliquot of slurried HC328(attHKHC583)/pHC534 [Δ<i>slmA</i> P<sub>sbs</sub>::<i>lacZ</i> (P<sub>lac-m3</sub>::<i>slmA</i>)/pUC-2xSBS] cells with PCR-mutagenized <i>slmA</i> was thawed, diluted to 10<sup>−7</sup>, and 100 µl of the dilution was spread on the indicated agar plates. The plates were incubated overnight at 30°C for two days and photographed. As shown, induction of mutagenized <i>slmA</i> with IPTG in the parental strain resulted in a plating defect of approximately an order of magnitude.</p