DrpB (YedR) Is a Nonessential Cell Division Protein in Escherichia coli.

We report that the small Escherichia coli membrane protein DrpB (formerly YedR) is involved in cell division. We discovered DrpB in a screen for multicopy suppressors of a ΔftsEX mutation that prevents divisome assembly when cells are plated on low ionic strength medium, such as lysogeny broth without NaCl. Characterization of DrpB revealed that (i) translation initiates at an ATG annotated as codon 22 rather than the GTG annotated as codon 1, (ii) DrpB localizes to the septal ring when cells are grown in medium of low ionic strength but localization is greatly reduced in medium of high ionic strength, (iii) overproduction of DrpB in a ΔftsEX mutant background improves recruitment of the septal peptidoglycan synthase FtsI, implying multicopy suppression works by rescuing septal ring assembly, (iv) a ΔdrpB mutant divides quite normally, but a ΔdrpB ΔdedD double mutant has a strong division and viability defect, albeit only in medium of high ionic strength, and (v) DrpB homologs are found in E. coli and a few closely related enteric bacteria, but not outside this group. In sum, DrpB is a poorly conserved nonessential division protein that improves the efficiency of cytokinesis under suboptimal conditions. Proteins like DrpB are likely to be a widespread feature of the bacterial cell division apparatus, but they are easily overlooked because mutants lack obvious shape defects.IMPORTANCE A thorough understanding of bacterial cell division requires identifying and characterizing all of the proteins that participate in this process. Our discovery of DrpB brings us one step closer to this goal in E. coli.

High-throughput screening for novel inhibitors of Neisseria gonorrhoeae penicillin-binding protein 2.

The increasing prevalence of N. gonorrhoeae strains exhibiting decreased susceptibility to third-generation cephalosporins and the recent isolation of two distinct strains with high-level resistance to cefixime or ceftriaxone heralds the possible demise of ß-lactam antibiotics as effective treatments for gonorrhea. To identify new compounds that inhibit penicillin-binding proteins (PBPs), which are proven targets for ß-lactam antibiotics, we developed a high-throughput assay that uses fluorescence polarization (FP) to distinguish the fluorescent penicillin, Bocillin-FL, in free or PBP-bound form. This assay was used to screen a 50,000 compound library for potential inhibitors of N. gonorrhoeae PBP 2, and 32 compounds were identified that exhibited >50% inhibition of Bocillin-FL binding to PBP 2. These included a cephalosporin that provided validation of the assay. After elimination of compounds that failed to exhibit concentration-dependent inhibition, the antimicrobial activity of the remaining 24 was tested. Of these, 7 showed antimicrobial activity against susceptible and penicillin- or cephalosporin-resistant strains of N. gonorrhoeae. In molecular docking simulations using the crystal structure of PBP 2, two of these inhibitors docked into the active site of the enzyme and each mediate interactions with the active site serine nucleophile. This study demonstrates the validity of a FP-based assay to find novel inhibitors of PBPs and paves the way for more comprehensive high-throughput screening against highly resistant strains of N. gonorrhoeae. It also provides a set of lead compounds for optimization of anti-gonococcal agents.

Species-specific and inhibitor-dependent conformations of LpxC: implications for antibiotic design.

LpxC is an essential enzyme in the lipid A biosynthetic pathway in gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC orthologs, and structural analysis reveals large conformational differences that mostly reflect inherent molecular features of distinct LpxC orthologs, whereas ligand-induced structural adaptations occur at a smaller scale. These observations highlight the need for a molecular understanding of inherent structural features and conformational plasticity of LpxC enzymes for optimizing LpxC inhibitors as broad-spectrum antibiotics against gram-negative infections.