Hongbaek Cho

Nucleoid occlusion factor SlmA is a DNA-activated FtsZ polymerization antagonist

The tubulin-like FtsZ protein initiates assembly of the bacterial cytokinetic machinery by polymerizing into a ring structure, the Z ring, at the prospective site of division. To block Z-ring formation over the nucleoid and help coordinate cell division with chromosome segregation, Escherichia coli employs the nucleoid-associated division inhibitor, SlmA. Here, we investigate the mechanism by which SlmA regulates FtsZ assembly. We show that SlmA disassembles FtsZ polymers in vitro. In addition, using chromatin immunoprecipitation (ChIP), we identified 24 SlmA-binding sequences (SBSs) on the chromosome. Remarkably, SlmA binding to SBSs dramatically enhanced its ability to interfere with FtsZ polymerization, and ChIP studies indicate that SlmA regulates FtsZ assembly at these sites in vivo. Because of the dynamic and highly organized nature of the chromosome, coupling SlmA activation to specific DNA binding provides a mechanism for the precise spatiotemporal control of its anti-FtsZ activity within the cell.

Beta-Lactam Antibiotics Induce a Lethal Malfunctioning of the Bacterial Cell Wall Synthesis Machinery

Penicillin and related beta-lactams comprise one of our oldest and most widely used antibiotic therapies. These drugs have long been known to target enzymes called penicillin-binding proteins (PBPs) that build the bacterial cell wall. Investigating the downstream consequences of target inhibition and how they contribute to the lethal action of these important drugs, we demonstrate that beta-lactams do more than just inhibit the PBPs as is commonly believed. Rather, they induce a toxic malfunctioning of their target biosynthetic machinery involving a futile cycle of cell wall synthesis and degradation, thereby depleting cellular resources and bolstering their killing activity. Characterization of this mode of action additionally revealed a quality control function for enzymes that cleave bonds in the cell wall matrix. The results thus provide insight into the mechanism of cell wall assembly and suggest how best to interfere with the process for future antibiotic development.

Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously

Multi-protein complexes organized by cytoskeletal proteins are essential for cell wall biogenesis in most bacteria. Current models of the wall assembly mechanism assume that class A penicillin-binding proteins (aPBPs), the targets of penicillin-like drugs, function as the primary cell wall polymerases within these machineries. Here, we use an in vivo cell wall polymerase assay in Escherichia coli combined with measurements of the localization dynamics of synthesis proteins to investigate this hypothesis. We find that aPBP activity is not necessary for glycan polymerization by the cell elongation machinery, as is commonly believed. Instead, our results indicate that cell wall synthesis is mediated by two distinct polymerase systems, shape, elongation, division, sporulation (SEDS)-family proteins working within the cytoskeletal machines and aPBP enzymes functioning outside these complexes. These findings thus necessitate a fundamental change in our conception of the cell wall assembly process in bacteria.

Reconstitution of the Biochemical Activities of the AttJ Repressor and the AttK, AttL, and AttM Catabolic Enzymes of Agrobacterium tumefaciens

The attKLM operon encodes a lactonase (AttM) that hydrolyzes acylhomoserine lactone autoinducers, as well as two putative dehydrogenases (AttK and AttL). Here we show that AttK, AttL, and AttM collectively covert gamma-butyrolactone to succinate. Two metabolic intermediates, gamma-hydroxybutyrate and succinic semialdehyde, inactivated the AttJ repressor in vitro and induced attKLM transcription in vivo.

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.

Identification of MltG as a potential terminase for peptidoglycan polymerization in bacteria.

Bacterial cells are fortified against osmotic lysis by a cell wall made of peptidoglycan (PG). Synthases called penicillin‐binding proteins (PBPs), the targets of penicillin and related antibiotics, polymerize the glycan strands of PG and crosslink them into the cell wall meshwork via attached peptides. The average length of glycan chains inserted into the matrix by the PBPs is thought to play an important role in bacterial morphogenesis, but polymerization termination factors controlling this process have yet to be discovered. Here, we report the identification of Escherichia coli MltG (YceG) as a potential terminase for glycan polymerization that is broadly conserved in bacteria. A clone containing mltG was initially isolated in a screen for multicopy plasmids generating a lethal phenotype in cells defective for the PG synthase PBP1b. Biochemical studies revealed that MltG is an inner membrane enzyme with endolytic transglycosylase activity capable of cleaving at internal positions within a glycan polymer. Radiolabeling experiments further demonstrated MltG‐dependent nascent PG processing in vivo, and bacterial two‐hybrid analysis identified an MltG‐PBP1b interaction. Mutants lacking MltG were also shown to have longer glycans in their PG relative to wild‐type cells. Our combined results are thus consistent with a model in which MltG associates with PG synthetic complexes to cleave nascent polymers and terminate their elongation.

Transsexuality in the Rhizosphere: Quorum Sensing Reversibly Converts Agrobacterium tumefaciens from Phenotypically Female to Male

Conjugative plasmids generally encode proteins that block the conjugative entry of identical or similar plasmids into the host cell, a phenomenon known as entry exclusion. Here, we demonstrate that two Ti plasmids of Agrobacterium tumefaciens encode robust entry exclusion functions. Two proteins, TrbJ and TrbK, can each mediate entry exclusion and act synergistically. The trbJ and trbK genes are included within the trb operon, which is tightly regulated by the quorum-sensing regulator TraR and the cognate acylhomoserine lactone. In the absence of quorum-sensing signals, these proteins are not significantly expressed, and cells lacking TrbJ and TrbK are efficient Ti plasmid recipients. In the presence of these signals, these strains block the entry of Ti plasmids and instead become efficient conjugal donors.

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.

A viral protein antibiotic inhibits lipid II flippase activity

For bacteriophage infections, the cell walls of bacteria, consisting of a single highly polymeric molecule of peptidoglycan (PG), pose a major problem for the release of progeny virions. Phage lysis proteins that overcome this barrier can point the way to new antibacterial strategies1, especially small lytic single-stranded DNA (the microviruses) and RNA phages (the leviviruses) that effect host lysis using a single non-enzymatic protein2. Previously, the A2 protein of levivirus Qβ and the E protein of the microvirus ϕX174 were shown to be ‘protein antibiotics’ that inhibit the MurA and MraY steps of the PG synthesis pathway2–4. Here, we investigated the mechanism of action of an unrelated lysis protein, LysM, of the Escherichia coli levivirus M5. We show that LysM inhibits the translocation of the final lipid-linked PG precursor called lipid II across the cytoplasmic membrane by interfering with the activity of MurJ. The finding that LysM inhibits a distinct step in the PG synthesis pathway from the A2 and E proteins indicates that small phages, particularly the single-stranded RNA (ssRNA) leviviruses, have a previously unappreciated capacity for evolving novel inhibitors of PG biogenesis despite their limited coding potential.LysM, the lysis protein of the Escherichia coli levivirus M, represents a new ‘protein antibiotic’ that interferes with the synthesis of peptidoglycan by inhibiting lipid II flipping.

An activation pathway governs cell wall polymerization by a bacterial morphogenic machine

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall (CW) assembly. In Escherichia coli, alterations in a CW synthase component of the system called PBP2 were identified that overcome other inactivating defects. Rod system activity was stimulated in the suppressors in vivo, and purified synthase complexes with these changes showed more robust CW synthesis in vitro. Polymerization of the actin-like MreB component of the Rod system was also found to be enhanced in cells with the activated synthase. The results suggest an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both CW glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient CW assembly. An analogous activation pathway involving similar enzymatic components is likely responsible for controlling CW synthesis by the division machinery.