Daniel P. Haeusser

Splitsville: structural and functional insights into the dynamic bacterial Z ring

Bacteria must divide to increase in number and colonize their niche. Binary fission is the most widespread means of bacterial cell division, but even this relatively simple mechanism has many variations on a theme. In most bacteria, the tubulin homologue FtsZ assembles into a ring structure, termed the Z ring, at the site of cytokinesis and recruits additional proteins to form a large protein machine — the divisome — that spans the membrane. In this Review, we discuss current insights into the regulation of the assembly of the Z ring and how the divisome drives membrane invagination and septal cell wall growth while flexibly responding to various cellular inputs.

The Kil Peptide of Bacteriophage λ Blocks Escherichia coli Cytokinesis via ZipA-Dependent Inhibition of FtsZ Assembly

Assembly of the essential, tubulin-like FtsZ protein into a ring-shaped structure at the nascent division site determines the timing and position of cytokinesis in most bacteria and serves as a scaffold for recruitment of the cell division machinery. Here we report that expression of bacteriophage λ kil, either from a resident phage or from a plasmid, induces filamentation of Escherichia coli cells by rapid inhibition of FtsZ ring formation. Mutant alleles of ftsZ resistant to the Kil protein map to the FtsZ polymer subunit interface, stabilize FtsZ ring assembly, and confer increased resistance to endogenous FtsZ inhibitors, consistent with Kil inhibiting FtsZ assembly. Cells with the normally essential cell division gene zipA deleted (in a modified background) display normal FtsZ rings after kil expression, suggesting that ZipA is required for Kil-mediated inhibition of FtsZ rings in vivo. In support of this model, point mutations in the C-terminal FtsZ-interaction domain of ZipA abrogate Kil activity without discernibly altering FtsZ-ZipA interactions. An affinity-tagged-Kil derivative interacts with both FtsZ and ZipA, and inhibits sedimentation of FtsZ filament bundles in vitro. Together, these data inspire a model in which Kil interacts with FtsZ and ZipA in the cell to prevent FtsZ assembly into a coherent, division-competent ring structure. Phage growth assays show that kil+ phage lyse ∼30% later than kil mutant phage, suggesting that Kil delays lysis, perhaps via its interaction with FtsZ and ZipA.

A mutation in Escherichia coli ftsZ bypasses the requirement for the essential division gene zipA and confers resistance to FtsZ assembly inhibitors by stabilizing protofilament bundling.

The earliest step in Escherichia coli cell division consists of the assembly of FtsZ protein into a proto‐ring structure, tethered to the cytoplasmic membrane by FtsA and ZipA. The proto‐ring then recruits additional cell division proteins to form the divisome. Previously we described an ftsZ allele, ftsZL169R, which maps to the side of the FtsZ subunit and confers resistance to FtsZ assembly inhibitory factors including Kil of bacteriophage λ. Here we further characterize this allele and its mechanism of resistance. We found that FtsZL169R permits the bypass of the normally essential ZipA, a property previously observed for FtsA gain‐of‐function mutants such as FtsA* or increased levels of the FtsA‐interacting protein FtsN. Similar to FtsA*, FtsZL169R also can partially suppress thermosensitive mutants of ftsQ or ftsK, which encode additional divisome proteins, and confers strong resistance to excess levels of FtsA, which normally inhibit FtsZ ring function. Additional genetic and biochemical assays provide further evidence that FtsZL169R enhances FtsZ protofilament bundling, thereby conferring resistance to assembly inhibitors and bypassing the normal requirement for ZipA. This work highlights the importance of FtsZ protofilament bundling during cell division and its likely role in regulating additional divisome activities.

Molecular Bases Determining Daptomycin Resistance-Mediated Resensitization to β-Lactams (Seesaw Effect) in Methicillin-Resistant Staphylococcus aureus.

ABSTRACT Antimicrobial resistance is recognized as one of the principal threats to public health worldwide, yet the problem is increasing. Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains are among the most difficult to treat in clinical settings due to the resistance of MRSA to nearly all available antibiotics. The cyclic anionic lipopeptide antibiotic daptomycin (DAP) is the clinical mainstay of anti-MRSA therapy. The decreased susceptibility to DAP (DAP resistance [DAPr]) reported in MRSA is frequently accompanied by a paradoxical decrease in β-lactam resistance, a process known as the “seesaw effect.” Despite the observed discordance in resistance phenotypes, the combination of DAP and β-lactams has been proven to be clinically effective for the prevention and treatment of infections due to DAPr MRSA strains. However, the mechanisms underlying the interactions between DAP and β-lactams are largely unknown. In the study described here, we studied the role of mprF with DAP-induced mutations in β-lactam sensitization and its involvement in the effective killing by the DAP-oxacillin (OXA) combination. DAP-OXA-mediated effects resulted in cell wall perturbations, including changes in peptidoglycan insertion, penicillin-binding protein 2 (PBP 2) delocalization, and reduced membrane amounts of PBP 2a, despite the increased transcription of mecA through mec regulatory elements. We have found that the VraSR sensor-regulator is a key component of DAP resistance, triggering mutated mprF-mediated cell membrane (CM) modifications that result in impairment of PrsA location and chaperone functions, both of which are essential for PBP 2a maturation, the key determinant of β-lactam resistance. These observations provide for the first time evidence that synergistic effects between DAP and β-lactams involve PrsA posttranscriptional regulation of CM-associated PBP 2a.

Bacteriophage Tubulins: Carrying Their Own Cytoskeleton Key

Cytoskeletal elements are well known to be widespread in eukaryotes and prokaryotes, providing important, diverse functions for cells large and small. Two new studies report that some bacteriophages encode their own tubulin homologs to facilitate phage reproduction within the host cell.

Corrigendum: Splitsville: structural and functional insights into the dynamic bacterial Z ring

Splitsville: structural and functional insights into the dynamic bacterial Z ring Daniel P. Haeusser & William Margolin Nature Reviews Microbiology 14, 305–319 (2016) In the sixth paragraph of the section ‘FtsA and the dynamics of proto-ring assembly’, the sentence “Finally, an FtsZ mutant with decreased self-bundling in vitro (FtsZ-E93R) has reduced function in cell division .” should have read “However, an FtsZ mutant with increased self-bundling in vitro (FtsZ-E93R) has reduced function in cell division” The authors apologize to the readers for any misunderstanding caused. C O R R E C T I O N

Erratum: Splitsville: structural and functional insights into the dynamic bacterial Z ring (Nature Reviews Microbiology (2016) 14 (305-319))

Splitsville: structural and functional insights into the dynamic bacterial Z ring Daniel P. Haeusser & William Margolin Nature Reviews Microbiology 14, 305–319 (2016) In the sixth paragraph of the section ‘FtsA and the dynamics of proto-ring assembly’, the sentence “Finally, an FtsZ mutant with decreased self-bundling in vitro (FtsZ-E93R) has reduced function in cell division .” should have read “However, an FtsZ mutant with increased self-bundling in vitro (FtsZ-E93R) has reduced function in cell division” The authors apologize to the readers for any misunderstanding caused. C O R R E C T I O N

Prokaryotic Cytokinesis: Little Rings Bring Big Cylindrical Things

At the division site, most bacteria assemble filaments of the tubulin homolog FtsZ that recruit other proteins into a functional divisome. A recent study describes the in vitro assembly of the divisome component SepF into small rings that organize FtsZ filaments into microtubule-like structures, possibly facilitating efficient septal growth and cytokinesis.