Competing Substrates for the Bifunctional Diaminopimelic Acid Epimerase/Glutamate Racemase Modulate Peptidoglycan Synthesis in Chlamydia trachomatis

ABSTRACTThe Chlamydia trachomatis genome encodes multiple bifunctional enzymes, such as DapF, which is capable of both diaminopimelic acid (DAP) epimerase and glutamate racemase activity. Our previous work demonstrated the bifunctional activity of chlamydial DapF in vitro and in a heterologous system (Escherichia coli). In the present study, we employed a substrate competition strategy to demonstrate DapFCt function in vivo in C. trachomatis. We reasoned that, because DapFCt utilizes a shared substrate-binding site for both racemase and epimerase activities, only one activity can occur at a time. Therefore, an excess of one substrate relative to another must determine which activity is favored. We show that the addition of excess l-glutamate or meso-DAP (mDAP) to C. trachomatis resulted in 90% reduction in bacterial titers, compared to untreated controls. Excess l-glutamate reduced in vivo synthesis of mDAP by C. trachomatis to undetectable levels, thus confirming that excess racemase substrate led to inhibition of DapFCt DAP epimerase activity. We previously showed that expression of dapFCt in a murI (racemase) ΔdapF (epimerase) double mutant of E. coli rescues the d-glutamate auxotrophic defect. Addition of excess mDAP inhibited growth of this strain, but overexpression of dapFCt allowed the mutant to overcome growth inhibition. These results confirm that DapFCt is the primary target of these mDAP and l-glutamate treatments. Our findings demonstrate that suppression of either the glutamate racemase or epimerase activity of DapF compromises the growth of C. trachomatis. Thus, a substrate competition strategy can be a useful tool for in vivo validation of an essential bifunctional enzyme.

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Chlamydia trachomatis Oligopeptide Transporter Performs Dual Functions of Oligopeptide Transport and Peptidoglycan Recycling

Infection and Immunity ◽

10.1128/iai.00086-20 ◽

2020 ◽

Vol 88 (5) ◽

Author(s):

Raghuveer Singh ◽

George Liechti ◽

Jessica A. Slade ◽

Anthony T. Maurelli

Keyword(s):

Chlamydia Trachomatis ◽

Host Cell ◽

De Novo ◽

Diaminopimelic Acid ◽

Intracellular Pathogens ◽

Oligopeptide Transporter ◽

Content Type ◽

E Coli ◽

Oligopeptide Transport

ABSTRACT Peptidoglycan, the sugar-amino acid polymer that composes the bacterial cell wall, requires a significant expenditure of energy to synthesize and is highly immunogenic. To minimize the loss of an energetically expensive metabolite and avoid host detection, bacteria often recycle their peptidoglycan, transporting its components back into the cytoplasm, where they can be used for subsequent rounds of new synthesis. The peptidoglycan-recycling substrate binding protein (SBP) MppA, which is responsible for recycling peptidoglycan fragments in Escherichia coli, has not been annotated for most intracellular pathogens. One such pathogen, Chlamydia trachomatis, has a limited capacity to synthesize amino acids de novo and therefore must obtain oligopeptides from its host cell for growth. Bioinformatics analysis suggests that the putative C. trachomatis oligopeptide transporter OppABCDF (OppABCDFCt) encodes multiple SBPs (OppA1Ct, OppA2Ct, and OppA3Ct). Intracellular pathogens often encode multiple SBPs, while only one, OppA, is encoded in the E. coli opp operon. We hypothesized that the putative OppABCDF transporter of C. trachomatis functions in both oligopeptide transport and peptidoglycan recycling. We coexpressed the putative SBP genes (oppA1Ct, oppA2Ct, oppA3Ct) along with oppBCDFCt in an E. coli mutant lacking the Opp transporter and determined that all three chlamydial OppA subunits supported oligopeptide transport. We also demonstrated the in vivo functionality of the chlamydial Opp transporter in C. trachomatis. Importantly, we found that one chlamydial SBP, OppA3Ct, possessed dual substrate recognition properties and is capable of transporting peptidoglycan fragments (tri-diaminopimelic acid) in E. coli and in C. trachomatis. These findings suggest that Chlamydia evolved an oligopeptide transporter to facilitate the acquisition of oligopeptides for growth while simultaneously reducing the accumulation of immunostimulatory peptidoglycan fragments in the host cell cytosol. The latter property reflects bacterial pathoadaptation that dampens the host innate immune response to Chlamydia infection.

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A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression

Applied and Environmental Microbiology ◽

10.1128/aem.00509-20 ◽

2020 ◽

Vol 86 (24) ◽

Author(s):

Erin M. Nawrocki ◽

Hillary M. Mosso ◽

Edward G. Dudley

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Microbial Interactions ◽

Severe Illness ◽

Content Type ◽

E Coli ◽

Wide Range ◽

Lambdoid Prophage

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo. Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

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Role of F1C Fimbriae, Flagella, and Secreted Bacterial Components in the Inhibitory Effect of Probiotic Escherichia coli Nissle 1917 on Atypical Enteropathogenic E. coli Infection

Infection and Immunity ◽

10.1128/iai.01431-13 ◽

2014 ◽

Vol 82 (5) ◽

pp. 1801-1812 ◽

Cited By ~ 23

Author(s):

Sylvia Kleta ◽

Marcel Nordhoff ◽

Karsten Tedin ◽

Lothar H. Wieler ◽

Rafal Kolenda ◽

...

Keyword(s):

Escherichia Coli ◽

Molecular Mechanisms ◽

Inhibitory Effect ◽

Host Cells ◽

Single Infection ◽

Content Type ◽

E Coli ◽

Initial Adhesion

ABSTRACTEnteropathogenicEscherichia coli(EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probioticE. colistrain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While bothin vitroandin vivostudies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenicE. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenicE. coli(aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression.

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MSMEG_2432 of Mycobacterium smegmatis mc2155 is a dual function enzyme that exhibits DD-carboxypeptidase and β-lactamase activities

Microbiology ◽

10.1099/mic.0.000902 ◽

2020 ◽

Vol 166 (6) ◽

pp. 546-553 ◽

Cited By ~ 1

Author(s):

Satya Deo Pandey ◽

Diamond Jain ◽

Neeraj Kumar ◽

Anwesha Adhikary ◽

Ganesh Kumar N. ◽

...

Keyword(s):

Mycobacterium Smegmatis ◽

Type Species ◽

Complex Structure ◽

Ectopic Expression ◽

Dual Function ◽

Content Type ◽

Penicillin Binding Proteins ◽

E Coli

Mycobacterial peptidoglycan (PG) is an unsolved puzzle due to its complex structure and involvement of multiple enzymes in the process of its remodelling. dd-Carboxypeptidases are low molecular mass penicillin-binding proteins (LMM-PBPs) that catalyzes the cleavage of terminal d-Ala of muramyl pentapeptide branches and thereby helps in the PG remodelling process. Here, we have assigned the function of a putative LMM-PBP, MSMEG_2432 of Mycobacterium smegmatis , by showing that it exhibits both dd-CPase and β-lactamase activities. Like conventional dd-CPase (PBP5 from E. coli), upon ectopic complementation in a deformed seven PBP deletion mutant of E. coli, MSMEG_2432 has manifested its ability to restore ~75 % of the cell population to their normal rod shape. Further, in vitro dd-CPase assay has confirmed its ability to release terminal d-Ala from the synthetic tripeptide and the peptidoglycan mimetic pentapeptide substrates ending with d-Ala-d-Ala. Also, elevated resistance against penicillins and cephalosporins upon ectopic expression of MSMEG_2432 suggests the presence of β-lactamase activity, which is further confirmed in vitro through nitrocefin hydrolysis assay. Moreover, it is found apparent that D169A substitution in MSMEG_2432 influences both of its in vivo and in vitro dd-CPase and β-lactamase activities. Thus, we infer that MSMEG_2432 is a dual function enzyme that possesses both dd-CPase and β-lactamase activities.

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Evolutionary Adaptation of an AraC-Like Regulatory Protein in Citrobacter rodentium and Escherichia Species

Infection and Immunity ◽

10.1128/iai.02697-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1384-1395 ◽

Cited By ~ 9

Author(s):

Aimee Tan ◽

Nicola K. Petty ◽

Dianna Hocking ◽

Vicki Bennett-Wood ◽

Matthew Wakefield ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Regulatory Protein ◽

Gene Repertoire ◽

Citrobacter Rodentium ◽

Environmental Strain ◽

Content Type ◽

E Coli ◽

Genes Encoding

The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogenCitrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and thegrlRAoperon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genusEscherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated thatregAhas been horizontally transferred toEscherichiaspp. andC. rodentium. Comparative studies of two RegA homologues, namely, those fromC. rodentiumandE. coliSMS-3-5, a multiresistant environmental strain ofE. coli, showed that the two regulators acted similarlyin vitrobut differed in terms of their abilities to activate the virulence ofC. rodentiumin vivo, which evidently was due to their differential activation ofgrlRA. Our data indicate that RegA fromC. rodentiumhas strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence byC. rodentiumand on the evolution of virulence-regulatory genes of bacterial pathogens in general.

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Escherichia coli CFT073 Fitness Factors during Urinary Tract Infection: Identification Using an Ordered Transposon Library

Applied and Environmental Microbiology ◽

10.1128/aem.00691-20 ◽

2020 ◽

Vol 86 (13) ◽

Cited By ~ 1

Author(s):

Allyson E. Shea ◽

Juan Marzoa ◽

Stephanie D. Himpsl ◽

Sara N. Smith ◽

Lili Zhao ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Human Urine ◽

Transposon Mutagenesis ◽

Content Type ◽

E Coli ◽

Tract Infections

ABSTRACT Urinary tract infections (UTI), the second most diagnosed infectious disease worldwide, are caused primarily by uropathogenic Escherichia coli (UPEC), placing a significant financial burden on the health care system. High-throughput transposon mutagenesis combined with genome-targeted sequencing is a powerful technique to interrogate genomes for fitness genes. Genome-wide analysis of E. coli requires random libraries of at least 50,000 mutants to achieve 99.99% saturation; however, the traditional murine model of ascending UTI does not permit testing of large mutant pools due to a bottleneck during infection. To address this, an E. coli CFT073 transposon mutant ordered library of 9,216 mutants was created and insertion sites were identified. A single transposon mutant was selected for each gene to assemble a condensed library consisting of 2,913 unique nonessential mutants. Using a modified UTI model in BALB/c mice, we identified 36 genes important for colonizing the bladder, including purB, yihE, and carB. Screening of the condensed library in vitro identified yigP and ubiG to be essential for growth in human urine. Additionally, we developed a novel quantitative PCR (qPCR) technique to identify genes with fitness defects within defined subgroups of related genes (e.g., genes encoding fimbriae, toxins, etc.) following UTI. The number of mutants within these subgroups circumvents bottleneck restriction and facilitates validation of multiple mutants to generate individual competitive indices. Collectively, this study investigates the bottleneck effects during UTI, provides two techniques for evading those effects that can be applied to other disease models, and contributes a genetic tool in prototype strain CFT073 to the field. IMPORTANCE Uropathogenic Escherichia coli strains cause most uncomplicated urinary tract infections (UTI), one of the most common infectious diseases worldwide. Random transposon mutagenesis techniques have been utilized to identify essential bacterial genes during infection; however, this has been met with limitations when applied to the murine UTI model. Conventional high-throughput transposon mutagenesis screens are not feasible because of inoculum size restrictions due to a bottleneck during infection. Our study utilizes a condensed ordered transposon library, limiting the number of mutants while maintaining the largest possible genome coverage. Screening of this library in vivo, and in human urine in vitro, identified numerous candidate fitness factors. Additionally, we have developed a novel technique using qPCR to quantify bacterial outputs following infection with small subgroups of transposon mutants. Molecular approaches developed in this study will serve as useful tools to probe in vivo models that are restricted by anatomical, physiological, or genetic bottleneck limitations.

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Effective Control of Salmonella Infections by Employing Combinations of Recombinant Antimicrobial Human β-Defensins hBD-1 and hBD-2

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03628-14 ◽

2014 ◽

Vol 58 (11) ◽

pp. 6896-6903 ◽

Cited By ~ 19

Author(s):

Soumitra Maiti ◽

Sunita Patro ◽

Sukumar Purohit ◽

Sumeet Jain ◽

Shantibhusan Senapati ◽

...

Keyword(s):

Bacterial Population ◽

Antibacterial Activities ◽

Effective Control ◽

Content Type ◽

E Coli ◽

Salmonella Infections ◽

Functional Peptides ◽

Lethal Doses

ABSTRACTWe successfully produced two human β-defensins (hBD-1 and hBD-2) in bacteria as functional peptides and tested their antibacterial activities againstSalmonella entericaserovar Typhi,Escherichia coli, andStaphylococcus aureusemploying both spectroscopic and viable CFU count methods. Purified peptides showed approximately 50% inhibition of the bacterial population when used individually and up to 90% when used in combination. The 50% lethal doses (LD50) of hBD-1 againstS.Typhi,E. coli, andS. aureuswere 0.36, 0.40, and 0.69 μg/μl, respectively, while those for hBD-2 against the same bacteria were 0.38, 0.36, and 0.66 μg/μl, respectively. Moreover, we observed that bacterium-derived antimicrobial peptides were also effective in increasing survival time and decreasing bacterial loads in the peritoneal fluid, liver, and spleen of a mouse intraperitoneally infected withS.Typhi. The 1:1 hBD-1/hBD-2 combination showed maximum effectiveness in challenging theSalmonellainfectionin vitroandin vivo. We also observed less tissue damage and sepsis formation in the livers of infected mice after treatment with hBD-1 and hBD-2 peptides individually or in combination. Based on these findings, we conclude that bacterium-derived recombinant β-defensins (hBD-1 and hBD-2) are promising antimicrobial peptide (AMP)-based substances for the development of new therapeutics against typhoid fever.

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Restrictive Streptomycin Resistance Mutations Decrease the Formation of Attaching and Effacing Lesions in Escherichia coli O157:H7 Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00709-13 ◽

2013 ◽

Vol 57 (9) ◽

pp. 4260-4266 ◽

Cited By ~ 2

Author(s):

Chun Chen ◽

Carla A. Blumentritt ◽

Meredith M. Curtis ◽

Vanessa Sperandio ◽

Alfredo G. Torres ◽

...

Keyword(s):

Escherichia Coli ◽

Ribosomal Subunit ◽

Streptomycin Resistance ◽

Resistance Mutations ◽

Content Type ◽

E Coli ◽

Enterocyte Effacement ◽

Translocated Proteins

ABSTRACTStreptomycin binds to the bacterial ribosome and disrupts protein synthesis by promoting misreading of mRNA. Restrictive mutations on the ribosomal subunit protein S12 confer a streptomycin resistance (Strr) phenotype and concomitantly increase the accuracy of the decoding process and decrease the rate of translation. Spontaneous Strrmutants ofEscherichia coliO157:H7 have been generated forin vivostudies to promote colonization and to provide a selective marker for this pathogen. The locus of enterocyte effacement (LEE) ofE. coliO157:H7 encodes a type III secretion system (T3SS), which is required for attaching and effacing to the intestinal epithelium. In this study, we observed decreases in both the expression and secretion levels of the T3SS translocated proteins EspA and EspB inE. coliO157:H7 Strrrestrictive mutants, which have K42T or K42I mutations in S12. However, mildly restrictive (K87R) and nonrestrictive (K42R) mutants showed slight or indistinguishable changes in EspA and EspB secretion. Adherence and actin staining assays indicated that restrictive mutations compromised the formation of attaching and effacing lesions inE. coliO157:H7. Therefore, we suggest thatE. coliO157:H7 strains selected for Strrshould be thoroughly characterized beforein vivoandin vitroexperiments that assay for LEE-directed phenotypes and that strains carrying nonrestrictive mutations such as K42R make better surrogates of wild-type strains than those carrying restrictive mutations.

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Antibiotic Trapping by Plasmid-Encoded CMY-2 β-Lactamase Combined with Reduced Outer Membrane Permeability as a Mechanism of Carbapenem Resistance in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02459-12 ◽

2013 ◽

Vol 57 (8) ◽

pp. 3941-3949 ◽

Cited By ~ 28

Author(s):

Wil H. F. Goessens ◽

Akke K. van der Bij ◽

Ria van Boxtel ◽

Johann D. D. Pitout ◽

Peter van Ulsen ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Permeability ◽

Outer Membrane ◽

Carbapenem Resistance ◽

Covalent Modification ◽

Content Type ◽

E Coli ◽

Outer Membrane Permeability

ABSTRACTA liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS)Escherichia colistrain, but during admission, a carbapenem-resistant (CR)E. colistrain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CRE. colilacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CRE. colipossessedblaCTX-M-15andblaOXA-1. The CRE. colistrain additionally harboredblaCMY-2and demonstrated a >15-fold increase in β-lactamase activity against nitrocefin, but no hydrolysis of meropenem was detected. However, nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation eitherin vitroorin vivowith meropenem, indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into variousE. colistrains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance. In conclusion, carbapenem resistance emerged by a combination of elevated β-lactamase production and lack of porin expression. Due to the reduced outer membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the large amount of the β-lactamase. This study, therefore, provides evidence that the mechanism of “trapping” by CMY-2 β-lactamase plays a role in carbapenem resistance.

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Escherichia coliZipA Organizes FtsZ Polymers into Dynamic Ring-Like Protofilament Structures

mBio ◽

10.1128/mbio.01008-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 15

Author(s):

Marcin Krupka ◽

Marta Sobrinos-Sanguino ◽

Mercedes Jiménez ◽

Germán Rivas ◽

William Margolin

Keyword(s):

Cell Division ◽

High Surface ◽

Bacterial Cells ◽

Content Type ◽

E Coli ◽

Membrane Attachment ◽

Lipid Surface ◽

Confocal Fluorescence Imaging

ABSTRACTZipA is an essential cell division protein inEscherichia coli. Together with FtsA, ZipA tethers dynamic polymers of FtsZ to the cytoplasmic membrane, and these polymers are required to guide synthesis of the cell division septum. This dynamic behavior of FtsZ has been reconstituted on planar lipid surfacesin vitro, visible as GTP-dependent chiral vortices several hundred nanometers in diameter, when anchored by FtsA or when fused to an artificial membrane binding domain. However, these dynamics largely vanish when ZipA is used to tether FtsZ polymers to lipids at high surface densities. This, along with somein vitrostudies in solution, has led to the prevailing notion that ZipA reduces FtsZ dynamics by enhancing bundling of FtsZ filaments. Here, we show that this is not the case. When lower, more physiological levels of the soluble, cytoplasmic domain of ZipA (sZipA) were attached to lipids, FtsZ assembled into highly dynamic vortices similar to those assembled with FtsA or other membrane anchors. Notably, at either high or low surface densities, ZipA did not stimulate lateral interactions between FtsZ protofilaments. We also usedE. colimutants that are either deficient or proficient in FtsZ bundling to provide evidence that ZipA does not directly promote bundling of FtsZ filamentsin vivo. Together, our results suggest that ZipA does not dampen FtsZ dynamics as previously thought, and instead may act as a passive membrane attachment for FtsZ filaments as they treadmill.IMPORTANCEBacterial cells use a membrane-attached ring of proteins to mark and guide formation of a division septum at midcell that forms a wall separating the two daughter cells and allows cells to divide. The key protein in this ring is FtsZ, a homolog of tubulin that forms dynamic polymers. Here, we use electron microscopy and confocal fluorescence imaging to show that one of the proteins required to attach FtsZ polymers to the membrane duringE. colicell division, ZipA, can promote dynamic swirls of FtsZ on a lipid surfacein vitro. Importantly, these swirls are observed only when ZipA is present at low, physiologically relevant surface densities. Although ZipA has been thought to enhance bundling of FtsZ polymers, we find little evidence for bundlingin vitro. In addition, we present several lines ofin vivoevidence indicating that ZipA does not act to directly bundle FtsZ polymers.

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