Identification of a NovelN-Acetylmuramic Acid Transporter in Tannerella forsythia

ABSTRACTTannerella forsythiais a Gram-negative periodontal pathogen lacking the ability to undergode novosynthesis of amino sugarsN-acetylmuramic acid (MurNAc) andN-acetylglucosamine (GlcNAc) that form the disaccharide repeating unit of the peptidoglycan backbone.T. forsythiarelies on the uptake of these sugars from the environment, which is so far unexplored. Here, we identified a novel transporter system ofT. forsythiainvolved in the uptake of MurNAc across the inner membrane and characterized a homolog of theEscherichia coliMurQ etherase involved in the conversion of MurNAc-6-phosphate (MurNAc-6-P) to GlcNAc-6-P. The genes encoding these components were identified on a three-gene cluster spanning Tanf_08375 to Tanf_08385 located downstream from a putative peptidoglycan recycling locus. We show that the three genes, Tanf_08375, Tanf_08380, and Tanf_08385, encoding a MurNAc transporter, a putative sugar kinase, and a MurQ etherase, respectively, are transcriptionally linked. Complementation of the Tanf_08375 and Tanf_08380 genes together intrans, but not individually, rescued the inability of anE. colimutant deficient in the phosphotransferase (PTS) system-dependent MurNAc transporter MurP as well as that of a double mutant deficient in MurP and components of the PTS system to grow on MurNAc. In addition, complementation with this two-gene construct inE. colicaused depletion of MurNAc in the medium, further confirming this observation. Our results show that the products of Tanf_08375 and Tanf_08380 constitute a novel non-PTS MurNAc transporter system that seems to be widespread among bacteria of theBacteroidetesphylum. To the best of our knowledge, this is the first identification of a PTS-independent MurNAc transporter in bacteria.IMPORTANCEIn this study, we report the identification of a novel transporter for peptidoglycan amino sugarN-acetylmuramic acid (MurNAc) in the periodontal pathogenT. forsythia. It has been known since the late 1980s thatT. forsythiais a MurNAc auxotroph relying on environmental sources for this essential sugar. Most sugar transporters, and the MurNAc transporter MurP in particular, require a PTS phosphorelay to drive the uptake and concurrent phosphorylation of the sugar through the inner membrane in Gram-negative bacteria. Our study uncovered a novel type of PTS-independent MurNAc transporter, and although so far, it seems to be unique toT. forsythia, it may be present in a range of bacteria both of the oral cavity and gut, especially of the phylumBacteroidetes.

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Heterogeneous and Flexible Transmission ofmcr-1in Hospital-AssociatedEscherichia coli

mBio ◽

10.1128/mbio.00943-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 23

Author(s):

Yingbo Shen ◽

Zuowei Wu ◽

Yang Wang ◽

Rong Zhang ◽

Hong-Wei Zhou ◽

...

Keyword(s):

Escherichia Coli ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Fluoroquinolone Resistance ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Genes Encoding

ABSTRACTThe recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route ofmcr-1amongEnterobacteriaceaespecies in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis ofEscherichia coliisolates collected in a hospital in Hangzhou, China. We found thatmcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread ofmcr-1. Themcr-1gene was found on either chromosomes or plasmids, but in most of theE. coliisolates,mcr-1was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission ofmcr-1and the coexistence ofmcr-1with other genes encoding β-lactamases and fluoroquinolone resistance in theE. coliisolates. These findings indicate thatmcr-1is heterogeneously disseminated in both commensal and pathogenic strains ofE. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology ofmcr-1among hospital-associatedE. colistrains.IMPORTANCEColistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergentmcr-1colistin resistance gene threatens the clinical utility of colistin and has gained global attention. Howmcr-1spreads in hospital settings remains unknown and was investigated by whole-genome sequencing ofmcr-1-carryingEscherichia coliin this study. The findings revealed extraordinary flexibility ofmcr-1in its spread among genetically diverseE. colihosts and plasmids, nosocomial transmission ofmcr-1-carryingE. coli, and the continuous emergence of novel Inc types of plasmids carryingmcr-1and newmcr-1variants. Additionally,mcr-1was found to be frequently associated with other genes encoding β-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology ofmcr-1and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat.

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Peptidoglycan Salvage Enables the Periodontal Pathogen Tannerella forsythia to Survive within the Oral Microbial Community

Microbial Physiology ◽

10.1159/000516751 ◽

2021 ◽

pp. 1-12

Author(s):

Isabel Hottmann ◽

Marina Borisova ◽

Christina Schäffer ◽

Christoph Mayer

Keyword(s):

Cell Wall ◽

Cell Walls ◽

Tooth Loss ◽

De Novo ◽

Periodontal Pathogen ◽

Tannerella Forsythia ◽

Gram Negative ◽

Salvage Pathways ◽

Axenic Growth ◽

Nutrition Source

Tannerella forsythia is an anaerobic, fusiform Gram-negative oral pathogen strongly associated with periodontitis, a multibacterial inflammatory disease that leads to the destruction of the teeth-supporting tissue, ultimately causing tooth loss. To survive in the oral habitat, T. forsythia depends on cohabiting bacteria for the provision of nutrients. For axenic growth under laboratory conditions, it specifically relies on the external supply of N-acetylmuramic acid (MurNAc), which is an essential constituent of the peptidoglycan (PGN) of bacterial cell walls. T. forsythia comprises a typical Gram-negative PGN; however, as evidenced by genome sequence analysis, the organism lacks common enzymes required for the de novo synthesis of precursors of PGN, which rationalizes its MurNAc auxotrophy. Only recently insights were obtained into how T. forsythia gains access to MurNAc in its oral habitat, enabling synthesis of the own PGN cell wall. This report summarizes T. forsythia’s strategies to survive in the oral habitat by means of PGN salvage pathways, including recovery of exogenous MurNAc and PGN-derived fragments but also polymeric PGN, which are all derived from cohabiting bacteria either via cell wall turnover or decay of cells. Salvage of polymeric PGN presumably requires the removal of peptides from PGN by an unknown amidase, concomitantly with the translocation of the polymer across the outer membrane. Two recently identified exo-lytic N-acetylmuramidases (Tf_NamZ1 and Tf_NamZ2) specifically cleave the peptide-free, exogenous (nutrition source) PGN in the periplasm and release the MurNAc and disaccharide substrates for the transporters Tf_MurT and Tf_AmpG, respectively, whereas the peptide-containing, endogenous (the self-cell wall) PGN stays unattached. This review also outlines how T. forsythia synthesises the PGN precursors UDP-MurNAc and UDP-N-acetylglucosamine (UDP-GlcNAc), involving homologs of the Pseudomonas sp. recycling enzymes AmgK/MurU and a monofunctional uridylyl transferase (named Tf_GlmU*), respectively.

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Correct Sorting of Lipoproteins into the Inner and Outer Membranes ofPseudomonas aeruginosaby theEscherichia coliLolCDE Transport System

mBio ◽

10.1128/mbio.00194-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 4

Author(s):

Christian Lorenz ◽

Thomas J. Dougherty ◽

Stephen Lory

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Transport Systems ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Outer Membranes

ABSTRACTBiogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. InEnterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that inPseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those fromEscherichia coli. TheP. aeruginosalipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either theE. coliorP. aeruginosaLolCDE. We further demonstrate that an inhibitor ofE. coliLolCDE is active againstP. aeruginosaonly when expressing theE. coliorthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCEGram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways ofEscherichia coliandPseudomonas aeruginosaare interchangeable, with theE. coliorthologues correctly sorting theP. aeruginosalipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified inE. colias aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.

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A Novel Nonantibiotic,lgt-Based Selection System for Stable Maintenance of Expression Vectors inEscherichia coliandVibrio cholerae

Applied and Environmental Microbiology ◽

10.1128/aem.02143-17 ◽

2017 ◽

Vol 84 (4) ◽

Author(s):

Manuela Terrinoni ◽

Stefan L. Nordqvist ◽

Susanne Källgård ◽

Jan Holmgren ◽

Michael Lebens

Keyword(s):

Antibiotic Resistance ◽

Recombinant Proteins ◽

Expression Vectors ◽

Cholera Toxin B Subunit ◽

Free System ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Genes Encoding ◽

Expression Plasmids

ABSTRACTAntibiotic selection for the maintenance of expression plasmids is discouraged in the production of recombinant proteins for pharmaceutical or other human uses due to the risks of antibiotic residue contamination of the final products and the release of DNA encoding antibiotic resistance into the environment. We describe the construction of expression plasmids that are instead maintained by complementation of thelgtgene encoding a (pro)lipoprotein glyceryl transferase essential for the biosynthesis of bacterial lipoprotein. Mutations inlgtare lethal inEscherichia coliand other Gram-negative organisms. Thelgtgene was deleted fromE. coliand complemented by theVibrio cholerae-derived gene provided intranson a temperature-sensitive plasmid, allowing cells to grow at 30°C but not at 37°C. A temperature-insensitive expression vector carrying theV. cholerae-derivedlgtgene was constructed, whereby transformants were selected by growth at 39°C. The vector was successfully used to express two recombinant proteins, one soluble and one forming insoluble inclusion bodies. Reciprocal construction was done by deleting thelgtgene fromV. choleraeand complementing the lesion with the corresponding gene fromE. coli. The resulting strain was used to produce the secreted recombinant cholera toxin B subunit (CTB) protein, a component of licensed as well as newly developed oral cholera vaccines. Overall, thelgtsystem described here confers extreme stability on expression plasmids, and this strategy can be easily transferred to other Gram-negative species using theE. coli-derivedlgtgene for complementation.IMPORTANCEMany recombinant proteins are produced in bacteria from genes carried on autonomously replicating DNA elements called plasmids. These plasmids are usually inherently unstable and rapidly lost. This can be prevented by using genes encoding antibiotic resistance. Plasmids are thus maintained by allowing only plasmid-containing cells to survive when the bacteria are grown in medium supplemented with antibiotics. In the described antibiotic-free system for the production of recombinant proteins, an essential gene is deleted from the bacterial chromosome and instead provided on a plasmid. The loss of the plasmid becomes lethal for the bacteria. Such plasmids can be used for the expression of recombinant proteins. This broadly applicable system removes the need for antibiotics in recombinant protein production, thereby contributing to reducing the spread of genes encoding antibiotic resistance, reducing the release of antibiotics into the environment, and freeing the final products (often used in pharmaceuticals) from contamination with potentially harmful antibiotic residues.

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Characterization of lptA and lptB, Two Essential Genes Implicated in Lipopolysaccharide Transport to the Outer Membrane of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01126-06 ◽

2006 ◽

Vol 189 (1) ◽

pp. 244-253 ◽

Cited By ~ 149

Author(s):

Paola Sperandeo ◽

Rachele Cescutti ◽

Riccardo Villa ◽

Cristiano Di Benedetto ◽

Daniela Candia ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

De Novo ◽

Essential Genes ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

E Coli ◽

Σ Factor

ABSTRACT The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic σ factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.

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Endotoxemia as a Diagnostic Tool for Patients with Suspected Bacteremia Caused by Gram-Negative Organisms: a Meta-Analysis of 4 Decades of Studies

Journal of Clinical Microbiology ◽

10.1128/jcm.03531-14 ◽

2015 ◽

Vol 53 (4) ◽

pp. 1183-1191 ◽

Cited By ~ 13

Author(s):

James C. Hurley ◽

Piotr Nowak ◽

Lars Öhrmalm ◽

Charalambos Gogos ◽

Apostolos Armaganidis ◽

...

Keyword(s):

Confidence Interval ◽

Meta Analysis ◽

Inclusion Criteria ◽

Summary Receiver Operating Characteristic ◽

Detection Rates ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Patient Groups ◽

Gram Negative Organisms

The clinical significance of endotoxin detection in blood has been evaluated for a broad range of patient groups in over 40 studies published over 4 decades. The influences of Gram-negative (GN) bacteremia species type and patient inclusion criteria on endotoxemia detection rates in published studies remain unclear. Studies were identified after a literature search and manual reviews of article bibliographies, together with a direct approach to authors of potentially eligible studies for data clarifications. The concordance between GN bacteremia and endotoxemia expressed as the summary diagnostic odds ratios (DORs) was derived for three GN bacteremia categories across eligible studies by using a hierarchical summary receiver operating characteristic (HSROC) method. Forty-two studies met broad inclusion criteria, with between 2 and 173 GN bacteremias in each study. Among all 42 studies, the DORs (95% confidence interval) were 3.2 (1.7 to 6.0) and 5.8 (2.4 to 13.7) in association with GN bacteremias withEscherichia coliand those withPseudomonas aeruginosa, respectively. Among 12 studies of patients with sepsis, the proportion of endotoxemia positivity (95% confidence interval) among patients withP. aeruginosabacteremia (69% [57 to 79%];P= 0.004) or withProteusbacteremia (76% [51 to 91%];P= 0.04) was significantly higher than that among patients without GN bacteremia (49% [33 to 64%]), but this was not so for patients bacteremic withE. coli(57% [40 to 73%];P= 0.55). Among studies of the sepsis patient group, the concordance of endotoxemia with GN bacteremia was surprisingly weak, especially forE. coliGN bacteremia.

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A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00537-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 5995-6002 ◽

Cited By ~ 7

Author(s):

Kristin R. Baker ◽

Bimal Jana ◽

Henrik Franzyk ◽

Luca Guardabassi

Keyword(s):

Escherichia Coli ◽

High Throughput ◽

High Throughput Screening ◽

Gram Negative Bacteria ◽

Chromogenic Substrate ◽

Intrinsic Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

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Correlation of Intracellular Trehalose Concentration with Desiccation Resistance of Soil Escherichia coli Populations

Applied and Environmental Microbiology ◽

10.1128/aem.01904-12 ◽

2012 ◽

Vol 78 (20) ◽

pp. 7407-7413 ◽

Cited By ~ 37

Author(s):

Qian Zhang ◽

Tao Yan

Keyword(s):

Escherichia Coli ◽

De Novo ◽

Potassium Acetate ◽

Desiccation Stress ◽

Desiccation Resistance ◽

Content Type ◽

Soil Desiccation ◽

E Coli ◽

Organic Solute ◽

Trehalose Synthesis

ABSTRACTNaturalized soilEscherichia colipopulations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soilE. colistrains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day−1) than that of MG1655 (0.85 day−1). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among theE. colistrains. AllE. colistrains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman's ρ = −1.0;P= 0.02).De novotrehalose synthesis was further determined for 15E. colistrains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. MostE. colistrains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soilE. colistrains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).

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Organization of the Flagellar Switch Complex ofBacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.00626-18 ◽

2018 ◽

Vol 201 (8) ◽

Cited By ~ 13

Author(s):

Elizabeth Ward ◽

Eun A Kim ◽

Joseph Panushka ◽

Tayson Botelho ◽

Trevor Meyer ◽

...

Keyword(s):

Bacillus Subtilis ◽

Protein Interaction ◽

Protein Complex ◽

Cross Linking ◽

Gram Positive ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Middle Domain ◽

Flagellar Rotation

ABSTRACTWhile the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied inEscherichia coliandSalmonella, less is known about the switch complex inBacillus subtilisor other Gram-positive species. Two component proteins (FliG and FliM) are shared betweenE. coliandB. subtilis, but in place of the protein FliN found inE. coli, theB. subtiliscomplex contains the larger protein FliY. Notably, inB. subtilisthe signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action inE. coli. Here, we have examined the architecture and function of the switch complex inB. subtilisusing targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, theB. subtilisswitch complex appears to be organized similarly to that inE. coli. The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that ofE. coli, with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences fromE. coliinvolve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases.IMPORTANCEFlagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex inBacillus subtilisor other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas inE. coliorSalmonellaCheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of theB. subtilisswitch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control inB. subtilisand other Gram-positive species.

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YejM Modulates Activity of the YciM/FtsH Protease Complex To Prevent Lethal Accumulation of Lipopolysaccharide

mBio ◽

10.1128/mbio.00598-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 10

Author(s):

Randi L. Guest ◽

Daniel Samé Guerra ◽

Maria Wissler ◽

Jacqueline Grimm ◽

Thomas J. Silhavy

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Content Type ◽

Essential Protein ◽

Ftsh Protease ◽

Acyl Chains ◽

Lipid Balance

ABSTRACT Lipopolysaccharide (LPS) is an essential glycolipid present in the outer membrane (OM) of many Gram-negative bacteria. Balanced biosynthesis of LPS is critical for cell viability; too little LPS weakens the OM, while too much LPS is lethal. In Escherichia coli, this balance is maintained by the YciM/FtsH protease complex, which adjusts LPS levels by degrading the LPS biosynthesis enzyme LpxC. Here, we provide evidence that activity of the YciM/FtsH protease complex is inhibited by the essential protein YejM. Using strains in which LpxC activity is reduced, we show that yciM is epistatic to yejM, demonstrating that YejM acts upstream of YciM to prevent toxic overproduction of LPS. Previous studies have shown that this toxicity can be suppressed by deleting lpp, which codes for a highly abundant OM lipoprotein. It was assumed that deletion of lpp restores lipid balance by increasing the number of acyl chains available for glycerophospholipid biosynthesis. We show that this is not the case. Rather, our data suggest that preventing attachment of lpp to the peptidoglycan sacculus allows excess LPS to be shed in vesicles. We propose that this loss of OM material allows continued transport of LPS to the OM, thus preventing lethal accumulation of LPS within the inner membrane. Overall, our data justify the commitment of three essential inner membrane proteins to avoid toxic over- or underproduction of LPS. IMPORTANCE Gram-negative bacteria are encapsulated by an outer membrane (OM) that is impermeable to large and hydrophobic molecules. As such, these bacteria are intrinsically resistant to several clinically relevant antibiotics. To better understand how the OM is established or maintained, we sought to clarify the function of the essential protein YejM in Escherichia coli. Here, we show that YejM inhibits activity of the YciM/FtsH protease complex, which regulates synthesis of the essential OM glycolipid lipopolysaccharide (LPS). Our data suggest that disrupting proper communication between LPS synthesis and transport to the OM leads to accumulation of LPS within the inner membrane (IM). The lethality associated with this event can be suppressed by increasing OM vesiculation. Our research has identified a completely novel signaling pathway that we propose coordinates LPS synthesis and transport.

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