scholarly journals Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in Pseudomonas aeruginosa PAO1

mBio ◽  
2011 ◽  
Vol 2 (4) ◽  
Author(s):  
Angela M. DeLucia ◽  
David A. Six ◽  
Ruth E. Caughlan ◽  
Patricia Gee ◽  
Ian Hunt ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Lipid A ◽  
Inner Core ◽  
Expression System ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
Ms Analysis

ABSTRACTGram-negative outer membrane (OM) integrity is maintained in part by Mg2+cross-links between phosphates on lipid A and on core sugars of adjacent lipopolysaccharide (LPS) molecules. In contrast to other Gram-negative bacteria,waaP, encoding an inner-core kinase, could not be inactivated inPseudomonas aeruginosa. To examine this further, expression of the kinases WaaP or WapP/WapQ/PA5006 was placed under the control of the arabinose-regulated pBAD promoter. Growth of these strains was arabinose dependent, confirming that core phosphorylation is essential inP. aeruginosa. Transmission electron micrographs of kinase-depleted cells revealed marked invaginations of the inner membrane. SDS-PAGE of total LPS from WaaP-depleted cells showed accumulation of a fast-migrating band. Mass spectrometry (MS) analysis revealed that LPS from these cells exhibits a unique truncated core consisting of two 3-deoxy-d-manno-octulosonic acids (Kdo), twol-glycero-d-manno-heptoses (Hep), and one hexose but completely devoid of phosphates, indicating that phosphorylation by WaaP is necessary for subsequent core phosphorylations. MS analysis of lipid A from WaaP-depleted cells revealed extensive 4-amino-4-deoxy-l-arabinose modification. OM prepared from these cells by Sarkosyl extraction of total membranes or by sucrose density gradient centrifugation lacked truncated LPS. Instead, truncated LPS was detected in the inner membrane fractions, consistent with impaired transport/assembly of this species into the OM.IMPORTANCEGram-negative bacteria have an outer membrane (OM) comprised of a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The OM protects cells from toxic molecules and is important for survival during infection. The LPS core kinase genewaaPcan be deleted in several Gram-negative bacteria but not inPseudomonas aeruginosa. We used a controlled-expression system to deplete WaaP directly inP. aeruginosacells, which halted growth. WaaP depletion also caused gross changes in cell morphology and led to the accumulation of an aberrant LPS lacking several core sugars and all core phosphates. The aberrant LPS failed to reach the OM, suggesting that WaaP is essential inP. aeruginosabecause it is required to produce the full-length LPS that is recognized by the OM transport/assembly machinery in this organism. Therefore, WaaP may constitute a good target for the development of novel antipseudomonal agents.

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

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
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.

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

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
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.

scholarly journals Mutation and Suppressor Analysis of the Essential Lipopolysaccharide Transport Protein LptA Reveals Strategies To Overcome Severe Outer Membrane Permeability Defects in Escherichia coli

2017 ◽  
Vol 200 (2) ◽  
Author(s):  
Federica A. Falchi ◽  
Elisa A. Maccagni ◽  
Simone Puccio ◽  
Clelia Peano ◽  
Cristina De Castro ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Antibiotic Sensitivity ◽  
Major Constituent ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Outer Leaflet ◽  
Increased Sensitivity

ABSTRACTIn Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane (OM), preventing the entry of toxic molecules, such as detergents and antibiotics. LPS is transported from the inner membrane (IM) to the OM by the Lpt multiprotein machinery. Defects in LPS transport compromise LPS assembly at the OM and result in increased antibiotic sensitivity. LptA is a key component of the Lpt machine that interacts with the IM protein LptC and chaperones LPS through the periplasm. We report here the construction oflptA41, a quadruple mutant in four conserved amino acids potentially involved in LPS or LptC binding. Although viable, the mutant displays increased sensitivity to several antibiotics (bacitracin, rifampin, and novobiocin) and the detergent SDS, suggesting thatlptA41affects LPS transport. Indeed,lptA41is defective in Lpt complex assembly, and its lipid A carries modifications diagnostic of LPS transport defects. We also selected and characterized two phenotypic bacitracin-resistant suppressors oflptA41. One mutant, in which only bacitracin sensitivity is suppressed, harbors a small in-frame deletion inmlaA, which codes for an OM lipoprotein involved in maintaining OM asymmetry by reducing accumulation of phospholipids in the outer leaflet. The other mutant, in which bacitracin, rifampin, and SDS sensitivity is suppressed, harbors an additional amino acid substitution in LptA41 and a nonsense mutation inopgH, encoding a glycosyltransferase involved in periplasmic membrane-derived oligosaccharide synthesis. Characterization of the suppressor mutants highlights different strategies adopted by the cell to overcome OM defects caused by impaired LPS transport.IMPORTANCELipopolysaccharide (LPS) is the major constituent of the outer membrane (OM) of most Gram-negative bacteria, forming a barrier against antibiotics. LPS is synthesized at the inner membrane (IM), transported across the periplasm, and assembled at the OM by the multiprotein Lpt complex. LptA is the periplasmic component of the Lpt complex, which bridges IM and OM and ferries LPS across the periplasm. How the cell coordinates the processes involved in OM biogenesis is not completely understood. We generated a mutant partially defective inlptAthat exhibited increased sensitivity to antibiotics and selected for suppressors of the mutant. The analysis of two independent suppressors revealed different strategies adopted by the cell to overcome defects in LPS biogenesis.

scholarly journals Outer Membrane Targeting of Pseudomonas aeruginosa Proteins Shows Variable Dependence on the Components of Bam and Lol Machineries

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Hanh H. Hoang ◽  
Nicholas N. Nickerson ◽  
Vincent T. Lee ◽  
Anastasia Kazimirova ◽  
Mohamed Chami ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Protein Translocation ◽  
Lipid Vesicles ◽  
Structural Features ◽  
Gram Negative Bacteria ◽  
Variable Effect ◽  
Gram Negative ◽  
Content Type

ABSTRACT In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. IMPORTANCE Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect, but the severity (and therefore, Lol and Bam dependence), varies with individual proteins. XcpQ, the secretin component of the type II secretion apparatus, is translocated into the OM without the assistance of Bam or Lol machineries. The hypothesis that XcpQ, after secretion across the cytoplasmic membrane, does not utilize the OM targeting machineries was supported by demonstrating that in vitro-synthesized XcpQ (but not the other P. aeruginosa secretins) can spontaneously incorporate into lipid vesicles. Therefore, the requirement for ancillary factors appears to be, in certain instances, dictated by the intrinsic properties of individual OM proteins, conceivably reflecting their propensities to misfold during periplasmic transit.

scholarly journals Interplay ofKlebsiella pneumoniaefabZandlpxCMutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Mina Mostafavi ◽  
Lisha Wang ◽  
Lili Xie ◽  
Kenneth T. Takeoka ◽  
Daryl L. Richie ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Gram Negative Bacteria ◽  
Resistance Mutations ◽  
Cellular Mechanisms ◽  
Gram Negative ◽  
Content Type ◽  
Outer Leaflet ◽  
Chemical Inhibition ◽  
Dependent Growth

ABSTRACTTight coordination of inner and outer membrane biosynthesis is very important in Gram-negative bacteria. Biosynthesis of the lipid A moiety of lipopolysaccharide, which comprises the outer leaflet of the outer membrane has garnered interest for Gram-negative antibacterial discovery. In particular, several potent inhibitors of LpxC (the first committed step of the lipid A pathway) are described. Here we show that serial passaging ofKlebsiella pneumoniaein increasing levels of an LpxC inhibitor yielded mutants that grew only in the presence of the inhibitor. These strains had mutations infabZandlpxCoccurring together (encoding either FabZR121L/LpxCV37Gor FabZF51L/LpxCV37G).K. pneumoniaemutants having only LpxCV37Gor LpxCV37Aor various FabZ mutations alone were less susceptible to the LpxC inhibitor and did not require LpxC inhibition for growth. Western blotting revealed that LpxCV37Gaccumulated to high levels, and electron microscopy of cells harboring FabZR121L/LpxCV37Gindicated an extreme accumulation of membrane in the periplasm when cells were subcultured without LpxC inhibitor. Significant accumulation of detergent-like lipid A pathway intermediates that occur downstream of LpxC (e.g., lipid X and disaccharide monophosphate [DSMP]) was also seen. Taken together, our results suggest that redirection of lipid A pathway substrate by less active FabZ variants, combined with increased activity from LpxCV37Gwas overdriving the lipid A pathway, necessitating LpxC chemical inhibition, since native cellular maintenance of membrane homeostasis was no longer functioning.IMPORTANCEEmergence of antibiotic resistance has prompted efforts to identify and optimize novel inhibitors of antibacterial targets such as LpxC. This enzyme catalyzes the first committed step of lipid A synthesis, which is necessary to generate lipopolysaccharide and ultimately the Gram-negative protective outer membrane. Investigation of this pathway and its interrelationship with inner membrane (phospholipid) biosynthesis or other pathways is therefore highly important to the fundamental understanding of Gram-negative bacteria and by extension to antibiotic discovery. Here we exploited the availability of a novel LpxC inhibitor to engender the generation ofK. pneumoniaeresistant mutants whose growth depends on chemical inhibition of LpxC. Inhibitor dependency resulted from the interaction of different resistance mutations and was based on loss of normal cellular mechanisms required to establish membrane homeostasis. This study provides new insights into the importance of this process inK. pneumoniaeand how it may be linked to novel biosynthetic pathway inhibitors.

scholarly journals Pyocin S5 Import into Pseudomonas aeruginosa Reveals a Generic Mode of Bacteriocin Transport

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Hannah M. Behrens ◽  
Edward D. Lowe ◽  
Joseph Gault ◽  
Nicholas G. Housden ◽  
Renata Kaminska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Animal Infection ◽  
Inner Membrane Protein ◽  
Biochemical Analyses ◽  
Functional Analyses

ABSTRACT Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria. IMPORTANCE Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets Pseudomonas aeruginosa. In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.

scholarly journals PmrC (EptA) and CptA Negatively Affect Outer Membrane Vesicle Production inCitrobacter rodentium

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Anshul Sinha ◽  
Sammy Nyongesa ◽  
Charles Viau ◽  
Samantha Gruenheid ◽  
Frédéric J. Veyrier ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Membrane Vesicle ◽  
Enteric Bacteria ◽  
Outer Membrane Vesicles ◽  
Gram Negative Bacteria ◽  
Citrobacter Rodentium ◽  
Wild Type ◽  
Gram Negative ◽  
Content Type

ABSTRACTOuter membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria by a bulging of the outer membrane (OM) and subsequent release into the environment. By serving as vehicles for various cargos, including proteins, nucleic acids and small metabolites, OMVs are central to interbacterial interactions and both symbiotic and pathogenic host bacterial interactions. However, despite their importance, the mechanism of OMV formation remains unclear. Recent evidence indicates that covalent modifications of lipopolysaccharides (LPS) influence OMV biogenesis. Several enteric bacteria modify LPS with phosphoethanolamine (pEtN) using the iron-regulated PmrC (EptA) and CptA pEtN transferases. In wild-typeCitrobacter rodentium, the presence of increasing subtoxic concentrations of iron was found to stimulate OMV production 4- to 9-fold above baseline.C. rodentiumuses the two-component system PmrAB to sense and adapt to environmental iron. Compared to the wild type, theC. rodentiumΔpmrABstrain exhibited heightened OMV production at similar iron concentrations. PmrAB regulates transcription ofpmrC(also known aseptA) andcptA. OMV production in strains lacking eitherpmrC(eptA) orcptAwas similarly increased in comparison to that of the wild type. Importantly, plasmid complementation ofC. rodentiumstrains with eitherpmrC(eptA) orcptAresulted in a drastic inhibition of OMV production. Finally, we showed that β-lactamase and CroP, two enzymes found in theC. rodentiumperiplasm and outer membrane (OM), respectively, are associated with OMVs. These data suggest a novel mechanism by whichC. rodentiumand possibly other Gram-negative bacteria can negatively affect OMV production through the PmrAB-regulated genespmrC(eptA) andcptA.IMPORTANCEAlthough OMVs secreted by Gram-negative bacteria fulfill multiple functions, the molecular mechanism of OMV biogenesis remains ill defined. Our group has previously shown that PmrC (also known as EptA) and CptA maintain OM integrity and provide resistance to iron toxicity and antibiotics in the murine pathogenCitrobacter rodentium. In several enteric bacteria, these proteins modify the lipid A and core regions of lipopolysaccharide with phosphoethanolamine moieties. Here, we show that these proteins also repress OMV production in response to environmental iron inC. rodentium. These data support the emerging understanding that lipopolysaccharide modifications are important regulators of OMV biogenesis in Gram-negative bacteria.

scholarly journals A Novel Inhibitor of the LolCDE ABC Transporter Essential for Lipoprotein Trafficking in Gram-Negative Bacteria

2018 ◽  
Vol 62 (4) ◽  
Author(s):  
Nicholas N. Nickerson ◽  
Christine C. Jao ◽  
Yiming Xu ◽  
John Quinn ◽  
Elizabeth Skippington ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Outer Membrane ◽  
Abc Transporter ◽  
Protein Complexes ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Content Type ◽  
High Level

ABSTRACTThe outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral β-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors ofEscherichia coligrowth followed by selection of compounds that induced the extracytoplasmic σEstress response. Mutations inlolC,lolD, andlolEconferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment ofE. colicells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolCQ258K), which confers high-level resistance to G0507in vivobut no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.

scholarly journals The Lipid A 1-Phosphatase, LpxE, Functionally Connects Multiple Layers of Bacterial Envelope Biogenesis

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Jinshi Zhao ◽  
Jinsu An ◽  
Dohyeon Hwang ◽  
Qinglin Wu ◽  
Su Wang ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Outer Membrane ◽  
Lipid A ◽  
Host Immune System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Phosphatase Activities ◽  
O Antigen

ABSTRACT Although distinct lipid phosphatases are thought to be required for processing lipid A (component of the outer leaflet of the outer membrane), glycerophospholipid (component of the inner membrane and the inner leaflet of the outer membrane), and undecaprenyl pyrophosphate (C55-PP; precursors of peptidoglycan and O antigens of lipopolysaccharide) in Gram-negative bacteria, we report that the lipid A 1-phosphatases, LpxEs, functionally connect multiple layers of cell envelope biogenesis in Gram-negative bacteria. We found that Aquifex aeolicus LpxE structurally resembles YodM in Bacillus subtilis, a phosphatase for phosphatidylglycerol phosphate (PGP) with a weak in vitro activity on C55-PP, and rescues Escherichia coli deficient in PGP and C55-PP phosphatase activities; deletion of lpxE in Francisella novicida reduces the MIC value of bacitracin, indicating a significant contribution of LpxE to the native bacterial C55-PP phosphatase activity. Suppression of plasmid-borne lpxE in F. novicida deficient in chromosomally encoded C55-PP phosphatase activities results in cell enlargement, loss of O-antigen repeats of lipopolysaccharide, and ultimately cell death. These discoveries implicate LpxE as the first example of a multifunctional regulatory enzyme that orchestrates lipid A modification, O-antigen production, and peptidoglycan biogenesis to remodel multiple layers of the Gram-negative bacterial envelope. IMPORTANCE Dephosphorylation of the lipid A 1-phosphate by LpxE in Gram-negative bacteria plays important roles in antibiotic resistance, bacterial virulence, and modulation of the host immune system. Our results demonstrate that in addition to removing the 1-phosphate from lipid A, LpxEs also dephosphorylate undecaprenyl pyrophosphate, an important metabolite for the synthesis of the essential envelope components, peptidoglycan and O-antigen. Therefore, LpxEs participate in multiple layers of biogenesis of the Gram-negative bacterial envelope and increase antibiotic resistance. This discovery marks an important step toward understanding the regulation and biogenesis of the Gram-negative bacterial envelope.

scholarly journals MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

2021 ◽  
Vol 22 (10) ◽  
pp. 5328
Author(s):  
Miao Ma ◽  
Margaux Lustig ◽  
Michèle Salem ◽  
Dominique Mengin-Lecreulx ◽  
Gilles Phan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

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