scholarly journals Structure and Stoichiometry of the Ton Molecular Motor

2020 ◽  
Vol 21 (2) ◽  
pp. 375 ◽  
Author(s):  
Herve Celia ◽  
Nicholas Noinaj ◽  
Susan K Buchanan
Keyword(s):  
Outer Membrane ◽  
Mode Of Action ◽  
Molecular Motor ◽  
Proton Gradient ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Inner Membrane ◽  
Gram Negative ◽  
X Ray ◽  
X Ray Crystallography

The Ton complex is a molecular motor that uses the proton gradient at the inner membrane of Gram-negative bacteria to generate force and movement, which are transmitted to transporters at the outer membrane, allowing the entry of nutrients into the periplasmic space. Despite decades of investigation and the recent flurry of structures being reported by X-ray crystallography and cryoEM, the mode of action of the Ton molecular motor has remained elusive, and the precise stoichiometry of its subunits is still a matter of debate. This review summarizes the latest findings on the Ton system by presenting the recently reported structures and related reports on the stoichiometry of the fully assembled complex.

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 Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial β-Defensin 1

2018 ◽  
Vol 86 (4) ◽  
Author(s):  
J. Wendler ◽  
D. Ehmann ◽  
L. Courth ◽  
B. O. Schroeder ◽  
N. P. Malek ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Outer Membrane ◽  
Specific Activity ◽  
Redox System ◽  
Gram Negative Bacteria ◽  
Periplasmic Space ◽  
Redox Systems ◽  
Native Form ◽  
Gram Negative ◽  
Content Type

ABSTRACTThe antimicrobial peptide human β-defensin 1 (hBD1) is continuously produced by epithelial cells in many tissues. Compared to other defensins, hBD1 has only minor antibiotic activity in its native state. After reduction of its disulfide bridges, however, it becomes a potent antimicrobial agent against bacteria, while the oxidized native form (hBD1ox) shows specific activity against Gram-negative bacteria. We show that the killing mechanism of hBD1ox depends on aerobic growth conditions and bacterial enzymes. We analyzed the different activities of hBD1 using mutants ofEscherichia colilacking one or more specific proteins of their outer membrane, cytosol, or redox systems. We discovered that DsbA and DsbB are essential for the antimicrobial activity of hBD1ox but not for that of reduced hBD1 (hBD1red). Furthermore, our results strongly suggest that hBD1ox uses outer membrane protein FepA to penetrate the bacterial periplasm space. In contrast, other bacterial proteins in the outer membrane and cytosol did not modify the antimicrobial activity. Using immunogold labeling, we identified the localization of hBD1ox in the periplasmic space and partly in the outer membrane ofE. coli. However, in resistant mutants lacking DsbA and DsbB, hBD1ox was detected mainly in the bacterial cytosol. In summary, we discovered that hBD1ox could use FepA to enter the periplasmic space, where its activity depends on presence of DsbA and DsbB. HBD1ox concentrates in the periplasm in Gram-negative bacteria, which finally leads to bleb formation and death of the bacteria. Thus, the bacterial redox system plays an essential role in mechanisms of resistance against host-derived peptides such as hBD1.

Nitric Oxide Inhibitory Activity of Xanthones from the Green Fruits of Cratoxylum formosum ssp. pruniflorum

2010 ◽  
Vol 63 (11) ◽  
pp. 1550 ◽  
Author(s):  
Nawong Boonnak ◽  
Achjana Khamthip ◽  
Chatchanok Karalai ◽  
Suchada Chantrapromma ◽  
Chanita Ponglimanont ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Antibacterial Activity ◽  
Inhibitory Activity ◽  
Spectroscopic Methods ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
X Ray ◽  
X Ray Crystallography ◽  
Ic50 Values ◽  
Mic Value

Three new xanthones, pruniflorone M-O (1–3), and a new xanthonolignoid, 3-methoxy-5′-demethoxycadensin G (4), were isolated from the green fruits of Cratoxylum formosum ssp. pruniflorum along with three known xanthones (5–7) and a known flavonoid (8). Their structures were elucidated by spectroscopic methods and the structure of 1 was also determined by X-ray crystallography. Compounds 2 and 7 showed potent nitric oxide inhibitory activity with IC50 values of 4.4 and 4.3 μM, respectively. Moreover, 7 also showed strong antibacterial activity against both Gram-positive and Gram-negative bacteria with an MIC value of 4.67 μg mL–1.

scholarly journals The essential inner membrane protein YejM is a metalloenzyme

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Uma Gabale ◽  
Perla Arianna Peña Palomino ◽  
HyunAh Kim ◽  
Wenya Chen ◽  
Susanne Ressl
Keyword(s):  
Antibiotic Resistance ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Critical Role ◽  
Membrane Properties ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Periplasmic Domain ◽  
Inner Membrane Protein

Abstract Recent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One such strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability. How the inner membrane protein YejM with its periplasmic domain changes membrane properties remains unknown. Despite overwhelming structural similarity between the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been previously reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its function of regulating outer membrane properties. Understanding the molecular mechanism by which YejM is involved in outer membrane remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

scholarly journals Depolarization, Bacterial Membrane Composition, and the Antimicrobial Action of Ceragenins

2010 ◽  
Vol 54 (9) ◽  
pp. 3708-3713 ◽  
Author(s):  
Raquel F. Epand ◽  
Jake E. Pollard ◽  
Jonathan O. Wright ◽  
Paul B. Savage ◽  
Richard M. Epand
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Bacterial Species ◽  
Membrane Depolarization ◽  
Membrane Lipid ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Cytoplasmic Membranes

ABSTRACT Ceragenins are cholic acid-derived antimicrobial agents that mimic the activity of endogenous antimicrobial peptides. Ceragenins target bacterial membranes, yet the consequences of these interactions have not been fully elucidated. The role of the outer membrane in allowing access of the ceragenins to the cytoplasmic membrane of Gram-negative bacteria was studied using the ML-35p mutant strain of Escherichia coli that has been engineered to allow independent monitoring of small-molecule flux across the inner and outer membranes. The ceragenins CSA-8, CSA-13, and CSA-54 permeabilize the outer membrane of this bacterium, suggesting that the outer membrane does not play a major role in preventing the access of these agents to the cytoplasmic membrane. However, only the most potent of these ceragenins, CSA-13, was able to permeabilize the inner membrane. Interestingly, neither CSA-8 nor CSA-54 caused inner membrane permeabilization over a 30-min period, even at concentrations well above those required for bacterial toxicity. To further assess the role of membrane interactions, we measured membrane depolarization in Gram-positive bacteria with different membrane lipid compositions, as well as in Gram-negative bacteria. We found greatly increased membrane depolarization at the minimal bactericidal concentration of the ceragenins for bacterial species containing a high concentration of phosphatidylethanolamine or uncharged lipids in their cytoplasmic membranes. Although membrane lipid composition affected bactericidal efficiency, membrane depolarization was sufficient to cause lethality, providing that agents could access the cytoplasmic membrane. Consequently, we propose that in targeting bacterial cytoplasmic membranes, focus be placed on membrane depolarization as an indicator of potency.

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 Synthesis, Characterization, and Bioactivity of Schiff Bases and TheirCd2+,Zn2+,Cu2+, andNi2+Complexes Derived from Chloroacetophenone Isomers with S-Benzyldithiocarbazate and the X-Ray Crystal Structure of S-Benzyl-β-N-(4-chlorophenyl)methylenedithiocarbazate

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Mohammed Khaled bin Break ◽  
M. Ibrahim M. Tahir ◽  
Karen A. Crouse ◽  
Teng-Jin Khoo
Keyword(s):  
Crystal Structure ◽  
Phenyl Ring ◽  
Crystallographic Analysis ◽  
Gram Negative Bacteria ◽  
Schiff Base Ligands ◽  
Gram Negative ◽  
X Ray ◽  
X Ray Crystallography ◽  
Gram Positive Bacteria ◽  
Antimicrobial Assay

Two bidentate Schiff base ligands having nitrogen sulphur donor sequence were derived from the condensation of S-benzyldithiocarbazate (SBDTC) with 2-chloroacetophenone and 4-chloroacetophenone to give S-benzyl-β-N-(2-chlorophenyl)methylenedithiocarbazate (NS2) and S-benzyl-β-N-(4-chlorophenyl)methylenedithiocarbazate (NS4) isomers. Each of the ligands was then chelated with Cd2+, Zn2+, Cu2+, and Ni2+. The compounds were characterized via IR spectroscopy and melting point while the structure of NS4 was revealed via X-ray crystallography. Finally, the compounds were screened for antimicrobial activity to investigate the effect that is brought by the introduction of the chlorine atom to the benzene ring. X-ray crystallographic analysis showed that the structure of NS4 is planar with a phenyl ring that is nearly perpendicular to the rest of the molecules. The qualitative antimicrobial assay results showed that NS4 and its complexes lacked antifungal activity while Gram-positive bacteria were generally inhibited more strongly than Gram-negative bacteria. Furthermore, NS4 metal complexes were inhibited more strongly than the ligand while the opposite was seen with NS2 ligand and its complexes due to the partial solubility in dimethyl sulfoxide (DMSO). It was concluded that generally NS2 derivatives have higher bioactivity than that of NS4 derivatives and that the Cd complexes of both ligands have pronounced activity specifically onK. rhizophila.

scholarly journals Structural insights into a novel family of integral membrane siderophore reductases

2021 ◽  
Author(s):  
Inokentijs Josts ◽  
Katharina Veith ◽  
Vincent Normant ◽  
Isabelle J. Schalk ◽  
Henning Tidow
Keyword(s):  
Crystal Structure ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Bacterial Species ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Functional Studies ◽  
Inner Membrane Protein ◽  
Structural Insights

AbstractGram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from P. aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a novel fold with two tightly-bound heme molecules. In combination with functional studies these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.

scholarly journals Lipopolysaccharide transport to the cell surface: biosynthesis and extraction from the inner membrane

2015 ◽  
Vol 370 (1679) ◽  
pp. 20150029 ◽  
Author(s):  
Brent W. Simpson ◽  
Janine M. May ◽  
David J. Sherman ◽  
Daniel Kahne ◽  
Natividad Ruiz
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Gram Negative ◽  
Final Destination ◽  
Outer Leaflet ◽  
Hydrophobic Compounds ◽  
Animal Hosts

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370 , 20150027. ( doi:10.1098/rstb.2015.0027 )) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.

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