scholarly journals BamA β16C strand and periplasmic turns are critical for outer membrane protein insertion and assembly

2017 ◽  
Vol 474 (23) ◽  
pp. 3951-3961 ◽  
Author(s):  
Yinghong Gu ◽  
Yi Zeng ◽  
Zhongshan Wang ◽  
Changjiang Dong
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Membrane Insertion ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Protein Insertion ◽  
E Coli ◽  
Functional Studies ◽  
Functional Assays ◽  
Membrane Protein Insertion

Outer membrane (OM) β-barrel proteins play important roles in importing nutrients, exporting wastes and conducting signals in Gram-negative bacteria, mitochondria and chloroplasts. The outer membrane proteins (OMPs) are inserted and assembled into the OM by OMP85 family proteins. In Escherichia coli, the β-barrel assembly machinery (BAM) contains four lipoproteins such as BamB, BamC, BamD and BamE, and one OMP BamA, forming a ‘top hat’-like structure. Structural and functional studies of the E. coli BAM machinery have revealed that the rotation of periplasmic ring may trigger the barrel β1C–β6C scissor-like movement that promote the unfolded OMP insertion without using ATP. Here, we report the BamA C-terminal barrel structure of Salmonella enterica Typhimurium str. LT2 and functional assays, which reveal that the BamA's C-terminal residue Trp, the β16C strand of the barrel and the periplasmic turns are critical for the functionality of BamA. These findings indicate that the unique β16C strand and the periplasmic turns of BamA are important for the outer membrane insertion and assembly. The periplasmic turns might mediate the rotation of the periplasmic ring to the scissor-like movement of BamA β1C–β6C, triggering the OMP insertion. These results are important for understanding the OMP insertion in Gram-negative bacteria, as well as in mitochondria and chloroplasts.

scholarly journals The crystal structure of the TonB-dependent transporter YncD reveals a positively charged substrate-binding site

2020 ◽  
Vol 76 (5) ◽  
pp. 484-495
Author(s):  
Rhys Grinter ◽  
Trevor Lithgow
Keyword(s):  
Binding Site ◽  
Outer Membrane ◽  
Substrate Binding ◽  
Outer Membrane Proteins ◽  
Ferric Citrate ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Substrate Binding Site ◽  
E Coli ◽  
Positively Charged

The outer membrane of Gram-negative bacteria is highly impermeable to hydrophilic molecules of larger than 600 Da, protecting these bacteria from toxins present in the environment. In order to transport nutrients across this impermeable membrane, Gram-negative bacteria utilize a diverse family of outer-membrane proteins called TonB-dependent transporters. The majority of the members of this family transport iron-containing substrates. However, it is becoming increasingly clear that TonB-dependent transporters target chemically diverse substrates. In this work, the structure and phylogenetic distribution of the TonB-dependent transporter YncD are investigated. It is shown that while YncD is present in some enteropathogens, including Escherichia coli and Salmonella spp., it is also widespread in Gammaproteobacteria and Betaproteobacteria of environmental origin. The structure of YncD was determined, showing that despite a distant evolutionary relationship, it shares structural features with the ferric citrate transporter FecA, including a compact positively charged substrate-binding site. Despite these shared features, it is shown that YncD does not contribute to the growth of E. coli in pure culture under iron-limiting conditions or with ferric citrate as an iron source. Previous studies of transcriptional regulation in E. coli show that YncD is not induced under iron-limiting conditions and is unresponsive to the ferric uptake regulator (Fur). These observations, combined with the data presented here, suggest that YncD is not responsible for the transport of an iron-containing substrate.

scholarly journals The crystal structure of the TonB-dependent transporter YncD reveals a positively charged substrate binding site

2020 ◽  
Author(s):  
Rhys Grinter ◽  
Trevor Lithgow
Keyword(s):  
Binding Site ◽  
Outer Membrane ◽  
Substrate Binding ◽  
Outer Membrane Proteins ◽  
Evolutionary Relationship ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Substrate Binding Site ◽  
E Coli ◽  
Positively Charged

AbstractThe outer membrane of Gram-negative bacteria is highly impermeable to hydrophilic molecules larger than 600 Da, protecting these bacteria from toxins present in the environment. In order to transport nutrients across this impermeable membrane, Gram-negative bacteria utilise a diverse family of outer-membrane proteins called TonB-dependent transporters. The majority of this family transport iron-containing substrates. However, it is becoming increasingly clear that TonB-dependent transporters target chemically diverse substrates. In this work, we investigate the structure and phylogenetic distribution of the TonB-dependent transporter YncD. We show that while YncD is present in some enteropathogens including E. coli and Salmonella spp., it is also widespread in Gamma and Betaproteobacteria of environmental origin. We determine the structure of YncD, showing that despite a distant evolutionary relationship, it shares structural features with the ferriccitrate transporter FecA, including a compact positively-charged substrate-binding site. Despite these shared features, we show that YncD does not contribute to the growth of E. coli in pure culture under-iron limiting conditions or with ferric-citrate as an iron source. Previous studies on transcriptional regulation in E. coli show that YncD is not induced under iron-limiting conditions and is unresponsive to the Ferric uptake regulator (Fur). These observations combined with the data we present, suggest that YncD is not responsible for the transport of an iron-containing substrate.

scholarly journals The outer membrane proteins OmpA, FhuA, OmpF, EstA, BtuB and OmpX have unique lipopolysaccharide fingerprints

2018 ◽  
Author(s):  
Jonathan Shearer ◽  
Damien Jefferies ◽  
Syma Khalid
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Coarse Grain ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Unique Pattern ◽  
E Coli ◽  
Outer Leaflet ◽  
Wide Range

AbstractThe outer membrane of Gram-negative bacteria has a highly complex asymmetrical architecture, containing a mixture of phospholipids in the inner leaflet and in the outer leaflet they contain almost exclusively lipopolysaccharide (LPS) molecules. In E. coli, the outer membrane contains a wide range proteins with a beta barrel architecture, that vary in size from the smallest having eight strands to larger barrels composed of twenty-two strands. Here we report coarse-grain molecular dynamics simulations of six proteins from the E. coli outer membrane OmpA, OmpX, BtuB, FhuA, OmpF and EstA in a range of membrane environments, which are representative of the in vivo for different strains of E. coli. We show that each protein has a unique pattern of interaction with the surrounding membrane, which is influenced by the composition of the protein, the level of LPS in the outer leaflet and the differing mobilities of the lipids in the two leaflets of the membrane. Overall we present analyses from over 200 microseconds of simulation for each protein.Author summaryWe present data from over 200 microseconds of coarse-grain simulations that show the complexities of protein-lipid interactions within the outer membranes of Gram-negative bacteria. We show that the slow movement of lipolysaccharide molecules necessitate simulations of over 30 microsecond duration to achieve converged properties such as protein tilt angle. Each of the six proteins studied here shows a unique pattern of interactions with the outer membrane and thus constitute a ‘fingerprint’ or ‘signature’.

scholarly journals The gain-of-function allelebamAE470Kbypasses the essential requirement for BamD in β-barrel outer membrane protein assembly

2020 ◽  
Vol 117 (31) ◽  
pp. 18737-18743 ◽  
Author(s):  
Elizabeth M. Hart ◽  
Meera Gupta ◽  
Martin Wühr ◽  
Thomas J. Silhavy
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Protein Assembly ◽  
Gram Negative Bacteria ◽  
Gain Of Function ◽  
Essential Requirement ◽  
Gram Negative ◽  
Selective Permeability ◽  
Catalytic Role ◽  
Global Defect

The outer membrane (OM) of gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral β-barrel outer membrane proteins (OMPs) function to establish and maintain the selective permeability of the OM. OMPs are assembled into the OM by the β-barrel assembly machine (BAM), which is composed of one OMP—BamA—and four lipoproteins—BamB, C, D, and E. BamB, C, and E can be removed individually with only minor effects on barrier function; however, depletion of either BamA or BamD causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation,bamAE470K, that bypasses the requirement for BamD. AlthoughbamD::kanbamAE470Kcells exhibit growth and OM barrier defects, they assemble OMPs with surprising robustness. Our results demonstrate that BamD does not play a catalytic role in OMP assembly, but rather functions to regulate the activity of BamA.

scholarly journals A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli

2015 ◽  
Vol 291 (4) ◽  
pp. 1921-1932 ◽  
Author(s):  
Matthias Urfer ◽  
Jasmina Bogdanovic ◽  
Fabio Lo Monte ◽  
Kerstin Moehle ◽  
Katja Zerbe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Permeability Barrier ◽  
Gram Negative ◽  
Fluorescence Studies ◽  
E Coli ◽  
Interaction Sites ◽  
External Stresses ◽  
Antibiotic Discovery

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.

scholarly journals Functions of the BamBCDE Lipoproteins Revealed by Bypass Mutations in BamA

2020 ◽  
Vol 202 (21) ◽  
Author(s):  
Elizabeth M. Hart ◽  
Thomas J. Silhavy
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative ◽  
Bam Complex ◽  
Essential Function ◽  
The Individual

ABSTRACT The heteropentomeric β-barrel assembly machine (BAM complex) is responsible for folding and inserting a diverse array of β-barrel outer membrane proteins (OMPs) into the outer membrane (OM) of Gram-negative bacteria. The BAM complex contains two essential proteins, the β-barrel OMP BamA and a lipoprotein BamD, whereas the auxiliary lipoproteins BamBCE are individually nonessential. Here, we identify and characterize three bamA mutations, the E-to-K change at position 470 (bamAE470K), the A-to-P change at position 496 (bamAA496P), and the A-to-S change at position 499 (bamAA499S), that suppress the otherwise lethal ΔbamD, ΔbamB ΔbamC ΔbamE, and ΔbamC ΔbamD ΔbamE mutations. The viability of cells lacking different combinations of BAM complex lipoproteins provides the opportunity to examine the role of the individual proteins in OMP assembly. Results show that, in wild-type cells, BamBCE share a redundant function; at least one of these lipoproteins must be present to allow BamD to coordinate productively with BamA. Besides BamA regulation, BamD shares an additional essential function that is redundant with a second function of BamB. Remarkably, bamAE470K suppresses both, allowing the construction of a BAM complex composed solely of BamAE470K that is able to assemble OMPs in the absence of BamBCDE. This work demonstrates that the BAM complex lipoproteins do not participate in the catalytic folding of OMP substrates but rather function to increase the efficiency of the assembly process by coordinating and regulating the assembly of diverse OMP substrates. IMPORTANCE The folding and insertion of β-barrel outer membrane proteins (OMPs) are conserved processes in mitochondria, chloroplasts, and Gram-negative bacteria. In Gram-negative bacteria, OMPs are assembled into the outer membrane (OM) by the heteropentomeric β-barrel assembly machine (BAM complex). In this study, we probe the function of the individual BAM proteins and how they coordinate assembly of a diverse family of OMPs. Furthermore, we identify a gain-of-function bamA mutant capable of assembling OMPs independently of all four other BAM proteins. This work advances our understanding of OMP assembly and sheds light on how this process is distinct in Gram-negative bacteria.

scholarly journals TP0453, a Concealed Outer Membrane Protein of Treponema pallidum, Enhances Membrane Permeability

2005 ◽  
Vol 187 (18) ◽  
pp. 6499-6508 ◽  
Author(s):  
Karsten R. O. Hazlett ◽  
David L. Cox ◽  
Marc Decaffmeyer ◽  
Michael P. Bennett ◽  
Daniel C. Desrosiers ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
Nutrient Transport ◽  
Treponema Pallidum ◽  
Protein S ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Membrane Spanning

ABSTRACT The outer membrane of Treponema pallidum, the noncultivable agent of venereal syphilis, contains a paucity of protein(s) which has yet to be definitively identified. In contrast, the outer membranes of gram-negative bacteria contain abundant immunogenic membrane-spanning β-barrel proteins mainly involved in nutrient transport. The absence of orthologs of gram-negative porins and outer membrane nutrient-specific transporters in the T. pallidum genome predicts that nutrient transport across the outer membrane must differ fundamentally in T. pallidum and gram-negative bacteria. Here we describe a T. pallidum outer membrane protein (TP0453) that, in contrast to all integral outer membrane proteins of known structure, lacks extensive β-sheet structure and does not traverse the outer membrane to become surface exposed. TP0453 is a lipoprotein with an amphiphilic polypeptide containing multiple membrane-inserting, amphipathic α-helices. Insertion of the recombinant, nonlipidated protein into artificial membranes results in bilayer destabilization and enhanced permeability. Our findings lead us to hypothesize that TP0453 is a novel type of bacterial outer membrane protein which may render the T. pallidum outer membrane permeable to nutrients while remaining inaccessible to antibody.

Genetics of the (gram-negative) bacterial surface

1978 ◽  
Vol 202 (1146) ◽  
pp. 5-30 ◽  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Capsular Polysaccharide ◽  
Repeat Unit ◽  
Outer Membrane Proteins ◽  
Missense Mutations ◽  
Protein Antigens ◽  
Gram Negative ◽  
E Coli ◽  
S Genes

The surface of a gram-negative bacterium is made up of the lipopolysaccharide (l. p. s.) and protein components of the outer leaflet of its outer membrane, and of capsular polysaccharide, flagella and fimbriae if present. In Salmonella all the special genes needed for synthesis of the O-specific oligosaccharide repeat unit (different in each O group) of the l. p. s. sidechains are found in the rfb cluster, near his . Nearly all so-far identified rfa genes, for synthesis of l. p. s. core, are clustered between cysE and pyrE . Genes for polymerization and modification of O units are scattered: some are part of prophage genomes and some show ‘form variation’ – spontaneous alternation between expression and non-expression, mechanism unknown. Escherichia coli differs by frequent presence of capsular polysaccharides (K antigens), some determined by kps genes, unlinked to l. p. s. genes, others by his -linked genes perhaps homologous with rfb . Expression of some non-l. p. s. polysaccharide genes, but not of l. p. s. genes, is greatly influenced by the environment. Major outer membrane proteins (more than 10 5 molec. /bacterium) include: a lipoprotein, in part covalently joined to the cell wall, perhaps anchoring the outer membrane; and several proteins of molec. mass 30000–40000 (one of them phage-determined), some of which serve to make the outer membrane permeable to small hydrophilic molecules. Genes affecting sensitivity (adsorbing capacity) to various phages and colicins (e. g. tonA, bfe ) specify various ‘minor’ outer membrane proteins concerned with uptake of nutrients (e. g. iron ferrichrome, vitamin B 12 ) when present at very low concentrations. Neither the ‘major’ nor the ‘minor’ protein genes are clustered: their expression is subject to conspicuous regulation by environmental conditions. In E. coli the flagellin and hook protein structural genes are located in different clusters of motility-related genes. Missense mutations in the flagellin gene may cause alteration in flagellar shape or in serological character, which in Salmonella is also affected by gene nml , for methylation of the free amino groups of some lysines of flagellin. Electron microscopy of re-annealed DNA from the relevant region indicates that change of flagellar antigenic phase in Salmonella results from a reversible inversion of a 750 base-pair segment, probably constituting the phase-determinant gene. Production of fimbriae (pili) requires function of several linked pil genes, and is subject to a kind of ‘form variation’ of unknown mechanism. Genes in conjugative plasmids when derepressed cause production of sex pili. E. coli protein antigens K88 and K99, apparently fimbrial, concerned with adhesion to intestinal mucosa and so with enteropathogenicity, are plasmid-determined.

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