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 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.

scholarly journals Antiproliferative effects of porins extracted from Klebsiella pneumoniae on HL-60, NIH/3T3 and Human foreskin fibroblast cell (HFFC) lines measured by ELISA method

2010 ◽  
Vol 4 (1) ◽  
pp. 28-35
Author(s):  
Amir H. Al–Shammary ◽  
Essam F. Al-Jumaily ◽  
Nidhal Abdulmohymen
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Primary Source ◽  
Gram Negative Bacteria ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Gram Negative ◽  
Isolation And Purification ◽  
Antiproliferative Effects

Approximately, 50% of the dry mass of the outer membrane of gram-negative bacteria consists of proteins, and more than 20 immunochemically distinct proteins (termed outer membrane proteins [OMPs]) have been identified. An identified local strain of Klebsiella pneumoniae was used as a primary source for the isolation and purification of porins. Multiple concentrations of purified porins (5, 10, 15, 20, 25) g/ml were incubated with three different cell lines for (24, 72 , 120) hrs, after the end of the incubation periods, the cells were treated with Cell proliferation ELISA, BrdU (colorimetric) kit to evaluate the antiproliferative effects of porins. The results revealed that porins are potent antiproliferative agent in a time and concentration dependent manner and thus could greatly affect prokaryote-eukaryote interaction as well as the whole inflammatory process resulted after infection with gram negative bacteria.

scholarly journals Gram-negative outer-membrane proteins with multiple β-barrel domains

2021 ◽  
Vol 118 (31) ◽  
pp. e2104059118
Author(s):  
Ron Solan ◽  
Joana Pereira ◽  
Andrei N. Lupas ◽  
Rachel Kolodny ◽  
Nir Ben-Tal
Keyword(s):  
Outer Membrane ◽  
De Novo ◽  
Outer Membrane Proteins ◽  
Gram Negative Bacteria ◽  
Single Chain ◽  
Gram Negative ◽  
Complementary Function ◽  
Beta Barrels ◽  
Β Sheets ◽  
The Individual

Outer-membrane beta barrels (OMBBs) are found in the outer membrane of gram-negative bacteria and eukaryotic organelles. OMBBs fold as antiparallel β-sheets that close onto themselves, forming pores that traverse the membrane. Currently known structures include only one barrel, of 8 to 36 strands, per chain. The lack of multi-OMBB chains is surprising, as most OMBBs form oligomers, and some function only in this state. Using a combination of sensitive sequence comparison methods and coevolutionary analysis tools, we identify many proteins combining multiple beta barrels within a single chain; combinations that include eight-stranded barrels prevail. These multibarrels seem to be the result of independent, lineage-specific fusion and amplification events. The absence of multibarrels that are universally conserved in bacteria with an outer membrane, coupled with their frequent de novo genesis, suggests that their functions are not essential but rather beneficial in specific environments. Adjacent barrels of complementary function within the same chain may allow for functions beyond those of the individual barrels.

Immunogenic cross-reaction among outer membrane proteins of Gram-negative bacteria

2005 ◽  
Vol 5 (7-8) ◽  
pp. 1151-1163 ◽  
Author(s):  
Changxin Xu ◽  
Sanying Wang ◽  
Zhang Zhaoxia ◽  
Xuanxian Peng
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Cross Reaction ◽  
Gram Negative Bacteria ◽  
Gram Negative

scholarly journals Classifying β-Barrel Assembly Substrates by Manipulating Essential Bam Complex Members

2016 ◽  
Vol 198 (14) ◽  
pp. 1984-1992 ◽  
Author(s):  
Tara F. Mahoney ◽  
Dante P. Ricci ◽  
Thomas J. Silhavy
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Mechanistic Model ◽  
Outer Membrane Proteins ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Bam Complex ◽  
Periplasmic Chaperones

ABSTRACTThe biogenesis of the outer membrane (OM) ofEscherichia coliis a conserved and vital process. The assembly of integral β-barrel outer membrane proteins (OMPs), which represent a major component of the OM, depends on periplasmic chaperones and the heteropentameric β-barrel assembly machine (Bam complex) in the OM. However, not all OMPs are affected by null mutations in the same chaperones or nonessential Bam complex members, suggesting there are categories of substrates with divergent requirements for efficient assembly. We have previously demonstrated two classes of substrates, one comprising large, low-abundance, and difficult-to-assemble substrates that are heavily dependent on SurA and also Skp and FkpA, and the other comprising relatively simple and abundant substrates that are not as dependent on SurA but are strongly dependent on BamB for assembly. Here, we describe novel mutations inbamDthat lower levels of BamD 10-fold and >25-fold without altering the sequence of the mature protein. We utilized these mutations, as well as a previously characterized mutation that lowers wild-type BamA levels, to reveal a third class of substrates. These mutations preferentially cause a marked decrease in the levels of multimeric proteins. This susceptibility of multimers to lowered quantities of Bam machines in the cell may indicate that multiple Bam complexes are needed to efficiently assemble multimeric proteins into the OM.IMPORTANCEThe outer membrane (OM) of Gram-negative bacteria, such asEscherichia coli, serves as a selective permeability barrier that prevents the uptake of toxic molecules and antibiotics. Integral β-barrel proteins (OMPs) are assembled by the β-barrel assembly machine (Bam), components of which are conserved in mitochondria, chloroplasts, and all Gram-negative bacteria, including many clinically relevant pathogenic species. Bam is essential for OM biogenesis and accommodates a diverse array of client proteins; however, a mechanistic model that accounts for the selectivity and broad substrate range of Bam is lacking. Here, we show that the assembly of multimeric OMPs is more strongly affected than that of monomeric OMPs when essential Bam complex components are limiting, suggesting that multiple Bam complexes are needed to assemble multimeric proteins.

scholarly journals Synergism between Outer Membrane Proteins and Antimicrobials

2011 ◽  
Vol 55 (5) ◽  
pp. 2206-2211 ◽  
Author(s):  
P. Veiga-Crespo ◽  
E. Fusté ◽  
T. Vinuesa ◽  
M. Viñas ◽  
T. G. Villa
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Bactericidal Effect ◽  
Resistant Bacteria ◽  
Logarithmic Phase ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Antibiotic Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Gram Negative

ABSTRACTAntibiotic-resistant bacteria are becoming one of the most important problems in health care because of the number of resistant strains and the paucity of new effective antimicrobials. Since antibiotic-resistant bacteria will continue to increase, it is necessary to look for new alternative strategies to fight against them. It is generally accepted that Gram-negative bacteria are intrinsically less susceptible than Gram-positive bacteria to antimicrobials. The main reason is that Gram-negative bacteria are surrounded by a permeability barrier known as the outer membrane (OM). Hydrophilic solutes most often cross the OM through water-filled channels formed by a particular family of proteins known as porins. This work explores the possibility of using exogenous porins to lower the required amounts of antibiotics (ampicillin, ciprofloxacin, cefotaxime, clindamycin, erythromycin, and tetracycline). Porins had a bactericidal effect onEscherichia colicultures, mainly in the logarithmic phase of growth, when combined with low antibiotic concentrations. The use of different antibiotic-porin mixtures showed a bactericidal effect greater than those of antibiotics and porins when used separately. It was possible to observe different behaviors according to the antibiotic type used.

scholarly journals Outer Membrane Permeability of Cyanobacterium Synechocystis sp. Strain PCC 6803: Studies of Passive Diffusion of Small Organic Nutrients Reveal the Absence of Classical Porins and Intrinsically Low Permeability

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
Hikaru Kowata ◽  
Saeko Tochigi ◽  
Hideyuki Takahashi ◽  
Seiji Kojima
Keyword(s):  
Membrane Permeability ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Inorganic Ions ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Organic Nutrients ◽  
Outer Membrane Permeability ◽  
Pcc 6803

ABSTRACT The outer membrane of heterotrophic Gram-negative bacteria plays the role of a selective permeability barrier that prevents the influx of toxic compounds while allowing the nonspecific passage of small hydrophilic nutrients through porin channels. Compared with heterotrophic Gram-negative bacteria, the outer membrane properties of cyanobacteria, which are Gram-negative photoautotrophs, are not clearly understood. In this study, using small carbohydrates, amino acids, and inorganic ions as permeation probes, we determined the outer membrane permeability of Synechocystis sp. strain PCC 6803 in intact cells and in proteoliposomes reconstituted with outer membrane proteins. The permeability of this cyanobacterium was >20-fold lower than that of Escherichia coli. The predominant outer membrane proteins Slr1841, Slr1908, and Slr0042 were not permeable to organic nutrients and allowed only the passage of inorganic ions. Only the less abundant outer membrane protein Slr1270, a homolog of the E. coli export channel TolC, was permeable to organic solutes. The activity of Slr1270 as a channel was verified in a recombinant Slr1270-producing E. coli outer membrane. The lack of putative porins and the low outer membrane permeability appear to suit the cyanobacterial autotrophic lifestyle; the highly impermeable outer membrane would be advantageous to cellular survival by protecting the cell from toxic compounds, especially when the cellular physiology is not dependent on the uptake of organic nutrients. IMPORTANCE Because the outer membrane of Gram-negative bacteria affects the flux rates for various substances into and out of the cell, its permeability is closely associated with cellular physiology. The outer membrane properties of cyanobacteria, which are photoautotrophic Gram-negative bacteria, are not clearly understood. Here, we examined the outer membrane of Synechocystis sp. strain PCC 6803. We revealed that it is relatively permeable to inorganic ions but is markedly less permeable to organic nutrients, with >20-fold lower permeability than the outer membrane of Escherichia coli. Such permeability appears to fit the cyanobacterial lifestyle, in which the diffusion pathway for inorganic solutes may suffice to sustain the autotrophic physiology, illustrating a link between outer membrane permeability and the cellular lifestyle.

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