scholarly journals Revelation of a Novel Protein Translocon in Bacterial Plasma Membrane

2017 ◽  
Author(s):  
Feng Jin ◽  
Zengyi Chang
Keyword(s):  
Plasma Membrane ◽  
Outer Membrane ◽  
Protein Interactions ◽  
Living Cell ◽  
Outer Membrane Proteins ◽  
Bacterial Cells ◽  
Bam Complex ◽  
Gxxxg Motif ◽  
Novel Protein

Many proteins are translocated across biomembranes via protein translocons in targeting to their subcellular destinations. Hitherto, the SecYEG/Sec61 translocon, existing in prokaryotes and eukaryotes, represents the most intensively studied one. According to the current perception, both periplasmic and β-barrel outer membrane proteins (β-barrel OMPs) are translocated via the SecYEG translocon in bacterial cells, although direct living cell evidences remain lacking. Here, mainly viain vivoprotein photo-crosslinking analysis, we revealed that the never reported membrane-integrated SecANprotein apparently functions as the translocon for β-barrel OMPs. Additionally, SecANcontains a GXXXG motif known for mediating protein interactions in biomembranes, and processing of β-barrel OMP precursors was severely affected in cells producing an assembly-defective SecANvariant resulted from the GXXXG motif mutations. Furthermore, SecANwas demonstrated to directly interact with the Bam complex, thus likely be a part of the supercomplex that we revealed earlier to be responsible for β-barrel OMP biogenesis.

scholarly journals The CpxQ sRNA Negatively Regulates Skp To Prevent Mistargeting of β-Barrel Outer Membrane Proteins into the Cytoplasmic Membrane

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Marcin Grabowicz ◽  
Daria Koren ◽  
Thomas J. Silhavy
Keyword(s):  
Stress Response ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Protein Misfolding ◽  
Outer Membrane Proteins ◽  
Inner Membrane ◽  
Bam Complex ◽  
Envelope Stress ◽  
Periplasmic Chaperone

ABSTRACT The promoter most strongly induced upon activation of the Cpx two-component envelope stress response is the cpxP promoter. The 3′ untranscribed region (UTR) of the cpxP transcript is shown to produce a small RNA (sRNA), CpxQ. We investigated the role of CpxQ in combating envelope stress. Remarkably, the two effectors specified by the transcript are deployed to combat distinct stresses in different cellular compartments. CpxP acts in both a regulatory negative-feedback loop and as an effector that combats periplasmic protein misfolding. We find that CpxQ combats toxicity at the inner membrane (IM) by downregulating the synthesis of the periplasmic chaperone Skp. Our data indicate that this regulation prevents Skp from inserting β-barrel outer membrane proteins (OMPs) into the IM, a lethal event that likely collapses the proton motive force. Our findings suggest that Skp can fold and directly insert OMPs into a lipid bilayer in vivo without the aid of the Bam complex. IMPORTANCE Skp is a well-characterized periplasmic chaperone that binds unfolded OMPs. Surprisingly, we find that Skp can catalyze the folding and mistargeting of OMPs into the inner membrane without the aid of the other cellular proteins that normally assemble OMPs. Several OMPs function as diffusion pores. Accordingly, their mistargeting is lethal because it depolarizes the inner membrane. We show that the most highly expressed transcript of the Cpx stress response produces an sRNA from the 3′ UTR, CpxQ, which combats this potential toxicity by downregulating Skp production. Defects in OMP assembly trigger the σ E response to upregulate factors, including Skp, that promote OMP folding. The Cpx response downregulates σ E . Our findings reveal that this heretofore puzzling hierarchy exists to protect the inner membrane.

scholarly journals Role of the lipid bilayer in outer membrane protein folding in Gram-negative bacteria

2020 ◽  
Vol 295 (30) ◽  
pp. 10340-10367 ◽  
Author(s):  
Jim E. Horne ◽  
David J. Brockwell ◽  
Sheena E. Radford
Keyword(s):  
Outer Membrane ◽  
Cell Structure ◽  
Outer Membrane Proteins ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
External Energy ◽  
Gram Negative ◽  
Cellular Environment

β-Barrel outer membrane proteins (OMPs) represent the major proteinaceous component of the outer membrane (OM) of Gram-negative bacteria. These proteins perform key roles in cell structure and morphology, nutrient acquisition, colonization and invasion, and protection against external toxic threats such as antibiotics. To become functional, OMPs must fold and insert into a crowded and asymmetric OM that lacks much freely accessible lipid. This feat is accomplished in the absence of an external energy source and is thought to be driven by the high thermodynamic stability of folded OMPs in the OM. With such a stable fold, the challenge that bacteria face in assembling OMPs into the OM is how to overcome the initial energy barrier of membrane insertion. In this review, we highlight the roles of the lipid environment and the OM in modulating the OMP-folding landscape and discuss the factors that guide folding in vitro and in vivo. We particularly focus on the composition, architecture, and physical properties of the OM and how an understanding of the folding properties of OMPs in vitro can help explain the challenges they encounter during folding in vivo. Current models of OMP biogenesis in the cellular environment are still in flux, but the stakes for improving the accuracy of these models are high. OMP folding is an essential process in all Gram-negative bacteria, and considering the looming crisis of widespread microbial drug resistance it is an attractive target. To bring down this vital OMP-supported barrier to antibiotics, we must first understand how bacterial cells build it.

scholarly journals The role of BamA in the biogenesis of beta-barrel membrane proteins

2014 ◽  
Vol 70 (a1) ◽  
pp. C578-C578
Author(s):  
Nicholas Noinaj ◽  
Adam Kuszak ◽  
Curtis Balusek ◽  
JC Gumbart ◽  
Petra Lukacik ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Crystal Structures ◽  
Outer Membrane Proteins ◽  
Hydrophobic Surface ◽  
Md Simulations ◽  
Structural Features ◽  
Bam Complex ◽  
Beta Barrel

Beta-barrel membrane proteins are essential for nutrient import, signaling, motility, and survival. In Gram-negative bacteria, the beta-barrel assembly machinery (BAM) complex is responsible for the biogenesis of beta-barrel outer membrane proteins (OMPs), with homologous complexes found in mitochondria and chloroplasts. Despite their essential roles, exactly how these OMPs are formed remains unknown. The BAM complex consists of a central and essential component called BamA (an OMP itself) and four lipoproteins called BamB-E. While the structure of the lipoproteins have been reported, the structure of full length BamA has been elusive. Recently though, we described the structure of BamA from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane beta-barrel domain. Together, our crystal structures and molecule dynamics (MD) simulations revealed several structural features which gave clues to the mechanism by which BamA catalyzes beta-barrel assembly. The first is that the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the beta-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. Third, the beta-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane. And fourth, a surface exposed exit pore positioned above the lateral opening site which may play a role in the biogenesis of extracellular loops. In this presentation, the crystal structures and MD simulations of BamA will be presented along with our work looking at the role of these four structural features in the role of BamA within the BAM complex.

scholarly journals Outer membrane characteristics of Salmonella enteritidis phage type 4 growing in chickens

1993 ◽  
Vol 111 (3) ◽  
pp. 449-454 ◽  
Author(s):  
H. Chart ◽  
D. Conway ◽  
B. Rowe
Keyword(s):  
Outer Membrane ◽  
Salmonella Enteritidis ◽  
Outer Membrane Proteins ◽  
Phage Type ◽  
Virulence Plasmid ◽  
Uptake System ◽  
Ferric Ions ◽  
Phenotypic Characteristics ◽  
A Subunit

SummaryStrains of Salmonella enteritidis belonging to phage type 4 (SE4) were grown in the peritoneal cavities of chickens, and without subculture on laboratory media examined for inducible in vivo phenotypic characteristics. These bacteria expressed three major outer membrane proteins (OMPs) of 33, 35 and 36 kilodaltons (kDa), and iron regulated OMPs of 74, 78 and 81 kDa. Bacteria growing in vivo did not express flagella, or fimbriae with a subunit molecular mass of 14 kDa (14 kDa fimbriae). Two OMPs of 55 and 23 kDa, expressed during culture in nutrient broth, were repressed during growth in chickens. Possession of a 38 MDa ‘mouse virulence’ plasmid did not influence the expression of OMPs, flagella or fimbriae. It was concluded that strains of SE4 growing in chicken tissues, use an enterobactin mediated iron uptake system to obtain ferric ions, do not express flagella or 14 kDa fimbriae and appear not to express novel OMPs involved in survival in vivo.

scholarly journals Localized adherence by enteropathogenic Escherichia coli is an inducible phenotype associated with the expression of new outer membrane proteins.

1991 ◽  
Vol 174 (5) ◽  
pp. 1167-1177 ◽  
Author(s):  
J Vuopio-Varkila ◽  
G K Schoolnik
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
De Novo ◽  
Outer Membrane Proteins ◽  
Temporal Correlation ◽  
Enteropathogenic Escherichia Coli ◽  
E Coli

Enteropathogenic Escherichia coli grow as discrete colonies on the mucous membranes of the small intestine. A similar pattern can be demonstrated in vitro; termed localized adherence (LA), it is characterized by the presence of circumscribed clusters of bacteria attached to the surfaces of cultured epithelial cells. The LA phenotype was studied using B171, an O111:NM enteropathogenic E. coli (EPEC) strain, and HEp-2 cell monolayers. LA could be detected 30-60 min after exposure of HEp-2 cells to B171. However, bacteria transferred from infected HEp-2 cells to fresh monolayers exhibited LA within 15 min, indicating that LA is an inducible phenotype. Induction of the LA phenotype was found to be associated with de novo protein synthesis and changes in the outer membrane proteins, including the production of a new 18.5-kD polypeptide. A partial NH2-terminal amino acid sequence of this polypeptide was obtained and showed it to be identical through residue 12 to the recently described bundle-forming pilus subunit of EPEC. Expression of the 18.5-kD polypeptide required the 57-megadalton enteropathogenic E. coli adherence plasmid previously shown to be required for the LA phenotype in vitro and full virulence in vivo. This observation, the correspondence of the 18.5-kD polypeptide to an EPEC-specific pilus protein, and the temporal correlation of its expression with the development of the LA phenotype suggest that it may contribute to the EPEC colonial mode of growth.

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.

In vivo expression of iron regulated outer-membrane proteins in Pasteurella haemolytica-A1

1991 ◽  
Vol 11 (5) ◽  
pp. 373-378 ◽  
Author(s):  
Douglas W. Morck ◽  
Brian D. Ellis ◽  
P.A.Gilbert Domingue ◽  
Merle E. Olson ◽  
J.William Costerton
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Pasteurella Haemolytica ◽  
In Vivo Expression

Structural basis for the interaction of BamB with the POTRA3–4 domains of BamA

2016 ◽  
Vol 72 (2) ◽  
pp. 236-244 ◽  
Author(s):  
Zhen Chen ◽  
Li-Hong Zhan ◽  
Hai-Feng Hou ◽  
Zeng-Qiang Gao ◽  
Jian-Hua Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Outer Membrane ◽  
Essential Role ◽  
Biochemical Analysis ◽  
Hydrophobic Interactions ◽  
Outer Membrane Proteins ◽  
Structural Basis ◽  
Conserved Residues ◽  
Bam Complex

InEscherichia coli, the Omp85 protein BamA and four lipoproteins (BamBCDE) constitute the BAM complex, which is essential for the assembly and insertion of outer membrane proteins into the outer membrane. Here, the crystal structure of BamB in complex with the POTRA3–4 domains of BamA is reported at 2.1 Å resolution. Based on this structure, the POTRA3 domain is associated with BamBviahydrogen-bonding and hydrophobic interactions. Structural and biochemical analysis revealed that the conserved residues Arg77, Glu127, Glu150, Ser167, Leu192, Leu194 and Arg195 of BamB play an essential role in interaction with the POTRA3 domain.

scholarly journals Docking and Assembly of the Type II Secretion Complex of Vibrio cholerae

2009 ◽  
Vol 191 (9) ◽  
pp. 3149-3161 ◽  
Author(s):  
Suzanne R. Lybarger ◽  
Tanya L. Johnson ◽  
Miranda D. Gray ◽  
Aleksandra E. Sikora ◽  
Maria Sandkvist
Keyword(s):  
Vibrio Cholerae ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Outer Membrane Proteins ◽  
Subcellular Location ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Protein Fusion ◽  
Green Fluorescent

ABSTRACT Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.

Sign in / Sign up

Export Citation Format

Share Document