Improved enrichment and proteomic identification of outer membrane proteins from a Gram-negative bacterium: Focus on Caulobacter crescentus

PROTEOMICS ◽  
2011 ◽  
Vol 12 (2) ◽  
pp. 251-262 ◽  
Author(s):  
Yuan Cao ◽  
Helen M. Johnson ◽  
Carthene R. Bazemore-Walker
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Caulobacter Crescentus ◽  
Outer Membrane Proteins ◽  
Negative Bacterium ◽  
Gram Negative ◽  
Proteomic Identification

scholarly journals Septic Shock by Gram-Negative Infections: Role of Outer Membrane Proteins

10.5772/29110 ◽  
2012 ◽  
Author(s):  
Marilena Galdiero ◽  
Marco Cantisani ◽  
Rossella Tarallo ◽  
Annarita Falanga ◽  
Stefania Galdiero
Keyword(s):  
Septic Shock ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Gram Negative

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.

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.

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 DegP primarily functions as a protease for the biogenesis of β-barrel outer membrane proteins in the Gram-negative bacteriumEscherichia coli

FEBS Journal ◽  
2014 ◽  
Vol 281 (4) ◽  
pp. 1226-1240 ◽  
Author(s):  
Xi Ge ◽  
Rui Wang ◽  
Jing Ma ◽  
Yang Liu ◽  
Anastasia N. Ezemaduka ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Gram Negative

scholarly journals Release of Gram‐Negative Outer‐Membrane Proteins into Human Serum and Septic Rat Blood and Their Interactions with Immunoglobulin in Antiserum toEscherichia coliJ5

2000 ◽  
Vol 181 (3) ◽  
pp. 1034-1043 ◽  
Author(s):  
Judith Hellman ◽  
Paul M. Loiselle ◽  
Emily M. Zanzot ◽  
Jennifer E. Allaire ◽  
Megan M. Tehan ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Human Serum ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Gram Negative ◽  
Rat Blood

scholarly journals Establishment of a Protein Concentration Gradient in the Outer Membrane Requires Two Diffusion-Limiting Mechanisms

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Luis David Ginez ◽  
Aurora Osorio ◽  
Laura Camarena ◽  
Sebastian Poggio
Keyword(s):  
Cell Wall ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Concentration Gradient ◽  
Protein Concentration ◽  
Caulobacter Crescentus ◽  
Outer Membrane Proteins ◽  
Structural Domains ◽  
Content Type ◽  
Terminal Domain

ABSTRACTOmpA-like proteins are involved in the stabilization of the outer membrane, resistance to osmotic stress, and pathogenesis. InCaulobacter crescentus, OmpA2 forms a physiologically relevant concentration gradient that forms by an uncharacterized mechanism, in which the gradient orientation depends on the position of the gene locus. This suggests that OmpA2 is synthesized and translocated to the periplasm close to the position of the gene and that the gradient forms by diffusion of the protein from this point. To further understand how the OmpA2 gradient is established, we determined the localization and mobility of the full protein and of its two structural domains. We show that OmpA2 does not diffuse and that both domains are required for gradient formation. The C-terminal domain binds tightly to the cell wall and the immobility of the full protein depends on the binding of this domain to the peptidoglycan; in contrast, the N-terminal membrane β-barrel diffuses slowly. Our results support a model in which once OmpA2 is translocated to the periplasm, the N-terminal membrane β-barrel is required for an initial fast restriction of diffusion until the position of the protein is stabilized by the binding of the C-terminal domain to the cell wall. The implications of these results on outer membrane protein diffusion and organization are discussed.IMPORTANCEProtein concentration gradients play a relevant role in the organization of the bacterial cell. TheCaulobacter crescentusprotein OmpA2 forms an outer membrane polar concentration gradient. To understand the molecular mechanism that determines the formation of this gradient, we characterized the mobility and localization of the full protein and of its two structural domains an integral outer membrane β-barrel and a periplasmic peptidoglycan binding domain. Each domain has a different role in the formation of the OmpA2 gradient, which occurs in two steps. We also show that the OmpA2 outer membrane β-barrel can diffuse, which is in contrast to what has been reported previously for several integral outer membrane proteins inEscherichia coli, suggesting a different organization of the outer membrane proteins.

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