Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins

Konovalova, Anna; Perlman, David H.; Cowles, Charles E.; Silhavy, Thomas J.

doi:10.1073/pnas.1417138111

Cited by 113 publications

(179 citation statements)

References 70 publications

(86 reference statements)

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“…The Rcs two-component phosphorelay uses the OM lipoprotein RcsF to detect defects throughout the envelope (28)(29)(30). Stress conditions are thought to enable RcsF to reach across the periplasm to promote activation of the RcsC IM histidine kinase, which then phosphorylates the RcsB response regulator, allowing it to control expression of Rcs regulon genes (28,30,31). Mislocalizing RcsF to the IM by mutating its +2 residue causes strong activation of the Rcs response even in the absence of stress (32).…”

Section: Abundant Lolb Is Required To Prevent Mislocalization Of An Amentioning

confidence: 99%

Redefining the essential trafficking pathway for outer membrane lipoproteins

Grabowicz

Silhavy

2017

Proc. Natl. Acad. Sci. U.S.A.

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104

142

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The outer membrane (OM) of Gram-negative bacteria is a permeability barrier and an intrinsic antibiotic resistance factor. Lipoproteins are OM components that function in cell wall synthesis, diverse secretion systems, and antibiotic efflux pumps. Moreover, each of the essential OM machines that assemble the barrier requires one or more lipoproteins. This dependence is thought to explain the essentiality of the periplasmic chaperone LolA and its OM receptor LolB that traffic lipoproteins to the OM. However, we show that in strains lacking substrates that are toxic when mislocalized, both LolA and LolB can be completely bypassed by activating an envelope stress response without compromising trafficking of essential lipoproteins. We identify the Cpx stress response as a monitor of lipoprotein trafficking tasked with protecting the cell from mislocalized lipoproteins. Moreover, our findings reveal that an alternate trafficking pathway exists that can, under certain conditions, bypass the functions of LolA and LolB, implying that these proteins do not perform any truly essential mechanistic steps in lipoprotein trafficking. Instead, these proteins' key function is to prevent lethal accumulation of mislocalized lipoproteins.

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Section: Abundant Lolb Is Required To Prevent Mislocalization Of An Amentioning

confidence: 99%

Redefining the essential trafficking pathway for outer membrane lipoproteins

Grabowicz

Silhavy

2017

Proc. Natl. Acad. Sci. U.S.A.

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104

142

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“…We have highly specific polyclonal anti-BamC antibodies [121] which do not bind to whole cells [100] and we could not detect BamC biotinylation [85]. Therefore, we concluded that BamC is a periplasmic lipoprotein, and we have used BamC as a negative-control for lipoprotein surface exposure [85,100]. However, independently raised polyclonal antibodies against BamC can recognize the protein on the cell surface, indicating that at least part of BamC is surface exposed [88].…”

Section: Common Methods To Study Topology Of Outer Membrane Lipoproteinsmentioning

confidence: 85%

“…The size exclusion limit of porins in E. coli was determined to be around 600 Da [111], so using water-soluble labels of much larger molecular weight will result in selective surface labelling as well. In our laboratory, we used a hydrophobic biotin label to study Lpp surface exposure [85] as well as for the initial discovery of surface-exposed RcsF [100]. We also used a hydrophilic maleimide with high molecular weight to study the conformation change in extracellular loop 6 of BamA [112].…”

Section: Common Methods To Study Topology Of Outer Membrane Lipoproteinsmentioning

confidence: 99%

Outer membrane lipoprotein biogenesis: Lol is not the end

Konovalova¹,

Silhavy²

2015

Phil. Trans. R. Soc. B

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117

111

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Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology.

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“…The RcsCDB phos-phorelay system is involved in the overproduction of CA (49). The membrane lipoprotein RcsF senses cell envelope stress and conveys the information to the histidine kinases RcsC and RcsD in the cytoplasmic membrane (50)(51)(52)(53). To further investigate whether this system is related to the excessive CA production in ⌬waaF cells, double mutant strains (⌬waaF ⌬rcsA, ⌬waaF ⌬rcsB, ⌬waaF ⌬rcsC, ⌬waaF ⌬rcsD, and ⌬waaF ⌬rcsF strains) were constructed, and levels of CA production in these strains were determined.…”

Section: Resultsmentioning

confidence: 99%

Effects of Lipopolysaccharide Core Sugar Deficiency on Colanic Acid Biosynthesis in Escherichia coli

Ren

Wang

et al. 2016

J Bacteriol

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When 10 Escherichia coli mutant strains with defects in lipopolysaccharide (LPS) core biosynthesis were grown on agar medium at 30°C, four of them, the ⌬waaF, ⌬waaG, ⌬waaP, and ⌬waaB strains, formed mucoid colonies, while the other six, the ⌬waaU, ⌬waaR, ⌬waaO, ⌬waaC, ⌬waaQ, and ⌬waaY strains, did not. Using light microscopy with tannin mordant staining, the presence of exopolysaccharide around the cells of the mutants that formed mucoid colonies could be discerned. The ⌬waaF mutant produced the largest amounts of exopolysaccharide, regardless of whether it was grown on agar or in liquid medium. The exopolysaccharide was isolated from the liquid growth medium of ⌬waaF cells, hydrolyzed, and analyzed by high-performance liquid chromatography with an ion-exchange column, and the results indicated that the exopolysaccharide was consistent with colanic acid. When the key genes related to the biosynthesis of colanic acid, i.e., wza, wzb, wzc, and wcaA, were deleted in the ⌬waaF background, the exopolysaccharide could not be produced any more, further confirming that it was colanic acid. Colanic acid could not be produced in strains in which rcsA, rcsB, rcsD, or rcsF was deleted in the ⌬waaF background, but a reduced level of colanic acid production was detected when the rcsC gene was deleted, suggesting that a change of lipopolysaccharide structure in ⌬waaF cells might be sensed by the RcsCDB phosphorelay system, leading to the production of colanic acid. The results demonstrate that E. coli cells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural deficiency of lipopolysaccharide. IMPORTANCELipopolysaccharide and colanic acid are important forms of exopolysaccharide for Escherichia coli cells. Their metabolism and biological significance have been investigated, but their interrelation with the cell stress response process is not understood. This study demonstrates, for the first time, that E. coli cells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural change of lipopolysaccharide, suggesting that bacterial cells can monitor the outer membrane integrity, which is essential for cell survival and damage repair. Bacteria produce a wide range of exopolysaccharides, which have specific monomer compositions, substituent decorations, and biosynthetic pathways. Genes involved in biosynthesis of these exopolysaccharides are often clustered in specific loci within the genome and encode glycosyltransferases, polymerizing and branching enzymes, and enzymes responsible for addition of substituents (1). Escherichia coli K-12 cells contain several gene clusters related to biosynthesis of polysaccharides (2-7), such as lipopolysaccharide (LPS) and colanic acid (CA) (Fig. 1). Understanding the fundamental processes involved in exopolysaccharide biosynthesis and the regulation of these processes is important.As a major molecule in the outer membrane of E. coli K-12 cells (8, 9), LPS consists of a hydrophobic lipid A domai...

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Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins

Cited by 113 publications

References 70 publications

Redefining the essential trafficking pathway for outer membrane lipoproteins

Redefining the essential trafficking pathway for outer membrane lipoproteins

Outer membrane lipoprotein biogenesis: Lol is not the end

Effects of Lipopolysaccharide Core Sugar Deficiency on Colanic Acid Biosynthesis in Escherichia coli

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