Unlabelled: In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively.
Importance: Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect, but the severity (and therefore, Lol and Bam dependence), varies with individual proteins. XcpQ, the secretin component of the type II secretion apparatus, is translocated into the OM without the assistance of Bam or Lol machineries. The hypothesis that XcpQ, after secretion across the cytoplasmic membrane, does not utilize the OM targeting machineries was supported by demonstrating that in vitro-synthesized XcpQ (but not the other P. aeruginosa secretins) can spontaneously incorporate into lipid vesicles. Therefore, the requirement for ancillary factors appears to be, in certain instances, dictated by the intrinsic properties of individual OM proteins, conceivably reflecting their propensities to misfold during periplasmic transit.
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http://dx.doi.org/10.1128/mBio.00246-11 | DOI Listing |
Antibiotics (Basel)
September 2019
Microbiology and Molecular Genetics Program, Graduate Division of Biological and Biomedical Sciences, Laney Graduate School, Emory University, Atlanta, GA 30322, USA.
mBio
May 2019
Emory Antibiotic Resistance Center, Emory University School of Medicine, Atlanta, Georgia, USA
Gram-negative bacteria produce lipid-anchored lipoproteins that are trafficked to their outer membrane (OM). These lipoproteins are essential components in each of the molecular machines that build the OM, including the Bam machine that assembles β-barrel proteins and the Lpt pathway that transports lipopolysaccharide. Stress responses are known to monitor Bam and Lpt function, yet no stress system has been found that oversees the fundamental process of lipoprotein trafficking.
View Article and Find Full Text PDFEcoSal Plus
March 2019
Emory Antibiotic Resistance Center, Emory University School of Medicine, Atlanta, GA 30322.
Lipoproteins are produced by both Gram-positive and Gram-negative bacteria. Once secreted, lipoproteins are quickly acylated, anchoring them into the plasma membrane. Recent work has shown that Gram-positive bacteria are able to generate considerable diversity in the acylation of their lipoproteins, though the mechanisms involved are only just beginning to emerge.
View Article and Find Full Text PDFJ Microbiol Biotechnol
January 2019
Department of Bioscience and Bioinformatics, Myongji University, Yongin 17058, Republic of Korea.
Gram-negative pathogens, such as , , and , pose a serious threat to public health worldwide, due to high rates of antibiotic resistance and the lack of development of novel antimicrobial agents targeting Gram-negative bacteria. The outer membrane (OM) of Gram-negative bacteria is a unique architecture that acts as a potent permeability barrier against toxic molecules, such as antibiotics. The OM is composed of phospholipids, lipopolysaccharide (LPS), outer membrane β-barrel proteins (OMP), and lipoproteins.
View Article and Find Full Text PDFAdv Exp Med Biol
April 2016
Department of Biochemistry, University of Toronto, Medical Science Building, Rm. 5366, 1 King's College Circle, Toronto, ON, Canada, M5S 1A8.
The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane.
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