Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump

Yum, Sabrina W.; Xu, Yongbin; Piao, Shunfu; Sim, Se-Hoon; Kim, Hong-Man; Jo, Wol-Soon; Kim, Kyungjin; Kweon, Hee‐Seok; Jeong, Min-Ho; Jeon, Hyesung; Lee, Kangseok; Ha, Nam‐Chul

doi:10.1016/j.jmb.2009.02.048

Cited by 104 publications

(221 citation statements)

References 46 publications

(74 reference statements)

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“…4). Like the Acr system, the Emr system nonspecifically effluxes multiple substrates (66), whereas the Mac system specifically effluxes macrolides (67). All 3 systems, however, require the outer membrane channel TolC to function (66,67).…”

Section: Discussionmentioning

confidence: 99%

Defining Genetic Fitness Determinants and Creating Genomic Resources for an Oral Pathogen

Narayanan

Ramsey

Stacy

et al. 2017

Appl Environ Microbiol

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Periodontitis is a microbial infection that destroys the structures that support the teeth. Although it is typically a chronic condition, rapidly progressing, aggressive forms are associated with the oral pathogen Aggregatibacter actinomycetemcomitans. One of this bacterium's key virulence traits is its ability to attach to surfaces and form robust biofilms that resist killing by the host and antibiotics. Though much has been learned about A. actinomycetemcomitans since its initial discovery, we lack insight into a fundamental aspect of its basic biology, as we do not know the full set of genes that it requires for viability (the essential genome). Furthermore, research on A. actinomycetemcomitans is hampered by the field's lack of a mutant collection. To address these gaps, we used rapid transposon mutant sequencing (Tn-seq) to define the essential genomes of two strains of A. actinomycetemcomitans, revealing a core set of 319 genes. We then generated an arrayed mutant library comprising Ͼ1,500 unique insertions and used a sequencing-based approach to define each mutant's position (well and plate) in the library. To demonstrate its utility, we screened the library for mutants with weakened resistance to subinhibitory erythromycin, revealing the multidrug efflux pump AcrAB as a critical resistance factor. During the screen, we discovered that erythromycin induces A. actinomycetemcomitans to form biofilms. We therefore devised a novel Tnseq-based screen to identify specific factors that mediate this phenotype and in follow-up experiments confirmed 4 mutants. Together, these studies present new insights and resources for investigating the basic biology and disease mechanisms of a human pathogen. IMPORTANCE Millions suffer from gum disease, which often is caused by Aggregatibacter actinomycetemcomitans, a bacterium that forms antibiotic-resistant biofilms. To fully understand any organism, we should be able to answer: what genes does it require for life? Here, we address this question for A. actinomycetemcomitans by determining the genes in its genome that cannot be mutated. As for the genes that can be mutated, we archived these mutants into a library, which we used to find genes that contribute to antibiotic resistance, leading us to discover that antibiotics cause A. actinomycetemcomitans to form biofilms. We then devised an approach to find genes that mediate this process and confirmed 4 genes. These results illuminate new fundamental traits of a human pathogen.KEYWORDS Aggregatibacter, Tn-seq, antibiotics, biofilms, essential genome O ne of the longest-studied human microbiomes is the "animalcules" (bacteria), first observed by van Leeuwenhoek (1), that inhabit the oral cavity. Oral bacteria readily attach to tooth surfaces and develop into spatially organized, multispecies biofilms (2). These communities also persist beneath the gum line in the subgingival pocket, where they are kept in check by host immune cells (3). Though normally benign, subgingival communities can accumulate opportunistic oral p...

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Section: Discussionmentioning

confidence: 99%

Defining Genetic Fitness Determinants and Creating Genomic Resources for an Oral Pathogen

Narayanan

Ramsey

Stacy

et al. 2017

Appl Environ Microbiol

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“…A "bridging model" was proposed for the TolC-DevBCA homologue TolC-MacAB, with a MacA hexamer fitting to the tip of TolC in a cogwheel-like manner (9,40,41). It was derived from in silico protein models based on MacA crystals resolved to hexamers and electron micrographs showing a barrel-like hexameric assembly.…”

Section: Discussionmentioning

confidence: 99%

Novel ATP-driven Pathway of Glycolipid Export Involving TolC Protein

Staron

Forchhammer

Maldener

2011

Journal of Biological Chemistry

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Background:The ABC exporter DevBCA and the outer membrane protein TolC are necessary for maturation of heterocysts in filamentous cyanobacteria. Results: DevBCA-TolC form an ATP-driven efflux pump and export heterocyst-specific glycolipids. Conclusion: DevBCA-TolC provide a novel pathway for glycolipid export. Significance: Mechanistic details of efflux pumps/ABC exporters are important for understanding various bacterial processes such as cell differentiation, acclimatization processes, or drug resistance.

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confidence: 97%

“…The crystal structure of the MFP MacA revealed a funnel-like hexameric assembly, where the six ␣-hairpin domains from the hexamer pack to form a hollow cylinder (29). The functional importance of the hexameric funnel structure also has been demonstrated (29,30). A chimeric protein containing the TolC ␣-barrel periplasmic end region formed a tight complex with MacA in an intermeshing cogwheel-to-cogwheel manner (30,31).…”

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confidence: 98%

Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria

Moeller

Jun

et al. 2012

Journal of Biological Chemistry

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Background: Pseudomonas aeruginosa mainly achieves multidrug resistance by use of the MexAB-OprM pump. Results: We determined the crystal structure of MexA. Electron microscopy work using MexA and OprM reveals that MexA makes a tip-to-tip interaction with OprM. Conclusion:We suggest an assembly and channel opening model for the pump. Significance: This study provides a better understanding of multidrug resistance in Gram-negative bacteria.

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Crystal Structure of the Periplasmic Component of a Tripartite Macrolide-Specific Efflux Pump

Cited by 104 publications

References 46 publications

Defining Genetic Fitness Determinants and Creating Genomic Resources for an Oral Pathogen

Defining Genetic Fitness Determinants and Creating Genomic Resources for an Oral Pathogen

Novel ATP-driven Pathway of Glycolipid Export Involving TolC Protein

Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria

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