Point of
VIEW
Crossing a Biological Velvet Rope Dewey G. McCafferty*
Department of Chemistry, B219 Levine Science Research Center, Box 90354, Duke University, Durham, North Carolina 27708-0354
I
n Gram-negative bacteria, the outer membrane (OM) serves as a selective permeability barrier, governing the influx and efflux of solutes essential for sustaining life while protecting against deleterious agents in the extracellular milieu. Recently, using chemical genetic methods, Silhavy and Kahne (1, 2) discovered that toxic small molecules can be used in selections employing strains with OM permeability defects to create particular chemical conditions that demand specific suppressor mutations to restore OM function in Escherichia coli. This “chemical conditionality” approach (2) was used to identify a multiprotein complex that is required for OM biogenesis. On page 385 of this issue of ACS Chemical Biology (ACS CB), Silhavy and Kahne (3) use chemical conditionality to identify YaeT as part of the OM assembly complex. They also report that the suppressor mutation in yaeT confers resistance to a specific structural subset of bile acids, thus demonstrating that structurally diverse toxic small molecules select different and specific genetic solutions for correcting permeability defects. This novel application of chemical genetics points to a molecular basis for OM barrier restoration by the OM assembly complex, provides molecular-level identification of potential targets for antimicrobial chemotherapy, and offers a mechanism for identifying factors involved in the assembly of other organelles. Gram-negative bacteria possess a dualmembrane architecture consisting of a phospholipid and protein inner membrane (IM), a periplasmic space containing peptidoglycan
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and soluble proteins, and an unusual OM largely composed of OM proteins (OMPs), lipopolysaccharides (LPSs), and phospholipids. The OM bilayer is an unusual asymmetric structure with the outer leaflet composed largely of highly compact LPS and the inner leaflet made of phospholipids (4). In addition, integral OMPs such as -barrel proteins span the bilayer, and lipoproteins are attached to the inner leaflet through covalent lipid modifications. Also, other components such as LPSs can be produced as an additional extracellular layer. The OM functions as a protective barrier to toxic materials, yet it is selectively permissive for solute import and waste disposal required for sustaining life in varying environments. The critical importance of OM function for Gram-negative pathogens also makes its assembly an antimicrobial target. The molecular components of the OM are biosynthesized in the cytoplasm or inner leaflet of the IM and exported across the periplasmic space and into the inner or outer leaflets of the OM (5). OM lipoproteins, biosynthesized and post-translationally modified in the IM and transported via the ATP-binding cassette (ABC) transporter LolCDE, are escorted through the periplasm by the LolA chaperone en route to the OM. Similar chaperone-assisted mechanisms are suggested for transport of Pili proteins to the OM. Integral -barrel OMPs are produced in the cytoplasm, targeted to the IM, and secreted into the periplasm in a secretory protein-dependent mechanism, where they interact with chaperones and protein folding factors. However, once OMPs
A B S T R A C T In contrast to our understanding of the composition of the outer membrane (OM) of Gram-negative bacteria, the biogenesis of this organelle has remained elusive. This is in part because factors involved in OM assembly have been refractive to chemical and biological analyses. A recent study shows how small molecules and chemical conditionality can be used to probe the biogenesis of the OM at the molecular level and suggests that similar techniques can be used to identify factors involved in the assembly of other organelles.
*Corresponding author,
[email protected].
Published online July 21, 2006 10.1021/cb6002948 CCC: $33.50 © 2006 by American Chemical Society
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Structurally different toxic small molecules select different and specific genetic solutions for correcting permeability defects.
are in the periplasm, less is known about their trafficking to the OM, bilayer insertion, penultimate organization within the OM, and impact on permeability. Analysis of the biogenesis of the OM has been hampered for decades because factors involved in OM assembly have not been amenable to identification and genetic analyses. Although “leaky” OM-defective mutants have been generated, generally these mutants have nonspecific permeability defects, precluding identification of factors responsible for OM biogenesis. However, several discoveries now may pave the way to a molecular-level understanding of OM barrier function. First is the discovery of the Cpx and E cell stress responses that led to the subsequent identification of potential OMbiogenesis candidates (6). The latter stress response is extracytoplasmic and specifically activated by misfolded OMPs and disruption of LPS structure. Identification of this regulon suggested possible involvement of a family of candidate OM factors. A second is the identification of OM-defective E. coli mutants of a candidate OM-biogenesis gene, imp. Genetic and biochemical analyses subsequently confirmed an essential role for imp in LPS assembly in the OM. Silhavy and Kahne (3) added an important chapter to our understanding of OM biogenesis by demonstrating an additional capability of forward chemical genetics. Armed with an E. coli strain carrying a defective imp allele (imp4213) and thus a leaky OM barrier susceptible to the effects of toxic molecules, Silhavy and Kahne established a forward genetic screen designed to identify mutants of imp4213 that restored OM barrier function. Standard genetic techniques were used to map the suppressor mutations that restored total or partial OM function. Unlike the typical forward chemical genetic screens which are designed to reveal the interaction between a molecule and its target or to illuminate downstream events caused by this interaction, this novel screen was designed to reveal mutants that governed the entry of 340
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the small molecule into the cell. This application has the potential to elucidate many aspects of organelle biogenesis. Silhavy and Kahne identified an interesting continuum of suppressor mutation behavior. For example, intragenic suppressors carrying an additional mutation in the imp4213 restored nearwild-type levels of OM barrier function and afforded protection from all small-molecule toxins. At the other continuum, some of the suppressors obtained by selection afforded protection only for bile salts and yet were susceptible to antibiotics. In the middle of this continuum was the yfgL suppressor, which restored the barrier function for bile salts and chlorobiphenyl vancomycin (CBP-V) but not for erythromycin or vancomycin. Intriguingly, yfgL suppressors exhibit remarkable chemical specificity: they remain sensitive to vancomycin but not the closely related CBP-V. Subsequently, the researchers determined that the yfgL suppressor gains its protective effects by altering the OM permeability barrier. Lipoprotein YfgL, a component of the cellular machinery that assembles -barrel OMPs in Gram-negative bacteria, is part of a -barrel assembly protein complex that includes the integral membrane protein YaeT and two other lipoproteins, YfiO and NlpB. In this issue of ACS CB, Silhavy and Kahne (3) have expanded the continuum of OM barrier function analysis, and a new OM barrier selectivity function has been revealed by another protein component of the OMP -barrel protein assembly complex. The authors demonstrate that structurally different toxic small molecules select different and specific genetic solutions for correcting permeability defects. Most intriguingly, the imp4213 suppressor mutation in yaeT results in partial restoration of OM barrier function and interestingly confers resistance to a specific structural subset of dihydroxylated bile acid regioisomers sodium deoxycholate and chenodeoxycholate vs the related trihydroxylated bile acid sodium cholate. Significant steps toward a plausible mechanism for the observed suppressor selectivity MCCAFFERTY
by components of the OMP -barrel assembly complex are presented. Different suppressors obtained via chemical conditionality exhibit chemical specificity, because the entry of each chemical into the cell is determined by its physicochemical properties. The most obvious difference between the structures of these three bile acids as well as the vancomycin/CBP-V derivatives is hydrophobicity. The authors suggest that yfgL– and yaeT6 mutations may cause a reduction in the phopholipid content of the outer leaflet of the imp4213 cells. The result is the reduction of the surface area of phospholipid bilayer patches and thereby the local concentration at the cell surface of hydrophobic and amphipathic compounds that partition into these patches. That yfgL– and yaeT6 mutations may affect OM phospholipids transport is possible as well. Thus chemical conditionality has been used to examine OM biogenesis and to discover a novel membrane protein complex that plays an important role in OM barrier assembly and function. This approach is clearly a powerful tool to identify factors involved in organelle and membrane biogenesis and to deconstruct assembly and regulatory events in such seemingly intractable biological environments. REFERENCES 1. Wu, T., Malinverni, J., Ruiz, N., Kim, S., Silhavy, T. J., and Kahne, D. (2005) Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli. Cell 121, 235–245. 2. Ruiz, N., Falcone, B., Kahne, D., and Silhavy, T. J. (2005) Chemical conditionality: a genetic strategy to probe organelle assembly. Cell 121, 307–317. 3. Ruiz, N., Wu, T., Kahne, D., and Silhavy, T. J. (2006) Probing the Barrier Functionality of the Outer Membrane with Chemical Conditionality, ACS Chem. Biol. 1, 385–395. 4. Ruiz, N., Kahne, D., and Silhavy, T. J. (2006) Advances in understanding bacterial outer-membrane biogenesis, Nat. Rev. Microbiol. 4, 57–66. 5. Tokuda, H., and Matsuyama, S. (2004) Sorting of lipoproteins to the outer membrane in E. coli. Biochim. Biophys. Acta 1694, IN1–9. 6. Ruiz, N., and Silhavy, T. J. (2005) Sensing external stress: watchdogs of the Escherichia coli cell envelope. Curr. Opin. Microbiol. 8, 122–126.
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