A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier.

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the ß-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

Inhibitor of intramembrane protease RseP blocks the σE response causing lethal accumulation of unfolded outer membrane proteins.

The outer membrane (OM) of Gram-negative bacteria forms a robust permeability barrier that blocks entry of toxins and antibiotics. Most OM proteins (OMPs) assume a ß-barrel fold, and some form aqueous channels for nutrient uptake and efflux of intracellular toxins. The Bam machine catalyzes rapid folding and assembly of OMPs. Fidelity of OMP biogenesis is monitored by the σE stress response. When OMP folding defects arise, the proteases DegS and RseP act sequentially to liberate σE into the cytosol, enabling it to activate transcription of the stress regulon. Here, we identify batimastat as a selective inhibitor of RseP that causes a lethal decrease in σE activity in Escherichia coli, and we further identify RseP mutants that are insensitive to inhibition and confer resistance. Remarkably, batimastat treatment allows the capture of elusive intermediates in the OMP biogenesis pathway and offers opportunities to better understand the underlying basis for σE essentiality.

Dual-Targeting Small-Molecule Inhibitors of the Staphylococcus aureus FMN Riboswitch Disrupt Riboflavin Homeostasis in an Infectious Setting.

Riboswitches are bacterial-specific, broadly conserved, non-coding RNA structural elements that control gene expression of numerous metabolic pathways and transport functions essential for cell growth. As such, riboswitch inhibitors represent a new class of potential antibacterial agents. Recently, we identified ribocil-C, a highly selective inhibitor of the flavin mononucleotide (FMN) riboswitch that controls expression of de novo riboflavin (RF, vitamin B2) biosynthesis in Escherichia coli. Here, we provide a mechanistic characterization of the antibacterial effects of ribocil-C as well as of roseoflavin (RoF), an antimetabolite analog of RF, among medically significant Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis. We provide genetic, biophysical, computational, biochemical, and pharmacological evidence that ribocil-C and RoF specifically inhibit dual FMN riboswitches, separately controlling RF biosynthesis and uptake processes essential for MRSA growth and pathogenesis. Such a dual-targeting mechanism is specifically required to develop broad-spectrum Gram-positive antibacterial agents targeting RF metabolism.

Selective small-molecule inhibition of an RNA structural element.

Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

Folding LacZ in the periplasm of Escherichia coli.

Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematically characterize the nonjamming fusions and determine their ultimate localization. We report that LacZ can be fully translocated into the periplasm, where it is toxic. We show that this toxicity is likely due to LacZ misfolding and that, in the absence of the periplasmic disulfide bond catalyst DsbA, LacZ folds in the periplasm. Using the novel phenotype of periplasmic ß-galactosidase activity, we show that the periplasmic chaperone FkpA contributes to LacZ folding in this nonnative compartment. We propose that targeted, nonjamming LacZ fusions may be used to further study folding and targeting in the periplasm of Escherichia coli.

Assembly of Outer Membrane ß-Barrel Proteins: the Bam Complex.

The major class of integral proteins found in the outer membrane (OM) of E. coli and Salmonella adopt a ß-barrel conformation (OMPs). OMPs are synthesized in the cytoplasm with a typical signal sequence at the amino terminus, which directs them to the secretion machinery (SecYEG) located in the inner membrane for translocation to the periplasm. Chaperones such as SurA, or DegP and Skp, escort these proteins across the aqueous periplasm protecting them from aggregation. The chaperones then deliver OMPs to a highly conserved outer membrane assembly site termed the Bam complex. In E. coli, the Bam complex is composed of an essential OMP, BamA, and four associated OM lipoproteins, BamBCDE, one of which, BamD, is also essential. Here we provide an overview of what we know about the process of OMP assembly and outline the various hypotheses that have been proposed to explain how proteins might be integrated into the asymmetric OM lipid bilayer in an environment that lacks obvious energy sources. In addition, we describe the envelope stress responses that ensure the fidelity of OM biogenesis and how factors, such as phage and certain toxins, have coopted this essential machine to gain entry into the cell.

An ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane.

The outer membranes (OMs) of gram-negative bacteria have an asymmetric lipid distribution with lipopolysaccharides at the outer leaflet and phospholipids (PLs) at the inner leaflet. This lipid arrangement is essential for the barrier function of the OM and for the viability of most gram-negative bacteria. Cells with OM assembly defects or cells exposed to harsh chemical treatments accumulate PLs in the outer leaflet of the OM and this disrupts lipopolysaccharide organization and increases sensitivity to small toxic molecules. We have identified an ABC transport system in Escherichia coli with predicted import function that serves to prevent PL accumulation in the outer leaflet of the OM. This highly conserved pathway, which we have termed the Mla pathway for its role in preserving OM lipid asymmetry, is composed of at least 6 proteins and contains at least 1 component in each cellular compartment. We propose that the Mla pathway constitutes a bacterial intermembrane PL trafficking system.

Contact-dependent growth inhibition requires the essential outer membrane protein BamA (YaeT) as the receptor and the inner membrane transport protein AcrB.

Contact-dependent growth inhibition (CDI) is a phenomenon by which bacterial cell growth is regulated by direct cell-to-cell contact via the CdiA/CdiB two-partner secretion system. Characterization of mutants resistant to CDI allowed us to identify BamA (YaeT) as the outer membrane receptor for CDI and AcrB as a potential downstream target. Notably, both BamA and AcrB are part of distinct multi-component machines. The Bam machine assembles outer membrane beta-barrel proteins into the outer membrane and the Acr machine exports small molecules into the extracellular milieu. We discovered that a mutation that reduces expression of BamA decreased binding of CDI+ inhibitor cells, measured by flow cytometry with fluorescently labelled bacteria. In addition, alpha-BamA antibodies, which recognized extracellular epitopes of BamA based on immunofluorescence, specifically blocked inhibitor-target cells binding and CDI. A second class of CDI-resistant mutants identified carried null mutations in the acrB gene. AcrB is an inner membrane component of a multidrug efflux pump that normally forms a cell envelope-spanning complex with the membrane fusion protein AcrA and the outer membrane protein TolC. Strikingly, the requirement for the BamA and AcrB proteins in CDI is independent of their multi-component machines, and thus their role in the CDI pathway may reflect novel, import-related functions.

Cloning and characterization of monofunctional catalase from photosynthetic bacterium Rhodospirillum rubrum S1.

In this study, an approx. 2.5-kb gene fragment including the catalase gene from Rhodospirillum rubrum S1 was cloned and characterized. The determination of the complete nucleotide sequence revealed that the cloned DNA fragment was organized into three open reading frames, designated as ORF1, catalase, and ORF3 in that order. The catalase gene consisted of 1,455 nucleotides and 484 amino acids, including the initiation and stop codons, and was located 326 bp upstream in the opposite direction of ORF1. The catalase was overproduced in Escherichia coli UM255, a catalase-deficient mutant, and then purified for the biochemical characterization of the enzyme. The purified catalase had an estimated molecular mass of 189 kDa, consisting of four identical subunits of 61 kDa. The enzyme exhibited activity over a broad pH range from pH 5.0 to pH 11.0 and temperature range from 20 degrees C to 60 degrees C. The catalase activity was inhibited by 3-amino-1,2,4-triazole, cyanide, azide, and hydroxylamine. The enzyme's K(m) value and V(max) of the catalase for H2O2 were 21.8 mM and 39,960 U/mg, respectively. Spectrophotometric analysis revealed that the ratio of A406 to A280 for the catalase was 0.97, indicating the presence of a ferric component. The absorption spectrum of catalase-4 exhibited a Soret band at 406 nm, which is typical of a heme-containing catalase. Treatment of the enzyme with dithionite did not alter the spectral shape and revealed no peroxidase activity. The combined results of the gene sequence and biochemical characterization proved that the catalase cloned from strain S1in this study was a typical monofunctional catalase, which differed from the other types of catalases found in strain S1.

Structure and function of an essential component of the outer membrane protein assembly machine.

Integral beta-barrel proteins are found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. The machine that assembles these proteins contains an integral membrane protein, called YaeT in Escherichia coli, which has one or more polypeptide transport-associated (POTRA) domains. The crystal structure of a periplasmic fragment of YaeT reveals the POTRA domain fold and suggests a model for how POTRA domains can bind different peptide sequences, as required for a machine that handles numerous beta-barrel protein precursors. Analysis of POTRA domain deletions shows which are essential and provides a view of the spatial organization of this assembly machine.

Lipoprotein SmpA is a component of the YaeT complex that assembles outer membrane proteins in Escherichia coli.

A major role of the outer membrane (OM) of Gram-negative bacteria is to provide a protective permeability barrier for the cell, and proper maintenance of the OM is required for cellular viability. OM biogenesis requires the coordinated assembly of constituent lipids and proteins via dedicated OM assembly machineries. We have previously shown that, in Escherichia coli, the multicomponent YaeT complex is responsible for the assembly of OM beta-barrel proteins (OMPs). This complex contains the OMP YaeT and three OM lipoproteins. Here, we report another component of the YaeT complex, the OM lipoprotein small protein A (SmpA). Strains carrying loss-of-function mutations in smpA are viable but exhibit defects in OMP assembly. Biochemical experiments show that SmpA is involved in maintaining complex stability. Taken together, these experiments establish an important role for SmpA in both the structure and function of the YaeT complex.

YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli.

Recent advances in the study of bacterial membranes have led to the identification of a multicomponent YaeT complex in the outer membrane (OM) of Gram-negative bacteria that is involved in the targeting and folding of beta-barrel outer membrane proteins (OMPs). In Escherichia coli, this complex consists of an essential OMP, YaeT, and three OM lipoproteins, YfgL, NlpB and YfiO. YfiO is the only essential lipoprotein component of the complex. We show that this lipoprotein is required for the proper assembly and/or targeting of OMPs to the OM but not the assembly of lipopolysaccharides (LPS). Depletion of YfiO causes similar phenotypes as does the depletion of YaeT, and we conclude that YfiO plays a critical role in YaeT-mediated OMP folding. We demonstrate that YfiO and YfgL directly interact with YaeT in vitro, while NlpB interacts directly with YfiO. Genetic analysis verifies the importance of YfiO and its interactions with NlpB in maintaining the functional integrity of the YaeT complex.