Lipoprotein Sorting Research Articles

Borrelia burgdorferi BB0346 is an Essential, Structurally Variant LolA Homolog that is Primarily Required for Homeostatic Localization of Periplasmic Lipoproteins.

In diderm bacteria, the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane (IM) to the outer membrane (OM) and therefore plays an essential role in bacterial envelope homeostasis. After extrusion of modified lipoproteins from the IM via the LolCDE complex, the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB, itself a lipoprotein with a LolA-like fold. Yet, LolB homologs appear restricted to γ-proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi, suggesting a different hand-off mechanism at the OM. Here, we solved the crystal structure of the B. burgdorferi LolA homolog BB0346 (LolABb) at 1.9 Å resolution. We identified multiple structural deviations in comparative analyses to other solved LolA structures, particularly a unique LolB-like protruding loop domain. LolABb failed to complement an Escherichia coli lolA knockout, even after codon optimization, signal I peptide adaptation, and a C-terminal chimerization which had allowed for complementation with an α-proteobacterial LolA. Analysis of a conditional B. burgdorferi lolA knockout strain indicated that LolABb was essential for growth. Intriguingly, protein localization assays indicated that initial depletion of LolABb led to an emerging mislocalization of both IM and periplasmic OM lipoproteins, but not surface lipoproteins. Together, these findings further support the presence of two separate primary secretion pathways for periplasmic and surface OM lipoproteins in B. burgdorferi and suggest that the distinct structural features of LolABb allow it to function in a unique LolB-deficient lipoprotein sorting system.

Rab30 facilitates lipid homeostasis during fasting

To facilitate inter-tissue communication and the exchange of proteins, lipoproteins, and metabolites with the circulation, hepatocytes have an intricate and efficient intracellular trafficking system regulated by small Rab GTPases. Here, we show that Rab30 is induced in the mouse liver by fasting, which is amplified in liver-specific carnitine palmitoyltransferase 2 knockout mice (Cpt2L−/−) lacking the ability to oxidize fatty acids, in a Pparα-dependent manner. Live-cell super-resolution imaging and in vivo proximity labeling demonstrates that Rab30-marked vesicles are highly dynamic and interact with proteins throughout the secretory pathway. Rab30 whole-body, liver-specific, and Rab30; Cpt2 liver-specific double knockout (DKO) mice are viable with intact Golgi ultrastructure, although Rab30 deficiency in DKO mice suppresses the serum dyslipidemia observed in Cpt2L−/− mice. Corresponding with decreased serum triglyceride and cholesterol levels, DKO mice exhibit decreased circulating but not hepatic ApoA4 protein, indicative of a trafficking defect. Together, these data suggest a role for Rab30 in the selective sorting of lipoproteins to influence hepatocyte and circulating triglyceride levels, particularly during times of excessive lipid burden.

Cryo-EM structures of LolCDE reveal the molecular mechanism of bacterial lipoprotein sorting in Escherichia coli.

Bacterial lipoproteins perform a diverse array of functions including bacterial envelope biogenesis and microbe–host interactions. Lipoproteins in gram-negative bacteria are sorted to the outer membrane (OM) via the localization of lipoproteins (Lol) export pathway. The ATP-binding cassette (ABC) transporter LolCDE initiates the Lol pathway by selectively extracting and transporting lipoproteins for trafficking. Here, we report cryo-EM structures of LolCDE in apo, lipoprotein-bound, and AMPPNP-bound states at a resolution of 3.5 to 4.2 Å. Structure-based disulfide crosslinking, photo-crosslinking, and functional complementation assay verify the apo-state structure and reveal the molecular details regarding substrate selectivity and substrate entry route. Our studies snapshot 3 functional states of LolCDE in a transport cycle, providing deep insights into the mechanisms that underlie LolCDE-mediated lipoprotein sorting in E. coli.

The lolB gene in Xanthomonas campestris pv. campestris is required for bacterial attachment, stress tolerance, and virulence

BackgroundXanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium that can cause black rot disease in crucifers. The lipoprotein outer membrane localization (Lol) system is involved in the lipoprotein sorting to the outer membrane. Although Xcc has a set of annotated lol genes, there is still little known about the physiological role in this phytopathogen. In this study, we aimed to characterize the role of LolB of Xcc in bacterial attachment, stress tolerance, and virulence.ResultsTo characterize the role of LolB, lolB mutant was constructed and phenotypic evaluation was performed. The lolB mutant revealed reductions in bacterial attachment, extracellular enzyme production, and virulence. Mutation of lolB also resulted in reduced tolerance to a myriad of stresses, including heat and a range of membrane-perturbing agents. Trans-complementation of lolB mutant with intact lolB gene reverted these altered phenotypes to the wild-type levels. From subsequent reporter assay and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, the expression of genes that encode the major extracellular enzymes and the stress-related proteins was reduced after lolB mutation.ConclusionsThe results in this work contribute to the functional understanding of lolB in Xanthomonas for the first time, and provide new insights into the function of lolB in bacteria.

Disorder is a critical component of lipoprotein sorting in Gram-negative bacteria.

Gram-negative bacteria express structurally diverse lipoproteins in their cell envelope. Here, we find that approximately half of lipoproteins destined to the Escherichia coli outer membrane display an intrinsically disordered linker at their N terminus. Intrinsically disordered regions are common in proteins, but establishing their importance in vivo has remained challenging. As we sought to unravel how lipoproteins mature, we discovered that unstructured linkers are required for optimal trafficking by the Lol lipoprotein sorting system, whereby linker deletion re-routes three unrelated lipoproteins to the inner membrane. Focusing on the stress sensor RcsF, we found that replacing the linker with an artificial peptide restored normal outer-membrane targeting only when the peptide was of similar length and disordered. Overall, this study reveals the role played by intrinsic disorder in lipoprotein sorting, providing mechanistic insight into the biogenesis of these proteins and suggesting that evolution can select for intrinsic disorder that supports protein function.

Structural basis for bacterial lipoprotein relocation by the transporter LolCDE.

Lipoproteins in the outer membrane of Gram-negative bacteria are involved in various vital physiological activities, including multidrug resistance. Synthesized in the cytoplasm and matured in the inner membrane, lipoproteins must be transported to the outer membrane through the Lol pathway mediated by the ATP-binding cassette transporter LolCDE in the inner membrane via an unknown mechanism. Here, we report cryo-EM structures of Escherichia coli LolCDE in apo, lipoprotein-bound, LolA-bound, ADP-bound and AMP-PNP-bound states at a resolution of 3.2-3.8 Å, covering the complete lipoprotein transport cycle. Mutagenesis and in vivo viability assays verify features of the structures and reveal functional residues and structural characteristics of LolCDE. The results provide insights into the mechanisms of sorting and transport of outer-membrane lipoproteins and may guide the development of novel therapies against multidrug-resistant Gram-negative bacteria.

Protein Secretion in Spirochetes.

Spirochetes form a separate phylum of bacteria with two membranes but otherwise unusual morphologies and envelope structures. Distinctive common features of Borrelia, Leptospira, and Treponema include the sequestration of flagella to the periplasm and thin peptidoglycan cell walls that are more closely associated with the inner membrane. Outer membrane compositions differ significantly between the genera. Leptospira most closely track Gram-negative bacteria due to the incorporation of lipopolysaccharides. Treponema and Borrelia outer membranes lack lipopolysaccharide, with treponemes expressing only a few outer membrane proteins and Borrelia displaying a dizzying diversity of abundant surface lipoproteins instead. Phylogenetic and experimental evidence indicates that spirochetes have adapted various modules of bacterial export and secretion pathways to build and maintain their envelopes. Export and insertion pathways in the inner membrane appear conserved, while spirochetal experimentation with various envelope architectures over time has led to variations in secretion pathways in the periplasm and outer membrane. Classical type I to III secretion systems have been identified, with demonstrated roles in drug efflux and export of flagellar proteins only. Unique activities of periplasmic proteases, including a C-terminal protease, are involved in maturation of some periplasmic proteins. Proper lipoprotein sorting within the periplasm appears to be dependent on functional Lol pathways that lack the outer membrane lipoprotein insertase LolB. The abundance of surface lipoproteins in Borrelia and detailed protein sorting studies suggest a lipoprotein secretion pathway that either extends Lol through the outer membrane or bypasses it altogether. Proteins can be released from cells in outer membrane vesicles or, rarely, as soluble proteins.

The Lipoprotein NlpE Is a Cpx Sensor That Serves as a Sentinel for Protein Sorting and Folding Defects in the Escherichia coli Envelope.

The envelope of Gram-negative bacteria is a complex compartment that is essential for viability. To ensure survival of the bacterial cells in fluctuating environments, several signal transduction systems, called envelope stress response systems (ESRSs), exist to monitor envelope biogenesis and homeostasis. The Cpx two-component system is an extensively studied ESRS in Escherichia coli that is active during exposure to a vast array of stresses and protects the envelope under those harmful circumstances. Overproduction of NlpE, a two-domain outer membrane lipoprotein of unclear function, has been used in numerous studies as a molecular trigger to turn on the system artificially. However, the mechanism of Cpx activation by NlpE, as well as its physiological relevance, awaited further investigation. In this paper, we provide novel insights into the role played by NlpE in the Cpx system. We found that, among all outer membrane lipoproteins in E. coli, NlpE is sufficient to induce Cpx when lipoprotein trafficking is perturbed. Under such conditions, fitness is increased by the presence of NlpE. Moreover, we show that NlpE, through its N-terminal domain, physically interacts with the Cpx sensor kinase CpxA. Our data suggest that NlpE also serves to activate the Cpx system during oxidative folding defects in the periplasm and that its C-terminal domain is involved in the sensing mechanism. Overall, our data demonstrate that NlpE acts as a sentinel for two important envelope biogenesis processes, namely, lipoprotein sorting and oxidative folding, and they further establish NlpE as a bona fide member of the Cpx two-component system.IMPORTANCE Bacteria rely on a sophisticated envelope to shield them against challenging environmental conditions and therefore need to ensure correct envelope assembly and integrity. A major signaling pathway that performs this role in Gram-negative species is the Cpx system. An outer membrane lipoprotein of unclear function, NlpE, has long been exploited as a research tool to study Cpx in E. coli, since it triggers this system when overproduced or mislocalized; however, the mechanism and physiological relevance of the NlpE-Cpx connection have awaited further investigation. We elucidate a new function for NlpE by showing that it physically interacts with the Cpx sensor CpxA and acts as a sentinel that specifically monitors two essential envelope biogenesis processes, namely, lipoprotein sorting and oxidative folding.

Correct Sorting of Lipoproteins into the Inner and Outer Membranes of Pseudomonas aeruginosa by the Escherichia coli LolCDE Transport System.

Biogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. In Enterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that in Pseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those from Escherichia coli The P. aeruginosa lipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either the E. coli or P. aeruginosa LolCDE. We further demonstrate that an inhibitor of E. coli LolCDE is active against P. aeruginosa only when expressing the E. coli orthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCE Gram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways of Escherichia coli and Pseudomonas aeruginosa are interchangeable, with the E. coli orthologues correctly sorting the P. aeruginosa lipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified in E. coli as aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.

Lipoprotein sorting as a potential target of resazomycins, a novel family of antibiotics

The prevalence of antibiotic-resistant bacterial infection has increased, promoting a global health concern. The significance of this resistance pertains to the limitation it places on effective and available microbial agents, resulting in a predicament worsened by the small number of antimicrobial agents introduced over the last couple of years. Therefore, new antibiotics must be developed with unique targets that are not susceptible to rapid antimicrobial resistance. With this stated, it is known that the Lol system is responsible for transporting lipoproteins to the outer membrane in Gram-negative bacteria which play essential roles in a wide variety of bacterial physiological processes. We have identified a novel family of compounds, resazomycins, which exhibit antimicrobial activity against select Gram-negative bacteria including the human pathogens Francisella tularensis and Neisseria gonorrhoeae. A common feature of resazomycin-sensitive bacteria is possession of a unique lipoprotein sorting system, LolDF. This complex differs from the LolCDE sorting complex found in most Gram-negative bacteria like Escherichia coli that are resistant to resazomycins. We sought to determine whether LolDF is the target of resazomycins by cloning the genes that encode the E. coli LolCDE transporter into an F. tularensis plasmid and then electroporated this plasmid into an attenuated F. tularensis live vaccine strain (LVS) to see if these would render the bacterium resistant to resazomycins. Thus far, LolCDE-expressing LVS exhibits no difference in susceptibility to resazomycins compared to wild-type LVS. Therefore, we are investigating the role of other F. tularensis genes insusceptibility to resazomycins.

Susceptibility of Brucella ovis to resazurin and resorufin pentyl ether.

Antibiotic resistance is one of the world’s biggest threats to public health. In the United States alone, there are approximately 2 million new cases of antibiotic-resistant infections annually resulting in 23,000 deaths and billions of dollars in healthcare related costs. The development of new antibiotics is essential to prevent the loss of additional lives from once “curable” diseases. We recently identified a novel family of resazurin-based compounds, resazomycins, which exhibit antimicrobial activity against F. tularensis and N. gonorrhoeae in vitro and in vivo. A common feature of both these bacterial species is possession of a unique lipoprotein sorting system, LolDF. To investigate the relationship between LolDF and susceptibility to resazomycins, we propose performing antibiotic susceptibility testing on a diverse collection of medically important LolDF-possessing bacterial strains. This study focuses on characterizing the efficacy of two resazomycins, resazurin (Rz) and resorufin pentyl ether (RPE), against Brucella ovis. B.ovis infects sheep causing a clinical or subclinical disease characterized by epididymitis and orchitis resulting in reduced fertility in rams. Additionally, B. ovis is occasionally linked with placentitis and abortions in ewes and increased perinatal mortality in lambs. Its significant impact on ram fertility causes a critical economic impact on sheep-producing regions globally. We are currently working to determine the minimal inhibitory concentration (MIC) of Rz and RPE against B. ovis and if these antibiotics are effective at killing this bacterium within eukaryotic cells.

Efficacy of Resazomycins Against LolDF-Expressing Gram-Negative Bacteria

A growing number of human pathogens are becoming resistant to most common antibiotics. Each year, antibiotic-resistant infections are responsible for 23,000 deaths in the United States and billions of dollars in health care costs. To prevent the loss of additional lives from once “curable” diseases, new antibiotics must be developed. We recently identified a novel family of resazurin-based compounds, resazomycins, which exhibit antimicrobial activity against Francisella tularensis and Neisseria gonorrhoeae in vitro and in vivo. A common feature of both these bacteria is possession of a unique lipoprotein sorting system, LolDF. To investigate the relationship between LolDF and susceptibility to resazomycins, we propose performing antibiotic susceptibility testing on a diverse collection of medically important LolDF-possessing bacterial strains. My project focuses on characterizing the efficacy of two resazomycins, resazurin (Rz) and resorufin pentyl ether (RPE), against three different bacterial strains: Acinetobacter baumanii, Moraxella catarrhalis, and Chromobacterium violaceum. These bacterial species are human pathogens known to be multi-drug resistant and primarily infect immunocompromised individuals. Using the agar dilution antibiotic susceptibility test, we have determined that A. baumanii and M. catarrhalis are resistant to resazomycins, while C. violaceumis susceptible. These data suggest that LolDF may not be the sole target of resazomycins. Further investigation is needed to determine the precise mechanism of action of resazomycins. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence)

Inhibition of lipoprotein sorting in Francisella tularensis: a potential target of resazomycins, a novel family of antibiotics.

Tularemia is a potentially fatal illness caused by the bacterium Francisella tularensis. Inhalation of less than 10 bacteria results in an acute pneumonia with an associated mortality rate of 30-60% if left untreated. Due to the potential use of F. tularensis as a weapon of bioterrorism and development of antibiotic resistance, new antibiotics are being sought against this pathogen. We have identified a novel family of resazurin-based compounds named resazomycins which exhibit antimicrobial activity against F. tularensis and Neisseria gonorrhoeae. In order to proceed with in vivo testing of resazomycins, their mechanism of action must be determined. A recent study has shown that while F. tularensis and N. gonorrhoeae are taxonomically distinct from one another, they share a similar lipoprotein sorting system, LolDF. This complex differs from the LolCDE sorting complex commonly found in other Gram negative bacteria which indicates this system may be a target of resazomycins. To determine whether resazurin inhibits the function of LolDF we will express the genes that encode for the E. coli lipoprotein sorting system (lolCDE) in F. tularensis to see if we can confer resistance to resazurin. Experimentation thus far has proven to be unsuccessful in trying to generate this construct since overexpression of lolCDE in E. coli is lethal. To limit expression of these genes until the plasmid has been transferred into F. tularensis, we will explore different inducible expression systems. Future investigations will focus on cloning the E. coli lolCDE genes into a Francisella shuttle vector containing a tetracycline-regulatable promoter.

Determining resistance of Francisella- tularensis to resazomycins, a novel family of antibiotics

Because a significant number of bacteria are becoming increasingly more resistant to antibiotic treatments, the development of new antibacterial drugs is imperative. Recently, a new family of resazurin-based compounds, resazomycins, have exhibited promising antimicrobial activity against Francisella- tularensis and Neisseria- gonorrheae in vitro and in vivo. While the mechanism of action of resazomycins has yet to be determined, preliminary data suggests that the lipoprotein sorting machinery (LolDF) of these bacteria may be the target. Since the Lol complex is essential in all Gram-negative bacteria, we hypothesize that both F. tularensis and N. gonorrhoeae will develop little resistance to resazomycins. To address this, we first sought to determine the minimal inhibitory concentration (MIC) of resazurin against F. tularensis cultured on chocolate agar. Bacterial growth was observed up to 7.7 µg/ml resazurin, then completely abolished at 11 µg/ml, indicating this is the MIC. We then wanted to determine if resistant colonies of F. tularensis would emerge following continuous cultivation on chocolate agar containing 1 x MIC of resazurin. Over time, hundreds of F. tularensis colonies were observed on the chocolate plates containing resazurin, suggesting this bacterium can develop resistance to resazomycins. We are currently working to isolate the genomic DNA from resistant isolates to send to Marshall University’s Genomics Core for whole genome sequencing to identify single nucleotide polymorphisms associated with resazomycin resistance. Future investigations will focus on generating mutants of genes identified as possible resazurin resistance targets in F. tularensis and determining the sensitivity of the resulting strains. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence)

Antibacterial activity of resazurin-based compounds against Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the cause of the second most common sexually transmitted infection, with approximately 80 million new cases of gonorrhea reported annually. The recent emergence of clinical isolates resistant to the last monotherapy against this bacterium, the cephalosporins, illustrates the need for new anti-gonococcal agents. We recently identified resazurin, a commonly used viability dye, as a novel antibacterial agent against N. gonorrhoeae. While resazurin exhibited potent in vitro antimicrobial activity, in vivo, resazurin did not limit the colonization of mice with N. gonorrhoeae following vaginal infection. The ineffectiveness of resazurin in vivo is likely due to its interaction with serum albumin which completely diminishes its antimicrobial activity. Treatment of mice with a resazurin analog that maintains its antimicrobial activity in the presence of serum albumin, resorufin pentyl ether, however, approached a significant decrease in the percentage of mice vaginally colonized. This treatment also decreased vaginal colonization by N. gonorrhoeae over time. Together, these data suggest resazurin derivatives have potential for the treatment of gonorrhea. Current investigations in the laboratory are focused on the mechanism of action of these compounds which appears to be linked to the lipoprotein sorting system of this bacterium. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence, a grant from NIH-NIAID [5K22AI087703], and funding from WV-NASA).

The Journey of Lipoproteins Through the Cell: One Birthplace, Multiple Destinations.

Bacterial lipoproteins are a very diverse group of proteins characterized by the presence of an N-terminal lipid moiety that serves as a membrane anchor. Lipoproteins have a wide variety of crucial functions, ranging from envelope biogenesis to stress response. In Gram-negative bacteria, lipoproteins can be targeted to various destinations in the cell, including the periplasmic side of the cytoplasmic or outer membrane, the cell surface or the external milieu. The sorting mechanisms have been studied in detail in Escherichia coli, but exceptions to the rules established in this model bacterium exist in other bacteria. In this chapter, we will present the current knowledge on lipoprotein sorting in the cell. Our particular focus will be on the surface-exposed lipoproteins that appear to be much more common than previously assumed. We will discuss the different targeting strategies, provide numerous examples of surface-exposed lipoproteins and discuss the techniques used to assess their surface exposure.

Roles of the Protruding Loop of Factor B Essential for the Localization of Lipoproteins (LolB) in the Anchoring of Bacterial Triacylated Proteins to the Outer Membrane

The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed.

Folding and Lipid Membrane Interactions of BamD, An Essential Component of the β-Barrel Assembly Machine from Escherichia Coli

BamD (YfiO) [1] is an essential component of the β-barrel assembly machine of the outer membrane of Escherichia coli. BamD is synthesized in the cytosol and translocated across the cytopasmic membrane in unfolded form for processing and transport to the outer membrane by the LOL system [2].To examine the interactions of BamD with lipid membranes and BamD folding, we have over-expressed BamD into the periplasm and isolated it from membrane fractions in unfolded form in 8 M urea. Circular dichorism spectroscopy indicated secondary structure formation in BamD upon rapid dilution of the denaturant urea in aqeuous buffer. In the absence of lipid bilayers, BamD displayed a lower content of α-helix structure. The CD-data indicated that native-like folding of BamD required the presence of lipid-membranes of phosphatidylglycerol and phosphatidylcholine. An increased content of phosphatidylglycerol facilitated BamD folding. Fluorescence spectroscopy demonstrated BamD binding to both zwitterionic and negatively charged lipid. The lipid/BamD stoichiometry was estimated to 17 phosphatidylglycerol/BamD and to 31 phosphatidylcholine/BamD.[1] Malinverni JC, Werner J, Kim S, Sklar JG, Kahne D, Misra R, Silhavy TJ (2006) YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli. Mol Microbiol 61:151-164.[2] Narita S, Tokuda H (2010) Sorting of bacterial lipoproteins to the outer membrane by the Lol system. Methods Mol Biol 619:117-129.

Importance of the proline-rich region for the regulatory function of RcsF, an outer membrane lipoprotein component of the Escherichia coli Rcs signal transduction system

The outer membrane lipoprotein RcsF is an essential component of the Rcs phosphorelay signal transduction system in Escherichia coli. It senses stresses imposed on the cell envelope and conveys the information to histidine kinase RcsC in the cytoplasmic membrane. Mislocalization of RcsF to the periplasm, effected by fusing it to the periplasmic maltose-binding protein, or to the cytoplasmic membrane, brought about by changing the lipoprotein sorting signal, leads to high activation of the Rcs system, suggesting that RcsF functions as a ligand for RcsC in activating the system. Here, we focus on the proline-rich region (PRR) in the N-terminal half of RcsF, a region which also contains many basic amino acid residues. Deletion of the PRR in the mislocalized RcsF resulted in even higher activation of the Rcs system. The same deletion in wild-type RcsF lipoprotein that is correctly localized to the outer membrane, however, blocked activation of the system under stresses that normally should activate it. It is highly likely that the PRR plays an important role in the regulation of the function of RcsF in activating the Rcs system.

The targeting, docking and anti-proteolysis functions of the secretin chaperone PulS

The Klebsiella oxytoca lipoprotein PulS might function as either or both a pilot and a docking factor in the outer membrane targeting and assembly of the Type II secretion system secretin PulD. In the piloting model, PulS binds to PulD monomers and targets them to the outer membrane via the lipoprotein sorting pathway components LolA and LolB. In this model, PulS also protects the PulD monomers from proteolysis during transit through the periplasm. In the docking model, PulS is targeted alone to the outer membrane, where it acts as a receptor for PulD monomers, allowing them to accumulate and assemble specifically in this membrane. PulS was shown to dissociate from and/or re-associate freely with PulD multimers in zwitterionic detergent, making it difficult to determine whether PulS remains associated with PulD dodecamers in the outer membrane by co-purification. However, PulD protomers in the dodecamer were shown to be stable in the absence of PulS, indicating that PulS is only required to protect the protease-susceptible monomer. DegP was identified as one of the proteases that could contribute to PulD degradation in the absence of PulS. Studies on the in vitro assembly and targeting of PulD into Escherichia coli membrane vesicles demonstrated its strong preference to insert into the inner membrane, as is the case in vivo in the absence of PulS. However, PulD could be targeted to outer membrane fragments in vitro if they were preloaded with PulS, indicating the technical feasibility of the docking model. We conclude that both modes of action might contribute to efficient outer membrane targeting of PulD in vivo, although the piloting function is likely to predominate.