Direct Interaction of the EpsL and EpsM Proteins of the General Secretion Apparatus in Vibrio cholerae

ABSTRACT The general secretion pathway of gram-negative bacteria is responsible for extracellular secretion of a number of different proteins, including proteases and toxins. This pathway supports secretion of proteins across the cell envelope in two distinct steps, in which the second step, involving translocation through the outer membrane, is assisted by at least 13 different gene products. Two of these components, the cytoplasmic membrane proteins EpsL and EpsM ofVibrio cholerae, have been purified and characterized. Based on gel filtration analysis, both purified EpsM(His)6 and wild-type EpsL present in anEscherichia coli Triton X-100 extract are dimeric proteins. EpsL and EpsM were also found to interact directly and form a Triton X-100 stable complex that could be precipitated with either anti-EpsL or anti-EpsM antibodies. In addition, when the L and M proteins were coexpressed in E. coli, they formed a stable complex and protected each other from proteolytic degradation, indicating that these two proteins interact in vivo and that no other Eps protein is required for their association. Since EpsL is predicted to contain a large cytoplasmic domain, while EpsM is predominantly exposed on the periplasmic side, we speculate that these components might be part of a structure that is involved in bridging the inner and outer membranes. Furthermore, since EpsL has previously been shown to interact with the autophosphorylating cytoplasmic membrane protein EpsE, we hypothesize that this trimolecular complex might be involved in regulating the opening and closing of the secretion pore and/or transducing energy to the site of outer membrane translocation.

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Structure and cell envelope associations of flagellar basal complexes of Vibrio cholerae and Campylobacter fetus

Canadian Journal of Microbiology ◽

10.1139/m84-048 ◽

1984 ◽

Vol 30 (3) ◽

pp. 322-333 ◽

Cited By ~ 23

Author(s):

F. G. Ferris ◽

T. J. Beveridge ◽

M. L. Marceau-Day ◽

A. D. Larson

Keyword(s):

Outer Membrane ◽

Vibrio Cholerae ◽

Cell Envelope ◽

Sodium Dodecyl ◽

Three Dimensions ◽

Thin Sections ◽

Campylobacter Fetus ◽

Basal Complex ◽

Nonionic Detergent ◽

Triton X

To isolate intact flagella with basal complexes from Vibrio cholerae, a rhamnolipid hemolysin from Pseudomonas aeruginosa was used to disrupt the cell envelope and flagellar sheath. The nonionic detergent, Triton X-100, provided similar results for Campylobacter fetus. Each of these basal complexes possessed, in addition to the four classical rings, concentric membrane rings (CMR's) similar to those found in Aquaspirillum serpens. Through the use of stereo imaging (which allows structures to be visualized in three dimensions) of thin sections of cells which had been sequentially treated with a number of envelope perturbants (i.e., ethylenediaminetetraacetate, lysozyme, Triton X-100, rhamnolipid hemolysin, and sodium dodecyl sulfate), we have progressively exposed the component parts of the basal organelles in V. cholerae and C. fetus. Since the action of these envelope perturbants has been well documented, we have been able to determine the associations of the exposed portions of the flagellar basal complex and the layer of the cell envelope in which they would normally reside. From our observations we have concluded that in both V. cholerae and C. fetus the L ring is embedded in the outer membrane and the P ring is associated with the peptidoglycan. The CMR's are bracketed by the L and P rings and are sandwiched between the outer membrane and the peptidoglycan. Elements of both the S and M rings appear to be associated with the plasma membrane.

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Vibrio cholerae VibF Is Required for Vibriobactin Synthesis and Is a Member of the Family of Nonribosomal Peptide Synthetases

Journal of Bacteriology ◽

10.1128/jb.182.6.1731-1738.2000 ◽

2000 ◽

Vol 182 (6) ◽

pp. 1731-1738 ◽

Cited By ~ 38

Author(s):

Joan R. Butterton ◽

Michael H. Choi ◽

Paula I. Watnick ◽

Patricia A. Carroll ◽

Stephen B. Calderwood

Keyword(s):

Outer Membrane ◽

Vibrio Cholerae ◽

Consensus Sequence ◽

Membrane Receptor ◽

Transcriptional Level ◽

Outer Membrane Receptor ◽

Chromosomal Dna ◽

E Coli ◽

Binding Consensus Sequence

ABSTRACT A 7.5-kbp fragment of chromosomal DNA downstream of theVibrio cholerae vibriobactin outer membrane receptor,viuA, and the vibriobactin utilization gene,viuB, was recovered from a Sau3A lambda library of O395 chromosomal DNA. By analogy with the genetic organization of the Escherichia coli enterobactin gene cluster, in which the enterobactin biosynthetic and transport genes lie adjacent to the enterobactin outer membrane receptor, fepA, and the utilization gene, fes, the cloned DNA was examined for the ability to restore siderophore synthesis to E. coli entmutants. Cross-feeding studies demonstrated that an E. coli entF mutant complemented with the cloned DNA regained the ability to synthesize enterobactin and to grow in low-iron medium. Sequence analysis of the cloned chromosomal DNA revealed an open reading frame downstream of viuB which encoded a deduced protein of greater than 2,158 amino acids, homologous to Yersinia sp. HMWP2, Vibrio anguillarum AngR, and E. coliEntF. A mutant with an in-frame deletion of this gene, namedvibF, was created with classical V. choleraestrain O395 by in vivo marker exchange. In cross-feeding studies, this mutant was unable to synthesize ferric vibriobactin but was able to utilize exogenous siderophore. Complementation of the mutant with a cloned vibF fragment restored vibriobactin synthesis to normal. The expression of the vibF promoter was found to be negatively regulated by iron at the transcriptional level, under the control of the V. cholerae fur gene. Expression ofvibF was not autoregulatory and neither affected nor was affected by the expression of irgA or viuA. The promoter of vibF was located by primer extension and was found to contain a dyad symmetric nucleotide sequence highly homologous to the E. coli Fur binding consensus sequence. A footprint of purified V. cholerae Fur on the vibFpromoter, overlapping the Fur binding consensus sequence, was observed using DNase I footprinting. The protein product of vibF is homologous to the multifunctional nonribosomal protein synthetases and is necessary for the biosynthesis of vibriobactin.

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Clearance and binding of native and defucosylated lactoferrin

Biochemical Journal ◽

10.1042/bj2120249 ◽

1983 ◽

Vol 212 (2) ◽

pp. 249-257 ◽

Cited By ~ 20

Author(s):

M J Imber ◽

S V Pizzo

Keyword(s):

Bovine Serum Albumin ◽

Serum Albumin ◽

Bovine Serum ◽

Specific Binding ◽

Gel Filtration ◽

Peritoneal Macrophages ◽

Mononuclear Phagocyte ◽

Stable Complex

These studies explore the role of carbohydrate recognition systems and the direct involvement of terminal alpha 1-3-linked fucose in the clearance of lactoferrin from the murine circulation and in the specific binding of lactoferrin to receptors on murine peritoneal macrophages. As previously reported, radiolabelled lactoferrin cleared very rapidly (t1/2 less than 1 min) after intravenous injection into mice. However, competing levels of ligands specific for the hepatic galactose receptor (asialo-orosomucoid), the hepatic fucose receptor (fucosyl-bovine serum albumin), and the mononuclear-phagocyte system pathway recognizing mannose, N-acetylglucosamine and fucose (mannosyl-, N-acetylglucosaminyl- and fucosyl-bovine serum albumin) did not block radiolabelled lactoferrin clearance in vivo or binding to mouse peritoneal macrophage monolayers in vitro. Almond emulsin alpha 1-3-fucosidase was used to prepare defucosylated lactoferrin in which 88% of the alpha 1-3-linked fucose was hydrolysed. No difference in clearance or receptor binding was observed between radiolabelled native and defucosylated lactoferrin. Fucoidin, a fucose-rich algal polysaccharide, completely inhibits the clearance in vivo and macrophage binding in vitro of lactoferrin. This effect, however, is probably not the result of competition for binding to the fucose receptor, since gel-filtration studies demonstrated formation of a stable complex between lactoferrin and fucoidin. The present results indicate that the lactoferrin-clearance pathway is distinct from several pathways mediating glycoprotein clearance through recognition of terminal galactose, fucose, N-acetylglucosamine or mannose. Furthermore, alpha 1-3-linked fucose on lactoferrin is not essential for lactoferrin clearance in vivo or specific binding to macrophage receptors in vitro.

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Arrangement of the Translocator of the Autotransporter Adhesin Involved in Diffuse Adherence on the Bacterial Surface

Infection and Immunity ◽

10.1128/iai.73.7.3851-3859.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 3851-3859 ◽

Cited By ~ 20

Author(s):

Daniel Müller ◽

Inga Benz ◽

Damini Tapadar ◽

Christian Buddenborg ◽

Lilo Greune ◽

...

Keyword(s):

Outer Membrane ◽

Protein Translocation ◽

Gel Filtration ◽

Analytical Techniques ◽

Cross Linking ◽

Secretion Systems ◽

Bacterial Surface ◽

Passenger Proteins

ABSTRACT Autotransporters of gram-negative bacteria are single-peptide secretion systems that consist of a functional N-terminal α-domain (“passenger”) fused to a C-terminal β-domain (“translocator”). How passenger proteins are translocated through the outer membrane has not been resolved, and at present essentially three different models are discussed. In the widely accepted “hairpin model” the passenger proteins are translocated through a channel formed by the β-barrel of the translocator that is integrated in the outer membrane. This model has been challenged by a recent proposal for a general autotransporter model suggesting that there is a hexameric translocation pore that is generated by the oligomerization of six β-domains. A third model suggests that conserved Omp85 participates in autotransporter integration and passenger protein translocation. To examine these models, in this study we investigated the presence of putative oligomeric structures of the translocator of the autotransporter adhesin involved in diffuse adherence (AIDA) in vivo by cross-linking techniques. Furthermore, the capacity of isolated AIDA fusion proteins to form oligomers was studied in vitro by several complementary analytical techniques, such as analytical gel filtration, electron microscopy, immunogold labeling, and cross-linking of recombinant autotransporter proteins in which different passenger proteins were fused to the AIDA translocator. Our results show that the AIDA translocator is mostly present as a monomer. Only a fraction of the AIDA autotransporter was found to form dimers on the bacterial surface and in solution. Higher-order structures, such as hexamers, were not detected either in vivo or in vitro and can therefore be excluded as functional moieties for the AIDA autotransporter.

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Binding of Ferric Enterobactin by theEscherichia coli Periplasmic Protein FepB

Journal of Bacteriology ◽

10.1128/jb.182.19.5359-5364.2000 ◽

2000 ◽

Vol 182 (19) ◽

pp. 5359-5364 ◽

Cited By ~ 53

Author(s):

Cathy Sprencel ◽

Zhenghua Cao ◽

Zengbiao Qi ◽

Daniel C. Scott ◽

Marjorie A. Montague ◽

...

Keyword(s):

Specific Activity ◽

Solid Phase ◽

Cell Envelope ◽

Binding Protein ◽

Gel Filtration ◽

Periplasmic Protein ◽

Left Handed ◽

Fluorescence Measurements ◽

Rate Limiting Step

ABSTRACT The periplasmic protein FepB of Escherichia coli is a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the Kd of the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (Kd = 30 nM) and ferric enantioenterobactin (Kd = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (Kd = 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.

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Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.00092-09 ◽

2009 ◽

Vol 191 (17) ◽

pp. 5398-5408 ◽

Cited By ~ 33

Author(s):

Aleksandra E. Sikora ◽

Sinem Beyhan ◽

Michael Bagdasarian ◽

Fitnat H. Yildiz ◽

Maria Sandkvist

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Outer Membrane ◽

Vibrio Cholerae ◽

Iron Homeostasis ◽

Cell Envelope ◽

Membrane Integrity ◽

Genetic Alterations ◽

Iron Storage ◽

Study Gene Expression

ABSTRACT The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.

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Direct Interaction between DNA Methyltransferase DIM-2 and HP1 Is Required for DNA Methylation in Neurospora crassa

Molecular and Cellular Biology ◽

10.1128/mcb.00823-08 ◽

2008 ◽

Vol 28 (19) ◽

pp. 6044-6055 ◽

Cited By ~ 89

Author(s):

Shinji Honda ◽

Eric U. Selker

Keyword(s):

Dna Methylation ◽

Neurospora Crassa ◽

Dna Methyltransferase ◽

Direct Interaction ◽

Stable Complex ◽

Heterochromatin Protein 1 ◽

Heterochromatin Protein ◽

Chromo Shadow Domain ◽

Two Hybrid

ABSTRACT DNA methylation is involved in gene silencing and genomic stability in mammals, plants, and fungi. Genetics studies of Neurospora crassa have revealed that a DNA methyltransferase (DIM-2), a histone H3K9 methyltransferase (DIM-5), and heterochromatin protein 1 (HP1) are required for DNA methylation. We explored the interrelationships of these components of the methylation machinery. A yeast two-hybrid screen revealed that HP1 interacts with DIM-2. We confirmed the interaction in vivo and demonstrated that it involves a pair of PXVXL-related motifs in the N-terminal region of DIM-2 and the chromo shadow domain of HP1. Both regions are essential for proper DNA methylation. We also determined that DIM-2 and HP1 form a stable complex independently of the trimethylation of histone H3K9, although the association of DIM-2 with its substrate sequences depends on trimethyl-H3K9. The DIM-2/HP1 complex does not include DIM-5. We conclude that DNA methylation in Neurospora is largely or exclusively the result of a unidirectional pathway in which DIM-5 methylates histone H3K9 and then the DIM-2/HP1 complex recognizes the resulting trimethyl-H3K9 mark via the chromo domain of HP1.

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Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a UropathogenicEscherichia coliIsolate

mBio ◽

10.1128/mbio.00603-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 11

Author(s):

Jingyu Diao ◽

Catrien Bouwman ◽

Donghong Yan ◽

Jing Kang ◽

Anand K. Katakam ◽

...

Keyword(s):

Outer Membrane ◽

Capsular Polysaccharide ◽

Cell Envelope ◽

Bloodstream Infections ◽

Serum Resistance ◽

E Coli ◽

Serum Killing ◽

Group 2

ABSTRACTMurein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins inEscherichia coli. Their roles in cell-envelope integrity have been documented inE. colilaboratory strains, and while Lpp has been linked to serum resistancein vitro, the underlying mechanism has not been established. Here,lppandpalmutants of uropathogenicE. colistrain CFT073 showed reduced survival in a mouse bacteremia model, but only thelppmutant was sensitive to serum killingin vitro. The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysisin vitroand complement-mediated clearancein vivo. Compared to the wild-type strain, thelppmutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for “group 2” capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenicE. coliisolates.IMPORTANCEUropathogenicE. coli(UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistancein vitroand for complement-mediated bacterial clearancein vivo. This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of “group 2” capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for murein lipoprotein in complex interactions between different outer membrane biogenesis pathways and further highlight the importance of lipoprotein assembly and transport in bacterial pathogenesis.

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Characterization of Escherichia coli ExbD protein modification in vivo

10.1101/2020.01.29.925446 ◽

2020 ◽

Author(s):

Aruna Kumar ◽

Kathleen Postle

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Active Transport ◽

Protein Modification ◽

Cytoplasmic Membrane ◽

Physical Contact ◽

Protonmotive Force ◽

Iron Starvation ◽

Tonb System

ABSTRACTThe TonB system of Escherichia coli couples the protonmotive force of the cytoplasmic membrane to active transport of nutrients across the outer membrane. In the cytoplasmic membrane, this system consists of three known proteins, TonB, ExbB, and ExbD. ExbB and ExbD appear to harvest the protonmotive force and transmit it to TonB, which then makes direct physical contact with TonB-dependent active transport proteins in the outer membrane. Using two-dimensional gel electrophoresis, we found that ExbD exists as two different species with the same apparent molecular mass but with different pIs. The more basic ExbD species was consistently present, while the more acidic species arose when cells were starved for iron by the addition of iron chelators. The cause of the modification was, however, more complex than simple iron starvation. Absence of either TonB or ExbB protein also gave rise to modified ExbD under iron-replete conditions where the wild-type strain exhibited no ExbD modification. The effect of the tonB or exbB mutations were not entirely due to iron limitation since an equally iron-limited aroB mutation did not replicate the ExbD modification. This constitutes the first report of in vivo modification for any of the TonB system proteins.

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YraP Contributes to Cell Envelope Integrity and Virulence ofSalmonella entericaSerovar Typhimurium

Infection and Immunity ◽

10.1128/iai.00829-17 ◽

2018 ◽

Vol 86 (11) ◽

Cited By ~ 5

Author(s):

Faye C. Morris ◽

Timothy J. Wells ◽

Jack A. Bryant ◽

Anna E. Schager ◽

Yanina R. Sevastsyanovich ◽

...

Keyword(s):

Outer Membrane ◽

Cell Envelope ◽

Systemic Infection ◽

Eukaryotic Cell ◽

Infection Model ◽

Content Type ◽

Protein Levels ◽

K 12

ABSTRACTMutations in σE-regulated lipoproteins have previously been shown to impact bacterial viability under conditions of stress and duringin vivoinfection. YraP is conserved across a number of Gram-negative pathogens, includingNeisseria meningitidis, where the homolog is a component of the Bexsero meningococcal group B vaccine. Investigations using laboratory-adaptedEscherichia coliK-12 have shown thatyraPmutants have elevated sensitivity to a range of compounds, including detergents and normally ineffective antibiotics. In this study, we investigate the role of the outer membrane lipoprotein YraP in the pathogenesis ofSalmonella entericaserovar Typhimurium. We show that mutations inS. TyphimuriumyraPresult in a defective outer membrane barrier with elevated sensitivity to a range of compounds. This defect is associated with attenuated virulence in an oral infection model and during the early stages of systemic infection. We show that this attenuation is not a result of defects in lipopolysaccharide and O-antigen synthesis, changes in outer membrane protein levels, or the ability to adhere to and invade eukaryotic cell linesin vitro.

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