scholarly journals Triflic acid treatment enables LC-MS/MS analysis of insoluble bacterial biomass

2018 ◽  
Author(s):  
Ana Y. Wang ◽  
Peter S. Thuy-Boun ◽  
Gregory S. Stupp ◽  
Andrew I. Su ◽  
Dennis W. Wolan
Keyword(s):  
Acid Treatment ◽  
Cell Envelope ◽  
Human Microbiome ◽  
Bacterial Biomass ◽  
Shotgun Proteomics ◽  
Triflic Acid ◽  
Bacterial Proteins ◽  
Left Behind ◽  
Associated Proteins ◽  
Complementary Subset

ABSTRACTThe lysis and extraction of soluble bacterial proteins from cells is a common practice for proteomics analyses, but insoluble bacterial biomasses are often left behind. Here, we show that with triflic acid treatment, the insoluble bacterial biomass of Gram- and Gram+ bacteria can be rendered soluble. We use LC-MS/MS shotgun proteomics to show that bacterial proteins in the soluble and insoluble post-lysis fractions differ significantly. Additionally, in the case of Gram-Pseudomonas aeruginosa, triflic acid treatment enables the enrichment of cell envelope-associated proteins. Finally, we apply triflic acid to a human microbiome sample to show that this treatment is robust and enables the identification of a new, complementary subset of proteins from a complex microbial mixture.

scholarly journals Triflic Acid Treatment Enables LC-MS/MS Analysis of Insoluble Bacterial Biomass

2018 ◽  
Vol 17 (9) ◽  
pp. 2978-2986 ◽  
Author(s):  
Ana Y. Wang ◽  
Peter S. Thuy-Boun ◽  
Gregory S. Stupp ◽  
Andrew I. Su ◽  
Dennis W. Wolan
Keyword(s):  
Acid Treatment ◽  
Bacterial Biomass ◽  
Triflic Acid ◽  
Ms Analysis

scholarly journals Glycosylation and biogenesis of a family of serine-rich bacterial adhesins

Microbiology ◽  
2009 ◽  
Vol 155 (2) ◽  
pp. 317-327 ◽  
Author(s):  
Meixian Zhou ◽  
Hui Wu
Keyword(s):  
Biosynthetic Pathway ◽  
Protein Glycosylation ◽  
Second Step ◽  
Complex Nature ◽  
Bacterial Physiology ◽  
Bacterial Proteins ◽  
New Family ◽  
Bacterial Adhesins ◽  
Platelet Binding ◽  
Associated Proteins

Glycosylation of bacterial proteins is an important process for bacterial physiology and pathophysiology. Both O- and N-linked glycan moieties have been identified in bacterial glycoproteins. The N-linked glycosylation pathways are well established in Gram-negative bacteria. However, the O-linked glycosylation pathways are not well defined due to the complex nature of known O-linked glycoproteins in bacteria. In this review, we examine a new family of serine-rich O-linked glycoproteins which are represented by fimbriae-associated adhesin Fap1 of Streptococcus parasanguinis and human platelet-binding protein GspB of Streptococcus gordonii. This family of glycoproteins is conserved in streptococcal and staphylococcal species. A gene cluster coding for glycosyltransferases and accessory Sec proteins has been implicated in the protein glycosylation. A two-step glycosylation model is proposed. Two glycosyltransferases interact with each other and catalyse the first step of the protein glycosylation in the cytoplasm; the cross-talk between glycosylation-associated proteins and accessory Sec components mediates the second step of the protein glycosylation, an emerging mechanism for bacterial O-linked protein glycosylation. Dissecting the molecular mechanism of this conserved biosynthetic pathway offers opportunities to develop new therapeutic strategies targeting this previously unrecognized pathway, as serine-rich glycoproteins have been shown to play a role in bacterial pathogenesis.

scholarly journals Compartmentalized Cell Envelope Biosynthesis in Mycobacterium tuberculosis

2022 ◽  
Author(s):  
Julia Puffal ◽  
Ian L. Sparks ◽  
James R. Brenner ◽  
Xuni Li ◽  
John D. Leszyk ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Proteomic Analysis ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Membrane Domain ◽  
Protein Fusion ◽  
Final Maturation ◽  
Associated Proteins ◽  
Gradient Fractionation ◽  
Membrane Compartmentalization

The intracellular membrane domain (IMD) is a metabolically active and laterally discrete membrane domain initially discovered in Mycobacterium smegmatis. The IMD correlates both temporally and spatially with the polar cell envelope elongation in M. smegmatis. Whether or not a similar membrane domain exists in pathogenic species remains unknown. Here we show that the IMD is a conserved membrane structure found in Mycobacterium tuberculosis. We used two independent approaches, density gradient fractionation of membrane domains and visualization of IMD-associated proteins through fluorescence microscopy, to determine the characteristics of the plasma membrane compartmentalization in M. tuberculosis. Proteomic analysis revealed that the IMD is enriched in metabolic enzymes that are involved in the synthesis of conserved cell envelope components such as peptidoglycan, arabinogalactan, and phosphatidylinositol mannosides. Using a fluorescent protein fusion of IMD-associated proteins, we demonstrated that this domain is concentrated in the polar region of the rod-shaped cells, where active cell envelope biosynthesis is taking place. Proteomic analysis further revealed the enrichment of enzymes involved in synthesis of phthiocerol dimycocerosates and phenolic glycolipids in the IMD. We validated the IMD association of two enzymes, α1,3-fucosyltransferase and fucosyl 4-O-methyltransferase, which are involved in the final maturation steps of phenolic glycolipid biosynthesis. Taken together, these data indicate that functional compartmentalization of membrane is an evolutionarily conserved feature found in both M. tuberculosis and M. smegmatis, and M. tuberculosis utilizes this membrane location for the synthesis of its surface-exposed lipid virulence factors.

Proteomic profiling of cell envelope-associated proteins fromStaphylococcus aureus

PROTEOMICS ◽  
2006 ◽  
Vol 6 (5) ◽  
pp. 1530-1549 ◽  
Author(s):  
Christine L. Gatlin ◽  
Rembert Pieper ◽  
Shih-Ting Huang ◽  
Emmanuel Mongodin ◽  
Elizabeth Gebregeorgis ◽  
...  
Keyword(s):  
Cell Envelope ◽  
Proteomic Profiling ◽  
Associated Proteins

The structure of SAV1646 fromStaphylococcus aureusbelonging to a new `ribosome-associated' subfamily of bacterial proteins

2015 ◽  
Vol 71 (2) ◽  
pp. 332-337 ◽  
Author(s):  
Yuri N. Chirgadze ◽  
Teresa E. Clarke ◽  
Vladimir Romanov ◽  
Gera Kisselman ◽  
Jean Wu-Brown ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Rotation Axis ◽  
Genomic Structure ◽  
Large Subunit ◽  
Functional Model ◽  
Homology Search ◽  
Bacterial Proteins ◽  
Bacterial Ribosome ◽  
Acid Protein ◽  
Associated Proteins

The crystal structure of the SAV1646 protein from the pathogenic microorganismStaphylococcus aureushas been determined at 1.7 Å resolution. The 106-amino-acid protein forms a two-layer sandwich with α/β topology. The protein molecules associate as dimers in the crystal and in solution, with the monomers related by a pseudo-twofold rotation axis. A sequence-homology search identified the protein as a member of a new subfamily of yet uncharacterized bacterial `ribosome-associated' proteins with at least 13 members to date. A detailed analysis of the crystal protein structure along with the genomic structure of the operon containing thesav1646gene allowed a tentative functional model of this protein to be proposed. The SAV1646 dimer is assumed to form a complex with ribosomal proteins L21 and L27 which could help to complete the assembly of the large subunit of the ribosome.

scholarly journals Heteromeric Interactions among Nucleoid-Associated Bacterial Proteins: Localization of StpA-Stabilizing Regions in H-NS of Escherichia coli

2001 ◽  
Vol 183 (7) ◽  
pp. 2343-2347 ◽  
Author(s):  
Jörgen Johansson ◽  
Sven Eriksson ◽  
Berit Sondén ◽  
Sun Nyunt Wai ◽  
Bernt Eric Uhlin
Keyword(s):  
Escherichia Coli ◽  
Stable Form ◽  
Lon Protease ◽  
Wild Type ◽  
Bacterial Proteins ◽  
Regulatory Systems ◽  
Associated Proteins ◽  
Gene Regulatory

ABSTRACT The nucleoid-associated proteins H-NS and StpA inEscherichia coli bind DNA as oligomers and are implicated in gene regulatory systems. There is evidence for both homomeric and heteromeric H-NS–StpA complexes. The two proteins show differential turnover, and StpA was previously found to be subject to protease-mediated degradation by the Lon protease. We investigated which regions of the H-NS protein are able to prevent degradation of StpA. A set of truncated H-NS derivatives was tested for their ability to mediate StpA stability and to form heteromers in vitro. The data indicate that H-NS interacts with StpA at two regions and that the presence of at least one of the H-NS regions is necessary for StpA stability. Our results also suggest that a proteolytically stable form of StpA, StpAF21C, forms dimers, whereas wild-type StpA in the absence of H-NS predominantly forms tetramers or oligomers, which are more susceptible to proteolysis.

scholarly journals The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA

2020 ◽  
Vol 48 (19) ◽  
pp. 10820-10831
Author(s):  
Kiyoto Kamagata ◽  
Kana Ouchi ◽  
Cheng Tan ◽  
Eriko Mano ◽  
Sridhar Mandali ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Binding Modes ◽  
Dna Structures ◽  
Bacterial Proteins ◽  
Associated Proteins ◽  
Diffusion Mechanisms ◽  
Dynamics Simulations

Abstract DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.

scholarly journals Identification of Genes Encoding Exported Mycobacterium tuberculosis Proteins Using a Tn552′phoA In Vitro Transposition System

2000 ◽  
Vol 182 (10) ◽  
pp. 2732-2740 ◽  
Author(s):  
Miriam Braunstein ◽  
Thomas J. Griffin ◽  
Jordan I. Kriakov ◽  
Sarah T. Friedman ◽  
Nigel D. F. Grindley ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protective Immunity ◽  
Cell Envelope ◽  
Open Reading Frames ◽  
Signal Peptides ◽  
Genomic Libraries ◽  
Target Dna ◽  
Genes Encoding ◽  
Associated Proteins

ABSTRACT Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552′phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552′phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosisdatabases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.

scholarly journals Okadaic Acid, a Bioactive Fatty Acid fromHalichondria okadai, Stimulates Lipolysis in Rat Adipocytes: The Pivotal Role of Perilipin Translocation

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Nen-Chung Chang ◽  
Aming Chor-Ming Lin ◽  
Cheng-Chen Hsu ◽  
Jung-Sheng Liu ◽  
Leo Tsui ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipid Droplet ◽  
Okadaic Acid ◽  
Acid Treatment ◽  
Tyrosine Phosphatase ◽  
Droplet Surface ◽  
Vanadyl Acetylacetonate ◽  
Beta Actin ◽  
Associated Proteins ◽  
Perilipin A

Lipid metabolism in visceral fat cells is correlated with metabolic syndrome and cardiovascular diseases. Okadaic-acid, a 38-carbon fatty acid isolated from the black spongeHalichondria okadai, can stimulate lipolysis by promoting the phosphorylation of several proteins in adipocytes. However, the mechanism of okadaic acid-induced lipolysis and the effects of okadaic acid on lipid-droplet-associated proteins (perilipins and beta-actin) remain unclear. We isolated adipocytes from rat epididymal fat pads and treated them with isoproterenol and/or okadaic acid to estimate lipolysis by measuring glycerol release. Incubating adipocytes with okadaic acid stimulated time-dependent lipolysis. Lipid-droplet-associated perilipins and beta-actin were analyzed by immunoblotting and immunofluorescence, and the association of perilipin A and B was found to be decreased in response to isoproterenol or okadaic acid treatment. Moreover, okadaic-acid treatment could enhance isoproterenol-mediated lipolysis, whereas treatment of several inhibitors such as KT-5720 (PKA inhibitor), calphostin C (PKC inhibitor), or KT-5823 (PKG inhibitor) did not attenuate okadaic-acid-induced lipolysis. By contrast, vanadyl acetylacetonate (tyrosine phosphatase inhibitor) blocked okadaic-acid-dependent lipolysis. These results suggest that okadaic acid induces the phosphorylation and detachment of lipid-droplet-associated perilipin A and B from the lipid droplet surface and thereby leads to accelerated lipolysis.

scholarly journals Usnic Acid Treatment Changes the Composition of Mycobacterium tuberculosis Cell Envelope and Alters Bacterial Redox Status

mSystems ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Elwira Sieniawska ◽  
Rafal Sawicki ◽  
Wieslaw Truszkiewicz ◽  
Andrey S. Marchev ◽  
Milen I. Georgiev
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Acid Treatment ◽  
Cell Envelope ◽  
Redox Homeostasis ◽  
Usnic Acid ◽  
Lipid Synthesis ◽  
Redox Balance ◽  
Resistance Mechanisms ◽  
Content Type

ABSTRACT Mycobacterium tuberculosis developed efficient adaptation mechanisms in response to different environmental conditions. This resulted in the ability to survive in human macrophages and in resistance to numerous antibiotics. To get insight into bacterial responses to potent antimycobacterial natural compounds, we tested how usnic acid, a lichen-derived secondary metabolite, would influence mycobacteria at transcriptomic and metabolomic levels. The analysis of expression of sigma factors revealed a profound impact of usnic acid on one of the primary genetic regulatory systems of M. tuberculosis. Combined liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses allowed us to observe the perturbations in metabolic pathways, as well as in lipid composition, which took place within 24 h of exposure. Early bacterial response was related to redox homeostasis, lipid synthesis, and nucleic acid repair. Usnic acid treatment provoked disturbances of redox state in mycobacterial cells and increased production of structural elements of the cell wall and cell membrane. In addition, to increase the number of molecules related to restoration of redox balance, the rearrangements of the cell envelope were the first defense mechanisms observed under usnic acid treatment. IMPORTANCE The evaluation of mechanisms of mycobacterial response to natural products has been barely studied. However, it might be helpful to reveal bacterial adaptation strategies, which are eventually crucial for the discovery of new drug targets and, hence, understanding the resistance mechanisms. This study showed that the first-line mycobacterial defense against usnic acid, a potent antimicrobial agent, is the remodeling of the cell envelope and restoring redox homeostasis. Transcriptomic data correlated with metabolomics analysis. The observed metabolic changes appeared similar to those exerted by antibiotics.

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