scholarly journals MmpA, a Conserved Membrane Protein Required for Efficient Surface Transport of Trehalose Lipids in Corynebacterineae

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1760
Author(s):  
Tamaryn J. Cashmore ◽  
Stephan Klatt ◽  
Rajini Brammananth ◽  
Arek K. Rainczuk ◽  
Paul K. Crellin ◽  
...  
Keyword(s):  
Membrane Transport ◽  
Cell Walls ◽  
Membrane Lipid ◽  
Global Changes ◽  
Long Chain Fatty Acids ◽  
Inner Membrane ◽  
Surface Transport ◽  
Mycolic Acids ◽  
Outer Membranes ◽  
Lipidomic Analysis

Cell walls of bacteria of the genera Mycobacterium and Corynebacterium contain high levels of (coryno)mycolic acids. These very long chain fatty acids are synthesized on the cytoplasmic leaflet of the inner membrane (IM) prior to conjugation to the disaccharide, trehalose, and transport to the periplasm. Recent studies on Corynebacterium glutamicum have shown that acetylation of trehalose monohydroxycorynomycolate (hTMCM) promotes its transport across the inner membrane. Acetylation is mediated by the membrane acetyltransferase, TmaT, and is dependent on the presence of a putative methyltransferase, MtrP. Here, we identify a third protein that is required for the acetylation and membrane transport of hTMCM. Deletion of the C. glutamicum gene NCgl2761 (Rv0226c in Mycobacterium tuberculosis) abolished synthesis of acetylated hTMCM (AcTMCM), resulting in an accumulation of hTMCM in the inner membrane and reduced synthesis of trehalose dihydroxycorynomycolate (h2TDCM), a major outer membrane glycolipid. Complementation with the NCgl2761 gene, designated here as mmpA, restored the hTMCM:h2TDCM ratio. Comprehensive lipidomic analysis of the ΔtmaT, ΔmtrP and ΔmmpA mutants revealed strikingly similar global changes in overall membrane lipid composition. Our findings suggest that the acetylation and membrane transport of hTMCM is regulated by multiple proteins: MmpA, MtrP and TmaT, and that defects in this process lead to global, potentially compensatory changes in the composition of inner and outer membranes.

Characterization of lipopolysaccharide transport protein complex

2014 ◽  
Vol 9 (2) ◽  
pp. 131-138
Author(s):  
Quanju Xiang ◽  
Haiyan Wang ◽  
Zhongshan Wang ◽  
Yizheng Zhang ◽  
Changjiang Dong
Keyword(s):  
Transport Mechanism ◽  
Transport Proteins ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
Toxic Compounds ◽  
Outer Membranes ◽  
Harsh Conditions ◽  
Cytoplasmic Side

AbstractLipopolysaccharide (LPS) is an essential component of the outer membranes (OM) of most Gram-negative bacteria, which plays a crucial role in protection of the bacteria from toxic compounds and harsh conditions. The LPS is biosynthesized at the cytoplasmic side of inner membrane (IM), and then transported across the aqueous periplasmic compartment and assembled correctly at the outer membrane. This process is accomplished by seven LPS transport proteins (LptA-G), but the transport mechanism remains poorly understood. Here, we present findings by pull down assays in which the periplasmic component LptA interacts with both the IM complex LptBFGC and the OM complex LptDE in vitro, but not with complex LptBFG. Using purified Lpt proteins, we have successfully reconstituted the seven transport proteins as a complex in vitro. In addition, the LptC may play an essential role in regulating the conformation of LptBFG to secure the lipopolysaccharide from the inner membrane. Our results contribute to the understanding of lipopolysaccharide transport mechanism and will provide a platform to study the detailed mechanism of the LPS transport in vitro.

scholarly journals MtrP, a putative methyltransferase in Corynebacteria, is required for optimal membrane transport of trehalose mycolates

2020 ◽  
Vol 295 (18) ◽  
pp. 6108-6119 ◽  
Author(s):  
Arek K. Rainczuk ◽  
Stephan Klatt ◽  
Yoshiki Yamaryo-Botté ◽  
Rajini Brammananth ◽  
Malcolm J. McConville ◽  
...  
Keyword(s):  
Cell Walls ◽  
Double Mutant ◽  
Pathogenic Bacteria ◽  
Genetic Locus ◽  
Methyltransferase Activity ◽  
Mycolic Acids ◽  
Bacterial Cell Membrane ◽  
The Individual ◽  
Cytoplasmic Side ◽  
Long Chain

Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis (Mycobacterium tuberculosis) and diphtheria (Corynebacterium diphtheriae). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 (Rv0224c in M. tuberculosis) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae.

Localization of carbonic anhydrase in the cytoplasmic membrane of Neisseria sicca (strain 19)

1981 ◽  
Vol 27 (1) ◽  
pp. 87-92 ◽  
Author(s):  
M. N. MacLeod ◽  
I. W. DeVoe
Keyword(s):  
Carbonic Anhydrase ◽  
Density Gradient ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Carbonic Anhydrase Activity ◽  
Protein Patterns ◽  
Inner Membrane ◽  
Outer Membranes ◽  
Neisseria Sicca

The carbonic anhydrase activity and the growth of Neisseria sicca 19 were inhibited by the sulfonamide acetazolamide (10−5 M). Such inhibition was completely overcome by the addition of exogenous bicarbonate. Some carbonic anhydrase activity associated with the membranous envelope fraction of the cell was released when cells were broken by sonic treatment but not during cell breakage by high-pressure extrusion. After the selective solubilization (4 °C) of the inner membrane of envelopes by treatment with 1% sodium lauroyl sarcosinate, all detectable carbonic anhydrase activity was found in the soluble (inner membrane) fraction. After fractionation of the cell envelope into inner and outer membranes by treatment with ethylenediaminetetraacetate (EDTA) followed by sucrose density gradient centrifugation, the total and specific activity of carbonic anhydrase paralleled that of succinate dehydrogenase, an inner membrane enzyme marker. The Coomassie blue stained protein patterns after polyacrylamide gel electrophoresis of the bands from the sucrose density gradient provided confirmation that the inner and outer membranes had indeed been separated.

Morphological stabilization of the glycocalyces of 23 strains of five Bacteroides species using specific antisera

1984 ◽  
Vol 30 (6) ◽  
pp. 809-819 ◽  
Author(s):  
Dwight W. Lambe Jr. ◽  
K. J. Mayberry-Carson ◽  
Kaethe P. Ferguson ◽  
J. William Costerton
Keyword(s):  
Cell Walls ◽  
Indirect Fluorescent Antibody Test ◽  
Fluorescent Antibody ◽  
Antibody Test ◽  
Ruthenium Red ◽  
Intercellular Spaces ◽  
Outer Membranes ◽  
Bacteroides Species ◽  
Heterologous Antiserum ◽  
Specific Antisera

Cells of five Bacteroides species were examined following treatment with homologous antisera and staining with ruthenium red. They were enveloped by glycocalyces and these extensive fibrous exopolysaccharide matrices were fully retained as an integral "capsule" by some cells, while other cells showed "capsule" as well as detached glycocalyx components forming an intercellular "slime." These extensive glycocalyces collapsed during dehydration for electron microscopy and formed electron-dense accretions on cell surfaces and electron-dense reticula in intercellular spaces when the cells were treated with heterologous antiserum or when antibody stabilization was omitted. The glycocalyces of all strains, both stabilized and unstabilized, were observed outside the outer membranes of cell walls that showed the "classic" gram-negative structural organization. Appropriate modifications of the indirect fluorescent antibody test demonstrated an integral "capsule" on all strains examined; detached glycocalyx and varying amounts of slime were demonstrated after stabilization with homologous, but not heterologous, antiserum.

scholarly journals The Type IV Pilus Assembly Complex: Biogenic Interactions among the Bundle-Forming Pilus Proteins of Enteropathogenic Escherichia coli

2002 ◽  
Vol 184 (13) ◽  
pp. 3457-3465 ◽  
Author(s):  
Sandra W. Ramer ◽  
Gary K. Schoolnik ◽  
Cheng-Yen Wu ◽  
Jaiweon Hwang ◽  
Sarah A. Schmidt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Cell Compartment ◽  
Inner Membrane ◽  
Outer Membranes ◽  
Type Iv ◽  
Pilin Subunit ◽  
Membrane Compartments

ABSTRACT Production of type IV bundle-forming pili (BFP) by enteropathogenic Escherichia coli (EPEC) requires the protein products of 12 genes of the 14-gene bfp operon. Antisera against each of these proteins were used to demonstrate that in-frame deletion of individual genes within the operon reduces the abundance of other bfp operon-encoded proteins. This result was demonstrated not to be due to downstream polar effects of the mutations but rather was taken as evidence for protein-protein interactions and their role in the stabilization of the BFP assembly complex. These data, combined with the results of cell compartment localization studies, suggest that pilus formation requires the presence of a topographically discrete assembly complex that is composed of BFP proteins in stoichiometric amounts. The assembly complex appears to consist of an inner membrane component containing three processed, pilin-like proteins, BfpI, -J, and -K, that localize with BfpE, -L, and -A (the major pilin subunit); an outer membrane, secretin-like component, BfpB and -G; and a periplasmic component composed of BfpU. Of these, only BfpL consistently localizes with both the inner and outer membranes and thus, together with BfpU, may articulate between the Bfp proteins in the inner membrane and outer membrane compartments.

scholarly journals The Synthesis of Modified Trehalose Glycolipids: Towards Understanding Mincle and MCL Binding

2021 ◽  
Author(s):  
◽  
Jessica Helen Bird
Keyword(s):  
Cell Walls ◽  
Biological Activities ◽  
Chain Fatty Acid ◽  
Wild Type ◽  
Mycobacterium Species ◽  
Immunostimulatory Activity ◽  
Long Chain Fatty Acid ◽  
Mycolic Acids ◽  
Adjuvant Therapies ◽  
Long Chain

<p>Trehalose glycolipids are a diverse family of long-chain fatty acid diesters isolated from the cell walls of bacteria, in particular Mycobacterium species including M. tuberculosis. These molecules possess an array of biological activities which contribute to the survival and virulence of the organism,however, it is their activity as potent stimulators of innate and early adaptive immunity for which they are of interest. In particular, trehalose glycolipids have an application as adjuvants in vaccines and immunotherapies, for diseases such as tuberculosis (TB) and cancer. Recently, the macrophage-inducible C-type lectin, Mincle, and the macrophage C-type lectin, MCL, were identified as receptors for trehalose glycolipids, however, the exact mechanisms by which these receptors recognise and bind glycolipids is, as yet, unknown.This thesis presents the synthesis of a variety of structurally diverse trehalose glycolipid analogues. As such, three mycolic acids bearing a C22 α-chain and diversified meromycolate branches were prepared from an epoxide intermediate, itself prepared in eight steps from commercially available starting materials. The mycolic acids were then coupled to TMS-trehalose and subsequently deprotected to give the mono-and diester derivatives, 1a-cand 2c, which will be assessed for their immunostimulatory activity through the activation of wild type and Mincle-/-murine macrophages. This work provides a first step towards determining how the structures of trehalose glycolipids influence Mincle and MCL binding and activity, and allow for the development of improved trehalose glycolipids for use in adjuvant therapies.</p>

scholarly journals Aim24 and MICOS modulate respiratory function, tafazzin-related cardiolipin modification and mitochondrial architecture

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Max Emanuel Harner ◽  
Ann-Katrin Unger ◽  
Toshiaki Izawa ◽  
Dirk M Walther ◽  
Cagakan Özbalci ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Respiratory Function ◽  
Structure And Function ◽  
Complete Loss ◽  
Mitochondrial Ultrastructure ◽  
Inner Membrane ◽  
Outer Membranes ◽  
Contact Sites ◽  
Inner Membrane Protein ◽  
And Function

Structure and function of mitochondria are intimately linked. In a search for components that participate in building the elaborate architecture of this complex organelle we have identified Aim24, an inner membrane protein. Aim24 interacts with the MICOS complex that is required for the formation of crista junctions and contact sites between inner and outer membranes. Aim24 is necessary for the integrity of the MICOS complex, for normal respiratory growth and mitochondrial ultrastructure. Modification of MICOS subunits Mic12 or Mic26 by His-tags in the absence of Aim24 leads to complete loss of cristae and respiratory complexes. In addition, the level of tafazzin, a cardiolipin transacylase, is drastically reduced and the composition of cardiolipin is modified like in mutants lacking tafazzin. In conclusion, Aim24 by interacting with the MICOS complex plays a key role in mitochondrial architecture, composition and function.

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