The isolation and characterisation of cytoplasmic and outer membranes from Micrococcus cryophilus

1984 ◽  
Vol 30 (11) ◽  
pp. 1357-1366 ◽  
Author(s):  
Geoffrey M. Lloyd ◽  
Nicholas J. Russell
Keyword(s):  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Phospholipid Composition ◽  
Marker Enzymes ◽  
Negative Bacterium ◽  
Gram Negative ◽  
Outer Membranes ◽  
Phospholipid Classes

The cytoplasmic and outer membranes of the Gram-negative bacterium, Micrococcus cryophilus, have been separated and purified. Both membrane preparations consist of a mixture of closed and apparently open vesicles, which vary in size but those of the outer membrane are on average 1.5 times the diameter of those of the cytoplasmic membrane. The membranes were characterised by their 2-keto-3-deoxyoctonate content and the activity of marker enzymes. There are gross differences in the protein and phospholipid composition of the two membranes. The outer membrane contains three major polypeptides, whereas the cytoplasmic membrane has many more. In addition the outer membrane is enriched in cardiolipin, at the expense of phosphatidylglycerol and phosphatidylethanolamine, relative to the cytoplasmic membrane. There are no significant differences in the fatty acid composition of the phospholipid classes, but all phospholipids of the outer membrane were slightly more saturated than those of the cytoplasmic membrane. Wax esters are present in both cytoplasmic and outer membranes. The significance of these findings is discussed in relation to known differences in the fluidity of the cytoplasmic and outer membranes in M. cryophilus and the specialized roles these membranes play.

scholarly journals An ExbD Disordered Domain Peptide Inhibits TonB System Activity

2020 ◽  
Author(s):  
Dale R. Kopp ◽  
Kathleen Postle
Keyword(s):  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Protonmotive Force ◽  
Gram Negative Bacteria ◽  
System Function ◽  
Gram Negative ◽  
Conserved Motif ◽  
Outer Membranes ◽  
Tonb System

ABSTRACTThe TonB system energizes transport of essential nutrients, such as iron siderophores, across unenergized outer membranes of Gram-negative bacteria. The integral cytoplasmic membrane proteins of the TonB system--ExbB, ExbD, and TonB--transduce the protonmotive force of the cytoplasmic membrane to TonB-dependent outer membrane transporters for active transport. ExbD protein is anchored in the cytoplasmic membrane, with the majority of it occupying the periplasm. We previously identified a conserved motif within a periplasmic disordered domain that is essential for TonB system function. Here we demonstrated that export of a peptide derived from that motif into the periplasm prevented TonB system function and inhibited all known ExbD interactions in vivo. Formaldehyde crosslinking captured the ExbD peptide in multiple ExbD and TonB complexes. Furthermore, peptides with mutations in the conserved motif not only had significantly reduced ability to inhibit TonB system activity, but they also altered interactions with ExbD and TonB, indicating the specificity of the interaction. Conserved motif peptide interactions with ExbD and TonB mostly occurred between Stage II and Stage III of the TonB energy transduction cycle, a transition that is characterized by the use of protonmotive force. Taken together, the data suggest that the ExbD disordered domain motif has multiple interactions with TonB and ExbD during between Stage II and III of the TonB energization cycle. Because of the essentiality of the motif, it may be a potential template for design of novel antibiotics that target the TonB system.IMPORTANCEGram-negative bacteria are intrinsically antibiotic-resistant due to the diffusion barrier posed by their outer membranes. The TonB system allows them to circumvent this barrier for their own nutritional needs, including iron. The ability of bacteria to acquire iron is a virulence factor for many Gram-negative pathogens. However, no antibiotics currently target the TonB system. Because TonB and ExbD must interact productively in the periplasm for transport across the outer membrane, they constitute attractive targets for potential antibiotic development where chemical characteristics need not accommodate the need to cross the hydrophobic cytoplasmic membrane. Here we show that a small ExbD-derived peptide can interfere with the TonB-ExbD interaction to inhibit the TonB system in vivo.

scholarly journals Phospholipid classes and fatty acid composition of ewe’s and goat’s milk

2013 ◽  
Vol 64 (3) ◽  
pp. 304-310 ◽  
Author(s):  
P. Hueso ◽  
L. Zancada ◽  
F. Pérez-Díez ◽  
F. Sánchez-Juanes ◽  
J. M. Alonso ◽  
...  
Keyword(s):  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Acid Composition ◽  
Goat’S Milk ◽  
Phospholipid Classes ◽  
Goat's Milk

scholarly journals Membrane fusion during phage lysis

2015 ◽  
Vol 112 (17) ◽  
pp. 5497-5502 ◽  
Author(s):  
Manoj Rajaure ◽  
Joel Berry ◽  
Rohit Kongari ◽  
Jesse Cahill ◽  
Ry Young
Keyword(s):  
Membrane Fusion ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Bacterial Host ◽  
Inner Membrane ◽  
Gram Negative ◽  
Encoded Proteins ◽  
Coliphage Lambda ◽  
Necessary And Sufficient

In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG.

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

2019 ◽  
Vol 116 (43) ◽  
pp. 21748-21757 ◽  
Author(s):  
Elizabeth M. Hart ◽  
Angela M. Mitchell ◽  
Anna Konovalova ◽  
Marcin Grabowicz ◽  
Jessica Sheng ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Small Molecule ◽  
Cytoplasmic Membrane ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bam Complex ◽  
Induced Aggregation

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 β-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.

scholarly journals The Outer Surface Lipoprotein VolA Mediates Utilization of Exogenous Lipids by Vibrio cholerae

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Aaron C. Pride ◽  
Carmen M. Herrera ◽  
Ziqiang Guan ◽  
David K. Giles ◽  
M. Stephen Trent
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Carbon Source ◽  
Outer Membrane ◽  
Vibrio Cholerae ◽  
Homeoviscous Adaptation ◽  
Gram Negative ◽  
Content Type ◽  
Enzymatic Function ◽  
Gram Negative Organisms

ABSTRACTPrevious work from our laboratory showed that the Gram-negative aquatic pathogenVibrio choleraecan take up a much wider repertoire of fatty acids than other Gram-negative organisms. The current work elaborated on the ability ofV. choleraeto exploit an even more diverse pool of lipid nutrients from its environment. We have demonstrated that the bacterium can use lysophosphatidylcholine as a metabolite for growth. Using a combination of thin-layer chromatography and mass spectrometry, we also showed that lysophosphatidylcholine-derived fatty acid moieties can be used for remodeling theV. choleraemembrane architecture. Furthermore, we have identified a lysophospholipase, VolA (Vibrioouter membrane lysophospholipase A), required for these activities. The enzyme is well conserved inVibriospecies, is coexpressed with the outer membrane fatty acid transporter FadL, is one of very few surface-exposed lipoprotein enzymes to be identified in Gram-negative bacteria and the first instance of a surface lipoprotein phospholipase. We propose a model whereby the bacterium efficiently couples the liberation of fatty acid from lysophosphatidylcholine to its subsequent metabolic uptake. An expanded ability to scavenge diverse environmental lipids at the bacterial surface increases overall bacterial fitness and promotes homeoviscous adaptation through membrane remodeling.IMPORTANCEOur understanding of how bacteria utilize environmental lipid sources has been limited to lipids such as fatty acids and cholesterol. This narrow scope may be attributed to both the intricate nature of lipid uptake mechanisms and the diversity of lipid substrates encountered within an ecological niche. By examining the ability of the pathogenVibrio choleraeto utilize exogenous lipids, we uncovered a surface-exposed lipoprotein (VolA) that is required for processing the prevalent host lipid lysophosphatidylcholine. VolA functions as a lipase liberating a fatty acid from exogenous lysophospholipids. The freed fatty acid is then transported into the cell, serving as a carbon source, or shunted into phospholipid synthesis for membrane assembly. A limited number of surface-exposed lipoproteins have been found in Gram-negative organisms, and few have enzymatic function. This work highlights the ability of bacteria to exploit exogenous lipids for both maintenance of the membrane and carbon source acquisition.

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