Subtilis Membranes Research Articles

In-Cell DEER Spectroscopy of Nanodisc-Delivered Membrane Proteins in Living Cell Membranes.

Membrane proteins are integral to numerous cellular processes, yet their conformational dynamics in native environments remains difficult to study. This study introduces a nanodelivery method using nanodiscs to transport spin-labeled membrane proteins into the membranes of living cells, enabling direct in-cell double electron-electron resonance (DEER) spectroscopy measurements. We investigated the membrane protein BsYetJ, incorporating spin labels at key positions to monitor conformational changes. Our findings demonstrate successful delivery and high-quality DEER data for BsYetJ in both Gram-negative E. coli and Gram-positive B. subtilis membranes. The delivered BsYetJ retains its ability to transport calcium ions. DEER analysis reveals distinct conformational states of BsYetJ in different membrane environments, highlighting the influence of lipid composition on the protein structure. This nanodelivery method overcomes traditional limitations, enabling the study of membrane proteins in more physiologically relevant conditions.

Biophysical investigation into the antibacterial action of modelin-5-NH2.

Modelin-5-CONH2 (M5-NH2) is a synthetic antimicrobial peptide, which was found to show potent activity against Bacillus subtilis (minimum lethal concentration = 8.47 μM) and to bind strongly to membranes of the organism (Kd = 10.44 μM). The peptide adopted high levels of amphiphilic α-helical structure in the presence of these membranes (>50%), which led to high levels of insertion (Δπ ≥ 8.0 mN m-1). M5-NH2 showed high affinity for anionic lipid (Kd = 7.46 μM) and zwitterionic lipid (Kd = 14.7 μM), which drove insertion into membranes formed from these lipids (Δπ = 11.5 and 3.5 mN m-1, respectively). Neutron diffraction studies showed that M5-NH2 inserted into B. subtilis membranes with its N-terminal residue, L16, located 5.5 Å from the membrane centre, in the acyl chain region of these membranes, and promoted a reduction in membrane thickness of circa 1.8 Å or 5% of membrane width. Insertion into B. subtilis membranes by the peptide also promoted other effects associated with membrane thinning, including increases in membrane surface area (Cs-1 decreases) and fluidity (ΔGmix > 0 to ΔGmix < 0). Membrane insertion and thinning by M5-NH2 induced high levels of lysis (>55%), and it is speculated that the antibacterial action of the peptide may involve the toroidal pore, carpet or tilted-type mechanism of membrane permeabilization.

A Lysine Cluster in Domain II of Bacillus subtilis PBP4a Plays a Role in the Membrane Attachment of This C1-PBP.

In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K) residues, K86, K114, K119, and K265, protrude from domain II. Replacement of these amino acids with glutamine (Q) residues by site-directed mutagenesis yielded Mut4KQ PBP4a. When produced in Escherichia coli without its predicted Sec-signal peptide, wild-type (WT) PBP4a was found mainly associated with the host cytoplasmic membrane, whereas Mut4KQ PBP4a remained largely unbound. After purification, the capacities of the two proteins to bind to B. subtilis membranes were compared. The results were similar to those obtained in E. coli: in vitro, a much higher percentage of WT PBP4a than of Mut4KQ PBP4a was found to interact with B. subtilis membranes. Immunodetection of PBP4a in B. subtilis membrane extracts revealed that a processed form of this PBP (as indicated by its size) associates with the B. subtilis cytoplasmic membrane. In the absence of any amphiphilic peptide in PBP4a, the crown of positive charges on the surface of domain II is likely responsible for the cellular localization of this PBP and its attachment to the cytoplasmic membrane.

A Systematic Approach Towards Elucidation of the Mode of Action of a Bacterial Thermosensor

The Bacillus subtilis membrane harbors the temperature sensing and signaling protein DesK. At low temperatures it triggers expression of a desaturase, which introduces double bonds into pre-existing phospholipids, thereby regulating membrane fluidity. Recently it was discovered [1] that both sensing and transmission of DesK, which has five transmembrane segments, can be captured into one single chimerical transmembrane segment, the so-called ‘minimal sensor’. This simple system offers excellent perspectives to study the molecular detail of a biologically very important mechanism. As a first step we analyzed membranes of Bacillus subtilis grown at different temperatures with several biophysical techniques including 31P-NMR and Differential Scanning Calorimetry. We analyzed the membrane lipid headgroup and acyl chain composition and we identified transition temperature fluctuations related to the growth temperature. We found significant differences in membrane lipid composition and phase behavior for Bacillus subtilis membranes depending on growth temperatures. Furthermore the transmembrane segment of the minimal sensor was synthesized and incorporated in the Bacillus membranes. These vesicles were analyzed with Tryptophan Fluorescence Spectroscopy to measure the position of the minimal sensor with respect to the membrane, which shows the switch behavior between high and low temperature of the minimal sensor.[1] Cybulski LE, Martin M, Mansilla MC, Fernandez A, de Mendoza D. Membrane thickness cue for cold sensing in a bacterium. Curr. Biol. 2010

A Systematic Approach Towards Elucidation of the Mode of Action of a Bacterial Thermosensor

The Bacillus subtilis membrane harbors the temperature sensing and signaling protein DesK. At low temperatures it triggers expression of a desaturase, which introduces double bonds into pre-existing phospholipids, thereby regulating membrane fluidity. Recently it was discovered [1] that both sensing and transmission of DesK, which has five transmembrane segments, can be captured into one single chimerical transmembrane segment, the so-called ‘minimal sensor’. This simple system offers excellent perspectives to study the molecular detail of a biologically very important mechanism. As a first step here we analyzed membranes of Bacillus subtilis grown at different temperatures with several biophysical techniques including 31P-NMR and Differential Scanning Calorimetry. We analyzed the membrane lipid headgroup and acyl chain composition and we identified transition temperature fluctuations related to the growth temperature. We found significant differences in membrane lipid composition and phase behavior for Bacillus subtilis membranes depending on growth temperatures. The next step is to synthesize the transmembrane segment of the minimal sensor and incorporate it in these membranes exploiting their properties to elucidate the molecular mechanism of thermosensing in Bacillus subtilis. [1] Cybulski LE, Martin M, Mansilla MC, Fernandez A, de Mendoza D. Membrane thickness cue for cold sensing in a bacterium. Curr Biol. 2010 20(17):1539-44

Investigations into the ability of the peptide, HAL18, to interact with bacterial membranes

Halocidin was isolated from hemocytes, Halocynthia aurantium as a heterodimeric peptide consisting of two alpha-helical subunits, Hal15 and Hal18. Hal18 was shown to have antibacterial properties against Bacillus subtilis (MLC = 15 microM) and Escherichia coli (MLC = 100 microM). The peptide was shown to produce stable monolayers, which were characteristic of alpha-helical peptides predicted to orientate parallel to the surface of the interface. Constant area assays showed that Hal18 was surface active (4 microM) inducing surface pressure changes >30 mN m(-1) characteristic of membrane interactive peptides. The peptide induced stable surface pressure changes in monolayers that were mimetic of B. subtilis membranes (circa 7 mN m(-1)) and E. coli membrane-mimics (circa 4 mN m(-1)). Hal18 inserted readily into zwitterionic DOPE and anionic DOPG monolayers inducing surface pressure changes circa 8 mN m(-1) in both cases, providing evidence that interaction is not headgroup specific. Thermodynamic analysis of compression isotherms showed that the presence of Hal18 destabilised B. subtilis membranes (DeltaG (Mix) > 0), which is in contrast to its stabilising effect on E. coli lipid extract implying the differential antimicrobial efficacy may be driven by lipid packing.

Purified, Recombinant TagF Protein from Bacillus subtilis 168 Catalyzes the Polymerization of Glycerol Phosphate onto a Membrane Acceptor in Vitro

We report the first characterization of a recombinant protein involved in the polymerization of wall teichoic acid. Previously, a study of the teichoic acid polymerase activity associated with membranes from Bacillus subtilis 168 strains bearing thermosensitive mutations in tagB, tagD, and tagF implicated TagF as the poly(glycerol phosphate) polymerase (Pooley, H. M., Abellan, F. X., and Karamata, D. (1992) J. Bacteriol. 174, 646-649). In the work reported here, we have demonstrated an unequivocal role for tagF in the thermosensitivity of one such mutant (tagF1) by conditional complementation at the restrictive temperature with tagF under control of the xylose promoter at the amyE locus. We have overexpressed and purified recombinant B. subtilis TagF protein, and we provide direct biochemical evidence that this enzyme is responsible for polymerization of poly(glycerol phosphate) teichoic acid in B. subtilis 168. Recombinant hexahistidine-tagged TagF protein was purified from Escherichia coli and was used to develop a novel membrane pelleting assay to monitor poly(glycerol phosphate) polymerase activity. Purified TagF was shown to incorporate radioactivity from its substrate CDP-[(14)C]glycerol into a membrane fraction in vitro. This activity showed a saturable dependence on the concentration of CDP-glycerol (K(m) of 340 microm) and the membrane acceptor (half-maximal activity at 650 microg of protein/ml of purified B. subtilis membranes). High pressure liquid chromatography analysis confirmed the polymeric nature of the reaction product, approximately 35 glycerol phosphate units in length.

DNA interpolyelectrolyte complexes as a tool for efficient cell transformation

A tool was developed for enhancement of plasmid penetration into an intact cell, based on increasing DNA hydrophobicity via inclusion into a soluble interpolyelectrolyte complex (IPC) with polycations. The characteristics of formation of DNA IPC with synthetic polycations [poly(N-ethyl-4-vinylpyridinium)bromide (PVP) and PVP modified with 3% of N-cetyl-4-vinylpyridinium units (PVP-C)] were studied using ultracentrifugation and polyacrylamide gel electrophoresis methods. The conditions were established under which the mixing of DNA and polycation aqueous solutions results in the self-assembly of soluble IPC species. Incorporation of DNA into IPC results in the enhancement of DNA binding with isolated Bacillus subtilis membranes. A considerable increase in the efficiency of transformation of B. subtilis cells with pBC16 plasmid resulted from incorporation of the plasmid into the IPC with PVP and CVP.

Structure and functions of linkage unit intermediates in the biosynthesis of ribitol teichoic acids in Staphylococcus aureus H and Bacillus subtilis W23.

The stepwise formation and characterization of linkage unit intermediates and their functions in ribitol teichoic acid biosynthesis were studied with membranes obtained from Staphylococcus aureus H and Bacillus subtilis W23. The formation of labeled polymer from CDP-[14C]ribitol and CDP-glycerol in each membrane system was markedly stimulated by the addition of N-acetylmannosaminyl(beta 1----4)N-acetylglucosamine (ManNAc-GlcNAc) linked to pyrophosphorylyisoprenol. Whereas incubation of S. aureus membranes with CDP-glycerol and ManNAc-[14C]GlcNAc-PP-prenol led to synthesis of (glycerol phosphate) 1-3-ManNAc-[14C]GlcNAc-PP-prenol, incubation of B. subtilis membranes with the same substrates yielded (glycerol phosphate)1-2-ManNAc-[14C]GlcNAc-PP-prenol. In S. aureus membranes, (glycerol phosphate)2-ManNAc-[14C]GlcNAc-PP-prenol as well as (glycerol phosphate)3-ManNAc-[14C]GlcNAc-PP-prenol served as an acceptor for ribitol phosphate units, but (glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol did not. In B. subtilis W23 membranes, (glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol served as a better acceptor for ribitol phosphate units than (glycerol phosphate)2-ManNAc-[14C]GlcNAc-PP-prenol. In this membrane system (ribitol phosphate)-(glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol was formed from ManNAc-[14C]GlcNAc-PP-prenol, CDP-glycerol and CDP-ribitol. The results indicate that (glycerol phosphate)1-3-ManNAc-GlcNAc-PP-prenol and (glycerol phosphate)1-2-ManNac-GlcNAc-PP-prenol are involved in the pathway for the synthesis of wall ribitol teichoic acids in S. aureus H and B. subtilis W23 respectively.

Electron-paramagnetic-resonance spectroscopy of Bacillus subtilis cytochrome b558 in Escherichia coli membranes and in succinate dehydrogenase complex from Bacillus subtilis membranes.

Cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex was studied by electron-paramagnetic-resonance (EPR) spectroscopy. The cytochrome amplified in Escherichia coli membranes by expression of the cloned cytochrome gene and in the succinate dehydrogenase complex immunoprecipitated from solubilized B. subtilis membranes, respectively, is shown to be low spin with a highly anisotropic (gmax approximately equal to 3.5) EPR signal. The amino acid residues most likely forming fifth and sixth axial ligands to heme in cytochrome b558 are discussed on the basis of the EPR signal and the recently determined gene sequence (K. Magnusson, M. Philips, J.R. Guest, and L. Rutberg, J. Bacteriol. 166:1067-1071, 1986) and in comparison with other b-type cytochromes.

Transformation in Bacillus subtilis: purification and partial characterization of a membrane-bound DNA-binding protein.

In DNA binding-deficient mutants of Bacillus subtilis a competence-specific protein with a subunit molecular weight of 18,000 was absent. The native protein containing this subunit was purified from B. subtilis membranes by chromatography on hydroxyapatite, DEAE-cellulose, and Sephacryl S-200. This protein appeared to be complexed with a second protein of slightly lower molecular weight (17,000) and a different isoelectric point. The native protein complex (apparent molecular weight, 75,000) contained approximately equal amounts of the two polypeptides and showed a strong DNA-binding activity. Incubation of the complex with plasmid and bacteriophage DNA revealed nuclease activity, specifically directed toward double-stranded DNA. Predominantly single-stranded nicks and a limited number of double-stranded breaks were introduced in the presence of Mg2+ ions. In the presence of Mn2+ ions the complex produced low-molecular-weight breakdown products from the DNA.

High-molecular-weight penicillin-binding proteins from membranes of bacilli.

Mixtures of high-molecular-weight, cephalosporin-sensitive penicillin-binding proteins (PBPs) can be purified from Bacillus subtilis membranes by cephalosporin affinity chromatography (G. Kleppe and J. L. Strominger, J. Biol. Chem. 254:4856-4862, 1979). By appropriate modification of this technique, B. subtilis PBP 1 was purified to homogeneity, and a mixture of Bacillus stearothermophilus PBPs 1, 2, and 4 was isolated. [14C]penicillin-PBP complexes of high-molecular-weight PBPs purified from membranes of these two bacilli, after denaturation, were found to have chemical reactivities typical of the penicilloyl-serine derivative formed by D-alanine carboxypeptidase from B. stearothermophilus. Although enzymatic activity catalyzed by these and several other high-molecular-weight PBPs from gram-positive organisms has not been detected with cell wall-related substrates, a slow, enzymatic acylation of B. subtilis PBPs 1, 2ab, and 4 by [14C]-diacetyl-L-lysyl-D-alanyl-D-lactate was demonstrated. Further study is necessary to clarify the physiological relevance of the slow acylation by this analog of a natural cell wall biosynthetic intermediate.

Cytochrome b reducible by succinate in an isolated succinate dehydrogenase-cytochrome b complex from Bacillus subtilis membranes.

In previous work with membranes of Bacillus subtilis, the succinate dehydrogenase complex was isolated by immunoprecipitation of Triton X-100-solubilized membranes. The complex included a polypeptide with an apparent molecular weight of 19,000, probably attributable to apocytochrome. This paper reports the further characterization of this cytochrome and its relation to the respiratory chain of B. subtilis. The cytochrome was identified as cytochrome b, and its difference absorption spectra showed maxima at 426, 529, and 558 nm at room temperature. The oxidized cytochrome had an absorption maximum at 413 nm. The cytochrome was reduced by succinate in the isolated succinate dehydrogenase complex and in Triton X-100-solubilized membranes. In whole membranes cytochromes b, c, and a were reduced by succinate. In membranes from a mutant containing normal cytochromes but lacking succinate dehydrogenase no reduction of cytochrome was seen with succinate. It was concluded that the isolated succinate dehydrogenase-cytochrome b complex is a functional unit in the intact B. subtilis membrane. An accompanying paper describes cytochrome b as a structural unit involved in the membrane binding of succinate dehydrogenase.

Studies of the high molecular weight penicillin-binding proteins of Bacillus subtilis.

Seven or eight penicillin-binding proteins (PBPs) were detected in Bacillus subtilis membranes. By introducing covalent affinity chromatography employing cephalosporins as ligands, milligram amounts of three high molecular weight PBPs (PBP 1 ab, Mr = 120,000; PBP 2b, Mr = 94,000; and PBP 4, Mr = 78,000) were obtained without any contamination of the major PBP 5, the D-alanine carboxypeptidase. Small amounts of pure PBP 2b could be isolated by manipulation of the affinity chromatography conditions. Structural and physical properties of these proteins as well as the generation of one major penicilloyl peptide from each PBP by digestion with pepsin suggest that each PBP is the product of a separate gene. No enzymatic activity could be found in mixtures of these high molecular weight PBPs employing substrates used for the transpeptidase and D-alanine carboxypeptidase assays in particulate membrane fractions.

Tunicamycin, an inhibitor of [formula omitted] peptidoglycan synthesis: A new site of inhibition

Bacillus subtilis membranes can transfer either N-acetylmuramyl-pentapeptide phosphate or N-acetylglucosaminyl phosphate from UMP directly onto undecaprenyl phosphate. Tunicamycin blocks only the latter transfer and inhibits peptidoglycan synthesis by toluenized cells of Bacillus megaterium utilizing added nucleotide sugar precursors or cell wall synthesis by intact cells of B. subtilis . Tunicamycin prevents formation of the cell wall disaccharide lipid intermediate by blocking transfer of N-acetylglucosamine onto undecaprenyl muramyl pentapeptidyl pyrophosphate.

The effects of some cations and anions on spin labeled cytoplasmic membranes of bacillus subtilis

The effects of CaCl 2, MgCl 2, LaCl 3 and some alkali halides on Bacillus subtilis cytoplasmic membranes were studied using stearic acid spin labels. The results indicate that two mechanisms are operating when these ions interact with B. subtilis membranes. At low ionic concentrations (0 to 0.1 M) there is direct cation binding to the anionic membranes which in the case of Ca 2+, Mg 2+ and La 3+, confers rigidity on the membranes and reaches saturation when the number of cations present equals the number of anionic sites on the lipids. At high concentrations there is a further effect on the membranes that parallels the known organizing/disorganizing effects that the ions studied have on water structure.

Five Penicillin-binding Components Occur in Bacillus subtilis Membranes

Abstract Membranes of Bacillus subtilis to which [14C]penicillin G had been bound were fractionated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Five labeled components were separated. These five components differed from each other in the rates at which they bound penicillins and cephalosporins by up to four orders of magnitude. The major penicillin-binding component, V, was identified as the d-alanine carboxypeptidase, whose activity is not vital for the cell. Three of the other components, in contrast, bound penicillins and cephalosporins at rates comparable to that of the penicillin killing site in this organism.