Article

The MureinLipoprotein Linkage in the Cell Wall of Escherichia coli

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Abstract

After digestion of the murein-lipoprotein complex (rigid layer) of the E. coli cell wall with lysozyme the solubilized lipoprotein contained 2 disaccharide units of the murein per 1 lipoprotein molecule. From purely enzymatic degradation of the murein-lipoprotein complex with pronase and lysozyme the peptide GlcNAc-MurNAc-l-Ala-d-Glu-Dpm-Lys-Arg was isolated, where MurNAc =N-acetyl-muramic acid and Dpm =meso-2,6-diaminopimelic acid. The missing d-alanine in this repeating unit of the murein is replaced by the lysyl-arginine dipeptide of the lipoprotein. The lipoprotein molecules are therefore linked to the carboxyl group of the optical l-center of the diaminopimelic acid of the murein.

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... A large component of the periplasm is Braun's lipoprotein, which is the most abundant source of protein in gram-negative bacteria. 36 This helical protein trimer is covalently bound to the cell wall at the C-terminus via a peptide bond of a terminal lysine and the N-terminus is lipidated, with a cysteine based palmitoyl moiety, where this lipid group rests in the OM, see The chemical structure of the cell wall is comprised of N -Acetylmuramic acid (NAM) and N -Acetylglucosamine (NAG) monomers, see Figure 1.3. 38 These repeating units are glycosidically bonded into strands and the strands are cross linked via three-four peptide linkages between the tetrapeptide side chains on the NAM molecules. ...
... This essentially forms a harmonic, where an energy penalty is applied in order to prevent the particles that are restrained from moving during a simulation. This can be expanded upon due to the nature of x,y and z coordinates, see Equation 36, for the expanded form of Equation 35. ...
... BLP is the most abundant source of protein that is present in E. coli. This lipoprotein consists of a 58 amino acid sequence followed by a lipid moiety at the N-terminus 36 . 137 It was the first discovered lipoprotein. ...
Thesis
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Given the current rise in antibiotic resistant bacteria, E. coli takes center stageas a bacteria that is used commonly as a model organism. Bacteria possess a complex, crowded cellular environment that appears to be chaotic and disordered at a first glance. Their cellular structure allows them to survive and adapt to harsh conditions and varied environments. Molecular dynamics is a widely used approach to study biomolecules in biological conditions. Using this the fine, atomic level details of the forces that hold these molecules together and as a sum drive biological function forward can be revealed. This enables the specific interactions of biological building blocks, such as proteins, lipids and other polymers, in the E. coli environment, to be studied. Chaperone proteins can transport small molecules in the cellular environment, where this is not limited to bacteria, such as in the case of human apolipoprotein D. Using a marriage of experimentally sourced data as an anchor to reality and approximations based on theory, the compartment of the bacteria known as the periplasm was studied. In Chapter 3 it was observed that Braun’s lipoprotein (BLP) acts as a staple that bends and tilts and can interact with outer membrane protein A (OmpA) and the cell wall. This was extended to a full periplasm, in Chapter 4, where BLP interacts with the cell wall in the presence of OmpA and TolR. In this it was shown that TolR and OmpA can bind with the cell wall simultaneously. Chapter 5 focuses on the lipid transport Mla proteins. These proteins, MlaC, MlaD and MlaA were shown to be a favourable environment for lipid binding, where the docking of the protein components is explored in tandem with modelling. The focus of Chapter 6 is chaperone behaviour. LolA was shown to bind the BLP lipid moiety and that the MAC13243 molecule inhibited interaction, but not binding. Apolipoprotein-D showed preference for arachidonic acid and cholesterol, displaying a similar theme of small hydrophobic ligand binding as LolA. These studies have provided insight into molecular I nteractions that occur on a microscopic level within biological simulation.
... the free form that is not (Braun and Rehn, 1969;Braun and Bosch, 1972;Braun and Wolff, 1970). Cowles et al. recently showed that the free form of Lpp is surface exposed (Cowles et al., 2011). ...
... Established pathways of lipoprotein translocation through the OM involve either a Type II or Type V secretion system (Pugsley et al., 1990;Pugsley, 1993;Francetic and Pugsley, 2005;Sauvonnet and Pugsley, 1996;Coutte et al., 2003;van Ulsen et al., 2003) Beyond the involvement of Braun's lipoprotein Lpp in bacterial cell envelope stability (Braun and Wolff, 1970), lipoproteins were shown to play roles in a variety of cellular and pathogenic processes most recently reviewed by (Kovacs-Simon et al., 2010). In Borrelia spirochetes, the etiologic agents of arthropod-borne Lyme disease and relapsing fever, surface lipoproteins are particularly abundant and constitute the predominant class of known virulence factors at the vector/host-pathogen interface (Brandt et al., 1990;Kudryashev et al., 2009;Bergström et al., 2010;Norris et al., 2010;Barbour and Travinsky, 2010). ...
... We used fusions that localized to the inner membrane and surface in B. burgdorferi and expressed them in E. coli. In a variation of this approach we also expressed and localized Braun's lipoprotein (Lpp) from E. coli (Braun and Rehn, 1969;Braun and Wolff, 1970) in B. burgdorferi and determined its subcellular localization. ...
... Lpp and Pal are both transported to the OM via the Lol-ABCDE system (10) and are anchored to the inner leaflet of the OM (11). Lpp, the first bacterial lipoprotein to be identified, is a small (~8-kDa) lipoprotein which has been demonstrated to play an essential role in membrane integrity and permeability mediated through a covalent linkage between the -amino group of the C-terminal lysine residue in Lpp and the meso-diaminopimelic acid residue on the peptidoglycan peptide stem (12)(13)(14). Lpp is the most abundant OM protein (OMP) in E. coli at~500,000 molecules per cell. It is found as a PG-bound periplasmic form and in a form that spans the outer membrane to become surface exposed (15). ...
... Lpp was discovered approximately 40 years ago and is one of the best-studied bacterial lipoproteins. It exists in both a free form and a PG-bound form (12,13,34). Bound-form Lpp trimers maintain the integrity of the bacterial cell surface by covalent interaction between the C-terminal lysine and a meso-diaminopimelic acid residue in the peptide chain of the PG layer (13,16,(35)(36)(37). Lpp-deficient E. coli strains possess significant OM defects, including release of periplasmic enzymes, increased levels of OM blebs, and increased sensitivity to certain compounds (16,17). ...
... Lpp was discovered approximately 40 years ago and is one of the best-studied bacterial lipoproteins. It exists in both a free form and a PG-bound form (12,13,34). Bound-form Lpp trimers maintain the integrity of the bacterial cell surface by covalent interaction between the C-terminal lysine and a meso-diaminopimelic acid residue in the peptide chain of the PG layer (13,16,(35)(36)(37). Lpp-deficient E. coli strains possess significant OM defects, including release of periplasmic enzymes, increased levels of OM blebs, and increased sensitivity to certain compounds (16,17). ...
Article
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Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli. Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro, the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro. The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo. Compared to the wild-type strain, the lpp mutant 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 uropathogenic E. coli isolates.
... The LPS content of this murein-lipoprotein complex was certainly below 1% as judged by the content of hydroxymyristic acid of this preparation. Muropeptide-containing lipoprotein was prepared by degradation of murein-lipoprotein complex with lysozyme (18) . Lipoprotein was freed from the enzyme and the murein-degradation products by chromatography on Sephadex G75 (18) . ...
... Muropeptide-containing lipoprotein was prepared by degradation of murein-lipoprotein complex with lysozyme (18) . Lipoprotein was freed from the enzyme and the murein-degradation products by chromatography on Sephadex G75 (18) . The resulting lipoprotein contains on the average three muropeptides covalently bound . ...
... Both kinds of lipoprotein differ only in the carboxyl-terminal end of the polypeptide chain. The "muropeptides containing lipoprotein" were released from murein by lysozyme and consists of the complete lipid-polypeptide chain to which two to three muropeptides remain attached (18) . The "murein-free lipoprotein" released by trypsin consists of the lipid-polypeptide chain lacking the carboxyl-terminal amino acid sequence tyrosyl-arginyl-lysine (9,17) . ...
Article
The lipoprotein of the outer membrane of Escherichia coli is a B-cell mitogen in mice. Polyclonal activation of B lymphocytes was measured by an increase in thymidine uptake, by the development of plaque-forming cells against densely coupled trinitrophenylated sheep red cells, and by selectively increased rates of synthesis and secretion of leucine-labeled IgM. Murein-free and muropeptides-containing lipoprotein are effective in B-cell activation, while free murein is inactive. Removal of ester-linked fatty acids from the amino-terminal end of the lipoprotein by alkaline hydrolysis abolishes the mitogenicity of the lipoprotein. B lymphocytes from high responder (C3H/Tif and BALB/c nu/nu) or from low responder (C3H/HeJ) mice to the mitogen lipopolysaccharide (LPS) both respond well to the lipoprotein. Anti-immunoglobulin antibodies inhibit the mitogenic stimulation of B cells by lipoprotein. A complex of structures including the Ig-receptor molecules, the LPS receptor, and the lipoprotein receptor appear involved in the regulation of mitogenic stimulation of B cells to proliferation and differentiation to IgM-secreting cells.
... PG analysis of Ldt mutant strains of E. coli identified that three of these enzymes (ErfK, YbiS, YcfS) function by covalently attaching Braun's lipoprotein to the PG (Magnet et al., 2007b. This type of cross-link requires L,Dtranspeptidation because the lipoprotein is attached through the e-amino group of a lysine residue in the C terminus of the protein to the a-carbon of meso-DAP at the third position in the peptide stem (Braun & Sieglin, 1970;Braun & Wolff, 1970;Magnet et al., 2007b). These findings suggest a role for these Ldt enzymes in cell envelope stability, as Braun's lipoprotein has previously been shown to be an important factor in the stabilization of the outer membrane (Cowles et al., 2011). ...
Article
E. coli has five genes encoding L,D-transpeptidases (Ldt) with varied functions. Three of these enzymes (YbiS, ErfK, YcfS) have been shown to crosslink Braun's lipoprotein (Lpp) to the peptidoglycan (PG), while the other two (YnhG, YcbB), form direct meso- diaminopimelate (DAP-DAP, or 3-3) crosslinks within the PG. In addition, Ldt enzymes can also incorporate non-canonical D-amino acids, such as D-methionine, into the PG. To further investigate the role of these enzymes and in particular, 3-3 linkages, in cell envelope physiology we constructed and phenotypically characterized a variety of multiple Ldt deletion mutants of E. coli. We report that a triple deletion mutant lacking ybiS, erfK, and ycfS, is hypersusceptible to the metal chelating agent EDTA, leaks periplasmic proteins, and is resistant to the toxic effect of D-methionine. A double ynhG ycbB mutant had no discernable phenotype, however, examination of the phenotypes of various Ldt mutants bearing an additional DAP auxotrophic mutation (dapA::Cm) showed that a quintuple mutant strain lacking all Ldt genes was severely impaired for growth on media with limited DAP. These data demonstrate that loss of the E. coli Ldt enzymes involved with coupling the PG to Braun's lipoprotein resulted in the loss of OM stability while loss of the Ldt enzymes involved with DAP-DAP linkages had no observable effect on the cell envelope. Loss all Ldt enzymes proved detrimental to growth when cells were starved for DAP indicating a combined role for both 3-3 and Braun's lipoprotein crosslinks in cell viability only under a specific PG stress.
... The E.coli K-12 genome encodes 86 putative lipoproteins [3], but for many years TraT was the only lipoprotein that was known to be localized on the cell surface. Lpp, which is probably the best-studied bacterial lipoprotein, was shown to exist more than 40 years ago in a 'bound' form that is tethered to the peptidoglycan layer and a more abundant 'free' form [38][39][40][41], but evidence that the free form is surface exposed emerged only in 2011 [42]. A subpopulation of another lipoprotein that binds to peptidoglycan (Pal) also appears to be exposed on the cell surface [43]. ...
Article
Bacterial lipoproteins are hydrophilic proteins that are anchored to a cell membrane by N-terminally linked fatty acids. It is widely believed that nearly all lipoproteins produced by Gram-negative bacteria are either retained in the inner membrane (IM) or transferred to the inner leaflet of the outer membrane (OM). Lipoproteins that are exposed on the cell surface have also been reported but are generally considered to be rare. Results from a variety of recent studies, however, now suggest that the prevalence of surface-exposed lipoproteins has been underestimated. In this review we describe the evidence that the surface exposure of lipoproteins in Gram-negative bacteria is a widespread phenomenon and discuss possible mechanisms by which these proteins might be transported across the OM.
... This bacterium and other Gram-negatives typically have a thin cell wall (~6 nm in E. coli and ~2.5 nm in Pseudomonas aeruginosa) composed of one to three layers of peptidoglycan that is contained within the double membrane structure unique to these organisms (Vollmer & Bertsche, 2007). The cell walls of these bacteria are also characterized by the fact that they contain large amounts of murein lipoprotein, much of which is covalently attached through its C-terminal amino acid residues via peptide bonds with m-A 2 pm residues in the peptide stem (Braun & Wolff, 1970, Holtje, 1998. As the lipoprotein is also attached to the outer membrane through its terminal lipid residues, this serves to link the peptidoglycan and the cell envelope. ...
Article
In order to inject their DNA into the bacterial cytoplasm and establish infection, bacteriophages must ensure their genetic material successfully traverses both the bacterial membrane(s) and the layer of peptidoglycan surrounding the host cell. Phages accomplish this in a variety of ways, and some have virion-associated murein hydrolase enzymes that facilitate this process, particularly in conditions where the peptidoglycan is highly cross-linked. Phages that infect the mycobacteria must also contend with these barriers to infection, as well an impermeable layer of mycolic acids that decorates the cell surface; however, the mechanisms by which they do this are mostly unknown. In this regard, three small sequence motifs have been identified within mycobacteriophage tape measure proteins (TMPs) − extended molecules that span the tail lumen and determine its length − at least two of which have similarity to host proteins with muralytic activity. This suggests that phages may utilize regions of the TMP, which because of its location within the tail might be uniquely primed for host interaction, to facilitate localized peptidoglycan hydrolysis and DNA injection. The focus of this study is the motif found in the TMPs of mycobacteriophages Barnyard and Giles that has identity to a group of bacterial proteins known as resuscitation promoting factors (Rpfs). These Rpf proteins stimulate growth of non-growing bacteria and seem to exert their activity by cleaving inert peptidoglycan in the cell wall. Notably, the Barnyard Rpf Motif is contained within a 70 kDa C-terminal cleavage product of TMP, which appears to be cell wall- and/or membrane-associated during infection. Further, mycobacteria expressing TMP fragments containing this motif show aberrant behavior in culture and on solid media, and hybrid proteins in which the Rpf domain of the Micrococcus luteus Rpf protein is replaced with either of the phage motifs have muralytic activity in vivo. A recombineering-based method for generating mutations on lytically replicating mycobacteriophages has been developed and utilized to make multiple mutations in the Giles TMP motifs. Mutant phages infect host cells in late-stationary phase with a reduced efficiency, an observation that further supports a role for these motifs in cell wall hydrolysis during infection.
... Lpp, also known as Braun's lipoprotein, is the major lipoprotein in E. coli. It is a small 6 kDa protein that exists in two forms: one known as the 'bound form' represents Lpp molecules that are covalently cross-linked to PG [78,79]; another fraction is not attached to the PG and is known as the 'free form' [80]. The function of the free form is not understood. ...
Article
Full-text available
Bacterial lipoproteins are lipid-anchored proteins that contain acyl groups covalently attached to the N-terminal cysteine residue of the mature protein. Lipoproteins are synthesized in precursor form with an N-terminal signal sequence (SS) that targets translocation across the cytoplasmic or inner membrane (IM). Lipid modification and SS processing take place at the periplasmic face of the IM. Outer membrane (OM) lipoproteins take the localization of lipoproteins (Lol) export pathway, which ends with the insertion of the N-terminal lipid moiety into the inner leaflet of the OM. For many lipoproteins, the biogenesis pathway ends here. We provide examples of lipoproteins that adopt complex topologies in the OM that include transmembrane and surface-exposed domains. Biogenesis of such lipoproteins requires additional steps beyond the Lol pathway. In at least one case, lipoprotein sequences reach the cell surface by being threaded through the lumen of a beta-barrel protein in an assembly reaction that requires the heteropentomeric Bam complex. The inability to predict surface exposure reinforces the importance of experimental verification of lipoprotein topology and we will discuss some of the methods used to study OM protein topology.
... This means that 65% of the surfacome represents Lpp. These proteins are distinguished by a lipid moiety at the N terminus by which they are anchored either in the outer leaflet of the cytoplasmic membrane or, in Gramnegative bacteria, also in the inner leaflet of the outer membrane (2,3). Lpp are synthesized as precursors and are processed into mature forms at the cytoplasmic membrane. ...
Article
SUMMARY Since the discovery in 1973 of the first of the bacterial lipoproteins (Lpp) in Escherichia coli , Braun's lipoprotein, the ever-increasing number of publications indicates the importance of these proteins. Bacterial Lpp belong to the class of lipid-anchored proteins that in Gram-negative bacteria are anchored in both the cytoplasmic and outer membranes and in Gram-positive bacteria are anchored only in the cytoplasmic membrane. In contrast to the case for Gram-negative bacteria, in Gram-positive bacteria lipoprotein maturation and processing are not vital. Physiologically, Lpp play an important role in nutrient and ion acquisition, allowing particularly pathogenic species to better survive in the host. Bacterial Lpp are recognized by Toll-like receptor 2 (TLR2) of the innate immune system. The important role of Lpp in Gram-positive bacteria, particularly in the phylum Firmicutes , as key players in the immune response and pathogenicity has emerged only in recent years. In this review, we address the role of Lpp in signaling and modulating the immune response, in inflammation, and in pathogenicity. We also address the potential of Lpp as promising vaccine candidates.
... The only known protein that provides a covalent link to PGN is Braun's lipoprotein (BLP, also known as ''Lpp'' and ''murein lipoprotein''), which is one of the most abundant proteins in E. coli (7,8). BLP is anchored in the OM via a lipidated N-terminus, whereas the C-terminus is covalently attached to the peptide chain of PGN. ...
Article
Full-text available
Gram-negative bacteria such as Escherichia coli are protected by a complex cell envelope. The development of novel therapeutics against these bacteria necessitates a molecular level understanding of the structure-dynamics-function relationships of the various components of the cell envelope. We use atomistic MD simulations to reveal the details of covalent and noncovalent protein interactions that link the outer membrane to the aqueous periplasmic region. We show that the Braun's lipoprotein tilts and bends, and thereby lifts the cell wall closer to the outer membrane. Both monomers and dimers of the outer membrane porin OmpA can interact with peptidoglycan in the presence of Braun's lipoprotein, but in the absence of the latter, only dimers of OmpA show a propensity to form contacts with peptidoglycan. Our study provides a glimpse of how the molecular components of the bacterial cell envelope interact with each other to mediate cell wall attachment in E. coli.
... B acterial lipoproteins (Lpp) possess a lipid moiety at their N terminus that enables their anchorage to the bacterial membranes 1,2 . The maturation and processing of Lpp involve three enzymes: diacylglyceryl transferase (Lgt), lipoprotein signal peptidase (Lsp), and an apolipoprotein N-acyltransferase (Lnt). ...
Article
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Lipoproteins (Lpp) of Gram-positive bacteria are major players in alerting our immune system. Here, we show that the TLR2 response induced by commensal species Staphylococcus aureus and Staphylococcus epidermidis is almost ten times lower than that induced by noncommensal Staphylococcus carnosus, and this is at least partially due to their different modifications of the Lpp lipid moieties. The N terminus of the lipid moiety is acylated with a long-chain fatty acid (C17) in S. aureus and S. epidermidis, while it is acylated with a short-chain fatty acid (C2) in S. carnosus. The long-chain N-acylated Lpp, recognized by TLR2-TLR1 receptors, silences innate and adaptive immune responses, while the short-chain N-acetylated Lpp, recognized by TLR2-TLR6 receptors, boosts it.
... The only known protein that provides a covalent link to PGN is Braun's lipoprotein (BLP, also known as ''Lpp'' and ''murein lipoprotein''), which is one of the most abundant proteins in E. coli (7,8). BLP is anchored in the OM via a lipidated N-terminus, whereas the C-terminus is covalently attached to the peptide chain of PGN. ...
... Lpp or murein-lipoprotein is the most abundant lipoprotein of E. coli and it is estimated to be numerically the most abundant protein with more than 500,000 molecules per cell (Vaara, 1992;Neidhardt and Umbarger, 1996). Lpp consists of 58 amino and exists in two forms, (i) the "bound form", in which Lpp is covalently bound to the peptidoglycan layer via the ε-amino group of the C-terminal lysine (Nikaido, 1996) and (ii) the "free form" (Braun and Rehn, 1969;Braun and Wolff, 1970;Braun and Bosch, 1972). The function of the free from is not understood, however it has been shown that the "free from" is exposed to the surface by its C-terminus (Cowles et al., 2011). ...
Article
Full-text available
Bacterial resistance to classical antibiotics is emerging worldwide. The number of infections caused by multidrug resistant bacteria is increasing and becoming a serious threat for human health globally. In particular, Gram-negative pathogens including multidrug resistant Escherichia coli are of serious concern being resistant to the currently available antibiotics. All Gram-negative bacteria are enclosed by an outer membrane which acts as an additional protection barrier preventing the entry of toxic compounds including antibiotics and antimicrobial peptides (AMPs). In this study we report that the outer membrane component lipopolysaccharide (LPS) plays a crucial role for the antimicrobial susceptibility of E. coli BW25113 against the cationic AMPs Cap18, Cap11, Cap11-1-18m2, melittin, indolicidin, cecropin P1, cecropin B, and the polypeptide antibiotic colistin, whereas the outer membrane protease OmpT and the lipoprotein Lpp only play a minor role for the susceptibility against cationic AMPs. Increased susceptibility toward cationic AMPs was found for LPS deficient mutants of E. coli BW25113 harboring deletions in any of the genes required for the inner part of core-oligosaccharide of the LPS, waaC, waaE, waaF, waaG, and gmhA. In addition, our study demonstrates that the antimicrobial activity of Cap18, Cap11, Cap11-1-18m2, cecropin B, and cecropin P1 is not only dependent on the inner part of the core oligosaccharide, but also on the outer part and its sugar composition. Finally, we demonstrated that the antimicrobial activity of selected Cap18 derivatives harboring amino acid substitutions in the hydrophobic interface, are non-active against wild-type E. coli ATCC29522. By deleting waaC, waaE, waaF, or waaG the antimicrobial activity of the non-active derivatives can be partially or fully restored, suggesting a very close interplay between the LPS core oligosaccharide and the specific Cap18 derivative. Summarizing, this study implicates that the nature of the outer membrane component LPS has a big impact on the antimicrobial activity of cationic AMPs against E. coli. In particular, the inner as well as the outer part of the core oligosaccharide are important elements determining the antimicrobial susceptibility of E. coli against cationic AMPs.
... Braun and co-workers (13,14) have isolated and characterized a low molecular weight lipoprotein which is associated with the murein layer of the cell wall of gram negative bacteria. More recently, Melchers et al. (15) have demonstrated that this lipoprotein is a potent mitogen for lymphocytes from C3H/HeJ as well as other strains. ...
Article
The experiments by Sultzer and Nilsson (1), and later by Watson and Riblet (2), established that spleen cells from the C3H/HeJ strain of mouse were refractory to the mitogenic effects of bacterial lipopolysaccharides (LPS). More recently, however, experiments from our laboratory (3) demonstrated that spleen cells from C3H/HeJ mice were in fact responsive to some preparations of LPS but not to others, and that the method of extraction played a critical role in determining activity. In particular, preparations of LPS prepared by extraction with aqueous butanol had potent mitogenic activity. Our data showed that the mitogenic activity of such positive preparations of LPS coisolated with the LPS during gel filtration chromatography and subsequent equilibrium banding on CsCl. In addition, lipid A isolated from positive preparations of LPS was also capable of stimulating C3H/HeJ spleen cells. Taken together, these experiments provided rather convincing data that it was the LPS (in particular the lipid A) itself, or some contaminant very tightly bound to the lipid A, which was responsible for its biological activity. We further demonstrated that treatment of positive preparations of LPS with hot phenol rendered such preparations nonmitogenic for C3H/HeJ spleens, yet activity for other strains was only moderately decreased. These experiments would suggest either that the phenol treatment chemically alters the lipid A region of the LPS molecule or that such treatment removes the putative tightly bound contaminant responsible for C3H/HeJ mitogenesis. In the experiments reported here, we have explored in greater detail the role of lipid A in the stimulation of C3H/HeJ spleen cells. For these experiments we have utilized our earlier observations that the antibiotic polymyxin B forms a highly stable molecular complex with the lipid A region of LPS (4), and that such polymyxin B-LPS complexes are unable to mitogenically stimulate B lymphocytes (5). In addition, we have attempted to distinguish between the two potential modes of action of phenol on LPS, namely, the chemical alteration of the lipid A or the removal of a tightly bound contaminant by phenol treatment. The results of the experiments we report here support the interpretation that mitogenic activity of positive preparations of LPS is associated with a low mol wt phenol soluble polypeptide of approximately 10,000 mol wt. After partial purification, this polypeptide intitiates a significant mitogenic response at concentrations as low as 10 μg/ml. We conclude that the C3H/HeJ strain of mouse is a true nonresponder to the stimulatory effects of the lipid A region of LPS.
... 32 Further, within the peptidoglycan the meso-DAP provides the anchor point for Lpp. 33 Due to the close relationship between DapF and Lpp, the expectation was that knocking out the genes coding for these proteins would result in largely similar changes in the cellular envelopes of those strains. This was not observed in this study. ...
Article
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) using a (CO2)6k+ gas cluster ion beam (GCIB) was used to analyze E. coli mutants previously identified as having impaired plasmid transfer capability. The particular sub-set of mutants were select-ed as the mutations were expected to result in changes in the bacterial envelope composition, through the deletion of genes encod-ing for FabF, DapF and Lpp, where the surface sensitivity of ToF-SIMS can be most useful. Analysis of arrays of spotted bacteria allowed changes in the lipid composition of the bacteria to be elucidated using multivariate analysis and confirmed through imag-ing of individual ion signals. Significant changes in chemical composition were observed, including a surprising loss of cyclopro-panated fatty acids in the fabF mutant where FabF is associated with the elongation of FA(16:1) to FA(18:1) and not cyclopropane formation. The ability of the GCIB to generate increased higher mass signals from biological samples allowed intact lipid A (m/z 1796) to be detected on the bacteria and, despite a 40 keV impact energy, depth profiled through the bacterial envelope along with other high mass ions including species at m/z 1820 and 2428, attributed to ECACYC, that were only observed below the surface of the bacteria and were notably absent in the depth profile of the Lpp mutant. The analysis provides new insights into the action of the specific pathways targeted in this study and paves the way for whole new avenues for the characterization of intact molecules within the bacterial envelope.
... providing mechanical strength to the envelope structure (3) or in the transport of OM constituents (4,5). A thin peptidoglycan layer beneath the OM is connected to it through a specific lipoprotein (Lpp) (6) and a ␤-barrel (OmpA) (7). ...
Article
Lipid homeostasis is critical for proper envelope functions. The level of LpxC, which catalyzes the first committed step of lipopolysaccharide (LPS) synthesis, is controlled by an essential protease complex comprised of FtsH and YciM. Work carried out here suggests YejM, an essential envelope protein, plays a central role in sensing the state of LPS synthesis and controls LpxC levels by regulating the activity of FtsH/YciM. All four essential proteins are attractive targets of therapeutic development.
... With these few exceptions the elution profile of the E. coli muropeptides under our conditions is the same as already known. We also found one anhydro Tetra-Tetra muropeptide (peak 11), but we did not find any structures still containing lysine and arginine, which would be remnants from Braun's Lipoprotein 21 . Instead we found masses corresponding to muropeptides which had lost the GlcNAc moiety (Tab. ...
Article
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Peptidoglycan (PGN) is ubiquitous in nearly all bacterial species. ThePGNsacculus protects the cells against their own internal turgor making PGN one of the most important targets for antibacterial treatment. Within the last sixty years PGN composition has been intensively studied by various methods. The breakthrough was the application of HPLC technology on the analysis of muropeptides. However, preparation of pure PGN relied on a very time consuming method of about one week. We established a purification protocol for both Gram-positive and Gram-negative bacteria which can be completely performed in plastic reaction tubes yielding pure muropeptides within 24 hours. The muropeptides can be analyzed by UPLC-MS, allowing their immediate determination. This new rapid method provides the feasibility to screen PGN composition even in high throughput, making it a highly useful tool for basic research as well as for the pharmaceutical industry.
... First, to identify elements that mediate OM-PG tethering, we collected PG from eight proteobacterial species grown to stationary phase and analysed PG using an unbiased tandem mass spectrometry (MS/MS) approach 25 . A general search of the PG MS/MS data using species-specific proteomic databases identified the signature lysyl-arginyl dipeptide from Lpp covalently attached to PG in E. coli 26 [1][2][3][4]. With a few exceptions, these β-barrel proteins exhibit little sequence similarity; however, invariant N-terminal residues are observed in a species-specific manner at the predicted signal peptide cleavage site. ...
Article
Full-text available
Gram-negative bacteria have a cell envelope that comprises an outer membrane (OM), a peptidoglycan (PG) layer and an inner membrane (IM)¹. The OM and PG are load-bearing, selectively permeable structures that are stabilized by cooperative interactions between IM and OM proteins2,3. In Escherichia coli, Braun’s lipoprotein (Lpp) forms the only covalent tether between the OM and PG and is crucial for cell envelope stability⁴; however, most other Gram-negative bacteria lack Lpp so it has been assumed that alternative mechanisms of OM stabilization are present⁵. We used a glycoproteomic analysis of PG to show that β-barrel OM proteins are covalently attached to PG in several Gram-negative species, including Coxiella burnetii, Agrobacterium tumefaciens and Legionella pneumophila. In C. burnetii, we found that four different types of covalent attachments occur between OM proteins and PG, with tethering of the β-barrel OM protein BbpA becoming most abundant in the stationary phase and tethering of the lipoprotein LimB similar throughout the cell cycle. Using a genetic approach, we demonstrate that the cell cycle-dependent tethering of BbpA is partly dependent on a developmentally regulated L,D-transpeptidase (Ldt). We use our findings to propose a model of Gram-negative cell envelope stabilization that includes cell cycle control and an expanded role for Ldts in covalently attaching surface proteins to PG.
... Amidases remove peptides to create "naked" glycan strands during PG turnover. Murein lipoprotein (Braun's lipoprotein) is covalently linked to about 5-9% of the DAP residues and is anchored to the outer membrane by the Nterminal lipid (Braun, 1970;Hantke, 1973). There is also variation in glycan strand length. ...
... T he outer membrane (OM) of Escherichia coli and other Gramnegative bacteria is kept close to the cell through the action of several proteins that simultaneously interact with the OM and the peptidoglycan cell wall, serving as bridges between these structures (1). In E. coli, the Braun (Lpp) and Pal lipoproteins together with OmpA have this function (2)(3)(4)(5)(6)(7)(8). Lpp and OmpA are two of the most abundant proteins in the cell (9)(10)(11). ...
Article
The outer membrane of Gram-negative bacteria is an essential structure involved in nutrient uptake, protection against harmful substances and cell growth. Different proteins keep the outer membrane from blebbing out by simultaneously interacting with it and with the cell wall. These proteins have been mainly studied in enterobacteria, where OmpA and the Braun and Pal lipoproteins stabilize the outer membrane. Some degree of functional redundancy exists between these proteins since none of them is essential but the absence of two of them results in a severe phenotype. Caulobacter crescentus has a different strategy to maintain its outer membrane since it lacks the Braun lipoprotein and Pal is essential. In this work we characterized OmpA2, an OmpA like protein in this bacterium. Our results show that this protein is required for normal stalk growth and that it plays a minor role in the stability of the outer membrane. An OmpA2 fluorescent fusion showed that the concentration of this protein decreases from the stalked to the new pole. This localization pattern is important for its function and it depends on the position of the gene locus in the chromosome, and in consequence in the cell. This result suggests that little diffusion occurs from the moment that the gene is transcribed until the mature protein attaches to the cell wall in the periplasm. This mechanism reveals the integration of different levels of information from protein function down to genome arrangement, to allow the cell to self-organize.
... In Gram-negative bacteria, the OM and peptidoglycan layer are intimately associated. For example, the Lpp OM lipoprotein covalently anchors the OM to the peptidoglycan layer (47,48), while the OM lipoproteins LpoA and LpoB are needed for the proper synthesis of the peptidoglycan layer (49,50). Consequently, defects in the biogenesis of one can affect the biogenesis of the other. ...
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The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function.
... Lpp is also anchored to the outer membrane, thus contributing to the integrity of the cell wall by providing a covalent link between peptidoglycan and the outer membrane (Asmar & Collet, 2018). Depletion of Lpp leads to instability of the outer membrane, which causes leakage of periplasmic proteins and hypersensitivity to various antibiotics (Braun & Wolff, 1970). ErfK, YcfS, and YbiS are able to independently catalyze the formation of an amide bond between A2pm 3 of a donor peptide and the side-chain amine of the C-terminal Lys 58 of Braun's lipoprotein. ...
Thesis
Antibiotic resistance is a growing and global threat to human health that has led to an acute need for the development of new antibiotics. Elucidating the mechanism of inhibition of antibiotic targets is crucial for the development of more potent drugs. The essentiality of peptidoglycan and more than seventy years of successful use of β-lactams have made polymerization of this major cell wall component an attractive and validated target for drug development. Active-site serine Penicillin-Binding Proteins (PBPs) have long been considered as the only enzymes catalyzing the essential cross-linking step of peptidoglycan polymerization. The thesis explores inhibition of a distinct family of enzymes, the active-site cysteine L,D-transpeptidases (LDTs), that have a preponderant role in peptidoglycan synthesis in Mycobacterium tuberculosis. We show that the efficacy of LDT inhibition by β-lactams is primarily governed by the reactivity of the four-membered ring. We propose that acylation of LDTs by β-lactams proceeds through formation of an amine anion intermediate, followed by a subsequent irreversible step that is essential for the antibacterial activity of the drugs. A fluorescence spectroscopy approach enabling kinetic analyses of the acylation steps was developed to explore inactivation mechanisms and to evaluate the efficacy of new synthetic drugs. We also identify diazabicyclooctanes (DBOs) as new pharmacophores that inactivate LDTs by formation of a thio-carbamoyl-enzyme. We discuss several mechanism-based strategies for rational optimization of LDT inhibitors belonging to the β-lactam and DBO families.
... Murein lipoprotein (Lpp) —originally discovered in Escherichia coli—is the largest protein substituent, by molarity, in many species of Gram-negative bacteria (Braun and Sieglin, 1970;Braun and Wolff, 1970;Neidhardt and Curtiss, 1996;Sha et al., 2004;van Lier et al., 2014). The characterized function of Lpp is to anchor the outer membrane to the bacterial cell wall, aiding in stability and durability of the bacterial cell as a whole. ...
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Escherichia coli lipoprotein (Lpp) is a major cellular component that exists in two distinct states, bound-form and free-form. Bound-form Lpp is known to interact with the periplasmic bacterial cell wall, while free-form Lpp is localized to the bacterial cell surface. A function for surface-exposed Lpp has yet to be determined. We hypothesized that the presence of C-terminal lysinses in the surface-exposed region of Lpp would facilitate binding to the host zymogen plasminogen (Plg), a protease commandeered by a number of clinically important bacteria. Recombinant Lpp was synthesized and the binding of Lpp to Plg, the effect of various inhibitors on this binding, and the effects of various mutations of Lpp on Lpp-Plg interactions were examined. Additionally, the ability of Lpp-bound Plg to be converted to active plasmin was analyzed. We determined that Lpp binds Plg via an atypical domain located near the center of mature Lpp that may not be exposed on the surface of intact E. coli according to the current localization model. Finally, we found that Plg bound by Lpp can be converted to active plasmin. While the consequences of Lpp binding Plg are unclear, these results prompt further investigation of the ability of surface exposed Lpp to interact with host molecules such as extracellular matrix components and complement regulators, and the role of these interactions in infections caused by E. coli and other bacteria.
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Pathogenic leptospires can infect wide spectrum of hosts and they can survive in the environment long time. The outer membrane is the cellular component participated in interaction of microorganisms and environment. In present time several proteins located in the outer membrane of leptospires which are responsible for colonization of host organism, protection from influence of immune system of host, transport of substances in to the cell and other processes have been described. The outer membrane contains proteins and lipopolysaccharide molecules which have citotoxic effect. It was shown that regulation of protein composition of membranes depends on several factors of environment such as temperature, osmolarity, presence of certain substances in environment. Lipopolysaccharide and protein molecules of outer membranes have antigenic properties. These molecules can be used in practice as the components of vaccine against leptospiroses and diagnostic tools. Current review summarize information concerning structural organization of the outer membrane of leptospires, diversities of incoming parts of molecules and regulation of their synthesis. Moreover, perspectives of practical using of the outer membrane components in diagnostics and prevention of leptospiroses are presented.
Article
Biogenesis and Membrane Targeting of Lipoproteins, Page 1 of 2 Abstract Bacterial lipoproteins represent a unique class of membrane proteins, which are anchored to membranes through triacyl chains attached to the amino-terminal cysteine. They are involved in various functions localized in cell envelope. Escherichia coli possesses more than 90 species of lipoproteins, most of which are localized in the outer membrane, with others being in the inner membrane. All lipoproteins are synthesized in the cytoplasm with an N-terminal signal peptide, translocated across the inner membrane by the Sec translocon to the periplasmic surface of the inner membrane, and converted to mature lipoproteins through sequential reactions catalyzed by three lipoprotein-processing enzymes: Lgt, LspA, and Lnt. The sorting of lipoproteins to the outer membrane requires a system comprising five Lol proteins. An ATP-binding cassette transporter, LolCDE, initiates the sorting by mediating the detachment of lipoproteins from the inner membrane. Formation of the LolA-lipoprotein complex is coupled to this LolCDE-dependent release reaction. LolA accommodates the amino-terminal acyl chain of lipoproteins in its hydrophobic cavity, thereby generating a hydrophilic complex that can traverse the periplasmic space by diffusion. Lipoproteins are then transferred to LolB on the outer membrane and anchored to the inner leaflet of the outer membrane by the action of LolB. In contrast, since LolCDE does not recognize lipoproteins possessing Asp at position +2, these lipoproteins remain anchored to the inner membrane. Genes for Lol proteins are widely conserved among gram-negative bacteria, and Lol-mediated outer membrane targeting of lipoproteins is considered to be the general lipoprotein localization mechanism.
Article
A detailed investigation of the amino acids of cell wall preparations obtained by various methods from the BCG strain is reported.1“Non-peptidoglycan” amino acids represent about 15% of the weight of crude, delipidated cell walls (Table 1); trypsin-chymotrypsin treated, delipidated cell walls contain mainly the peptidoglycan amino acid alanine, glutamic acid and meso-2,2′-diaminopimelic acid, the ratio Ala/A2pm being 1.5 and the ratio Glu/A2pm being 2.3, as compared with the expected values of 2 and 1 for a classical peptidoglycan; there are, however, equal molar amounts of d-glutamic acid and diaminopimelic acid and the excess of glutamic acid is the l-form.2After enzymatic solubilization of the peptidoglycan by lysozyme or by Myxobacter AL1 enzyme 90% of the peptidoglycan amino acids are found in the soluble part; this has been fractionated on Sephadex G-50 and in most of the fractions obtained the ratio Glu/A2pm is 1, as expect ed, but the ratio Ala/A2pm is closer to 1 than to 2, indicating some other, not yet defined cross link to diaminopimelic acid.3The insoluble residue obtained after lysozyme or Myxobacter AL1 enzyme digestion contains a poly (l-glutamic acid); partial acid hydrolysis allows the solubilization of various l-glutamic acid oligopeptides, which have been studied by mass spectrometry, after N-acetylation and per-methylation; Fig. 3 shows a partial mass spectrum obtained from a peptide containing 6 or 7 glutamic acid residues, proving the sequence Glu-Glu-Glu-Glu; pyroglutamic acid is observed in all spectra, indicating that at least the N-terminal glutamic acid residue is in α-linkage. The intact polymer could not be isolated, but the largest fragment obtained has an average chain length of eleven glutamic acid residues.4The presence of l-glutamic acid polymers seems to be restricted to human and bovine strains of Mycobacteria and to Mycobacterium kansasii.
Article
A new structure of a lipid and its covalent linkage to a protein (murein-lipoprotein of the Escherichia coli outer membrane) is described. Glycerylcysteine (S-(propane-2′,3′-diol)-3-thio-2-aminopropanic acid) at the N-terminal end of the polypeptide chain is the attachment site of two ester-bound fatty acids. An additional fatty acid is bound as amide to the N-terminal group. The diglyceride residue on the cysteine could be derived from the phospholipid pathway since the fatty acid composition is very similar to that of the phospholipids from the same cells. In contrast, 65% of the amide-linked fatty acid is palmitate. The main fatty acids are palmitic acid (53%), cis-vaccenic acid (20.7%), 9,10-methylene-hexadecanoic acid (10.6%) and palmitoleic acid (9.4%). The structure of glycerylcysteine was established by chemical degradation of the compound containing the fatty acids, by incorporation studies of [35S]sulfate, [35S]cysteine, [14C]cystine, [methyl-3H]methionine and [2-3H]glycerol and by chemical synthesis. Glycerylcysteine was isolated as a constituent of the peptide (Ser)-Ser-Am-Ala-Lys. The C-terminal part of the lipopeptide sequence overlaps with the N-terminal end of the known sequence of the polypeptide chain. Its N-terminal position was confirmed by isolation of the N-terminal polypeptide fragment (position 1–31) after cleavage with cyanogen bromide. This contained the lipid.
Chapter
Cohn suggested that the resistance of bacteria to attack by acids and alkalies was because of the presence of a rigid structure surrounding the cell. The phenomena of plasmolysis has further helped in observing the first visual evidence for a cell wall. During plasmolysis the cytoplasm loses water and retracts from a rigid cell wall. It was then suggested that the retracted cytoplasm is itself surrounded by a membrane responsive to osmotic changes. This membrane was variously termed “cytoplasmic membrane” or “plasma membrane.” An intimate connection between cell wall and cytoplasmic membrane is expected in view of recent findings that the cytoplasmic membrane is involved in the biosynthesis of some cell–wall components. Cell walls of Gram-positive bacteria consist principally of an insoluble polymer, the peptidoglycan together with one or more other macromolecular components, which can be protein, polysaccharide or teichoic acid. The cell walls of Gram-negative bacteria are more complex—containing relatively small amounts of peptidoglycan and large amounts of protein, lipid, and lipopolysaccharides. This chapter describes the intracytoplasmic and cytoplasmic membranes of gram positive and negative bacteria along with details about bacterial cell wall.
Article
A strategy for the characterization of bacterial lipoprotein-in this case Braun's lipoprotein (an outer membrane 7-ku lipoprotein) isolated from Escherichia coli —is described by time-of-flight mass spectrometric (TOF/MS) techniques [252Cf plasma desorption (PD) TOF/MS and matrix-assisted laser desorption-ionization (MALDI) TOF/MS]. Covalent linkage of lipid at the N-terminal cysteine (posttranslationally modified to a S-[2,3-bis(acyloxy)-propyl]-N-acylcysteine) and, therefore, strict insolubility in aqueous solution constitute common features for this class of proteins. Relative molecular mass determination of the major molecular species of Braun's lipoprotein was obtained by selection of an appropriate mixture of organic solvents compatible with matrix/support materials useful for the mass spectrometric techniques applied. Minor components of this lipoprotein that differ only in the fatty acid composition of the lipid anchor were detected by PD TOF/MS after enzymatic release of the extremely hydrophobic N-terminal amino acid followed by selective extraction with chloroform. Part of the primary sequence of this lipoprotein was confirmed based on peptide fragment ions observed in the positive ion PD mass spectra of cyanogen bromide-generated peptide fragments that had been isolated previously by reverse phase high-performance liquid chromatography (HPLC). Peptidoglycan fragments that represent the attachment sites of lipoprotein to peptidoglycan were enzymatically released, separated by reverse phase HPLC, and finally characterized by time-of-flight mass spectrometric techniques (252Cf-PD TOF/MS, MALDI TOF/MS). The results obtained with both techniques differed only in the better sensitivity obtained with MALDI TOF/MS, which consumed a factor of 100 to 1000 less material than with PD TOF/MS.
Article
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Acinetobacter baumannii is a clinically important, predominantly health care–associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii , we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii .
Chapter
This chapter discusses the action of hydrolytic enzymes on model systems of aggregated lipids. In view of their specificity of action and the generally mild conditions under which they work, hydrolytic enzymes are excellent tools for the selective modification of membrane structure by elimination or alteration of a specific component or group of components. A membrane thus modified is then subject to study by other methods. The various aspects of organization and topography, and the correlates of molecular processes, are probably best manifested in the rate and extent of hydrolysis of various membrane components by specific enzymes. The molecular aspects are best manifested while considering modification and modulation of various specific membrane functions following treatment of functional membrane with hydrolytic enzymes. The chapter also discusses the modification of gross morphological features of membranes, the alteration of features relating to the general system properties, and the perturbation of specific molecular features that give rise to specialized membrane functions.
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Despite increasing evidence suggesting that antibiotic heteroresistance can lead to treatment failure, the significance of this phenomena in the clinic is not well understood, because many clinical antibiotic susceptibility testing approaches lack the resolution needed to reliably classify heteroresistant strains. Here we present G0790, a new globomycin analog and potent inhibitor of the Escherichia coli type II signal peptidase LspA. We demonstrate that in addition to previously known mechanisms of resistance to LspA inhibitors, unstable genomic amplifications containing lspA can lead to modest yet biologically significant increases in LspA protein levels that confer a heteroresistance phenotype.
Article
Background Brucella species (B. spp.) are Gram-negative intracellular bacteria, causing severe inflammatory diseases in animals and humans. Two major lipoproteins (L19) and (L16) of Brucella outer membrane proteins (OMPs) were extensively explored in associating with inflammatory response of human monocytes (THP-1). Methods Activated THP-1 cells induced with recombinant L19 and L16 were analyzed in comparison with unlipidated forms (U19 and U16) and lipopolysaccharide (LPS) of B. melitensis, respectively. Results Secretion of inflammatory factors TNF-α, IL-6 and IL-1β was significantly increased from L19, L16 or both stimulated THP-1 cells. High secretion of IL-18 was detected only from L19-induced cells. Signaling of those cytokine responses was identified mainly through P38-MAPK pathway, and signaling of L19-induced IL-1β response was partly occurred via NF-κB. Exploration for different forms of IL-18 found that L19-induced production of active IL-18 (18 kD) was through up-regulating NLRP3 and activating caspase-1, while L16-induced production of inactive IL-18 fragments (15 kD and 16 kD) occurred through activating caspase-8/3. Additionally, L19 up-regulated phosphorylation of XIAP for inhibiting caspase-3 activity to cleave IL-18, while L16 activated caspase-3 for producing GSDME-N and leading to pyroptosis of THP-1 cells. Conclusion Brucella L19 and L16 differentially induce IL-18 response or pyroptosis in THP-1 cells, respectively.
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Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first described Lgt inhibitor that potently inhibits Lgt biochemical activity in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of the major outer membrane lipoprotein, lpp , leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. Importance As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last fifty years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance; namely, the deletion of the major Gram-negative outer membrane lipoprotein, lpp . Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway, and potentially points to new antibacterial targets in this pathway.
Preprint
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Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify the first ever described Lgt inhibitors that potently inhibit Lgt biochemical activity in vitro and are bactericidal against wild-type Acinetobacter baumannii and E. coli strains. Unlike inhibition of other steps in lipoprotein biosynthesis, deletion of the major outer membrane lipoprotein, lpp , is not sufficient to rescue growth after Lgt depletion or provide resistance to Lgt inhibitors. Our data validate Lgt as a novel druggable antibacterial target and suggest that inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of downstream steps of bacterial lipoprotein biosynthesis and transport.
Article
To withstand the high intracellular pressure, the cell wall of most bacteria is stabilized by a unique cross-linked biopolymer called murein or peptidoglycan. It is made of glycan strands [poly-(GlcNAc-MurNAc)], which are linked by short peptides to form a co-valently closed net. Completely surrounding the cell, the murein represents a kind of bacterial exoskeleton known as the murein sacculus. Not only does the sacculus endow bacteria with mechanical stability, but in addition it maintains the specific shape of the cell. Enlargement and division of the murein sacculus is a prerequisite for-growth of the bacterium. Two groups of enzymes, hydrolases and synthases, have to cooperate to allow the insertion of new subunits into the murein net. The action of these enzymes must be well coordinated to guarantee growth of the stress-bearing sacculus without risking bacteriolysis. Protein-protein interaction studies suggest that this is accomplished by the formation of ct multienzyme complex, a murein-synthesizing machinery combining murein hydrolases and synthases. Enlargement of both the multilayered murein of gram-positive and the thin, single-layered murein of gram-negative bacteria seems to follow an inside-to-outside growth strategy. New material is hooked in a relaxed state underneath the stress-bearing sacculus before it becomes inserted upon cleavage of covalent bonds in the layer(s) under tension. A model is presented that postulates that maintenance of bacterial shape is achieved by the enzyme complex copying the preexisting murein sacculus that plays the role of a template.
Article
Protein concentration gradients play a relevant role in the organization of the bacterial cell. The Caulobacter crescentus protein OmpA2 forms an outer membrane polar concentration gradient. To understand the molecular mechanism that determines the formation of this gradient, we characterized the mobility and localization of the full protein and of its two structural domains an integral outer membrane β-barrel and a periplasmic peptidoglycan binding domain. Each domain has a different role in the formation of the OmpA2 gradient, which occurs in two steps. We also show that the OmpA2 outer membrane β-barrel can diffuse, which is in contrast to what has been reported previously for several integral outer membrane proteins in Escherichia coli , suggesting a different organization of the outer membrane proteins.
Article
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The cell envelope of Gram-negative bacteria is synthesized and maintained via mechanisms that are targets for development of novel antibiotics. Here we focus on the process of moving Braun’s lipoprotein (BLP) from the periplasmic space to the outer membrane of E. coli, via the LolA protein. In contrast to current thinking, we show that binding of multiple inhibitor molecules inside the hydrophobic cavity of LolA does not prevent subsequent binding of BLP inside the same cavity. Rather, based on our atomistic simulations we propose the theory that once inhibitors and BLP are bound inside the cavity of LolA, driven by hydrophobic interactions, they become entangled with each other. Our umbrella sampling calculations show that on the basis of energetics, it is more difficult to dislodge BLP from the cavity of LolA when it is uncomplexed compared to complexed with inhibitor. Thus the inhibitor reduces the affinity of BLP for the LolA cavity.
Chapter
Since this volume is concerned primarily with the action of toxins on eukaryotic cell surfaces, the inclusion of a chapter on toxic proteins, active only against certain bacteria, requires some justification at the outset. Such justification is provided on the grounds that obstacles encountered by large colicin molecules, seeking to reach specific intracellular targets in sensitive cells, are likely to be fundamentally the same as those shared by many animal and plant cell toxins. In addition, the ease of handling and the ability to apply to complex problems, powerful techniques such as genetic analysis or specialized molecular biological methods, makes the study of colicin-bacteria interactions an excellent model system for studies in more complex organisms.
Chapter
Pseudomonas are typical gram-negative bacteria and have the unique envelope architecture comprising of two membranes, the outer membrane and inner (cytoplasmic) membrane, separated by thick viscous periplasmic space which houses thin layer of peptidoglycan, the cell wall. The multilayered cell envelope limits the cell size and protects from environmental stresses and performs important functions such as nutrient acquisition, adhesion, secretion, signaling, pathogenicity and efflux pumps for exclusion of antibiotics. The outer membrane has asymmetrical structure in which the inner leaflet is composed of phospholipid similar to the double-layered inner membrane which is universal. The outer leaflet is composed of lipopolysaccharide having three subunits, a glycolipid, lipid A which holds it in position, and a core polysaccharide which forms a link between the lipid A and the O-antigen which extends outwards. The outer membrane allows selective permeability through porins embedded in OM and is a host to several other proteins and enzymes. The peptidoglycan is thin and is held in position in the periplasm by lipoproteins which anchor it to the outer membrane, and some molecules extend all through the periplasm between IM and OM. Periplasm is the site for several biological activities such as polymerization of macromolecules and export of several surface proteins and other molecules. Precursors for these are synthesized in the cytoplasm or inner surface of the inner membrane and then transported to the periplasm for polymerization. The inner membrane is the typical phospholipid bilayer forming a mosaic of proteins for nutrient transport, energy generation, syntheses of precursors for cell wall, outer membrane, etc. Genomic and proteomic analyses show that envelope represents more than 1/3 of the ORFs and is host to a large number of enzymes and proteins involved in transport and enzymatic reactions and as structural proteins.
Chapter
The outer membrane of gram-negative bacteria contains abundant copies of a limited number of proteins, the so-called major outer membrane proteins. One of the major outer membrane proteins in E. coli and other gram-negative bacteria is the murein lipoprotein (Braun and Rehn, 1969). The NH2 terminus of this protein consists of a novel amino acid, glycerylcysteine. Two moles of fatty acid are attached to the glyceryl moiety through ester linkage, and one mole of fatty acid is amide-linked to the amino group of the cysteine moiety (Hantke and Braun, 1973).
Chapter
This chapter discusses the identification and the characterization of the chemical moiety of LPS responsible for C3H/HeJ spleen cell mitogenicity. Unlike phenol-extracted LPS (P-LPS), LPS prepared by aqueous butanol extraction (BLPS) has potent mitogenic activity toward C3H/HeJ and also the responder C3H/St spleen cells. Polymyxin B (PB) has been shown to block the mitogenic response of mouse spleen cells to LPS. In a study described in the chapter, gel filtration chromatography of the phenol extract in 8 M urea revealed the presence of two low molecular weight protein fractions of equivalent specific activity. The mitogenic response to B-LPS and isolated LAP is decreased by greater than 50% following short periods of mild alkaline hydrolysis suggesting that the mitogenic activity of LAP and B-LPS is not because of lipid A.
Chapter
Endotoxin or lipopolysaccharide (LPS) is one of the most extraordinary bacterial products because of the variety of biologic effect it engenders in susceptible animals. Some of these reactions are undesirable, since they may be associated with pathologic changes and even with death. Others are beneficial when the result is enhanced resistance or immunity to toxemia and/or infection. It must be emphasized, however, that, depending upon the degree of reaction and the particular circumstances, one and the same effect may be either beneficial or harmful. Thus, high fever may have undesirable results or, as utilized some time ago, may be curative when used in patients to cure certain diseases or to enhance resistance. Pyrogenicity has been studied for more than two centuries, as reviewed in depth by Westphal et al. (1977). Similarly, an immune response to a given antigenic determinant may have either beneficial, harmful, or indifferent effects.
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A particulate enzyme system from strains of Escherichia coli is described which catalyzes the utilization of the uridine nucleotides, UDP-N-acetylmuramyl-l-Ala d-Glu-α, ε-diaminopimelyl-d-Ala-d-Ala and UDP-N-acetylglucosamine, for peptidoglycan synthesis. Unlike the systems previously studied in gram-positive cocci, this particulate enzyme catalyzes the terminal cross-linking reaction in cell wall synthesis, a transpeptidation in which a d-alanine residue is lost from the end of one of the N-acetylmuramyl-pentapeptide residues incorporated into the product. The d-alanine residue is also lost from the end of the second unit involved in the cross-linking, apparently through the action of a d-alanine carboxypeptidase. Both the transpeptidase and the d-alanine carboxypeptidase are inhibited by various penicillins and cephalosporins.
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d-Alanine carboxypeptidase I, which removes the terminal d-alanine residue of the uridine nucleotide, UDP-N-acetylmuramyl pentapeptide, and several related compounds occurs in both the particulate and soluble fractions of Escherichia coli and several other gram-negative bacteria. The soluble enzyme has been purified 120-fold and some of its properties are reported. The enzyme is competitively inhibited by penicillins and cephalosporins at very low concentrations. d-Alanine carboxypeptidase II, which catalyzes the hydrolysis of the penultimate d-alanine residue of the uridine nucleotide substrate, has also been purified and separated from d-alanine carboxypeptidase I. This enzyme is not inhibited by penicillins and cephalosporins. The possible physiological substrate and function of these enzymes are discussed.
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The three concentric layers of the cell wall of E. coli B (the outer lipoprotein layer, the intermediate lipopolysaccharide layer and the inner protein- and mucopolymer containing rigid layer) contribute 60%, 12% and 21%, respectively, to the total weight of the cell wall. — Treatment of the rigid layer with proteolytic enzymes removes the bulk of its protein components but leaves it otherwise intact: still rigid and cell-shaped, it is found to contain glucose, lipid and the mucopeptide constituents muramic acid, glucosamine, diaminopimelic acid, glutamic acid and alanine in molar ratios corresponding to the chemical compositions of known enzymatic mucopolymer split-products from E. coli-walls. Quantitative balance sheets of these components are made up and their implications on the structural concept of the rigid layer are discussed.
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E. coli B contains enzymes which can be assigned as mucopeptidhydrolase. They degrade cell wall mucopeptides of the same organism specifically into smaller molecules and are therefore valuable tools for structural analysis of these compounds. According to their specificity the five mucopeptidehydrolysases described below are defined as D-alanincarboxypeptidase. mucoendopeptidase. muramylamidase, N-acetylglucosaminidase and E. coli lysozyme. Their possible role as autolysins in the living cell is discussed.
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A simple, rapid, and reliable technique for identifying small amounts of phenylthiohydantoin (PTH) derivatives has been developed for use in the analysis by Edman degradation of small amounts of peptides isolated from fingerprints (10). Solvent systems used in paper and column chromatography of PTH amino acids have been adapted for use in thin-layer chromatography (TLC) on precoated flexible TL sheets with an incorporated fluorescent indicator (7). Twenty PTH amino acids can be identified on one TL sheet by ascending chromatography in one direction by the repeated utilization of two different solvent systems.
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After pronase treatment of the murein-lipoprotein complex (rigid layer) of the cell wall of E. coli B or E. coli K12 (W 945), lysine and arginine remain as the sole amino acids covalently bound to the murein (peptidoglycan, glycopeptide). These amino acids occur in equimolar amounts, each equal to the amount of lysine remaining with the murein after trypsin digestion of the murein-lipoprotein complex. From partial acid hydrolysates of such a murein, prepared by pronase digestion of the mureinlipoprotein complex, the following peptides have been isolated: (1) diaminopimelyl-lysyl-arginine; (2) alanyl-glutamyl-diaminopimelyl-lysyl-arginine; (3) glucosaminyl-muramyl-alanyl-glutamyl-diaminopimelyl-lysyl-arginine. Peptide 1 shows that the lipoprotein is bound by the α-amino group of the presumbaly N-terminal lysine to the carboxyl group of diaminopimelic acid. Peptide 2 consists of a peptide side chain of the murein to which the two amino acids of the N-terminal end of the lipoprotein, lysine and arginine, are attached. Peptide 3 constitutes a repeating unit of the murein to which the peptide lysyl-arginine of the lipoprotein is bound. The lipoprotein cleaved from the murein by a short trypsin digestion had an amino acid composition similar to the lipoprotein in the untreated murein-lipoprotein complex and arginine as N-terminal amino acid. The following structure is proposed: murein-lysyl-arginyl-lipoprotein. In the rapid reaction of trypsin with the cell wall the enzyme apparently cleaves at the C-terminal end of the lysime of the murein-lipoprotein linkage which results in murein-lysine and arginyl-lipoprotein. Some cleavage at the C-terminal end of arginine gives rise to free arginine (14%). These results support the supramolecular structure of the murein-lipoprotein complex previously proposed. On the average one lipoprotein molecule is covalently bound to every tenth repeating unit of the murein from which an average distance of 103 Å between two lipoprotein molecules along the polysaccharide chains of the murein is deduced.
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An uncross-linked monomer (nascent peptidoglycan unit) accumulates in cells of Staphylococcus aureus treated with low concentrations of penicillin G or with ampicillin, methicillin, or cephalothin. The uncross-linked monomer has been isolated, and analyses indicate that it represents a prefabricated subunit of the wall bearing both of the d-alanine residues of its pentapeptide precursor as well as an open pentaglycine chain. Pulse labeling experiments indicate that this uncross-linked unit is a direct precursor of the cross-linked peptidoglycan and that its cross-linking is inhibited by penicillin G. At high concentrations of penicillin G, wall synthesis ceases abruptly and no accumulation of the nascent peptidoglycan units is observed. These data have been obtained in support of the hypothesis that penicillins are substrate analogues of the d-alanyl-d-alanine end of the nascent peptidoglycan units and that they acylate the transpeptidase which catalyzes the cross-linking reaction. The data may also provide an explanation of the paradoxical observation that the killing rate in S. aureus by penicillin G is higher at low concentrations than at high concentrations of the antibiotic. In the presence of low concentrations a weakened wall may be formed, thus rendering the organisms more susceptible to lysis than at high concentrations of penicillin where wall synthesis abruptly ceases.
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A particulate enzyme system from strains of Escherichia coli is described which catalyzes the utilization of the uridine nucleotides, UDP-N-acetylmuramyl-l-Ala d-Glu-α, ε-diaminopimelyl-d-Ala-d-Ala and UDP-N-acetylglucosamine, for peptidoglycan synthesis. Unlike the systems previously studied in gram-positive cocci, this particulate enzyme catalyzes the terminal cross-linking reaction in cell wall synthesis, a transpeptidation in which a d-alanine residue is lost from the end of one of the N-acetylmuramyl-pentapeptide residues incorporated into the product. The d-alanine residue is also lost from the end of the second unit involved in the cross-linking, apparently through the action of a d-alanine carboxypeptidase. Both the transpeptidase and the d-alanine carboxypeptidase are inhibited by various penicillins and cephalosporins.
Article
A decrease of the absorbance at 578 nm of a cell wall suspension of mid log phase E. coli occurs when the suspension is incubated with trypsin. The reaction is so rapid that 55% of the total decrease is obtained within the first 2 min [ratio of enzyme to total cell wall protein = 1: 50 (w/w), room temperature]. The rate of the reaction is specific for trypsin when compared with other proteases, different lipases, lysozyme and other glycosidases. A peptide bond especially sensitive to trypsin could be localized within the complex cell wall by the demonstration that the decrease of the absorbance is paralleled by the splitting of the protein from the murein.
Article
In 1929, Fleming discovered penicillins and observed that this group of substances kills gram-positive bacteria more effectively than gram-negative bacteria (thus implying some important physiological difference between the two groups of organisms). I Subsequent knowledge of the bacterial cell wall made it possible to establish both on physiological and chemical grounds that penicillins are specific and highly selective inhibitors of the biosynthesis of cell walls, both in gram-positive and in gram-negative bacteria.2-6 The reason for the relative insensitivity of gramnegative bacteria to most penicillins has remained obscure. More recent knowledge of the structure and biosynthesis of bacterial cell walls has suggested that, in a terminal reaction in cell wall synthesis, linear glycopeptide strands are cross-linked in a transpeptidation, accompanied by release of the terminal D-alanine of the pentapeptide precursor, with formationi of a two- or threedimensional network. Direct chemical analyses of cell walls prepared from cells treated with penicillinll 8 and isotopic studies of wall biosynthesis9' 10 suggested that penicillin was interfering with this hypothetical g]ycopeptide cross-linking reaction. In the presence of penicillin, nascent glycopeptide, an uncross-linked mlonomeric uniit of the wall, accumulated.8 Pulse-labeling experiments have established that this nascent unit is an immediate precursor of the final cross-linked glycopeptide; penicillin completely inhibited its integration into the iietwork.10 Moreover, molecular models of penicillin resembled the acyl-D-alanyl-D-alanine in the ]inear glycopeptide and it was possible to suggest a molecular mlechanism for the transpeptidation and its inhibition by penicillin.8 However, the actual reaction or reactions inhibited had not been demoonstrated. In this paper we wish to report that a cell-free enzymatic system has been obtained from ce]ls of Escherichia coli which catalyzes this cross-linking reactiorn. We call the enzyme which catalyzes this step glycopeptide transpeptidase, since it catalyzes a reaction in which the peptide chains of two linear glycopeptide strands are cross-linked by a transpeptidation, in which the terminal D-alanine residue of one of the strands is elimirnated; this peptide bond synthesis proceeds without any other source of energy and is reversible. The terminal D-alanine residue of the other strand is also removed by hydrolysis catalyzed by a D-alanine carboxypeptidase in the preparation (Fig. 1). Both of these reactions are inhibited by low levels of penici]lin G, other penicillins, and a cephalosporin. Materials and Methods.-Preparation of substrates: UDP-MtirNAc L-ala -D-glu H3-mesoDAP D-ala D-ala and UDP-MurNAc L-ala- D-glu meso-DAP C14-D-ala C14-D-ala were prepared enzymatically by sequential addition of amino acids to the appropriate uridine nucleotides. lOa UJDP-GlcNAc-C14 is the same preparation uised previously. Preparation of enzyme: Two particulate enzymes, obtained from cells of E. coli strain Y-1O or E. coli strain B, were employed. The cells were ground with alumina. The fraction of the dis
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The protein sequenator is an instrument for the automatic determination of amino acid sequences in proteins and peptides. It operates on the principle of the phenylisothiocyanate degradation scheme. The automated process embraces the formation of the phenylthiocarbamyl derivative of the protein and the splitting off of the N-terminal amino acid as thiazolinone. The degradation proceeds at a rate of 15.4 cycles in 24 hours and with a yield in the individual cycle in excess of 98%. The material requirements are approximately 0.25 μmoles of protein. The thiazolinones are converted to the corresponding phenylthiohydantoins in a separate operation, and the latter identified by thin layer chromatography. The process has been applied to the whole molecule of apomyoglobin from the humpback whale, and it has been possible to establish the sequence of the first 60 amino acids from the N-terminal end.
Article
T2-phage enzyme or lysozyme remove from the complex cell wall of Escherichia coli B an integral component of one of its layers. The material is released in the form of several chemically closely related split products which have been characterized as mucopeptides. Two of these fragments, quantitatively predominant, were isolated in pure and crystalline form. One of them, designated C5, is made up from one residue each of N-acetyl-glucosamine, N-acetyl-muramic acid, glutamic acid, α,ϵ-diaminopimelic acid, and alanine. The molecule of the other fragment, C6, comprises one additional alanine residue. Ultracentrifugal determination of molecular weights definitely established the size of the fragments C5 and C6 to be as indicated.The arrangement of amino sugars and amino acids within each of the two fragments was cleared up in part. A short peptide chain, comprising either one (C5) or two (C6) alanines, one glutamic acid and one α,ϵ-diaminopimelic acid must be linked by its amino endgroup to the carboxyl group of N-acetylmuramic acid which carries the N-acetylglucosaminyl-residue attached by a β-glucosidic link.
Article
Free di-saccharide (N-acetylglucosamine-N-acetylmuramic acid) is released from a purified amino sugar complex, probably tetra-saccharide, by the action of egg-white lysozyme and of a similar enzyme secreted by a Streptomyces. The di-saccharide is also released from a purified poly-acetylamino sugar-peptide-di-saccharide compound by the action of the same enzymes on its poly-acetylamino sugar moiety. Differences in the affinity of egg-white lysozyme and of the Streptomyces enzyme for their substrates are discussed.A second bacteriolytic enzyme, also secreted by the Streptomyces, liberates free disaccharide from the purified peptide-di-saccharide and poly-acetylamino sugar-peptide-di-saccharide complexes by splitting the bond between the carboxyl group of muramic acid and the amino group of the peptide moiety.
Article
The structure conferring rigidity and shape on the complex cell wall of Escherichia coli strain B has been isolated in a state virtually free from other wall material. It shows a characteristic surface pattern which, together with observations on its mode of disintegration by phage enzyme or lysozyme, indicates the fairly simple principles of its construction.
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