Morphogenetic Aspects of Murein Structure and Biosynthesis

The shape of Escherichia coli is fixed by the form of the sacculus. This sacculus is a macromolecule made up from the polymer murein. In an investigation of the possible factors determining the shape of the sacculus, we attempted to resolve between two fundamental alternatives. (i) Is the shape of the sacculus automatically fixed by its chemical composition? or (ii) does a special morphogenetic system exist which determines the shape of the sacculus? An analysis of sacculi from cells grown in poor and rich media and harvested at different stages of growth was made. Significant variations in the composition of murein were found, whereas the general shape of the cells remained unchanged. This finding stands opposed to the assumption of a strict correlation between chemistry and shape of the sacculus. The second alternative was investigated by attempting to change artificially the shape of the sacculus by modifying the form of the hypothetical morphogenetic system. Rod-shaped cells were converted into spherical spheroplasts which were subsequently allowed to reform a new spherical sacculus. In chemical composition this spherical sacculus was found to be indistinguishable from the rod-shaped sacculus. This finding is taken as evidence for the existence of a distinct morphogenetic apparatus in the cell wall whose form is reflected by the shape of the sacculus.     

The membranous labyrinth and its innervation inChaetodon trifasciatus (Pisces,Teleostei, Chaetodontidae)

Synopsis In the butterflyfishChaetodon trifasciatus, the labyrinth is characterized by its elevated form and especially the size of the vertical canals, the almost circular form of the horizontal canal and its posterior opening not directly in the utriculus but in the common pillar of the two vertical canals. There is an almost complete separation between utriculus and sacculus which are only linked by a virtual pore. The lagena, which is medially situated to the posterior part of the sacculus, is separated from it by an incomplete vertical wall. There are two maculae neglectae, the anterior macula being situated in the pore separating utriculus from sacculus and filling this pore, the posterior in a gutter of the floor of the utriculus. A long and narrow endolymphatic canal, originating from the sacculus close to the communication with the utriculus, follows the common pillar of the two vertical canals and widens into an endolymphatic sac at the top of the membranous labyrinth. The innervation of the labyrinth is made by the acoustic ganglion, which is connected to the brain by two roots and elongated into three parts: the anterior part innervates the anterior and horizontal cristae and the utricular and saccular maculae; the middle part innervates the macula sacculae and the macula neglecta 1; the posterior part innervates the macula neglecta II, the macula lagenae and the posterior crista. The important size of the vertical canals and the almost circular form of the horizontal canal may reflect very precise locomotory aptitudes.     

Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli

The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.     

Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli

The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.     

Interactions of outer membrane proteins O-8 and O-9 with peptidoglycan sacculus of Escherichia coli K-12.

The outer membrane proteins O-8 and O-9 were specifically bound to the peptidoglycan sacculus in sodium dodecyl sulfate (SDS) solution. Other cellular proteins failed to interact with the peptidoglycan sacculus under the same conditions. When the outer membrane was preheated in SDS solution, the binding did not take place. Optimum binding was observed at pH 8 in the presence of 5 mM Mg2+. A high concentration of sodium chloride strongly inhibited the binding. The effects of these factors on the bindings of O-8 and O-9 required neither the bound nor the free form of Braun's lipoprotein, nor was the binding of either protein necessary for the binding of the other. Proteins O-8 and O-9 were also found in the peptidoglycan sacculus when it was prepared from cells in SDS solution at 60 degrees. A dilution experiment showed that the complex was not an artifact. The mode of interaction between these proteins and peptidoglycan in the preparation was similar to that in the reassembled O-8-O-9-peptidoglycan complex, as judged from the sensitivity to sodium chloride and temperature. The physiological importance of the complex is discussed in relation to the assembly of the outer membrane on the cell surface.     

The lagena (the third otolith endorgan in vertebrates)

In this review, the structure and functions of the lagena (the third otolith organ) in an evolutionary lineage of the vertebrates are described and discussed. The lagenar macula appears first in the posterior part of the sacculus of elasmobranchs; in these animals, the lagena is considered to be involved in the balance support (orientation with respect to the gravitation force). The lagena as a separate endorgan has been described in teleost fishes; in some species, the lagena is connected with the sacculus, while in other species the interrelations of these structures can be dissimilar. The lagena supplements the functions of the sacculus; in fishes (animals with no special organ of hearing), it is involved in discrimination of sound oscillations, identification of the gravitation vector, and orientation in the course of movements within the vertical plane. In amphibians, the lagena is localized in the posterior part of the sacculus, near the auditory structures; it performs mostly vestibular and (to a much lesser extent) auditory functions. In amniotes, the lagena was first separated from the sacculus; it is localized in the cochlear canal, distally with respect to the hearing organ. Information on the functions of the lagena in amniotes is rather limited and contradictory. Central projections of this organ have been examined practically only in birds. Lagenar afferents project to the vestibular nuclei and cerebellum, while some fibers come to the auditory nuclei of the medulla. The lagena in birds can be related to their navigation abilities (birds are supposed to be capable of orienting within the magnetic field of the Earth due to the magnetic properties of the lagenar otoconia; this structure can also provide detection of movements along the vertical axis. The close proximity between the otolithic and auditory endorgans in the cochlear canal of amniotes can be indicative of the functional significance of these interrelations. This aspect, however, remains at present undiscovered. In mammals (except Monotremata), there is no lagena as an independent endorgan. Neirofiziologiya/Neurophysiology, Vol. 40, No. 2, pp. 160–178, March–April, 2008.     

Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli

The murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylase-transpeptidase murein synthase PBP1B was retained by PBP3-sepharose when a membrane fraction of E. coli was applied. The direct protein-protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two-hybrid system, and by cross-linking/co-immunoprecipitation. In the bacterial two-hybrid system, a truncated PBP3 comprising the N-terminal 56 amino acids interacted with PBP1B. Both synthases could be cross-linked in vivo in wild-type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo, indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division.     

Ultrastructure and Function of Cephalopod Chromatophores

SYNOPSIS. Each chromatophore organ consists of a pigment celland of several radial muscle fibers that represent separatecells. The pigment granules are contained within an elasticsacculus within the pigment cell. The sacculus is attached aroundthe equator of the chromatophore to the cell membrane by zonalhaptosomes. In turn, the cell membrane is attached to the radialmuscle fibers by a dense basal lamina. The cell membrane ofthe retracted chromatophore is highly folded. Contraction ofthe radial muscle fibers is initiated by (a) excitatory junctionpotentials, (b) miniature potentials, or (c) spike potentials.The latter arise spontaneously in the muscle fibers when thesehave undergone some internal (metabolic?) change. The contractionof the muscle fibers causes expansion of the pigment-containingsacculus. Relaxation of the muscle fibers permits the sacculusto assume its original lenticular or near-spherical shape; theenergy for this is stored within theexpanded elastic componentsof the sacculus. In normal skin the chromatophore organs areentirely under the control of the central nervous system, themuscle fibers being activated only by local, excitatory postsynapticpotentials initiated by motor nerve impulses. That postsynapticpotentials are non-propagating insures that individual motorfibers can be activated individually, thus permitting a delicatecontrol of skin color by recruitment as well as by frequency.Tonic contractions and pulsations, involving spontaneous releaseof transmitter from nerve terminals and spike generation withinthe muscle fibers, respectively, are the result of altered,abnormal conditions within the skin.     

Analysis of the mechanism of amplitude change in Pacinian corpuscle receptor potential in a solution with a decreased calcium ion concentration

The effect of Ca2+-free solution on the amplitude increase in the receptor potential (RP) of Pacinian corpuscles was studied using external perfusion technique. The RP amplitude increased in Ca2+-free solution. It was blocked after addition of 10-20 mM of tetraethylamonium. A temporary increase in the RP amplitude is seen in the solution with 0.2 mM of 2.4-dinitrophenol. Sensitivity of the receptor membrane to mechanical stimuli does not change in Ca2+-free solution. It is suggested that near the mechanosensitive ionic canal of Pacinian corpuscle receptor membrane the fixed negative charges which could influence the "gate" system state of the mechanosensitive canal are absent.     

Distribution of enzymes involved in mannan synthesis in plasma membranes and mesosomal vesicles of Micrococcus lysodeikticus.

The distribution of membrane-bound enzymes involved in mannan biosynthesis in plasma and mesosomal membranes of Micrococcus lysodeikticus has been investigated. Isolated mesosomal vesicles, unlike plasma membrane preparations, cannot catalyze the transfer of [14C]mannose from GDP-[14C]mannose into mannan. This appears to result from the inability of this membrane system to synthesize the carrier lipid [14C]mannosyl-1-phosphorylundecaprenol. In contrast, this is the major mannolipid synthesized from GDP-[14C]mannose by isolated plasma membranes. The possibility that substrate inaccessibility could account for the failure to detect the enzyme in isolated mesosomal vesicles appears unlikely from the lack of activity following disruption of the vesicles with ultrasound or with surface active agents. Both membrane preparations possessed the ability to catalyse the transfer of [14C]mannose from purified [14C]mannosyl-1-phosphorylundecaprenol into mannan. Furthermore, free mannan and mannan located on both unlabeled mesosomal and unlabeled plasma membranes could act as acceptors of [14C]mannosyl units from 14C-labeled carrier lipid located in prelabeled plasma membranes. The possibility that the juxtaposition of mesosomal vesicles and enveloping plasma membrane (i.e. the mesosomal sacculus) in vivo allows mannan, located on mesosomal vesicles, to accept mannosyl units from carrier lipid located in the sacculus membrane is discussed.     

Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain

Exponentially growing cells of Bacillus subtilis and Escherichia coli were Gram stained with potassium trichloro(eta 2-ethylene)platinum(II) (TPt) in place of the usual KI-I2 mordant. This electron-dense probe allowed the staining mechanism to be followed and compared with cellular perturbations throughout the staining process. A crystal violet (CV)-TPt chemical complex was formed within the cell substance and at the cell surface of B. subtilis when the dye and Pt mordant were added. The ethanol decolorization step dissolved the precipitate from the cell surface, but the internal complex was retained by the cell wall and remained within the cell. This was not the case for E. coli; the ethanol decolorization step removed both surface-bound and cellular CV-TPt. During its removal, the outer membrane was sloughed off the cells until only the murein sacculus and plasma membrane remained. We suspect that the plasma membrane was also perturbed, but that it was retained within the cell by the murein sacculus. Occasionally, small holes within the murein and plasma membrane could be distinguished through which leaked CV-TPt and some cellular debris. Biochemical identification of distinct envelope markers confirmed the accuracy of these images.     

Origin and differentiation of nephridia in the Onychophora provide no support for the Articulata

Comparative morphology currently permits no unambiguous decision on the primary homology of the nephridia of Annelida and Arthropoda. In order to obtain additional information on this subject, ultrastructure of morphogenesis and further differentiation of nephridia was studied in the onychophoran Epiperipatus biolleyi (Peripatidae). In this species, the nephridial anlage develops by reorganization of the lateral portion of the embryonic coelomic wall that initially gives rise to a ciliated canal. All other structural components, including the sacculus, merge after the nephridial anlage has been separated from the remaining mesodermal tissue. The nephridial sacculus does not represent a ‘persisting coelomic cavity’, since it arises de novo during embryogenesis. There is no evidence for ‘nephridioblast‘ cells participating in the nephridiogenesis of Onychophora, which is in contrast to the general mode of nephridial formation in Annelida. Available data on nephridiogenesis in euarthropods (Chelicerata, Myriapoda, Crustacea, and Hexapoda) also provide no evidence for nephridia of Annelida and Arthropoda being a synapomorphy of these taxa. These findings accordingly weaken the traditional Articulata hypothesis.     

Interactions of Escherichia coli membrane lipoproteins with the murein sacculus.

Bifunctional cross-linking reagents were used to identify cell envelope proteins that interacted with the murein sacculus. This revealed that a number of [3H]leucine-labeled proteins and [3H]palmitate-labeled lipoproteins were reproducibly cross-linked to the sacculus in plasmolyzed cells. The results suggested that most of the cell envelope lipoproteins, and not only the murein lipoprotein, mediate interactions between the murein sacculus and the inner and/or outer membrane of the cell.     

Peptidoglycan structure and architecture

The peptidoglycan (murein) sacculus is a unique and essential structural element in the cell wall of most bacteria. Made of glycan strands cross-linked by short peptides, the sacculus forms a closed, bag-shaped structure surrounding the cytoplasmic membrane. There is a high diversity in the composition and sequence of the peptides in the peptidoglycan from different species. Furthermore, in several species examined, the fine structure of the peptidoglycan significantly varies with the growth conditions. Limited number of biophysical data on the thickness, elasticity and porosity of peptidoglycan are available. The different models for the architecture of peptidoglycan are discussed with respect to structural and physical parameters.     

Interaction of bacteriophage T4 with reconstituted cell envelopes of Escherichia coli K-12.

The interaction with bacteriophage T4 of the cell surface of Escherichia coli K-12 reconstituted from outer membrane protein O-8, lipopolysaccharide, and the lipoprotein-bearing peptidoglycan sacculus was studied. The reconstituted cell surface was active as a receptor for the phage, resulting in the contraction of the tail sheath, a morphological change in the base plate which was accompanied by the extension of short tail pins down to the cell surface and the penetration of the needle through the cell surface. However, the ejection of phage deoxyribonucleic acid did not take place. Both O-8 and lipopolysaccharide were essential for the interaction. In the reconstitution, the wild-type lipopolysaccharide could not be replaced by either heptoseless lipopolysaccharide or lipid A. The lipoprotein-bearing peptidoglycan sacculus was also found to be an active component for the phage adsorption. The sacculus most likely functioned as a basal framework on which O-8 and lipopolysaccharide assembled to form a flat sheet which is large enough to interact with individual distal ends of long tail fibers of a single phage particle.     

Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coli K12

Bacterial peptidoglycan (PG or murein) is a single, large, covalently cross‐linked macromolecule and forms a mesh‐like sacculus that completely encases the cytoplasmic membrane. Hence, growth of a bacterial cell is intimately coupled to expansion of murein sacculus and requires cleavage of pre‐existing cross‐links for incorporation of new murein material. Although, conceptualized nearly five decades ago, the mechanism of such essential murein cleavage activity has not been studied so far. Here, we identify three new murein hydrolytic enzymes in Escherichia coli, two (Spr and YdhO) belonging to the NlpC/P60 peptidase superfamily and the third (YebA) to the lysostaphin family of proteins that cleave peptide cross‐bridges between glycan chains. We show that these hydrolases are redundantly essential for bacterial growth and viability as a conditional mutant lacking all the three enzymes is unable to incorporate new murein and undergoes rapid lysis upon shift to restrictive conditions. Our results indicate the step of cross‐link cleavage as essential for enlargement of the murein sacculus, rendering it a novel target for development of antibacterial therapeutic agents.     

Distribution of enzymes involved in mannan synthesis in plasma membranes and mesosomal vesicles of Micrococcus lysodeikticus

The distribution of membrane-bound enzymes involved in mannan biosynthesis in plasma and mesosomal membranes of Micrococcus lysodeikticus has been investigated.Isolated mesosomal vesicles, unlike plasma membrane preparations, cannot catalyze the transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose into mannan. This appears to result from the inability of this membrane system to synthesize the carrier lipid [¹⁴C]mannosyl-l-phosphorylundecaprenol. In contrast, this is the major manno-lipid synthesized from GDP-[¹⁴C]mannose by isolated plasma membranes. The possibility that substrate inaccessibility could account for the failure to detect the enzyme in isolated mesosomal vesicles appears unlikely from the lack of activity following disruption of the vesicles with ultrasound or with surface active agents.Both membrane preparations possessed the ability to catalyse the transfer of [¹⁴C]mannose from purified [¹⁴C]mannosyl-l-phosphorylundecaprenol into mannan. Furthermore, free mannan and mannan located on both unlabeled mesosomal and unlabeled plasma membranes could act as acceptors of [¹⁴C]mannosyl units from ¹⁴C-labeled carrier lipid located in prelabeled plasma membranes. The possibility that the juxtaposition of mesosomal vesicles and enveloping plasma membrane (i.e. the mesosomal sacculus) in vivo allows mannan, located on mesosomal vesicles, to accept mannosyl units from carrier lipid located in the sacculus membrane is discussed.     

Growth of the Stress-Bearing and Shape-Maintaining Murein Sacculus of Escherichia coli

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 covalently 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 a 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.     

Deformations in the cytoplasmic membrane of Escherichia coli direct the synthesis of peptidoglycan. The hernia model.

To explain the growth of the Gram-negative envelope and in particular how it could be strengthened where it is weakest, we propose in the hernia model that local weakening of the peptidoglycan sacculus allows turgor pressure to cause the envelope to bulge outwards in a hernia; the consequent local alteration in the radius of curvature of the cytoplasmic membrane causes local alterations in phospholipid structure and composition that determine both the synthesis and hydrolysis of peptidoglycan. This proposal is supported by evidence that phospholipid composition determines the activity of phospho-N-acetylmuramic acid pentapeptide translocase, UDP-N-acetylglucosamine:N-acetylmuramic acid-(pentapeptide)-P-P-bactoprenyl-N-acetylglucosamine transferase, bactoprenyl phosphate phosphokinase, and N-acetylmuramyl-L-alanine amidase. We also propose that the shape of Escherichia coli is maintained by contractile proteins acting at the hernia. Given the universal importance of membranes, these proposals have implications for the determination of shape in eukaryotic cells.