Demonstration and characterization of the cell wall carbohydrate and protein antigens from Clostridium botulinum type E Saroma

Two different cell wall antigens, carbohydrate (CHO) and protein (P), from Clostridium botulinum type E Saroma were extracted with sodium dodecyl sulfate (SDS) and purified by chromatography on DEAE-Sepharose CL-6B and Sephadex G-75 or G-100. The CHO antigen was composed of glucose, galactose, glucosamine, galactosamine, alanine and phosphorus with a molar ratio of 1.5:1.5:0.25:0.25:1:1. The P antigen was an acidic protein with a molecular weight of 60 kDa, in which the major amino acids were aspartate, glutamate and serine, while the minor ones were cysteine and methionine. Thin sections of the intact or SDS-extracted cells of the organism demonstrated that the cell wall was composed of a two-layered structure, an inner layer about 20 nm thick and an outer layer about 10 nm, and by the extraction with SDS, the outer layer disappeared from the cell surface, leaving the inner layer. Immunogel diffusion tests demonstrated that either CHO antigen or P antigen was common among the nonproteolytic strains of C. botulinum.     

Ultrastructure of bacteria and the proportion of Gram-negative bacteria in marine sediments

Bacteria in sediments from the surface aerobic layer (0–1 cm) and a deeper anaerobic layer (20–21 cm) of a seagrass bed were examined in section by transmission electron microscopy. Bacteria with a Gram-negative ultrastructure made up 90% of bacteria in the surface layer, and Gram-positive bacteria comprised 10%. In the anaerobic zone, Gram-negative bacteria comprised 70% and Gram-positive bacteria 30% of the bacterial population. These differences were highly significant and support predictions of these proportions made from muramic acid measurements and direct counting with fluorescence microscopy. Most cells were enveloped in extracellular slime layers or envelopes, some with considerable structural complexity. The trophic value to animals of these envelopes is discussed. A unique organism with spines was observed.     

In vitro activity of ramoplanin and comparator drugs against anaerobic intestinal bacteria from the perspective of potential utility in pathology involving bowel flora

Susceptibility of intestinal bacteria to various antimicrobial agents in vitro, together with levels of those agents achieved in the gut, provides information on the likely impact of the agents on the intestinal flora. Orally administered drugs that are poorly absorbed may be useful for treatment of intestinal infections and for certain other situations in which intestinal bacteria may play a role. The antimicrobial activity of ramoplanin (MDL 62,198) against 928 strains of intestinal anaerobic bacteria was determined using the NCCLS-approved Wadsworth brucella laked-blood agar dilution method. The activity of ramoplanin was compared with that of ampicillin, bacitracin, metronidazole, trimethoprim/sulfamethoxazole (TMP/SMX), and vancomycin. The organisms tested included Bacteroides fragilis group (n=89), other Bacteroides species (n=16), other anaerobic Gram-negative rods (n=56) anaerobic cocci (n=114), Clostridium species (n=426), and non-sporeforming anaerobic Gram-positive rods (n=227). The overall MIC(90)s of ramoplanin, ampicillin, bacitracin, metronidazole, and vancomycin were 256, 32, 128, 16, and 128 mcg/ml, respectively. Ramoplanin was almost always highly active vs. Gram-positive organisms and relatively poor in activity against Gram-negative organisms, particularly Bacteroides, Bilophila, Prevotella, and Veillonella. Vancomycin was quite similar to ramoplanin in its activity. Ampicillin was relatively poor in activity vs. organisms that often produce beta-lactamase, including most of the Gram-negative rods as well as Clostridium bolteae and C. clostridioforme. Bacitracin was relatively poor in activity against most anaerobic Gram-negative rods, but better vs. most Gram-positive organisms. Metronidazole was very active against all groups other than bifidobacteria and some strains of other types of non-sporeforming Gram-positive bacilli. TMP/SMX was very poorly active, with an MIC(90) of >2048 mcg/ml.     

Cell wall studies on budding bacteria of the Planctomyces/Pasteuria group and on a Prosthecomicrobium sp.

Seven strains of budding, non-prosthecate bacteria belonging to the Planctomyces/Pasteuria group and a Prosthecomicrobium sp. were examined for muramic and diaminopimelic acids. These typical components of Gram-negative murein were found only in Prosthecomicrobium strain IFAM 1314, but they could not be detected in seven budding bacteria. Electron micrographs of ultrathin cell wall sections of strains IFAM 1313 and 1317 showed a membrane with bilayer structure outside the cytoplasmic membrane. 10% sodium dodecylsulfate treatment (30 min, 100°C) allowed the isolation of highly stable cell sacculi which, upon chemical analysis, proved to be mainly proteinaceous. The budding bacteria also showed considerable resistance against penicillin G, ampicillin, cephalotin and D-cycloserin. Our data indicate that these bacteria lack an ordinary Gram-negative type of murein and, instead, carry a stable protein envelope.     

Epitope mapping of monoclonal antibodies specific for the directly cross-linked mesodiaminopimelic acid peptidoglycan found in the anaerobic beer spoilage bacterium Pectinatus cerevisiiphilus.

Nineteen monoclonal antibodies (Mabs) were isolated based on reactivity with disrupted Pectinatus cerevisiiphilus cells. All of the Mabs reacted with cells from which the outer membrane had been stripped by incubation with sodium dodecyl sulphate, suggesting the peptidoglycan (PG) layer was involved in binding. Mab reactivity with purified PG confirmed this. Epitope mapping revealed the Mabs in total recognize four binding sites on the PG. Mabs specific for each of the four sites also bound strongly to disrupted Pectinatus frisingensis, Selenomonas lacticifix, Zymophilus paucivorans, and Zymophilus raffinosivorans cells, but weakly to disrupted Megasphaera cerevisiae cells. No antibody reactivity was seen with disrupted cells of 11 other species of Gram-negative bacteria. These results confirm that a common PG structure is used by several species of anaerobic Gram-negative beer spoilage bacteria. These results also indicate that PG-specific Mabs can be used to rapidly detect a range of anaerobic Gram-negative beer spoilage bacteria, provided the bacterial outer membrane is first removed to allow antibody binding.     

Changes in the fine structure of microbial cells induced by chaotropic salts

The electron microscopic examination of the thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37 degrees C for 3-5 h or at 100 degrees C for 5-6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.     

Lipid trafficking to the outer membrane of Gram-negative bacteria

The envelope of Gram-negative bacteria is composed of two distinct lipid membranes: an inner membrane and outer membrane. The outer membrane is an asymmetric bilayer with an inner leaflet of phospholipids and an outer leaflet of lipopolysaccharide. Most of the steps of lipid synthesis occur within the cytoplasmic compartment of the cell. Lipids must then be transported across the inner membrane and delivered to the outer membrane. These topological features combined with the ability to apply the tools of biochemistry and genetics make the Gram-negative envelope a fascinating model for the study of lipid trafficking. In addition, as lipopolysaccharide is essential for growth of most strains and is a potent inducer of the mammalian innate immune response via activation of Toll-like receptors, Gram-negative lipid transport is also a promising target for the development of novel antibacterial and anti-inflammatory compounds. This review focuses on recent developments in our understanding of lipid transport across the inner membrane and to the outer membrane of Gram-negative bacteria.     

The structures of glycolipids isolated from the highly thermophilic bacterium Thermus thermophilus Samu-SA1

Thermophiles constitute a class of microorganisms able to grow at extremely elevated temperatures. Some of these species are classified as Gram-negative bacteria, because of the presence of an outer membrane in the cell envelope, which is located on the top of a thick murein layer. Unlike typical Gram-negative bacteria, the outer membranes of Thermus species are not composed of lipopolysaccharides but of peculiar glycolipids (GL), whose structures seem to be strictly involved in the adaptation to high temperatures. In this work, the complete structures of the major GL components from the cell envelope of the thermophilic bacterium Thermus thermophilus Samu-SA1 are presented. Protocols conventionally adopted for Gram-negative bacteria were used, and, for the first time, GL from Thermus were analyzed in their native form. Two GL and one phosphoglycolipid (PGL) were detected and characterized. The two GL, analyzed by nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry, possessed the same tetrasaccharide structure linked to a glycerol unit or, alternatively, to a long-chain diol. Moreover, a PGL from Thermus was characterized for the first time, in which N-glyceroyl-heptadecaneamine was present. These molecules are chemically related to other GL from thermophile bacteria, in which they play a crucial role in the adaptation of cell membranes to heat.     

The variable T model for gram-negative morphology

Gram-negative micro-organisms possess only a very thin murein sacculus to resist the stress caused by the internal hydrostatic pressure. The sacculus consists of at most one molecular layer of peptidoglycan in an extended conformation. It must grow by the insertion and cross-linking of new murein to the old before the selective cleavages of the stress-bearing murein are made which allow wall enlargement. Since insertion of new murein occurs all over the surface of Escherichia coli (even in completed poles), the internal pressure would tend to force the cells into a spherical shape and prevent both cylindrical elongation and cell division. Of course, Gram-negative bacteria do achieve a variety of shapes and do divide. Because prokaryote cells, unlike eukaryotic cells, do not have cytoskeletons and contractile proteins to transduce biochemical free energy into the mechanical work needed to achieve aspherical shapes and to divide, this paradox seems to be resolvable only by postulating that the details of the biochemical mechanism for wall growth vary in different regions of the surface, affecting the work required to enlarge the wall locally. Depending on the degree and rate of change in the biochemical energetics, it is possible to account for rod and the other more complex shapes of Gram-negative bacteria. Division occurs in Gram-negative organisms by the development of constrictions that progressively invade the cytoplasm. The work to cause these morphological processes must ultimately derive from the biochemical process of the stress-bearing wall formation. A biophysical basis for cell division in these prokaryotic organisms is proposed.     

Identification of the initial binding sites of alphas2-casein f(183-207) and effect on bacterial membranes and cell morphology

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide alphas2-casein f(183-207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of alphas2-casein f(183-207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the alphas2-casein peptide f(183-207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183-207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.     

Double hexameric ring assembly of the type III protein translocase ATPase HrcN

The specialized type III secretion (T3S) apparatus of pathogenic and symbiotic Gram-negative bacteria comprises a complex transmembrane organelle and an ATPase homologous to the F1-ATPase beta subunit. The T3S ATPase HrcN of Pseudomonas syringae associates with the inner membrane, and its ATP hydrolytic activity is stimulated by dodecamerization. The structure of dodecameric HrcN (HrcN12) determined to 1.6 nm by cryo-electron microscopy is presented. HrcN12 comprises two hexameric rings that are probably stacked face-to-face by the association of their C-terminal domains. It is 11.5 +/- 1.0 nm in diameter, 12.0 +/- 2.0 nm high and has a 2.0-3.8 nm wide inner channel. This structure is compared to a homology model based on the structure of the F1-beta-ATPase. A model for its incorporation within the T3S apparatus is presented.     

Correlation of the DNA nucleotide makeup with the physiological and cytological characteristics of spore-forming anaerobic bacteria]

The nucleotide composition of DNA from 12 studied species of anaerobic bacteria belongs to AT type, with G+C varying from 28.4 to 36.8 mole%. In the anaerobic group of Clostridium bifermentans, a correlation has been established between the nucleotide composition of DNA, the type of appendages on spores, and some physiologo-biochemical characteristics. The nucleotide composition of DNA in the spores of four anaerobic species is shifted toward GC type as compared to DNA in the vegetative cells. Data on the content of GC pairs in DNA of the spores may sometimes be of a higher taxonomic value than the corresponding evidence on DNA of the vegetative cells.     

Breaching the Barrier: Quantifying Antibiotic Permeability across Gram-negative Bacterial Membranes

The double-membrane cell envelope of Gram-negative bacteria is a sophisticated barrier that facilitates the uptake of nutrients and protects the organism from toxic compounds. An antibiotic molecule must find its way through the negatively charged lipopolysaccharide layer on the outer surface, pass through either a porin or the hydrophobic layer of the outer membrane, then traverse the hydrophilic peptidoglycan layer only to find another hydrophobic lipid bilayer before it finally enters the cytoplasm, where it typically finds its target. This complex uptake pathway with very different physico-chemical properties is one reason that Gram-negative are intrinsically protected against multiple classes of antibiotic-like molecules, and is likely the main reason that in vitro target-based screening programs have failed to deliver novel antibiotics for these organisms. Due to the lack of general methods available for quantifying the flux of drugs into the cell, little is known about permeation rates, transport pathways and accumulation at the target sites for particular molecules. Here we summarize the current tools available for measuring antibiotic uptake across the different compartments of Gram-negative bacteria.     

The fine structure of Eadie's ovals isolated from sheep rumen.

The structure of two strains of the Gram-negative rumen organism, Eadie's Oval, was examined with the electron microscope. Despite their large size, their fine structure indicated that they were bacteria. They had a cell envelope consisting of two membranes separated by a dense layer which could be solubilized by lysozyme. They possessed characteristic bacterial flagella, and lacked internal organization with ribosomes and DNA-like material dispersed throughout the cytoplasm. The outer membrane was corrugated and each strain had a characteristic pattern of corrugations. One strain had sheathed flagella, the other did not. Both strains were coated with fibrils up to 660 nm long, but which apparently contracted to give an unusual cross-banded layer when treated with lysozyme.     

The Cysts of Blastocrithidia triatomae Cerisola et al., 19711

ABSTRACT. Flagellar cysts of Blastocrithidia triatomae form from active flagellates by diminution in size. The pellicular microtubules disappear. The inner layer of the cell membrane thickens progressively as the organism shrinks. The fully formed cyst has an electrondense layer that corresponds to the outer layer of the unit membrane. An electron-lucent layer is approximately twice the thickness of the middle layer of the unit membrane. Inside that is a 92 nm layer that may represent the cytoplasm. The nuclear content is in the form of whorled bundles of 10–15 nm fibrils. The kinetoplast was not seen in electron micrographs of cysts.     

Effect of lysozyme or modified lysozyme fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli

Previously we have shown that chicken egg white lysozyme, an efficient bactericidal agent, affects both gram-positive and gram-negative bacteria independently of its muramidase activity. More recently we reported that the digestion of lysozyme by clostripain yielded a pentadecapeptide, IVSDGNGMNAWVAWR (amino acid 98-112 of chicken egg white lysozyme), with moderate bactericidal activity but without muramidase activity. On the basis of this amino acid sequence three polypeptides, in which asparagine 106 was replaced by arginine (IVSDGNGMRAWVAWR, RAWVAWR, RWVAWR), were synthesized which showed to be strongly bactericidal. To elucidate the mechanisms of action of lysozyme and of the modified antimicrobial polypeptides Escherichia coli strain ML-35p was used. It is an ideal organism to study the outer and the inner membrane permeabilization since it is cryptic for periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless the outer or inner membrane becomes damaged. For the first time we present evidence that lysozyme inhibits DNA and RNA synthesis and in contrast to the present view is able to damage the outer membrane of Escherichia coli. Blockage of macromolecular synthesis, outer membrane damage and inner membrane permeabilization bring about bacterial death. Ultrastructural studies indicate that lysozyme does not affect bacterial morphology but impairs stability of the organism. The bactericidal polypeptides derived from lysozyme block at first the synthesis of DNA and RNA which is followed by an increase of the outer membrane permeabilization causing the bacterial death. Inner membrane permeabilization, caused by RAWVAWR and RWVAWR, follows after the blockage of macromolecular synthesis and outer membrane damage, indicating that inner membrane permeabilization is not the deadly event. Escherichia coli bacteria killed by the substituted bactericidal polypeptides appeared, by electron microscopy, with a condensed cytoplasm and undulated bacterial membrane. So the action of lysozyme and its derived peptides is not identical.     

Factors Related to the Oxygen Tolerance of Anaerobic Bacteria

The effect of atmospheric oxygen on the viability of 13 strains of anaerobic bacteria, two strains of facultative bacteria, and one aerobic organism was examined. There were great variations in oxygen tolerance among the bacteria. All facultative bacteria survived more than 72 h of exposure to atmospheric oxygen. The survival time for anaerobes ranged from less than 45 min for Peptostreptococcus anaerobius to more than 72 h for two Clostridium perfringens strains. An effort was made to relate the degree of oxygen tolerance to the activities of superoxide dismutase, catalase, and peroxidases in cell-free extracts of the bacteria. All facultative bacteria and a number of anaerobic bacteria possessed superoxide dismutase. There was a correlation between superoxide dismutase activity and oxygen tolerance, but there were notable exceptions. Polyacrylamide gel electropherograms stained for superoxide dismutase indicated that many of the anaerobic bacteria contained at least two electrophoretically distinct enzymes with superoxide dismutase activity. All facultative bacteria contained peroxidase, whereas none of the anaerobic bacteria possessed measurable amounts of this enzyme. Catalase activity was variable among the bacteria and showed no relationship to oxygen tolerance. The ability of the bacteria to reduce oxygen was also examined and related to enzyme content and oxygen tolerance. In general, organisms that survived for relatively long periods of time in the presence of oxygen but demonstrated little superoxide dismutase activity reduced little oxygen. The effects of medium composition and conditions of growth were examined for their influence on the level of the three enzymes. Bacteria grown on the surface of an enriched blood agar medium generally had more enzyme activity than bacteria grown in a liquid medium. The data indicate that superoxide dismutase activity and oxygen reduction rates are important determinants related to the tolerance of anaerobic bacteria to oxygen.     

Isolation of differentiated membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction containing attachment sites between the inner and outer membranes and the murein skeleton of the cell envelope

Cell envelopes of Salmonella typhimurium and Escherichia coli were disrupted in a French pressure cell and fractionated by successive cycles of sedimentation and floatation density gradient centrifugation. This permitted the identification and isolation of several membrane fractions in addition to the major inner membrane and murein-outer membrane fractions. One of these fractions (fraction OML) accounted for about 10% of the total cell envelope protein, and is likely to include the murein-membrane adhesion zones that are seen in electron micrographs of plasmolyzed cells. Fraction OML contained inner membrane, murein, and outer membrane in an apparently normal configuration, was capable of synthesizing murein from UDP-[3H]N-acetylglucosamine and UDP-N-acetylmuramylpentapeptide and covalently linking it to the endogenous murein of the preparation, and showed a labeling pattern in [3H]galactose pulse-chase experiments that was consistent with its acting as an intermediate in the movement of newly synthesized lipopolysaccharide from inner membrane to outer membrane. The fractionation procedure also identified two new minor membrane fractions, with characteristic protein patterns, that are usually included in the region of the major inner membrane peak in other fractionation procedures but can be separated from the major inner membrane fraction and from contaminating flagellar fragments by the subsequent floatation centrifugation steps.     

Fine Structure of Spirochaeta stenostrepta, a Free-living, Anaerobic Spirochete

The fine structure of Spirochaeta stenostrepta strain Z1, a free-living anaerobic spirochete, was studied by electron microscopy. The organism possessed a coiled protoplasmic cylinder, an axial filament inserted subterminally, and a loosely fitting sheath which enclosed both the protoplasmic cylinder and the axial filament. The axial filament consisted of two fibrils partially overlapping in a 1-2-1 arrangement. The axial fibrils appeared to possess a sheath surrounding an inner core. Both inner core and sheath were apparently enclosed in a cross-striated tubular structure, which was itself surrounded by an outer sheath. The axial filament exhibited a basal hook. A disc- or mushroom-shaped structure, possibly consisting in part of cytoplasmic membrane, was observed at the insertion end of isolated filaments. The protoplasmic cylinder had a distinctive surface structure consisting of an array of tightly packed, longitudinally arranged helices measuring 2.0 to 2.5 nm in diameter. This layer of helices lay below the outer cell sheath and the axial filament. Ballistic disintegration loosened the helical array, causing individual helices or segments of helices to become separated from the cell. The function of this layer of helices is still obscure.     

Identification of the initial binding sites of αs2-casein f(183-207) and effect on bacterial membranes and cell morphology

The aim of this work was to identify the initial binding sites to the bacterial membranes of the antimicrobial peptide α_s2-casein f(183-207) and also to acquire further insight into membrane permeabilization of this peptide. Furthermore, cell morphology was studied by transmission electron microscopy. In all the experiments, bovine LFcin was employed as a comparison. Results showed that initial binding sites of α_s2-casein f(183-207) peptide were lipoteichoic acid in Gram-positive bacteria and lipopolysaccharide in Gram-negative. The peptide was able to permeabilize the outer and inner membranes. Moreover, the α_s2-casein peptide f(183-207) generated pores in the outer membrane of Gram-negative bacteria and in the cell wall of Gram-positive bacteria. In the Gram-negative bacteria, f(183-207) originated cytoplasm condensation, and in the Gram-positive bacteria the cytoplasmic content leaked into the extracellular medium. Furthermore, the experiments of inner and outer membrane permeabilization performed with LFcin-B showed that this peptide also has the ability to permeabilize both the inner and outer membranes.