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PubMed1983

Survival of cultured cells after functional and structural disorganization of plasma membrane by bacterial haemolysins and phospholipases.

Lesions were induced in the plasma membranes of cultured human fibroblasts by membrane damaging toxins of bacterial origin (haemolysins). Structural disorganization of the membrane was measured as leakage of a radiolabelled small cytoplasmic marker and functional membrane damage was measured as decreased uptake of aminoisobutyrate. Cell survival was scored 24 and 48 hr later by measuring uptake of Trypan Blue and by light microscopical evaluation of cell morphology and proliferation. The membrane damage induced by most bacterial toxins was reversible upon removal of the toxin, since toxin-treated cells recovered and excluded Trypan Blue although they had been permeable to the dye immediately after the toxin treatment. Among ten bacterial toxins tested, the only exception of this general behavior was the Aeromonas hydrophila beta-haemolysin, which irreversibly damaged human fibroblasts. Thus, the action of bacterial haemolysins on cultured cells generally seems restricted to a plasma membrane permeabilization, which is reversible regardless of the mechanism of membrane damaging action of the toxin or of the relative size of the structural lesions induced. Furthermore, the use of Trypan Blue uptake as a measure of cell death caused by membrane damaging agents appears to be of limited value.

Aeromonas↗
PubMedDate not supplied

Structural analysis shows five glycohydrolase families diverged from a common ancestor.

We have solved the X-ray structure of barley chitinase and bacterial chitosanase. Structural constraints predicted these would work by an inverting mechanism, which has been confirmed biochemically. The two enzymes were compared with lysozymes from goose (GEWL), phage (T4L), and hen (HEWL). Although the proteins share no significant amino acid similarities, they are shown to have a structurally invariant core containing two helices and a three-stranded beta sheet that from the substrate binding and catalytic cleft. These enzymes represent a superfamily of hydrolases arising from the divergent evolution of an ancient protein. The glycohydrolase superfamily can be structurally divided into a bacterial family (chitosanase and T4L), and a eucaryotic family represented by chitinase, GEWL, and HEWL. Both families contain the ancestral core but differ at the amino and carboxy termini. The eucaryotes have a small N terminal domain, while the procaryotes have none. The C terminal domain of the eucaryotic family contains a single alpha-helix, while the prokaryotic domain has three antiparallel helices.

Animals↗
PubMed2006

Identification of bacterial groups preferentially associated with mycorrhizal roots of Medicago truncatula.

The genetic structures of bacterial communities associated with Medicago truncatula Gaertn. cv. Jemalong line J5 (Myc+ Nod+) and its symbiosis-defective mutants TRV48 (Myc+ Nod-) and TRV25 (Myc- Nod-) were compared. Plants were cultivated in a fertile soil (Châteaurenard, France) and in soil from the Mediterranean basin showing a low fertility (Mas d'Imbert, France). Plant growth, root architecture, and the efficiency of root symbiosis of the three plant genotypes were characterized in the two soils. Structures of the bacterial communities were assessed by automated-ribosomal intergenic spacer analysis (A-RISA) fingerprinting from DNA extracted from the rhizosphere soil and root tissues. As expected, the TRV25 mutant did not develop endomycorrhizal symbiosis in any of the soils, whereas mycorrhization of line J5 and the TRV48 mutant occurred in both soils but at a higher intensity in the Mas d'Imbert (low fertility) than in the Châteaurenard soil. However, modifications of plant growth and root architecture, between mycorrhizal (J5 and TRV48) and nonmycorrhizal (TRV25) plants, were recorded only when cultivated in the Mas d'Imbert soil. Similarly, the genetic structures of bacterial communities associated with mycorrhizal and nonmycorrhizal plants differed significantly in the Mas d'Imbert soil but not in the Châteaurenard soil. Multivariate analysis of the patterns allowed the identification of molecular markers, explaining these differences, and markers were further sequenced. Molecular marker analysis allowed the delineation of 211 operational taxonomic units. Some of those belonging to the Comamonadaceae and Oxalobacteraceae (beta-Proteobacteria) families were found to be significantly more represented within bacterial communities associated with the J5 line and the TRV48 mutant than within those associated with the TRV25 mutant, indicating that these bacterial genera were preferentially associated with mycorrhizal roots in the Mas d'Imbert soil.

Betaproteobacteria↗
PubMed2005

Crystal structure of SmcL, a bacterial neutral sphingomyelinase C from Listeria.

Sphingomyelinases C are enzymes that catalyze the hydrolysis of sphingomyelin in biological membranes to ceramide and phosphorylcholine. Various pathogenic bacteria produce secreted neutral sphingomyelinases C that act as membrane-damaging virulence factors. Mammalian neutral sphingomyelinases C, which display sequence homology to the bacterial enzymes, are involved in sphingolipid metabolism and signaling. This article describes the first structure to be determined for a member of the neutral sphingomyelinase C family, SmcL, from the intracellular bacterial pathogen Listeria ivanovii. The structure has been refined to 1.9-A resolution with phases derived by single isomorphous replacement with anomalous scattering techniques from a single iridium derivative. SmcL adopts a DNase I-like fold, and is the first member of this protein superfamily to have its structure determined that acts as a phospholipase. The structure reveals several unique features that adapt the protein to its phospholipid substrate. These include large hydrophobic beta-hairpin and hydrophobic loops surrounding the active site that may bind and penetrate the lipid bilayer to position sphingomyelin in a catalytically competent position. The structure also provides insight into the proposed general base/acid catalytic mechanism, in which His-325 and His-185 play key roles.

Amino Acid Sequence↗
PubMed1991

Antibiotic GE2270 a: a novel inhibitor of bacterial protein synthesis. II. Structure elucidation.

GE2270 A, produced by Planobispora rosea ATCC 53773, inhibits Gram-positive bacteria and anaerobes by acting on the bacterial protein synthesis. The structure has been determined by physico-chemical methods applied to the intact molecule and to the main hydrolysis products. Characterization by UV, IR, NMR (double quantum filter COSY), acid-base ionization, elemental analysis and FAB-MS indicated that GE2270 A is a highly modified peptide having MW 1,289 and formula C56H55N15O10S6, and a weak basic function, and that it belongs to the thiazolyl peptide group of antibiotics. Acid hydrolysis yielded a main product (MW 634), responsible for the chromophoric absorption, and a number of hydrolyzed products of lower MW. 13C NMR inverse techniques and MS studies (EI, positive ion chemical ionization, and collision induced dissociation FAB-MS-MS experiments) on GE2270 A, the chromophoric compound, and the other hydrolysis products led to the complete identification of the various amino acid residues and their sequence. Two out of the six chiral centers have been determined. The structure is thought to originate from modification of a chain of 14 amino acids in a process which creates 6 thiazole rings and one pyridine. The modification process also closes the linear polypeptide to form a cyclic part with an attached side-chain. GE2270 A plausibly has a similar biosynthetic origin to that of other thiazolyl peptide antibiotics such as nosiheptide and micrococcin.

Actinomycetales↗
PubMed1993

The absence of correlation between allozyme and rrn RFLP analysis indicates a high gene flow rate within human clinical Pseudomonas aeruginosa isolates.

Two hundred and fifty seven human clinical Pseudomonas aeruginosa strains isolated between 1984 and 1990 in several regions of France, as well as two reference strains, were studied by computer-assisted statistical analysis of the data from their esterase electrophoretic patterns and rrn restriction fragment length polymorphisms. No correlation was found between the two sets of data except for some strains of serotype O12 which, thus, may constitute a distinct group within the species. This absence of correlation indicates a high gene flow rate within human isolates of the P. aeruginosa species. A possible explanation is that, because of an as yet unidentified selective advantage, the esterase loci are a major target for recombinational events. Alternatively, horizontal genetic transfers between strains may have occurred at so high a rate that the clonal structure usually observed in bacterial populations has been disrupted. This study highlights clearly the need for caution in inferring bacterial population structure from any single class of genetic markers.

Esterases↗
PubMed2000

Bacterial adhesins: function and structure.

Specific adhesion to host tissue cells is an essential virulence factor of most bacterial pathogens. The fundamental processes that determine bacterial attachment to host tissue surfaces are mediated by microbial adhesins. Host specificity and tissue tropism are characteristics exhibited by different bacteria and are determined (at least in part) by the interaction between adhesins and their complementary receptors on host cell surfaces. A detailed picture of how bacteria are able to target to various receptors is emerging. A large number of bacterial adhesins with individual receptor specificities have been identified. Furthermore, recent research has shown that individual adhesins are prone to rapid microevolution that results in changes in the receptor specificity of individual adhesins. Microbial adhesins are often assembled into complex polymeric organelle structures, however non-organelle adhesins linked to the cell surface as monomers or simple oligomers also exist. This review gives an overview of bacterial adhesins and focuses on some general aspects of their biogenesis and role in bacterial colonization of host cell surfaces and as virulence factors.

Adhesins, Bacterial↗
PubMedDate not supplied

Studies of the structural organization of a bacterial chemoreceptor by electron microscopy.

We used analysis by electron microscopy to obtain structural information about a transmembrane receptor that mediates chemotaxis in Escherichia coli. Two-dimensional arrays of regularly packed particles of the receptor Trg were obtained by reconstitution of purified, detergent-solubilized protein into lipid bilayers. Preliminary image processing of negatively stained arrays revealed an almost square 8.8 x 8.8-nm unit cell and resolved the particles into four peaks of density around a central depression. In certain conditions, reconstituted, Trg-containing bilayers associated into membrane stacks. The regular spacing of the stacks provided a value of 15 nm for the dimension of the receptor normal to the membrane. Using these dimensions, the estimated occupied volume of the structure would be sufficient to contain four monomers of Trg. This tetramer form may be a dimer of two antiparallel or parallel homodimers. Our analysis indicates that a receptor monomer is approximately 4.4 nm at the widest point and 15 nm long. Given the dimensions of the periplasmic domain of the closely related receptor Tars, determined by X-ray crystallography, and a minimum bilayer thickness of 3 nm, the cytoplasmic domain would be approximately 5.0 by 4.4 nm. Higher resolution analysis should reveal additional information about receptor structure.

Bacterial Proteins↗
PubMed2003

Chaperonin-mediated protein folding: fate of substrate polypeptide.

Chaperonins are megadalton ring assemblies that mediate essential ATP-dependent assistance of protein folding to the native state in a variety of cellular compartments, including the mitochondrial matrix, the eukaryotic cytosol, and the bacterial cytoplasm. Structural studies of the bacterial chaperonin, GroEL, both alone and in complex with its co-chaperonin, GroES, have resolved the states of chaperonin that bind and fold non-native polypeptides. Functional studies have resolved the action of ATP binding and hydrolysis in driving the GroEL-GroES machine through its folding-active and binding-active states, respectively. Yet the exact fate of substrate polypeptide during these steps is only poorly understood. For example, while binding involves multivalent interactions between hydrophobic side-chains facing the central cavity of GroEL and exposed hydrophobic surfaces of the non-native protein, the structure of any polypeptide substrate while bound to GroEL remains unknown. It is also unclear whether binding to an open GroEL ring is accompanied by structural changes in the non-native substrate, in particular whether there is an unfolding action. As a polypeptide-bound ring becomes associated with GroES, do the large rigid-body movements of the GroEL apical domains serve as another source of a potential unfolding action? Regarding the encapsulated folding-active state, how does the central cavity itself influence the folding trajectory of a substrate? Finally, how do GroEL and GroES serve, as recently recognized, to assist the folding of substrates too large to be encapsulated inside the machine? Here, such questions are addressed with the findings available to date, and means of further resolving the states of chaperonin-associated polypeptide are discussed.

Adenosine Triphosphate↗
PubMed1964

RELATIONSHIP OF STRUCTURE TO FUNCTION IN BACTERIAL O ANTIGENS. III. BIOLOGICAL PROPERTIES OF ENDOTOXOIDS.

Johnson, Arthur G. (The University of Michigan, Ann Arbor), and Alois Nowotny. Relationship of structure to function in bacterial O antigens. III. Biological properties of endotoxoids. J. Bacteriol. 87:809-814. 1964.-Endotoxin (somatic O antigen) was rendered much less lethal for mice and rabbits after treatment with boron trifluoride, potassium methylate, or pyridinium formate. In addition, the ability to induce the second fever peak of the biphasic febrile response characteristic of endotoxin was abolished. The capacity to prepare for the local Shwartzman reaction was also reduced after such treatments. On the other hand, stimulation of nonspecific resistance, and adjuvant action in mice was retained. The ability to elicit specific antibody was diminished after boron trifluoride treatment but only slightly retarded by the other two procedures.

Animals↗
PubMed2005

Ontogenetic development of the gastrointestinal microbiota in the marine herbivorous fish Kyphosus sydneyanus.

Molecular techniques were used to investigate the composition and ontogenetic development of the intestinal bacterial community in the marine herbivorous fish Kyphosus sydneyanus from the north eastern coast of New Zealand. Previous work showed that K. sydneyanus maintains an exclusively algivorous diet throughout post-settlement life and passes through an ontogenetic diet shift from a juvenile diet which is readily digestible to an adult diet high in refractory algal metabolites. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to investigate the relationship between bacterial community structure and fish size. Bacterial diversity was higher in posterior gut sections than anterior gut sections, and in larger fish than in smaller fish. Partial sequencing of bacterial 16S rDNA genes PCR amplified and cloned from intestine content samples was used to identify the phylogenetic affiliation of dominant gastrointestinal bacteria. Phylogenetic analysis of clones showed that most formed a clade within the genus Clostridium, with one clone associated with the parasitic mycoplasmas. No bacteria were specific to a particular intestinal section or size class of host, though some appeared more dominant than others and were established in smaller fishes. Clones closely related to C. lituseburense were particularly dominant in most intestine content samples. All bacteria identified in the intestinal samples were phylogenetically related to those possessing fermentative type metabolism. Short-chain fatty acids in intestinal fluid samples increased from 15.6 +/- 2.1 mM in fish <100 mm to 51.6 +/- 5.5 mM in fish >300 mm. The findings of this study support the hypothesis that the ontogenetic diet shift of K. sydneyanus is accompanied by an increase in the diversity of intestinal microbial symbionts capable of degrading refractory algal metabolites into short-chain fatty acids, which can then be assimilated by the host.

Age Factors↗
PubMed1983

Haemagglutinating, adhesive and physico-chemical surface properties of different Yersinia enterocolitica and Yersinia enterocolitica-like bacteria.

Fourteen strains of Yersinia enterocolitica, two strains of Yersinia kristensenii and one strain of Yersinia frederiksenii were investigated for haemagglutination, association with cultured cells, motility and physico-chemical surface properties. Three of the Y. enterocolitica strains, both Y. kristensenii and the Y. frederiksenii strain caused haemagglutination. Y. enterocolitica O-serotype 3 had the greatest tendency to associate with cultured cells. No correlation was seen between association and haemagglutination or motility. Bacterial cytotoxicity towards cultured cells was lost and haemagglutination acquired after repeated subcultivation. haemagglutinating strains were more hydrophobic than non-haemagglutinating strains. These results indicate that the bacterial surface structures, probably fimbriae, conferring bacterial haemagglutinating properties, are hydrophobic.

Adhesiveness↗
PubMed2002

A comparison of enzymatic and molecular approaches to characterize the cellulolytic microbial ecosystems of the rumen and the cecum.

We used RNA probes and enzyme activities to compare the cellulolytic microbial ecosystems of the rumen and the cecum. Four rumen- and cecum-cannulated wethers were fed a diet of barley plus hay (60:40). Digesta samples were collected 1 h before feeding and 3, 6, and 9 h after feeding for measurements on microbial populations, and 1 h before feeding and 3 and 6 h after feeding for digestion measurements, pH, and VFA. Polysaccharidase and glycosidase specific activities of solid-adherent microorganisms were measured respectively by the amount of reducing sugars released from xylan or avicel or p-nitrophenol from the p-nitrophenol derivatives of xylose and glucose. The distribution and amounts of the three main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) were determined by dot-blot hybridization using specific 16SrRNA-targeting probes. Enzyme activities were higher in the rumen than in the cecum and before feeding than at 3 h after feeding. The sum of the three cellulolytic bacterial species represented, on average, 4.5% of the total bacterial RNA in the two compartments and did not vary with sampling time. The cellulolytic bacterial community structure was different in the two compartments, with F. succinogenes as the main species in the rumen and R. flavefaciens in the cecum. The lower cellulolytic activity in the cecum than in the rumen could not be ascribed to any difference in the structure of the cellulolytic bacterial community between these two compartments, and other hypotheses related to digestion are proposed.

Animal Feed↗
PubMed2005

Beta-lactam antibiotic resistance: a current structural perspective.

Bacterial resistance to beta-lactam antibiotics can be achieved by any of three strategies: the production of beta-lactam-hydrolyzing beta-lactamase enzymes, the utilization of beta-lactam-insensitive cell wall transpeptidases, and the active expulsion of beta-lactam molecules from Gram-negative cells by way of efflux pumps. In recent years, structural biology has contributed significantly to the understanding of these processes and should prove invaluable in the design of drugs to combat beta-lactam resistance in the future.

Anti-Bacterial Agents↗
PubMed1963

Relation of structure to function in bacterial O antigens. I. Isolation methods.

Nowotny, Anna M. (City of Hope Medical Center, Duarte, Calif.), Scott Thomas, Olga S. Duron, and Alois Nowotny. Relation of structure to function in bacterial O antigens. I. Isolation methods. J. Bacteriol. 85:418-426. 1963.-Six isolation methods, four previously described and two reported herein, were compared for the extraction of endotoxic O antigens from three different enterobacteria: Serratia marcescens Bizio, Salmonella typhosa O901, and Escherichia coli K-12 lambda. Large variations were observed in the toxin yield, in the serologically reactive material yield, and in the nucleic acid-releasing abilities of the six methods when applied to the different bacterial strains. Chemical analyses of the toxic O antigens included quantitative determination of the amino acids, long- and short-chain carboxylic acids, and carbohydrates. Comparisons of the nucleic acid content were also made. Although the chemical composition of the preparations was quite different, no correlation could be found between the percentage of the above components and the lethal toxicity or the serological reactivity. It was concluded that the structural elements or properties responsible for these biological activities cannot be detected by gross chemical analyses.

Antigens↗
PubMed1991

Small and medium-angle X-ray analysis of bacterial lipoteichoic acid phase structure.

X-ray scattering analysis was performed on various types of bacterial lipoteichoic acid in solution. The X-ray data show that all samples investigated were characterized by a similar micellar ultrastructure (hydrophilic moiety on the outside) with a fatty acid chain conformation of the disordered, alpha-type at all temperatures between 5 degrees-53 degrees C. The size distribution of Staphylococcus aureus lipoteichoic acid micelles was sufficiently homogeneous to determine their size and some related molecular parameters by detailed small-angle X-ray scattering analysis. Nearly independent of the degree of D-alanine substitution and the ionic strength of the aqueous dispersion, an average micelle contained about 150 lipoteichoic acid molecules arranged in a spherical assembly with a diameter of about 22 nm, whereby the hydrophilic region occupied an outer shell of about 8.5 nm thickness. Based on the average chain length of lipoteichoic acid, it could be estimated that each glycerophosphate residue contributed by about 0.34 nm to the thickness of the hydrophilic shell as compared to a theoretical value of approximately 0.8 nm for a fully extended chain conformation, indicating a highly coiled conformation of the hydrophilic chain. The bearing of these findings on the properties of membrane-associated and secreted lipoteichoic acids is discussed.

Bifidobacterium↗
PubMed1996

Phylogenetic analysis of tmRNA secondary structure.

The bacterial tmRNA acts with dual tRNA-like and mRNA-like character to tag incomplete translation products for degradation. Comparative analysis of 17 tmRNA genes (including eight new sequences) has allowed us to deduce conserved features of the tmRNA secondary structure. Except in a segment that includes the first codon of the tag reading frame, tmRNA is highly structured, with four pseudoknots and a total of 11 conserved base pairing regions. The previously identified tRNA minihelix structure is connected by a long base paired region to a large structured domain composed of a pseudoknot, followed by the tag reading frame and a string of three rather similar pseudoknots. The conservation of numerous structural elements among diverse eubacterial species indicates that these elements have important function beyond simply forming an endonuclease-resistant link between the reading frame and the tRNA-like domain.

Bacteria↗