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The three-dimensional structure of the ligand-binding domain of a wild-type bacterial chemotaxis receptor. Structural comparison to the cross-linked mutant forms and conformational changes upon ligand binding.

The three-dimensional structures of the ligand-binding domain of the wild-type Salmonella typhimurium aspartate receptor have been determined in the absence (apo) and presence of bound aspartate (complex) and compared to a cross-linked mutant containing a cysteine at position 36 which does not change signaling behavior of the intact receptor. The structures of the wild-type forms were determined in order to assess the effects of cross-linking on the structure and its influence on conformational changes upon ligand binding. As in the case of the cross-linked mutant receptor, the non-cross-linked ligand-binding domain is dimeric and is composed of 4-alpha-helical bundle monomer subunits related by a crystallographic 2-fold axis in the unbound form and by a non-crystallographic axis in the aspartate-bound form. A comparative study between the non-cross-linked and cross-linked structures has led to the following observations: 1) The long N-terminal helices of the individual subunits in the cross-linked structures are bent toward each other to accommodate the disulfide bond. 2) The rest of the subunit conformation is very similar to that of the wild-type. 3) The intersubunit angle of the cross-linked apo structure is larger by about 13 degrees when compared to the wild-type apo structure. 4) The nature and magnitude of the aspartate-induced conformational changes in the non-cross-linked wild-type structures are very similar to those of the cross-linked structures.

Amino Acid Sequence↗

Unidentified bacterial microorganisms entrapped within blood capillary spaces of tissue from different epidemiological types of Kaposi's sarcoma.

Tissue specimens of different epidemiological types of Kaposis sarcoma (KS) from various geographical regions were investigated by transmission electron microscopy. Freshly fixed KS biopsies originated from 9 German patients: 3 classic KS cases, 5 AIDS-associated KS cases, and 1 atypical classic KS case. Additionally, KS autopsy material from the brain of a German AIDS patient was examined. Further biopsies came from 29 Ugandan patients: 16 endemic KS cases and 13 AIDS-associated KS cases. While investigating the ultrastructure, we discovered relatively small-sized bacterial microorganisms within blood capillary spaces of tumor tissue from 5 KS cases of different epidemiological type. The microorganisms often occurred in clusters. They were of coccoid-bacillary form and limited by a wrinkled multilayered cell wall. Many of them were encapsulated. They were not observed outside of the capillary lumen. The bacterial structures were often seen attached to capillary endothelial cells, which sometimes showed blistering into the capillary lumen. The observed bacterial microorganisms obviously represented agents of a bloodstream infection and must have been entrapped and accumulated within capillary spaces of KS tissue. The bacteria, which had an almost identical morphology in all 5 KS cases, could not be identified. If they are of pathogenic significance, it remains unknown.

Acquired Immunodeficiency Syndrome↗

Undulipodia, flagella and cilia.

The term flagella is ambiguous. It refers to bacterial structures composed of flagellin protein and to eukaryotic structures composed of microtubule proteins and ATPase (tubulin and dynein). The fact that cilia are nearly identical to eukaryotic flagella and have nothing in common with prokaryotic flagella is not apparent from the terminology. It is proposed that the 30-year old suggestion of Smagina and reiterated by Kuznicki and others, be adopted: that cilia and eukaryotic flagella be called "undulipodia." The term flagella ought to be restricted to prokaryotic organelles, bacterial flagella and spirochaete axial filaments: solid structures composed of flagellin which protrude through the plasma membrane and lack intrinsic motility throughout their length. Undulipodia are defined as intrinsically motile intracellular structures showing a 9-fold symmetry in the pattern of arrangement of 24 nm diameter microtubules. They are limited to eukaryotes, members of the protoctist, animal and plant kingdoms.

Animals↗

Crystal structure of a bacterial cocaine esterase.

Here we report the first structure of a cocaine-degrading enzyme. The bacterial esterase, cocE, hydrolyzes pharmacologically active (-)-cocaine to a non-psychoactive metabolite with a rate faster than any other reported cocaine esterase (kcat = 7.8 s-1 and KM = 640 nM). Because of the high catalytic proficiency of cocE, it is an attractive candidate for novel protein-based therapies for cocaine overdose. The crystal structure of cocE, solved by multiple anomalous dispersion (MAD) methods, reveals that cocE is a serine esterase composed of three domains: (i) a canonical alpha/beta hydrolase fold (ii) an alpha-helical domain that caps the active site and (iii) a jelly-roll-like beta-domain that interacts extensively with the other two domains. The active site was identified within the interface of all three domains by analysis of the crystal structures of transition state analog adduct and product complexes, which were refined at 1.58 A and 1.63 A resolution, respectively. These structural studies suggest that substrate recognition arises partly from interactions between the benzoyl moiety of cocaine and a highly evolved specificity pocket.

Acylation↗

Interaction of polyphemusin I and structural analogs with bacterial membranes, lipopolysaccharide, and lipid monolayers.

Three structural variants (PV5, PV7, and PV8) of the horseshoe crab cationic antimicrobial peptide polyphemusin I were designed with improved amphipathic profiles. Circular dichroism spectroscopy analysis indicated that in phosphate buffer polyphemusin I, PV7, and PV8 displayed the spectrum of a type II beta-turn-rich structure, but, like polyphemusin I, all three variants adopted a typical beta-sheet structure in an anionic lipid environment. Both polyphemusin I and variants were potent broad spectrum antimicrobials that were clearly bactericidal at their minimal inhibitory concentrations. The variants were moderately less active in vitro but more effective in animal models. Moreover, these variants exhibited delayed bacterial killing, whereas polyphemusin I killed Escherichia coli UB1005 within 5 min at 2.5 microg/mL. All the peptides showed similar abilities to bind to bacterial lipopolysaccharide (LPS) and permeabilize bacterial outer membranes. Consistent with this was the observation that all peptides significantly inhibited cytokine production by LPS-stimulated macrophages and penetrated polyanionic LPS monolayers to similar extents. None of the peptides had affinity for neutral lipids as evident from both tryptophan fluorescence spectroscopy and Langmuir monolayer analysis. As compared to polyphemusin I, all variants showed reduced ability to interact with anionic lipids, and the hemolytic activity of the variants was decreased by 2-4-fold. In contrast, polyphemusin I efficiently depolarized the cytoplasmic membrane of E. coli, as assessed using a membrane potential sensitive fluorescent dye 3,3-dipropylthiacarbocyanine (diSC(3)5) assay, but the variants showed a substantially delayed and decreased depolarizing ability. The coincident assessment of cell viability indicated that depolarization of the bacterial cytoplasmic membrane potential by polyphemusin I occurred prior to lethal damage to cells. Our data suggest that increase of amphipathicity of beta-sheet polyphemusin I generally resulted in variants with decreased activity for membranes. Interestingly, all variants showed an improved ability to protect mice both against infection by Pseudomonas aeruginosa and from endotoxaemia.

Animals↗

Functional and structural analyses of trichloroethylene-degrading bacterial communities under different phenol-feeding conditions: laboratory experiments.

The effects of different phenol-feeding conditions on trichloroethylene (TCE) biodegradation and bacterial population structure in an aquifer soil community were studied. The soil sample, minerals, phenol, and TCE were mixed in glass bottles, which were then incubated under three different phenol-feeding conditions. First, phenol was supplied only once at 0.2 mM (condition 0.2P); second, it was added at 2.0 mM (condition 2.0P); and third, it was periodically supplied ten times at 0.2 mM (condition 0.2PS). TCE concentrations remained stable under conditions 0.2P and 2.0P. In contrast, TCE was completely degraded under condition 0.2PS. TCE/phenol-degrading bacteria were enumerated indirectly and functionally by quantitative PCR. The low- K(s) (half saturation constant) group of phenol-degrading bacteria, exhibiting high TCE-degrading activity, yielded a 50-fold higher population under condition 0.2PS than under condition 2.0P. The bacterial community structure under condition 0.2PS was studied by denaturing gradient gel electrophoresis targeting the genes encoding 16S rRNA and the largest subunit of multicomponent phenol hydroxylase. Sequence analysis of the major bands detected indicated the predominance of the low- K(s) group of TCE/phenol-degrading bacteria belonging to beta-Proteobacteria. These results suggest that continuous supplementation with phenol at a low concentration increases the population of the low- K(s) group of TCE/phenol-degrading bacteria.

Bacteria↗

Dynamic structural determinants in bacterial microcompartment shells.

Bacterial microcompartments (BMCs) are polyhedral structures that segregate enzymatic cargo from the cytosol via encapsulation within a protein shell. Unlike other biological polyhedra, such as viral capsids and encapsulins, BMC shells can exhibit a highly advantageous structural and functional plasticity, conforming to a variety of anabolic (CO2 fixation in carboxysomes) and catabolic (nutrient assimilation in metabolosomes) roles. Consequently, understanding the subunit properties and associated protein-protein interaction processes that guide shell assembly and function is a necessary step to fully harness BMCs as modular, biotechnological nanomachines. Here, we describe the recent insights into the dynamics of structural features of the key BMC domain (Pfam00936)-containing proteins, which serve as a structural template for BMC-H and BMC-T shell building blocks.

Bacterial Proteins↗

The crystal structure of the bacterial chaperonin GroEL at 2.8 A.

The crystal structure of Escherichia coli GroEL shows a porous cylinder of 14 subunits made of two nearly 7-fold rotationally symmetrical rings stacked back-to-back with dyad symmetry. The subunits consist of three domains: a large equatorial domain that forms the foundation of the assembly at its waist and holds the rings together; a large loosely structured apical domain that forms the ends of the cylinder; and a small slender intermediate domain that connects the two, creating side windows. The three-dimensional structure places most of the mutationally defined functional sites on the channel walls and its outward invaginations, and at the ends of the cylinder.

Amino Acid Sequence↗

Fine structure of Calymmatobacterium granulomatis with particular reference to the surface structure.

Ultrastructural study of C. granulomatis, the causative organism of donovanosis (granuloma inguinale), in human tissue revealed the presence of a complex cell envelope. The cytoplasm of these organisms showed presence of electron dense polar material, in addition to regular bacterial structures like mesosome, ribosomes, and nuclear material. Surface appendages i.e., fimbriae and blebs were studied in detail. Origin of these structures was clearly endogenous to the cell wall. Morphology of fimbrium at the site of its attachment to the cell membrane has been described. A distinct layer of homogenous material of varying density surrounding the organism indicated the possibility of it being a capsule.

Cell Membrane↗

Molecular cloning and expression of a T-cell stimulating membrane protein of Francisella tularensis.

The isolation and expression in Escherichia coli of a gene encoding a T-cell stimulating 17 kiloDalton (kDa) membrane protein of Francisella tularensis is described. A genomic library of DNA from the live vaccine strain LVS of F. tularensis was constructed in the E. coli expression vector phage lambda gt11. The library was probed with antibodies directed against the 17 kDa protein. One recombinant phage was isolated, containing a 2.8 kilobase (kb) DNA insert. The insert was cleaved and a resulting 1.2 kb fragment was found to express the 17 kDa protein. The 1.2 kb fragment was inserted in the high copy number plasmid pUC18 and expressed in E. coli. Membrane preparations of these bacteria induced a response in T cells from F. tularensis-primed individuals but not in T cells from non-primed individuals. The cloned gene may become useful in studies on host interaction with F. tularensis and enable a precise identification of bacterial structures involved in the T-cell response.

Adult↗

[Physiopathologic mechanisms in bacterial infection and antibiotics].

There are basically three mains determinants of effective antimicrobial therapy: pharmacodynamic, pharmaceutical and pharmacokinetic. The pharmacodynamic characteristics of an antibiotic describe its mechanisms of action and its safety profile, that is largely defined in vitro. Its pharmaceutical aspects deal with the formulation of the drug. And the pharmacokinetic determinants deal with those process involved in drug absorption, distribution, metabolism and excretion. The fact that antimicrobial drugs are used in an infected host need to reconsider its possible interactions with the induced non specific and specific host defense reactions. Such inflammatory reactions could modify the pharmacodynamic characteristics, the tissue and cellular distribution of a given antibiotic. Moreover define antibiotics, acting themselves on bacterial structure which are proinflammatory, or cellular compartments involved in the defense mechanisms, such as macrophages, might also interfere indirectly or directly on the expression of the inflammatory reactions associated with bacterial infections. Better knowledges about interelationships between the effects of antimicrobial drugs on the pathophysiological mechanisms induced by bacterial, viral parasitic and fungal infection are warranted.

Anti-Bacterial Agents↗

[Proliferative response of murine lymphocytes caused by the activity of amphiphilic molecules from Propionibacterium acnes].

Splenic lymphocytes from mice treated with Propionibacterium acnes cells as well as with their cell walls were found to be variably active on the lymphoproliferative responsiveness. Furthermore, the effect of these bacterial agents on the ex vivo Con A response of the lymphocytes showed a certain stimulation that was higher with oral treatments. In the same conditions the influence of these agents on the LPS lymphocytes stimulation was almost without any statistical significance. In vitro blastogenesis experiments were undertaken in order to elucidate the influence of different amphiphilic molecules from peripheric bacterial structures on the lymphoproliferative response of murine splenocytes. Stimulation rates were also determined as a function of the (3H) thymidine incorporation. Combined effects of mitogens (Con A and LPS) with bacterial amphiphilic molecules were also evaluated as a function of the DNA synthesis variations. All cases resulted in a variable inhibition of the mitogenic response which appeared dose-dependent and more active for associations of Con A and amphiphilic molecules. The most effective intrinsic mitogenic activities were detected with teichoic acids and intracellular polysaccharides. These last molecules without purification, assayed as cytoplasmic fractions, appeared modified in the intensity of their action, depending on their carbohydrate/protein ratios.

Animals↗

Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex.

We have used systematic fluorescence resonance energy transfer and distance-constrained docking to define the three-dimensional structures of bacterial RNA polymerase holoenzyme and the bacterial RNA polymerase-promoter open complex in solution. The structures provide a framework for understanding sigma(70)-(RNA polymerase core), sigma(70)-DNA, and sigma(70)-RNA interactions. The positions of sigma(70) regions 1.2, 2, 3, and 4 are similar in holoenzyme and open complex. In contrast, the position of sigma(70) region 1.1 differs dramatically in holoenzyme and open complex. In holoenzyme, region 1.1 is located within the active-center cleft, apparently serving as a "molecular mimic" of DNA, but, in open complex, region 1.1 is located outside the active center cleft. The approach described here should be applicable to the analysis of other nanometer-scale complexes.

Bacteria↗

[The biomedical aspects of the persistence of bacteria].

A correlation between the structure and function of bacteria was analyzed in the process of their persistence in the host body. A variety of persistent forms of bacteria was shown to be based on the isolation of their morphological substrate, peptidoglycane, which a cell "masked" (screening by surface bacterial structures, antigenic mimicry), "lost" (L-forms of bacteria, mycoplasmas) or protected against the system of host immunity by secreted factors. A new group of secreted bacterial (antilysozyme, anti-interferon, anti-immunoglobulin, anticomplement) factors, permitting microbial persistence in the host body, was described. Different methodological approaches to their determination were developed on the basis of the principle of "delayed antagonism". Applied aspects of the problem of persistence of bacteria were reviewed. The efficacy of new methods developed for the isolation and identification of a causative agent under the control of persistence markers was demonstrated on facultative microflora in different surgical, obstetrical, gynecological, urological diseases and diseases of internal organs. The facts concerning the use of the factors of bacterial persistence were presented for the solution of therapeutic (selection of means for controlling cell parasites), prognostic (development of carrier state in convalescents) and ecological (microbiological monitoring of the environment) problems.

Animals↗

The structure of resting bacterial populations in soil and subsoil permafrost.

The structure of individual cells in microbial populations in situ of the Arctic and Antarctic permafrost was studied by scanning and transmission electron microscopy methods and compared with that of cyst-like resting forms generated under special conditions by the non-spore-forming bacteria Arthrobacter and Micrococcus isolated from the permafrost. Electron microscopy examination of microorganisms in situ revealed several types of bacterial cells having no signs of damage, including "dwarf" curved forms similar to nanoforms. Intact bacterial cells in situ and frozen cultures of the permafrost isolates differed from vegetative cells by thickened cell walls, the altered structure of cytoplasm, and the compact nucleoid, and were similar in these features to cyst-like resting forms of non-spore-forming "permafrost" bacterial strains of Arthrobacter and Micrococcus spp. Cyst-like cells, being resistant to adverse external factors, are regarded as being responsible for survival of the non-spore-formers under prolonged exposure to subzero temperatures and can be a target to search for living microorganisms in natural environments both on the Earth and on extraterrestrial bodies.

Arthrobacter↗

Innate immunity and mucosal bacterial interactions in the intestine.

PURPOSE OF REVIEW: Exciting progress has been made recently in identifying receptors and effector molecules of innate immunity. The review focuses on new insights and their applications to intestinal physiology in the areas of toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-containing proteins as microbial sensors, and defensins and cathelicidins as antimicrobial effectors. RECENT FINDINGS: Toll-like receptors recognize conserved bacterial structures including cell wall components and specific DNA motifs. Several TLRs are expressed constitutively or inducibly in the intestine, and contribute to immune defense against enteric pathogens such as Salmonella. NOD proteins are cytoplasmic sensors of bacterial components. NOD1 is expressed in intestinal epithelial cells and activates proinflammatory cytokine production in response to a peptidoglycan motif in gram-negative bacteria. NOD2 is present in macrophages, dendritic and Paneth cells, and can be induced in enterocytes. Its activation by bacterial muramyl dipeptide induces expression of proinflammatory mediators. Mutations in NOD2 are highly associated with the development of Crohn disease. The major groups of antimicrobial proteins in humans are defensins, with at least 8 alpha- and 10 beta-defensin genes, and cathelicidins, with only one known gene, LL-37/hCAP18. They all have broad-spectrum antimicrobial activity, but several also exhibit immunoregulatory and angiogenic functions. Their differential expression and regulation in the epithelium throughout the gastrointestinal tract suggests that the various antimicrobial peptides have distinct functional niches in mucosal innate defense. SUMMARY: More than 50 human genes have been identified to date that can sense and destroy enteric microbes. Elucidation of their physiologic functions will aid in developing new treatment and prevention strategies for inflammatory and infectious diseases in the intestine.

Journal Article↗

Small-Scale Distribution of Bacteria, Enzymatic Activities, and Organic Matter in Coastal Sediments.

The small-scale distribution of several structural (bacterial abundance, phytopigment, total and soluble protein, and carbohydrate content) and functional ecological variables (enzymatic activities, frequency of dividing cells) was investigated in coastal sediments during a spring bloom. For bacterial abundance, enzymatic activity, and organic matter determinations, samples were collected at 5-m depth from a sediment surface delimited by a 42 x 42-cm frame, divided into 49 squares. In order to test the influence of the bottom microtopography on the investigated variables, the size of this frame was defined to cover the distance between two subsequent ripples. As indicated by Fisher's index, benthic bacteria, enzymatic activities, proteins, carbohydrates, and their soluble fractions exhibited an aggregate distribution. Sampling size (i.e., sample unit of 36 cm2) was appropriate for all variables, except for chlorophyll a and frequency of dividing cells that displayed a contagious distribution. To estimate the reliability of the current sampling strategy, we compared the mean values from three randomly selected sample units with the average value of the entire sediment surface (i.e., 49 samples). For all variables reported in this study, the use of three replicates was representative of the mean values of the sampled area with a confidence limit within +/-20%. Bacterial population sizes did not correlate with their potential food sources (e.g., phytopigments, proteins, and carbohydrates), or with enzymatic activities, suggesting the presence of possible time lags between organic inputs and microbial response. Chlorophyll a during the spring bloom displayed much higher concentrations than phaeopigments and correlated with carbohydrates. Chlorophyll a distribution was autocorrelated and displayed a large patch size (1,134 cm2). Phaeopigments and proteins accumulated in the central depression of the ripple-mark structure, apparently depending upon a passive accumulation due to the reduced current action. In contrast, microphytobenthic biomass and bacterial numbers were highest in the two opposite ripples, suggesting that different driving forces operate selectively on the living components.

Journal Article↗