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Phenazines and other redox-active antibiotics promote microbial mineral reduction.

Natural products with important therapeutic properties are known to be produced by a variety of soil bacteria, yet the ecological function of these compounds is not well understood. Here we show that phenazines and other redox-active antibiotics can promote microbial mineral reduction. Pseudomonas chlororaphis PCL1391, a root isolate that produces phenazine-1-carboxamide (PCN), is able to reductively dissolve poorly crystalline iron and manganese oxides, whereas a strain carrying a mutation in one of the phenazine-biosynthetic genes (phzB) is not; the addition of purified PCN restores this ability to the mutant strain. The small amount of PCN produced relative to the large amount of ferric iron reduced in cultures of P. chlororaphis implies that PCN is recycled multiple times; moreover, poorly crystalline iron (hydr)oxide can be reduced abiotically by reduced PCN. This ability suggests that PCN functions as an electron shuttle rather than an iron chelator, a finding that is consistent with the observation that dissolved ferric iron is undetectable in culture fluids. Multiple phenazines and the glycopeptidic antibiotic bleomycin can also stimulate mineral reduction by the dissimilatory iron-reducing bacterium Shewanella oneidensis MR1. Because diverse bacterial strains that cannot grow on iron can reduce phenazines, and because thermodynamic calculations suggest that phenazines have lower redox potentials than those of poorly crystalline iron (hydr)oxides in a range of relevant environmental pH (5 to 9), we suggest that natural products like phenazines may promote microbial mineral reduction in the environment.

Anti-Bacterial Agents↗

Clostridial toxins: molecular probes of Rho-dependent signaling and apoptosis.

The Rho family small GTPases are members of the Ras superfamily of small GTPases. Rho proteins were first determined to act as key regulators of many types of actin cytoskeletal-dependent cellular functions. Recent work by several investigators indicates that Rho GTPases are also critical modulators of several important intracellular and nuclear signal transduction pathways. Certain clostridial toxins and exoenzymes covalently modify, and thereby inactivate, specific types of Rho family GTPases. As such, these microbial enzymes have proven invaluable in helping to identify structural and functional attributes of Rho GTPases.

Apoptosis↗

Microbial diversity and bioremediation of a hydrocarbon-contaminated aquifer (Vega Baja, Puerto Rico).

Hydrocarbon contamination of groundwater resources has become a major environmental and human health concern in many parts of the world. Our objectives were to employ both culture and culture-independent techniques to characterize the dynamics of microbial community structure within a fluidized bed reactor used to bioremediate a diesel-contaminated groundwater in a tropical environment. Under normal operating conditions, 97 to 99% of total hydrocarbons were removed with only 14 min hydraulic retention time. Over 25 different cultures were isolated from the treatment unit (96% which utilized diesel constituents as sole carbon source). Approximately 20% of the isolates were also capable of complete denitrification to nitrogen gas. Sequence analysis of 16S rDNA demonstrated ample diversity with most belonging to the infinity, beta and gamma subdivision of the Proteobacteria, Bacilli, and Actinobacteria groups. Moreover, the genetic constitution of the microbial community was examined at multiple time points with a Functional Gene Array (FGA) containing over 12,000 probes for genes involved in organic degradation and major biogeochemical cycles. Total community DNA was extracted and amplified using an isothermal phi29 polymerase-based technique, labeled with Cy5 dye, and hybridized to the arrays in 50% formimide overnight at 50 degrees C. Cluster analysis revealed comparable profiles over the course of treatment suggesting the early selection of a very stable microbial community. A total of 270 genes for organic contaminant degradation (including naphthalene, toluene [aerobic and anaerobic], octane, biphenyl, pyrene, xylene, phenanthrene, and benzene); and 333 genes involved in metabolic activities (nitrite and nitrous oxide reductases [nirS, nirK, and nosZ], dissimilatory sulfite reductases [dsrAB], potential metal reducing C-type cytochromes, and methane monooxygenase [pmoA]) were repeatedly detected. Genes for degradation of MTBE, nitroaromatics and chlorinated compounds were also present, indicating a broad catabolic potential of the treatment unit. FGA's demonstrated the early establishment of a diverse community with concurrent aerobic and anaerobic processes contributing to the bioremediation process.

Bacteria↗

Replacement of inorganic phosphorus by microbial phytase for young pigs fed on a maize-soyabean-meal diet.

Ninety-six crossbred young pigs (body weight 7.8 kg) were used in a 5-week trial to determine the effectiveness of microbial phytase (EC 3.1.3 26) in improving the bioavailabilities of P and other nutrients in maize-soyabean-meal diets and, thus, replacing inorganic P with phytase. A 2 x 5 factorial arrangement of treatments was employed with two available P (aP) levels (0.7 and 1.6 g/kg) and five phytase levels (0, 350, 700, 1050, 1400 U (the quantity of enzyme that liberates 1 mumol inorganic phosphate/min from 5.1 mm-sodium phytate at pH 5.5 and 37 degrees)/kg diet). In addition, two extra diets were formulated to supply the National Research Council (1988) recommended level of aP (3.2 g/kg) with 0 or 1400 U phytase. The addition of graded levels of phytase resulted in linear increases in average daily weight gain, average daily feed intake and weight gain:feed intake for pigs fed on diets containing 0.7 or 1.6 g aP/kg (P < 0.04). Also, the addition of phytase linearly increased apparent digestibilities of P and Ca (P < 0.01), whereas faecal P excretion was linearly decreased (P < 0.01). Linear increases in shear force, shear energy and ash content of both the metacarpal and tenth rib, and shear stress of the metacarpal were found to respond to added phytase (P < 0.01). These improvements in performance, apparent P absorption and bone measurements by phytase were also observed by increasing dietary aP levels for most measurements. Adding 1400 U phytase to the 3.2 g aP/kg diet further increased average daily weight gain, average daily feed intake, apparent absorption of P, Ca and N and metatarsal shear force and ash content (P < 0.01 to 0.05). Generally, maximum responses occurred at a phytase level of 1050 U/kg diet for the 0.7 g aP/kg diets and 700 U for the 1.6 g aP/kg diets. Based on non-linear and linear response equations generated for the phytase and aP levels, the average function of the equivalency of P (Y, g/kg) v. microbial phytase (X, U/kg) was developed across aP levels of 0.7 and 1.6 g/kg for average daily weight gain and apparent digestibility of P: Y = 2.622-2.559e 0.00185X. The replacement of 1 g inorganic P as defluorinated phosphate would require about 246 U microbial phytase. This represents 41% of released P from phytate.

6-Phytase↗

The relationship between microbial carbon and the resource quality of soil carbon.

The biological health of soil is an important aspect of soil quality because of the many critical functions performed by organisms in soil. Various indicators of soil quality have been proposed, but measurements of microbial biomass are most commonly used. During decomposition of plant residues in soil the relative intensities of the O-alkyl-C signal decreases and the alkyl-C signal increases in nuclear magnetic resonance (NMR) spectra. This leads to the suggestion that the alkyl-C to O-alkyl-C ratio of a soil may indicate the degree of decomposition. Consequently, the overall resource quality of soil C as a substrate for heterotrophic microorganisms may be inversely related to the alkyl-C to O-alkyl-C ratio. Our hypothesis is that a relationship exists between the size of the soil microbial community (microbial biomass) and the quality of soil carbon as a resource for microorganisms. New data have been combined with previously published data to show that there was a significant, negative correlation between the biomass C to total C (Cmic, to Corg) ratio and the alkyl-C to O-alkyl-C ratio (p < 0.01), which supports our hypothesis.

Biomass↗

Probing molecular interactions and mechanical properties of microbial cell surfaces by atomic force microscopy.

Knowledge of the surface properties of microbial cells is a key to gain a detailed understanding of their functions in the natural environment and to efficiently exploit them in biotechnological processes. In this paper, we present force-distance curves recorded, by atomic force microscopy (AFM) in aqueous solutions, on various microbial samples: reconstituted S-layers, whole fungal spores and several bacterial strains. The approach and retraction curves exhibited important differences--depending on the type of microorganism, on the physiological state (dormancy versus germination) and on the environmental conditions (ionic strength)--which were shown to reflect differences in long-range surface forces, adhesion forces and mechanical properties. These data illustrate the great potential of AFM force measurements to elucidate the physical properties of microbial cells and to understand, at the molecular level, biointerfacial phenomena such as cell adhesion and cell aggregation.

Cell Adhesion↗

The extracellular matrix protein mindin is a pattern-recognition molecule for microbial pathogens.

Microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. However, ECM components can also serve as an integral part of the innate immunity. Mice lacking expression of mindin (spondin 2), a highly conserved ECM protein, have an impaired ability to clear bacterial infection, and mindin-deficient macrophages show defective responses to a broad spectrum of microbial stimuli. Moreover, mindin binds directly to bacteria and their components and functions as an opsonin for macrophage phagocytosis of bacteria. Thus, mindin is essential in the initiation of the innate immune response and represents a unique pattern-recognition molecule in the ECM for microbial pathogens.

Amino Acid Sequence↗

Structure-activity relation of human beta-defensin 3: influence of disulfide bonds and cysteine substitution on antimicrobial activity and cytotoxicity.

Human beta-defensins form a group of cysteine-rich antimicrobial peptides which have been found in epithelial tissue and, more recently, in the male genital tract. They play a role in the defense against microbial pathogens in innate immunity and display additional chemotactic functions in the adaptive immune system. An important characteristic of antimicrobial peptides is that they also exhibit toxic potential on eukaryotic cells. Very little is known about the structure dependence of antimicrobial and cytotoxic effects. We investigated human beta-defensin 3 (hBD-3), a potent broad-spectrum antimicrobial effector peptide, regarding the influence of structural parameters on the antimicrobial and cytotoxic activity. We have established a structure-activity relation of the hBD-3 using synthetic derivatives differing in length, charge, disulfide connectivity, and overall hydrophobicity. The antimicrobial activity of the peptides was compared to the cyctotoxic effects on monocytic THP-1 cells and the hemolytic activity on human erythrocytes. We found that it is not important for antimicrobial and cytotoxic activity whether and how cysteine residues are arranged to form disulfide bonds. Substitution of half-cystinyl residues by tryptophan resulted in increased activities, while other substitutions did not change activity. Correlation of activities with the structural changes demonstrates that the activity on eukaryotic cells appears to depend strongly on the overall hydrophobicity. In contrast, the antimicrobial potency of hBD-3 peptides is determined by the distribution of positively charged amino acid residues and hydrophobic side chains. The results facilitate the understanding of beta-defensin interaction with different cell types and guide the design of antimicrobially active peptides.

Amino Acid Sequence↗

Infection rates in surgical neonates and infants receiving parenteral nutrition: a five-year prospective study.

We performed a prospective, observational, cohort study on 208 surgical neonates and infants between 1992 and 1997. Surveillance cultures of the oropharynx and rectum were obtained at the start of parenteral nutrition and thereafter twice weekly. Blood cultures were taken on clinical indication only. Microbial translocation was diagnosed when the micro-organisms in the blood were not distinguishable from those carried in the oropharynx and/or rectum. Liver function was monitored weekly and when septicaemia was suspected. The incidence of septicaemia was 15%. The predominant micro-organisms (86%) were the low-level pathogens, coagulase-negative staphylococci and enterococci. Potential pathogens, including aerobic Gram-negative bacilli, were responsible for the remainder. Microbial translocation was responsible for 84% of septicaemic episodes in 76% of patients. The potential pathogens caused septicaemia significantly later than coagulase-negative staphylococci, at a time when liver function was significantly more impaired. In neonates and infants receiving parenteral nutrition, septicaemia is mainly a gut-derived phenomenon and requires novel strategies for prevention.

Bacteremia↗

Nature and significance of microbial cometabolism of xenobiotics.

Microbial cometabolism, i.e. "transformation of a non-growth substrate in the obligate presence of a growth substrate or another transformable compound" (Dalton and Stirling 1982) is a whole-cell phenomenon physiologically based on coupling of different catabolic pathways at the cellular level. It is frequently observed in transformation of xenobiotic non-growth substrates by individual microbial species. Transformation processes of this type are usually mediated by appropriate non-specific enzymes of the peripheric cellular metabolism able to modify a variety of substances other than their natural substrates. The precise mechanisms of coupling between metabolism of xenobiotic non-growth substrates and of particular additional carbon substrates may be different depending on the substrates and the microbial species involved. However, experimental data indicate that the primary function of the respective additional carbon substrates is to supply either energy, cofactors or metabolites for the different cellular events involved in the transformation process (e.g. uptake of the xenobiotic non-growth substrate, functioning of appropriate degradative enzymes of the peripheric cellular metabolism). Cometabolism of xenobiotics involves nothing special or novel from the standpoint of biochemistry. On the contrary, there are numerous examples where the turnover of particular natural compounds by certain aerobic or anaerobic microorganisms is essentially based on coupling of different catabolic pathways at the cellular level by transfer of hydrogen (i.e. reducing power) and/or energy between two or more enzymatic reactions. Synthetic chemicals which resist total degradation by individual microbial species may undergo mineralization due to complementary catabolic sequences mediated by certain multispecies microbial associations with cometabolic transformations being the initial steps. Although taking place in certain natural habitats (e.g. rhizospheres, sewage), microbial cometabolism of xenobiotics in natural ecosystems occurs with slow rates since the respective cometabolizing populations are generally small and will not increase in number or biomass in response to the introduced chemicals. However, under conditions of axenic microbial cultures, high concentrations of biomass, and appropriate substrate mixtures cometabolism of synthetic chemicals may be a useful technique of considerable practical importance to accumulate biochemical products at high yields. In addition, cometabolic capabilities of wild-type microorganisms may serve as a tool for the construction of microbial strains with a new degradative potential for recalcitrant xenobiotic compounds.

Aerobiosis↗

Changes in the tear film and ocular surface from dry eye syndrome.

Dry eye syndrome (DES) refers to a spectrum of ocular surface diseases with diverse and frequently multiple aetiologies. The common feature of the various manifestations of DES is an abnormal tear film. Tear film abnormalities associated with DES are tear deficiency, owing to insufficient supply or excessive loss, and anomalous tear composition. These categorizations are artificial, as in reality both often coexist. DES disrupts the homeostasis of the tear film with its adjacent structures, and adversely affects its ability to perform essential functions such as supporting the ocular surface epithelium and preventing microbial invasion. In addition, whatever the initial trigger, moderate and severe DES is characterized by ocular surface inflammation, which in turn becomes the cause and consequence of cell damage, creating a self-perpetuating cycle of deterioration. Progress has been made in our understanding of the aetiology and pathogenesis of DES, and these advances have encouraged a proliferation of therapeutic options. This article aims to amalgamate prevailing ideas of DES development, and to assist in that, relevant aspects of the structure, function, and production of the tear film are reviewed. Additionally, a synopsis of therapeutic strategies for DES is presented, detailing treatments currently available, and those in development.

Animals↗

Novel approach to quantitative detection of specific rRNA in a microbial community, using catalytic DNA.

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities.

Base Sequence↗

[Function of neonatal neutrophilic granulocytes].

Phagocytic leukocytes especially neutrophils form an essential part of human host defense against infection by invading microorganisms. The principal activities exhibited by circulating neutrophils in response to inflammation include adherence to vascular endothelium, chemotaxis, phagocytosis and microbial killing. As reviewed in this article, each of these aspects of cell function has been studied in neutrophils of newborn and preterm infants. The most consistent finding has been abnormal chemotaxis. Whether the newborn infant who suffers from an increased susceptibility to serious infections is actually predisposed to infection because of deficiencies in neutrophil function remains questionable. It seems likely that subnormal phagocyte function combined with other defects in host defense such as deficient complement and opsonizing activity in neonatal sera could predispose the newborn infant to bacterial infections that rapidly become systemic.

Bacterial Infections↗

Hemocytes from the tobacco hornworm Manduca sexta have distinct functions in phagocytosis of foreign particles and self dead cells.

Phagocytosis is an important innate immune response against microbial infections and an effective mechanism to eliminate apoptotic cells. In vertebrates, phagocytes such as macrophages and dendritic cells are involved in phagocytosis. We demonstrate here that insect hemocytes have distinct functions in phagocytosis of foreign particles and self dead cells. Plasmatocytes from the tobacco hornworm Manduca sexta were major hemocytes involved in phagocytosis of non-self microsphere beads, whereas granulocytes were apparently the only hemocytes that phagocytose self dead cells. We also showed that M. sexta immulectin-2, a pattern recognition receptor that protects larvae from bacterial infection, has an opsonic activity in phagocytosis. Immulectin-2 bound to the surface of granulocytes from the naïve larvae, but more immulectin-2 bound to plasmatocytes when larvae were injected with microsphere beads. Coupling of immulectin-2 onto microsphere beads enhanced in vitro phagocytosis of the beads. Our results suggest that insect hemocytes can have specialized functions similar to vertebrate phagocytes in phagocytosis, and pattern recognition receptors may function as opsonins to enhance phagocytosis.

Animals↗

Expression patterns of E-cadherin, involucrin, and connexin gap junction proteins in the lining epithelia of inflamed gingiva.

The structural integrity and functional differentiation of the lining epithelium were studied in relation to inflammatory changes associated with destructive periodontitis. In the different regions of lining epithelia from clinically healthy gingiva and periodontitis, comparisons were made of the expression patterns of E-cadherin, which is critical in intercellular adhesion; of proteins associated with gap junction communication channels; and of involucrin, which is a key marker of differentiation in stratified epithelia. Filamentous actin (F-actin), which is important in cell structural integrity, attachment, and migration, was also examined. Semiquantitative immunohistochemical analysis revealed that in both clinically healthy gingiva and lesions of advanced periodontitis, expression patterns of E-cadherin, involucrin, and connexins 26 and 43 were similar, with a statistically significant reduction in staining intensity from the external oral epithelium, through the gingival sulcus, to the junctional epithelium or pocket epithelium, respectively. Furthermore, there was a striking reduction in staining for E-cadherin, involucrin, and both connexins in the pathological lining epithelium of the periodontal pocket. These changes were associated with marked alterations of filamentous actin expression, collectively indicating profound perturbation of the epithelial structure. The data reported support the concept that the ability of the pathological lining epithelium to function as an effective barrier against the ingress of microbial products into the tissues is severely compromised.

Actins↗

Defective costimulatory function is a striking feature of antigen-presenting cells in an HLA-B27-transgenic rat model of spondylarthropathy.

OBJECTIVE: A disease resembling the human spondylarthropathies develops in HLA-B27-transgenic rats. This disease in rats is mediated by CD4+ T cells, but antigen-presenting cells (APCs) may also play a role. Dendritic cells (DCs) have been reported to be defective in allogeneic mixed lymphocyte culture in this model. Here, we further investigated the functional defect of APCs. METHODS: DCs and B cells from nontransgenic, HLA-B27 (33-3)-transgenic, and HLA-B7 (120-4)-transgenic rats were used to stimulate T cells. Surface expression of HLA-B transgene and rat molecules on APCs and the formation of conjugates between DCs and T cells were monitored by flow cytometry. RESULTS: We observed a strikingly defective stimulation of allogeneic and syngeneic T lymphocytes by APCs from HLA-B27 but not HLA-B7 rats, even if stimulation was driven in the presence of anti-T cell receptor (TCR) antibody. We found no evidence that HLA-B27 DCs were immature, lacked production of some diffusible factor, or produced an inhibitory factor for T cells. When comparing the levels of expression of class II major histocompatibility complex, CD2, intercellular adhesion molecule 1, lymphocyte function-associated antigen 1, B7, and CD40 molecules at the surface of DCs from 33-3, 120-4, and nontransgenic rats, we found little difference. However, HLA-B27-transgenic DCs formed fewer conjugates with T cells than did nontransgenic DCs. Furthermore, the proportion of conjugates formed between DCs and T cells, as well as the difference between nontransgenic and HLA-B27-transgenic DCs, were in large part reduced by blocking CD86 on DCs. CONCLUSION: We confirmed defective stimulation of T cells by APCs in HLA-B27 rats, the mechanism of which appears to implicate APC/T cell contact, independent of TCR engagement. In addition, decreased use of the CD86 costimulatory molecule by B27 DCs was observed. Impaired costimulatory function could result in a loss of tolerance toward microbial flora in this model.

Animals↗

Compared tolerance to osmotic stress in various microorganisms: towards a survival prediction test.

The osmotic tolerance of microbial cells of different microorganisms was investigated as a function of glycerol concentration and temperatures. Cells displayed specific sensitivity to dehydration in glycerol solutions. The viability of Gram-negative strains (Escherichia coli, Bradyrhizobium japonicum), Gram-positive strains (Lactobacillus plantarum, L. bulgaricus), and yeasts (Saccharomyces cerevisiae, Candida utilis) decreased with increasing osmotic pressure. For each strain, a characteristic osmotic pressure threshold causing a loss of 40% of the population at the growth temperature was determined: 26-40 MPa for E. coli, 15-25 MPa for B. japonicum, 7-15 MPa for L. bulgaricus, 40-133 MPa for L. plantarum, 50-100 MPa for S. cerevisiae, and 15-26 MPa for C. utilis. Because this threshold varies with temperature, it was possible to construct a diagram that could be helpful to the determination of the sensitivity of each strain to osmotic stress as a function of osmotic pressure and temperature.

Gram-Negative Bacteria↗

Structure-function relations in flavodoxins.

Flavodoxins are low molecular weight, FMN containing, proteins which function as electron transfer agents in a variety of microbial metabolic processes, including nitrogen fixation. Utilizing structural information obtained from x-ray crystal analysis, it has been possible to derive some new and important insights into the relationships which exist between flavin properties and protein environment by comparing the spectroscopic, thermodynamic and kinetic behavior of the flavodoxins with that of free flavin. Thus, for example, a qualitative understanding of the contribution of the protein to flavin redox potentials, semiquinone reactivity and mechanism of electron transfer is beginning to emerge. The highly negative redox potential required for the biochemical activity of the flavodoxins is accomplished by stabilizing the semiquinone via a hydrogen bond to the N-5 position of the flavin and destabilizing the fully-reduced form by constraining it to assume an unfavorable planar conformation. The reactivity of the semiquinone form is lowered by the aforementioned hydrogen bond, as well as by an interaction with a tryptophan residue in the binding site. Electron transfer is accomplished through the exposed dimethylbenzene ring of the bound coenzyme. Although it is not possible at present to determine the extent to which this understanding can be generalized to other flavoproteins, it is clear that a study of the flavodoxins will provide us with at least some of the principles which biological systems have used to modify flavin properties to fulfill a biochemical need.

Amino Acid Sequence↗