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PubMed2004

Natural products from endophytic microorganisms.

Endophytic microorganisms are to be found in virtually every plant on earth. These organisms reside in the living tissues of the host plant and do so in a variety of relationships ranging from symbiotic to pathogenic. Endophytes may contribute to their host plant by producing a plethora of substances that provide protection and ultimately survival value to the plant. Ultimately, these compounds, once isolated and characterized, may also have potential for use in modern medicine, agriculture, and industry. Novel antibiotics, antimycotics, immunosuppressants, and anticancer compounds are only a few examples of what has been found after the isolation and culturing of individual endophytes followed by purification and characterization of some of their natural products. The prospects of finding new drugs that may be effective candidates for treating newly developing diseases in humans, plants, and animals are great. Other applications in industry and agriculture may also be discovered among the novel products produced by endophytic microbes.

Antioxidants↗
PubMed1998

Isolation and Identification of Two l-Azetidine-2-carboxylic Acid-Degrading Soil Microorganisms, Enterobacter agglomerans and Enterobacter amnigenus

Soil samples collected at several times during the growing season and at different locations within Convallaria majalis beds in Ann Arbor, MI, were screened for their ability to grow with the cyclic amino acid, l-azetidine-2-carboxylic acid (l-A-2-C), as their sole nitrogen source (i.e., metabolize l-A-2-C). Two different soil microorganisms were isolated, characterized, and identified using fundamental selection methods, the standard battery of biochemical characterization tests, and scanning electron microscopy. The assignment of the identity of these organisms as Enterobacter agglomerans and Enterobacter amnigenus was further verified by comparison with authentic microbial samples obtained from ATCC that were able to utilize l-A-2-C as their sole nitrogen source.

Journal Article↗
PubMed1997

Non-cultivable microorganisms from symbiotic associations of insects and other hosts.

Many symbiotic associations involve microorganisms which cannot be cultivated on laboratory media. These organisms remained little known until the recent advent of methods of recombinant DNA analysis and molecular phylogenetics. Applications of these methods to endosymbionts have resulted in substantial new insights concerning the genetics and evolution of these organisms. This communication provides a listing of recently studied associations involving non-cultivable symbionts. The associations involve a diverse set of host taxa and a wide range of effects, both favorable and deleterious, on host biology. Among beneficial endosymbionts, a variety of nutritional interactions have been documented. One type of association has been demonstrated for a number of animal hosts, namely endosymbioses that result from a single infection of an ancestral host by a prokaryote. In these associations, endosymbionts are transmitted maternally and are not exchanged between host lineages, resulting in a long-term pattern of codiversification of hosts and endosymbionts. The association between aphids and non-cultivable prokaryotic endosymbionts is a well studied example of such a symbiosis.

Animals↗
PubMed1998

Carbon dioxide as a regulator of gene expression in microorganisms.

CO2 regulates gene expression across a diverse group of microorganisms including fungi, and both photosynthetic and non photosynthetic bacteria. The processes that CO2 regulates are diverse. Several CO2-responsive random promoter lacZ fusions of unknown function have been isolated from a marine Synechococcus and a Pseudoalteromonas sp., highlighting the wide effect of CO2 control in these organisms. Regulatory proteins have been described that mediate the CO2 response at transcription level in Bacillus anthracis, the group A streptococci and two Rhodobacter spp. These regulatory proteins include: AcpA and AtxA that are involved in CO2 control of B. anthracis capsule and toxin production; Mga that regulates surface associated virulence factors in the group A streptococci; and RegB/A, a two component signal transduction system that responds to environmental stimuli including CO2, to regulate photosynthetic apparatus and CO2 fixation enzyme synthesis in Rhodobacter spp.

Bacteria↗
PubMed2000

Efficacy of chlorhexidine against some strains of cultured and clinically isolated microorganisms.

The efficacy of chlorhexidine digluconate was determined against some strains of collected and clinically isolated bacteria and fungi. The efficacy was evaluated either by calculating a minimum inhibitory concentration (MIC) or by efficacy trials according to the guidelines of the European Committee for Standardization. The MIC values of chlorhexidine for Staphylococcus aureus, Microsporum gypseum, Microsporum canis and Trichophyton mentagrophytes were 0.625 microg/ml, 12.5 microg/ml, 50 microg/ml and 6.25 microg/ml, respectively. The in vitro efficacy of chlorhexidine was higher against ATCC strains of S. aureus and P. aeruginosa (0.5 mg/ml for 5 min and 0.5 mg/ml for 10 min, respectively) than against clinical isolates (0.5 mg/ml for 15 min and 1 mg/ml for 10 min, respectively). The antiseptic activity of aqueous solutions of chlorhexidine against spores of Bacillus subtilis, Bacillis sfericus and Clostridium perfringens required longer contact times than against the vegetative forms. Nevertheless, 5 mg/ml of chlorhexidine in water-ethanol 20:80 v/v was totally effective against the vegetative forms or spores of these microorganisms.

Anti-Infective Agents, Local↗
PubMed2001

Survival of microorganisms under the extreme conditions of the Atacama Desert.

Spores of Bacillus subtilis, conidia of Aspergillus niger, versicolor and ochraceus and cells of Deinococcus radiodurans have been exposed in the dark at two locations (at about 23 degrees S and 24 degrees S) in the Atacama Desert for up to 15 months. B. subtilis spores (survival approximately 15%) and A. niger conidia (survival approximately 30%) outlived the other species. The survival of the conidia and spores species was only slightly poorer than that of the corresponding laboratory controls. However, the Deinococcus radiodurans cells did not survive the desert exposure, because they are readily inactivated at relative humidities between 40 and 80% which typically occur during desert nights. Cellular monolayers of the dry spores and conidia have in addition been exposed to the full sun light for up to several hours. The solar fluences causing 63% loss in viability (F37-values) have been determined. These F37-values are compared with those determined at other global locations such as Punta Arenas (53 degrees S), Key Largo (25 degrees N) or Mainz (50 degrees N) during the same season. The solar UVB radiation kills even the most resistant microorganisms within a few hours due to DNA damages. The data are also discussed with respect to possible similarities between the climatic conditions of the recent Atacama Desert and the deserts of early Mars.

Aspergillus↗
PubMed1994

Pharmacokinetic analysis of in vivo metabolism of amino acid or dipeptide conjugates of salicylic acid in rabbit intestinal microorganisms.

We analyzed the pharmacokinetics of salicylic acid (SA)-amino acid (alanine, glutamic acid, methionine, and tyrosine) or SA-dipeptide (glycylglycine) conjugates in rabbits, by using a model that takes into account the metabolism of prodrug to SA by intestinal microorganisms and, also, by model-independent analysis. The blood concentration profiles of these prodrugs and released SA following intracecal and oral administration to rabbits were obtained previously (Nakamura et al., J. Pharm. Pharmacol., 44, 295-299, 1992; Chem. Pharm. Bull., 40, 2164-2168, 1992; Int. J. Pharm., 87, 59-66, 1992; J. Pharm. Pharmacol., 44, 713-716, 1992). First, the overall in vivo behavior was evaluated by statistical moment analysis. Next, the blood concentration profiles of prodrug and SA following intracecal and oral administration were simultaneously fitted to the above model. In general, good agreement was observed between fitted lines and experimental data for every prodrug, suggesting the validity of this model. The obtained parameters characterized the difference in the rate of metabolism and absorption among the prodrugs. Lower absorbability and enhanced hydrolysis rate of the prodrug lead to prolonged blood concentration of SA.

Administration, Oral↗
PubMed2002

Origins and diversification of sulfate-respiring microorganisms.

If the diversification of microbial life can be depicted as a single tree, as inferred by comparative sequencing of ribosomal RNAs, this could provide a framework for defining the order of emergence of new metabolic pathways. However, recent recognition that lateral gene transfer has been a significant force in microbial evolution has created uncertainty about the interpretation of taxonomies based on gene sequences. In this context, the origins and evolution of sulfate respiration will be evaluated considering the evolutionary history of a central enzyme in this process, the dissimilatory sulfite reductase. These studies suggest at least two major lateral transfer events during the early diversification of sulfate respiring microorganisms. The high sequence conservation of this enzyme has also provided a mechanism to directly explore the natural diversity of sulfate-respiring organisms using molecular techniques, avoiding the bias of culture-based identification. These studies suggest that the habitat range and evolutionary diversity of this key functional group of organisms is greater than now appreciated.

Evolution, Molecular↗
PubMed2002

The molecular basis of sensing and responding to light in microorganisms.

Photon absorption in biological signal transfer is mediated by a limited number of photoreceptor families, each characterised by binding of chromophore from a particular class of chemical compounds. Most photosensors become activated upon light-induced E/Z (i.e., trans/cis) isomerization of a double bond in their chromophore. This change in configuration of the chromophore subsequently must be translated into a change in the conformation of the photosensor protein, and transmitted to the downstream partner in the signal transduction chain. Particularly in archaea the molecular mechanism of signal transfer from the photosensor all the way to its target, i.e., the motility machinery, specific promoters and/or specific enzymes, is well understood for selected examples. In most of these, this flow of information makes use of a mechanism that is based on the so-called 'two-component paradigm'. Best know among these are the light-induced behavioural responses in Halobacterium salinarum, i.e., attraction by green- and repulsion by blue light. Regarding eukaryotic microorganisms our understanding of light-induced signal transfer, beyond the photoreceptor proteins, is restricted. This is due to their much more complex motility apparatus, the involvement of various secondary messengers and their compartmentalisation. The latter may require translocation of transcriptional activators to the nucleus and may form the basis of the sensing of the direction of the light. For a limited number of photoreceptor proteins we begin to understand the intra-molecular transition required to bring about the change in conformation of the protein that initiates signal transfer, i.e., the structure of the so-called 'signalling state'. This insight is most advanced in the photoactive yellow protein from Ectothiorhodospira halophila, a photoreceptor initiating a repellent response upon blue light excitation. In some well-established examples of sensor proteins involved in the transmission of chemical signals, formation of the signalling state appears to be just a shift in the equilibrium between two states that both are already present in the absence of signals. In photoreceptor proteins, however, this situation appears to be much more complex.

Archaeal Proteins↗
PubMed2002

Understanding the diversity in catabolic potential of microorganisms for the development of bioremediation strategies.

Molecular ecological approaches have detected diverse microorganisms that occur in response to pollution and bioremediation; however, most of these organisms have not been isolated, and their physiological traits are poorly understood. One important objective in current bioremediation studies would therefore be an assessment of the physiology and functions of the diverse microbial population at a polluted site. Among the parameters relating to the diversity of the microbial catabolic potential, e.g., substrate specificity, inducer specificity, number of catabolic routes and kinetics of catabolic enzymes, our studies have focused on the kinetic diversity of phenol-degrading bacteria. In one example, a kinetic analysis allowed functionally important phenol-degrading bacteria to be identified in activated sludge; this information could be used to improve the performance of phenol-degrading activated sludge. In an analysis of phenol-degrading bacteria present in trichloroethylene (TCE)-contaminated aquifer soil, the kinetic data could be linked to group-specific monitoring of their phenol-hydroxylase genes. The results have suggested that one group of phenol-degrading bacteria can effectively contribute to TCE bioremediation, while other groups work poorly. Based on this information, we have succeeded in developing a high-performance TCE-degrading bioreactor. We suggest that a careful analysis of the diversity of microbial catabolic potential, particularly of the kinetic traits, may facilitate the development of new bioremediation strategies.

Biodegradation, Environmental↗
PubMed2004

Lipase-producing microorganisms from a Kenyan alkaline soda lake.

Lipolytic enzyme production of 150 isolated strains from samples of Lake Bogoria (Kenya) was examined. Among these, fifteen isolates were selected on the basis of their lipolytic activities and subjected to morphological and 16S rRNA gene sequencing analyses for their identification. All the microorganisms have been selected under culture conditions with pH ranges between 7-10 and temperatures of 37-55 degrees C. Most of them showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v). Ten of the isolates were Gram-negative, nine of which were closely related to the Pseudomonas cluster and one to the Halomonas cluster sharing high similarity profile with Halomonas desiderata. The remaining Gram-positive isolates were closely related to the Bacillus cluster, and were grouped with Bacillus halodurans, Bacillus alcalophilus and Bacillus licheniformis. Four members of the Bacillus cluster and the Halomonas sp. produced lipolytic activity under alkaline conditions, while others did so at neutral pH values.

Bacillus↗
PubMed2004

Casuarina cunninghamiana tissue extracts stimulate the growth of Frankia and differentially alter the growth of other soil microorganisms.

Aqueous extracts of host plant Casuarina cunninghamiana tissue altered the in vitro growth of its diazotrophic microsymbiont Frankia and a selection of other soil microorganisms. The growth of actinomycetous Frankia strains, 55005. AvcI1, CesI5, CjI82 001, and Cj was stimulated by aqueous extracts of C. cunninghamiana tissue. Green cladodes (photosynthetic branches), unsuberized roots, and suberized roots were more stimulatory than dry cladodes and seed tissue. Aqueous extracts of green cladodes of C. cunninghamiana most stimulated the growth of Casuarina-derived Frankia strains CjI82 001 and 55005. The growth of isolates of soil bacteria Bradyrhizobium japonicum, Arthrobacter globiformis and Bacillus subtillis and of the soil fungi Penicillium oxalicum and Arthroderma cookiellum was either inhibited or not affected by cladode extracts. Cladode extracts stimulated the growth of the actinomycete Streptomyces albus and the fungus Rhizopus homothallicus. The magnitude (as great as 100%) of the increase in growth caused by tissue extracts for the Casuarina-derived Frankia strains relative to other soil microbes suggests a host-specific enhancement of the microsymbiont.

Bacteria↗
PubMed2004

Polysaccharides from extremophilic microorganisms.

Several marine thermophilic strains were analyzed for exopolysaccharide production. The screening process revealed that a significant number of thermophilic microorganisms were able to produce biopolymers, and some of them also revealed interesting chemical compositions. We have identified four new polysaccharides from thermophilic marine bacteria, with complex primary structures and with different repetitive units: a galacto-mannane type from strain number 4004 and mannane type for the other strains. The thermophilic Bacillus thermantarcticus produces two exocellular polysaccharides (EPS 1, EPS 2) that give the colonies a typical mucous character. The exopolysaccharide fraction was produced with all substrates assayed, although a higher yield 400 mg liter(-1) was obtained with mannose as carbon and energy source. NMR spectra confirmed that EPS 1 was a heteropolysaccharide of which the repeating unit was constituted by four different alpha-D-mannoses and three different beta-D-glucoses. It seems to be close to some xantan polymers. EPS 2 was a mannan. Four different alpha-D-mannoses were found as the repeating unit. Production and chemical studies of biopolymers produced by halophilic archaea, Haloarcula species were also reported.

Bacillus↗
PubMed2003

Transformation of flavonoids by intestinal microorganisms.

Fruit, vegetables and cereals contain a wealth of secondary plan metabolites which have been implicated in the promotion of health. To understand the mechanism of their action it is necessary to gain more information on their fate in the body following ingestion. A certain proportion of ingested secondary plant constituents may escape absorption in the small intestine and therefore undergo transformation by intestinal microorganisms or enterohepatic circulation. To study the transformation of secondary plant metabolites by bacteria, Eubacterium ramulus was isolated from human feces and incubated with selected flavonoids. E. ramulus is a strictly anaerobic bacterium which was found to be present in the gastrointestinal tract of most individuals investigated. E. ramulus cleaves the ring system of several flavonols and flavones giving rise to the corresponding hydroxyphenylacetic and hydroxyphenylpropionic acids, respectively, as well as acetate and butyrate. Degradation pathways were proposed based on the intermediates detected by high performance liquid chromatography (HPLC) and HPLC coupled with mass spectrometry (LC-MS) and the detection of enzymes that catalyze reactions such as taxifolin isomerization, phloretin hydrolysis and phloroglucinol reduction. The dearomatizing phloroglucinol reductase, presumably part of all flavonoid degradation pathways, was purified and characterized. The gene encoding phloretin hydrolase was cloned from a E. ramulus gene library taking advantage of a newly developed fluorescence test for activity screening. Moreover, a new intermediate was discovered and identified by MS and 1H and 13C NMR analysis as alphitonin. To investigate the degradational potential of E. ramulus under in vivo conditions, germfree rats were associated with E. ramulus. Following the intragastric application of quercetin-3-glucoside, urine and feces of gnotobiotic rats were analyzed for degradational products originating from quercetin-3-glucoside. In feces of rats monoassociated with E. ramulus, 3,4-dihydroxyphenylacetic acid was found, indicating that this organism is able to cleave quercetin under in vivo conditions. To investigate in which way the dietary flavonoid content affects the cell counts of E. ramulus in the human intestinal tract, twelve human subjects consumed a flavonoid-free diet for one week and at one point during this period a large dose of flavonoids. Fecal samples from both phases of the study were analyzed by in-situ hybridization for total bacterial counts and counts of E. ramulus. Total cell counts and the cell counts of E. ramulus decreased significantly during the flavonoid-free period, while there was an increase in the E. ramulus counts of up to 10-fold during the flavonoid-rich period indicating that dietary secondary plant metabolites may have an influence on the intestinal microflora. E. ramulus is also capable of converting the isoflavonoids genistein and daidzein to the products 2-(4-hydroxyphenyl)-propionic acid and O-desmethylangolensin, respectively.

Animals↗
PubMed1999

Enhanced interleukin-8 production in THP-1 human monocytic cells by lipopolysaccharide from oral microorganisms and granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-8 (IL-8) plays an important role in macrophage mediated inflammatory processes including exacerbation of periodontal diseases, one of the most common complications in GM-CSF receiving cancer patients. The effect of GM-CSF supplementation on IL-8 production was investigated in a human monocyte cell line THP-1, stimulated with lipopolysaccharide extracted from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. Resting THP-1 cells were treated with lipopolysaccharide (1 microgram/ml) of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) for varying time periods. The production of IL-8 in THP-1 cells was measured by a solid-phase enzyme-linked immunosorbent assay (ELISA). A very low level of the cytokine IL-8 was produced constitutive in THP-1 cells. Starting from 8 h of treatment and afterwards GM-CSF alone significantly increased IL-8 production in THP-1 cells. Lipopolysaccharide (1 microgram/ml) extracts from either F. nucleatum or P. gingivalis amplified IL-8 production 500-800 times in comparison to resting THP-1 cells. When lipopolysaccharide of F. nucleatum or P. gingivalis was supplemented with 50 IU/ml of GM-CSF, there was a statistically significant enhanced production of IL-8 by THP-1 cells after 1 day to 7 days of treatment as compared with lipopolysaccharide treatment alone. GM-CSF (50 IU/ml) also significantly increased IL-8 production from 2-7 days of treatment of THP-1 cells when supplemented with a positive control, phorbol-12-myristate-13 acetate (PMA), as compared to PMA treatment alone. These investigations using the in vitro THP-1 human monocyte cell model indicate that there may be an increase in the response on a cellular level to oral endotoxin following GM-CSF therapy as evidenced by enhanced production of the tissue-reactive inflammatory cytokine, IL-8.

Cell Line↗
PubMed2003

Evaluation of root canal microorganisms isolated from teeth with endodontic failure and their antimicrobial susceptibility.

Studies of the microbiota from the canals of teeth with failure of endodontic therapy have revealed that it differs markedly from that of untreated necrotic dental pulps. This study aimed to evaluate the microbiota of 30 root-filled teeth with persisting periapical lesions and to test the antibiotic susceptibility of the most prevalent species. Microbial samples, isolation and speciation were done using advanced microbiologic techniques for anaerobic species. A total of 55 bacterial species were isolated, 80% were gram-positives and 58% facultative anaerobic microorganisms. The bacterial genera most frequently recovered were Enterococcus, Streptococcus, Peptostreptococcus and Actinomyces. Antibiotic sensitivity of Enterococcus faecalis and Peptostreptococcus spp. was accomplished with the E-test system. All species studied were susceptible to benzylpenicillin, amoxicillin, amoxicillin combined with clavulanate. However, 20% of the E.faecalis strains were resistant to erythromycin and 60% to azithromycin. It was concluded that microbial flora in canals after endodontic failure comprised predominantly facultative anaerobes and gram-positive species. E.faecalis was the species most frequently isolated and showed erythromycin and azithromycin resistance among the isolates.

Actinomyces↗
PubMed2000

B-cell activation by T-cell-independent type 2 antigens as an integral part of the humoral immune response to pathogenic microorganisms.

Antigens that are expressed on the surface of pathogens in an organized, highly repetitive form can activate specific B cells by cross-linking of antigen receptors in a multivalent fashion. B cells respond to these multivalent antigens in the absence of MHC class II-restricted T-cell help by a mechanism that depends on the expression of a functional Bruton's tyrosine kinase (Btk). Accordingly, this class of immunogens has been designated T-cell-independent type 2 (TI-2) antigens. The unique properties of the B-cell response to TI-2 antigens are critically dependent on the formation of a small number of antigen receptor clusters, each of which contains approximately 10 to 20 antigen-bound membrane Ig (mIg) molecules. These clusters induce local membrane association of multiple activated Btk molecules, which results in long-term mobilization of intracellular ionized calcium. Such persistent calcium fluxes efficiently recruit transcription factors and thereby induce T-cell-independent B-cell activation and proliferation. While this first signal of multivalent mIg cross-linking can induce B-cell proliferation, we propose that a second signal is required for a TI-2 Ig secretory response. We have found that engagement of members of the Toll-like receptor (TLR) family could provide second signals that selectively induce Ig secretion in B cells that were activated by multivalent, but not by bivalent, antigen receptor engagement. This finding demonstrates a general mechanism by which TLRs recognize molecular motifs on the surface of pathogens and provide the TI-2-activated B cell with a second signal. In addition, TLR-dependent recognition of these non-self motifs by cells of the innate immune system can induce these cells to provide alternative and/or additional second signals in the TI-2 response. The complement system provides another link between the B cell and the innate immune system, and facilitates the mIg signal transduction by recruitment of CD21 in the immune response. Thus, the TI-2 response provides the host with a combination of "the best of both worlds": the recruitment of the fine specificity of the adaptive immune response and the utilization of both the speed of the innate immune system and the wealth of cytokines produced by its member cells upon stimulation by pathogenic organisms or their products. By combining these two pathways, the TI-2 response enables the host to rapidly produce antigen-specific Ig effector molecules that can be secreted at a sufficient rate to keep up with the rapid multiplication of invading infectious microorganisms, and will also prevent the intracellular spreading of a significant part of this population.

Animals↗