Nontuberculous mycobacteria in the environment.

It is likely that the incidence of infection by environmental opportunistic mycobacteria will continue to rise. Part of the rise will be caused by the increased awareness of these microbes as human pathogens and improvements in methods of detection and culture. Clinicians and microbiologists will continue to be challenged by the introduction of new species to the already long list of mycobacterial opportunists (see Table 3).

Rapid detection of lytic antimicrobial activity against yeast and filamentous fungi.

A rapid method for assessing the lytic activity of antimicrobial agents against yeast and fungi has been developed. The assay is based on the release of the intracellular enzyme, maltase (alpha-glucosidase). The released maltase activity was measured colorimetrically by the production of p-nitrophenol from p-nitrophenyl-alpha-D-glucopyranoside (PNPG).

Fluorescent acid-fast microscopy for measuring phagocytosis of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum by Tetrahymena pyriformis and their intracellular growth.

Fluorescent acid-fast microscopy (FAM) was used to enumerate intracellular Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum in the ciliated phagocytic protozoan Tetrahymena pyriformis. There was a linear relationship between FAM and colony counts of M. avium cells both from cultures and within protozoa.

A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings.

Phytic acid (myo-inositol hexakisphosphate) is the major storage form of phosphorus in plant seeds. During germination, stored reserves are used as a source of nutrients by the plant seedling. Phytic acid is degraded by the activity of phytases to yield inositol and free phosphate.

A luciferase-based method for assessing chlorine-susceptibility of Mycobacterium avium.

A rapid and quantitative assay for the disinfection of the water-borne pathogen, Mycobacterium avium, was developed using firefly luciferase as a reporter gene. There was a correlation between the quantity of light produced and the number of colony-forming units. In chlorine-disinfection studies of a luciferase-carrying derivative of M.

Molecular characterization of At5PTase1, an inositol phosphatase capable of terminating inositol trisphosphate signaling.

The inositol triphosphate (IP(3))-signaling pathway has been associated with several developmental and physiological processes in plants, but we currently know little about the regulation of this pathway. Inositol 5' phosphatases (5PTases) are enzymes that remove a 5' phosphate from several potential second messengers, including IP(3). In catalyzing the removal of a 5' phosphate from second messenger substrates, 5PTases can act to terminate signal transduction events.

Use of methanolysis for the determination of total ellagic and gallic acid contents of wood and food products.

Anhydrous methanolic HCl has been found to be an excellent reagent for releasing ellagic acid and gallic acid (as methyl gallate) from biomass substrates. Optimization of both the reaction conditions and the gradient HPLC analysis has led to the development of a new protocol. The method provides ellagic acid yields significantly higher than those obtained previously, indicating total ellagic acid contents of several substrates have previously been underestimated.

Expression of D-myo-inositol-3-phosphate synthase in soybean. Implications for phytic acid biosynthesis.

Phytic acid, a phosphorylated derivative of myo-inositol, functions as the major storage form of phosphorus in plant seeds. Myo-inositol phosphates, including phytic acid, play diverse roles in plants as signal transduction molecules, osmoprotectants, and cell wall constituents. D-myo-inositol-3-phosphate synthase (MIPS EC 5.

Gene transfer to the desiccation-tolerant cyanobacterium Chroococcidiopsis.

The coccoid cyanobacterium Chroococcidiopsis dominates microbial communities in the most extreme arid hot and cold deserts. These communities withstand constraints that result from multiple cycles of drying and wetting and/or prolonged desiccation, through mechanisms which remain poorly understood. Here we describe the first system for genetic manipulation of Chroococcidiopsis.

Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other Mycobacteria in drinking water distribution systems.

Eight water distribution systems were sampled over an 18-month period (528 water and 55 biofilm samples) to measure the frequency of recovery and number of mycobacteria, particularly Mycobacterium avium and Mycobacterium intracellulare, in raw source waters before and after treatment and within the distribution system. The systems were chosen to assess the influence of source water, treatment, and assimilable organic carbon levels on mycobacterial numbers. Overall, mycobacterial recovery from the systems was low (15% of samples).

Fragilysin, the enterotoxin from Bacteroides fragilis, enhances the serum antibody response to antigen co-administered by the intranasal route.

Fragilysin, an extracellular zinc metalloprotease produced by enterotoxigenic strains of the anaerobic bacterium Bacteroides fragilis, disrupts the paracellular barrier by cleavage of the intercellular proteins between epithelial cells resulting in fluid secretion. Intranasal immunization of mice with fragilysin and co-administered ovalbumin (Ova) resulted in an Ova-specific serum IgG response that was over 18000-fold higher than Ova alone, as well as detectable levels of serum IgA. Serum IgG titers were comparable with those seen when whole cholera toxin was used as the adjuvant, although the responses obtained with fragilysin showed more variability between mice.

The use of the 2-aminobenzoic acid tag for oligosaccharide gel electrophoresis.

Gel electrophoresis of fluorophore labeled saccharides provides a rapid and reliable method to screen enzymatic and/or chemical treatments of polysaccharides and glycoconjugates, as well as a sensitive and efficient microscale method to separate and purify oligosaccharides for further analysis. A simple and inexpensive method of derivatization and analysis using 2-aminobenzoic acid (anthranilic acid, AA) is described and applied to the extracellular polysaccharide released by the desiccation tolerant cyanobacterium Nostoc commune DRH-1. The results of these analyses suggest a possible protective functionality of two pendent groups, as well as a potential relationship between these groups and the desiccation tolerance of the organism.

The role of the MoFe protein alpha-125Phe and beta-125Phe residues in Azotobacter vinelandii MoFe protein-Fe protein interaction.

Site-directed mutagenesis and gene-replacement techniques were used to substitute alanine for the MoFe protein alpha- and beta-subunit phenylalanine-125 residues both separately and in combination. These residues are located on the surface of the MoFe protein near the pseudosymmetric axis of symmetry between the alpha- and beta-subunits. Altered MoFe proteins that contain an alanine substitution at only one of the respective positions exhibit proton reduction activities of about 25-50% when compared to that of the wild-type protein.

Identification and characteristics of a novel Burkholderia strain with broad-spectrum antimicrobial activity.

A Burkholderia strain isolated from soil is capable of inhibiting the growth of bacteria, plant-pathogenic fungi, pathogenic yeasts, and protozoa. Inhibition does not involve cell contact or the presence of living cells, suggesting that at least a substantial portion of the antimicrobial activity is due to the excretion of extracellular compounds.

The effect of overliming on the toxicity of dilute acid pretreated lignocellulosics: the role of inorganics, uronic acids and ether-soluble organics.

Although the treatment of dilute acid pretreated lignocellulosics with calcium hydroxide or carbonate (overliming) is known to improve the fermentability of carbohydrate-rich hydrolyzate streams, a firm understanding of the chemistry behind the process is lacking. Quantitative evaluation of inorganics, uronic acids, and non-polar organics indicates that only a portion of the improvement can be ascribed to these materials. Upon overliming the concentrations of inorganics either increase (Ca, Mg), decrease (Fe, P, Zn, K) or remain relatively the same (Al, Na).

Chlorine, chloramine, chlorine dioxide, and ozone susceptibility of Mycobacterium avium.

Environmental and patient isolates of Mycobacterium avium were resistant to chlorine, monochloramine, chlorine dioxide, and ozone. For chlorine, the product of the disinfectant concentration (in parts per million) and the time (in minutes) to 99.9% inactivation for five M.

Engineering desiccation tolerance in Escherichia coli.

Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized in Escherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide.

Structural characterization of the released polysaccharide of desiccation-tolerant Nostoc commune DRH-1.

The structure of the viscous extracellular polysaccharide (glycan) of desiccation-tolerant Nostoc commune DRH-1 was determined through chromatographic and spectroscopic methods. The polysaccharide is novel in that it possesses a 1-4-linked xylogalactoglucan backbone with D-ribofuranose and 3-O-[(R)-1-carboxyethyl]-D-glucuronic acid (nosturonic acid) pendant groups. The presence of D-ribose and nosturonic acid as peripheral groups is unusual, and their potential roles in modulating the rheological properties of the glycan are discussed.

Catalytic and biophysical properties of a nitrogenase Apo-MoFe protein produced by a nifB-deletion mutant of Azotobacter vinelandii.

A Zn-immobilized metal-affinity chromatography technique was used to purify a poly-histidine-tagged, FeMo-cofactorless MoFe protein (apo-MoFe protein) from a nifB-deletion mutant of Azotobacter vinelandii. Apo-MoFe protein prepared in this way was obtained in sufficient concentrations for detailed catalytic, kinetic, and spectroscopic analyses. Metal analysis and electron paramagnetic resonance spectroscopy (EPR) were used to show that the apo-MoFe protein does not contain FeMo-cofactor.

Cloning and characterization of the gene for the metalloprotease enterotoxin of Bacteroides fragilis.

The Bacteroides fragilis enterotoxin is an extracellular zinc metalloprotease that has been implicated in diarrheal disease of humans and animals. This toxin causes fluid accumulation in intestinal loops and is cytotoxic for HT-29 cells, an intestinal carcinoma cell line. Here we report the cloning and sequencing of the toxin gene (bftP).