Hypothesis: a motor neuron toxin produced by a clostridial species residing in gut causes ALS.

We hypothesize that a yet-to-be-identified motor neuron toxin produced by a clostridial species causes sporadic amyotrophic lateral sclerosis (ALS) in susceptible individuals. This clostridial species would reside undetected in the gut and chronically produce a toxin that targets the motor system, like the tetanus and botulinum toxins. After gaining access to the lower motor neuron, the toxin would be transported back to the cell body, as occurs with the tetanus toxin, and destroy the lower motor neuron - the essential feature of ALS. Again like the tetanus toxin, some of the toxin would cross to neighboring cells and to the upper motor neuron and similarly destroy these motor neurons. Weakness would relentlessly progress until not enough motor neurons remained to sustain life. If this hypothesis were correct, treatment with appropriate antibiotics or antitoxins might slow or halt progression of disease, and immunization might prevent disease.

Global differential gene expression in response to growth temperature alteration in group A Streptococcus.

Pathogens are exposed to different temperatures during an infection cycle and must regulate gene expression accordingly. However, the extent to which virulent bacteria alter gene expression in response to temperatures encountered in the host is unknown. Group A Streptococcus (GAS) is a human-specific pathogen that is responsible for illnesses ranging from superficial skin infections and pharyngitis to severe invasive infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. GAS survives and multiplies at different temperatures during human infection. DNA microarray analysis was used to investigate the influence of temperature on global gene expression in a serotype M1 strain grown to exponential phase at 29 degrees C and 37 degrees C. Approximately 9% of genes were differentially expressed by at least 1.5-fold at 29 degrees C relative to 37 degrees C, including genes encoding transporter proteins, proteins involved in iron homeostasis, transcriptional regulators, phage-associated proteins, and proteins with no known homologue. Relatively few known virulence genes were differentially expressed at this threshold. However, transcription of 28 genes encoding proteins with predicted secretion signal sequences was altered, indicating that growth temperature substantially influences the extracellular proteome. TaqMan real-time reverse transcription-PCR assays confirmed the microarray data. We also discovered that transcription of genes encoding hemolysins, and proteins with inferred roles in iron regulation, transport, and homeostasis, was influenced by growth at 40 degrees C. Thus, GAS profoundly alters gene expression in response to temperature. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many unforeseen lines of pathogenesis investigation.

Identification of Rgg-regulated exoproteins of Streptococcus pyogenes.

Streptococcus pyogenes secretes many proteins that influence host-pathogen interactions. Despite their importance, relatively little is known about the regulation of these proteins. The rgg gene (also known as ropB) is required for the expression of streptococcal erythrogenic toxin B (SPE B), an extracellular cysteine protease that contributes to virulence. Proteomics was used to determine if rgg regulates the expression of additional exoproteins. Exponential- and stationary-phase culture supernatant proteins made by S. pyogenes NZ131 rgg and NZ131 speB were separated by two-dimensional electrophoresis. Differences were identified in supernatant proteins from both exponential- and stationary-phase cultures, although considerably more differences were detected among stationary-phase supernatant proteins. Forty-two proteins were identified by peptide fingerprinting with matrix-assisted laser desorption mass spectrometry. Mitogenic factor, DNA entry nuclease (open reading frame [ORF 226]), and ORF 953, which has no known function, were more abundant in the culture supernatants of the rgg mutant compared to the speB mutant. ClpB, lysozyme, and autolysin were detected in the culture supernatant of the speB mutant but not the rgg mutant. To determine if Rgg affected protein expression at the transcriptional level, real-time (TaqMan) reverse transcription (RT)-PCR was used to quantitate Rgg-regulated transcripts from NZ131 wild-type and speB and rgg mutant strains. The results obtained with RT-PCR correlated with the proteomic data. We conclude that Rgg regulates the transcription of several genes expressed primarily during the stationary phase of growth.

Spatial and Seasonal Variation in a Reservoir Sedimentary Microbial Community as Determined by Phospholipid Analysis.

Sediment samples were collected monthly from Acton Lake, a eutrophic reservoir located in an agricultural region of southwestern Ohio, from three stations (River, Middle, and Dam) during the period May 1995 through January 1997. Sedimentary microbial biomass and community structures from these stations were studied using phospholipid analysis. At the River and Middle stations, the water column remained aerobic throughout the year, whereas the water overlying the Dam station sediments became anaerobic during summer stratification. Sedimentary microbial biomass at the River and Middle stations, as measured by the phospholipid phosphate (PLP) method, ranged from 225 to 450 nmol PLP g?1 d.w. (dry weight). Sedimentary microbial biomass at the Dam station was typically greater and ranged from 500 to 1,500 nmol PLP g?1 d.w. Principal component analysis of phospholipid fatty acid (PLFA) profiles indicated that the sedimentary microbial communities at all three stations displayed seasonal patterns of change. Among these patterns of change was a shift from aerobic microorganisms during times of cold water to anaerobic microorganisms during times of warm water. The Dam station differed from the River and Middle stations in that sediments from this station had disproportionately more polyenoic fatty acids, whereas sediments from the River and Middle stations had disproportionately more bacterial fatty acids. These data suggest that the Dam station may be a depositional zone for microeukaryotic phytoplankton produced in the overlying water column. These findings have implications for the understanding of carbon flux in reservoirs and preservation of organic matter in aquatic systems.

Comparison of differential plating media and two chromatography techniques for the detection of histamine production in bacteria.

The bacterial enzyme histidine decarboxylase (Hdc) catalyses the conversion of histidine into histamine. This amine is essential for the biosynthesis of iron chelators (siderophores) and is an important cause of food poisoning after consumption of fish contaminated with histamine-producing bacteria. In this work we compared different methods for detecting histamine secreted by different bacterial strains. The presence of histamine in the culture supernatant of Vibrio anguillarum, which produces Hdc and secretes the histamine-containing siderophore anguibactin, was detected by thin-layer chromatography. Similar results were obtained using the culture supernatant of the Acinetobacter baumannii 19606 prototype strain that secretes the histamine-containing siderophore acinetobactin. Conversely, histamine was not detected in the culture supernatant of an isogenic V. anguillarum Hdc mutant and the A. baumannii 8399 strain that secretes a catechol siderophore different from anguibactin and acinetobactin. These results were confirmed by capillary gas chromatography/mass spectrometry. However, all these strains tested positive for histamine secretion when cultured on differential plating media containing histidine and a pH indicator, which were specifically designed for the detection of histamine-producing bacteria. The pH increase of the medium surrounding the bacterial colonies was however drastically reduced when the histidine-containing medium was supplemented with peptone, beef extract, and glucose. The histidine-containing culture supernatants of the A. baumannii and V. anguillarum strains showed an increase of about two units of pH, turned purple upon the addition of cresol red, and contained high amounts of ammonia. Escherichia coli strains, which are Hdc negative and do not use histidine as a carbon, nitrogen, and energy source, gave negative results with the differential solid medium and produced only moderate amounts of ammonia when cultured in the presence of excess histidine. This study demonstrates that, although more laborious and requiring some expensive equipment, thin-layer and gas chromatography/mass spectrometry are more accurate than differential media for detecting bacterial histamine secretion. The results obtained with these analytical methods are not affected by byproducts such as ammonia, which are generated during the degradation of histidine and produce false positive results with the differential plating media.

Plasmid-mediated histamine biosynthesis in the bacterial fish pathogen Vibrio anguillarum.

Histamine production in bacteria-contaminated fish is the result of the presence of bacterial histidine decarboxylase activity, which converts histidine present in muscle proteins to histamine. The fish pathogen Vibrio anguillarum harbors a plasmid-encoded histidine decarboxylase gene (angH) that is essential for biosynthesis of the siderophore anguibactin. However, the role of angH in histamine biosynthesis by this pathogen has not been fully determined. Thus, the objectives of this study were to monitor the production and release of histamine by the wild-type as well as by a plasmidless strain and angH isogenic mutants generated by allelic exchange. Reverse transcription polymerase chain reaction showed that only the wild-type strain expressed angH, while no angH message was detected in the mutants and the plasmidless derivative. The iron uptake-deficient phenotype of one of the angH mutants confirmed the location of the mutation and the unique role of this gene in iron acquisition. Thin-layer chromatography, gas chromatography, and mass spectrometry showed that histamine was released by the strain harboring a wild-type angH gene when grown in excess histidine. This biogenic amine was not detected in the culture supernatants of the plasmidless derivative and the angH mutant when cultured under the same experimental conditions. These results indicate that angH is essential for histamine biosynthesis in V. anguillarum, a compound responsible for food poisoning and potentially involved in bacterial virulence. Thin-layer chromatography of wild-type culture supernatants and beta-galactosidase assays using the isogenic angH mutant demonstrated that the expression of this gene is independent of the histidine concentration of the medium under both iron-rich and iron-limiting conditions.