Potential of the melanophore pigment response for detection of bacterial toxicity.

Chromatophore cells have been investigated as potential biodetectors for function-based detection of chemically and biologically toxic substances. Oncorhynchus tshawytscha (chinook salmon) melanophores, a chromatophore cell type containing brown pigment, rapidly detect the salmonid pathogens Aeromonas salmonicida, Yersinia ruckeri, and Flavobacterium psychrophilum and the human pathogen Bacillus cereus.

Conservation of the chromatophore pigment response.

Toxicant sensing technology has evolved to include biological sensors, such as cell-based biosensors, which rely on viable cells to convey a measurable physiological signal. Chromatophores are a class of pigment cells that have been investigated as cell-based biosensors. We report the characterization of Oncorhynchus tshawytscha melanophores and describe the melanophore pigment response to neurotransmitters in terms of pigment area occupied. Compared with the previously described model, Betta splendens erythrophores, O. tshawytscha melanophores responded similarly, indicating that pigment responses are biologically conserved between these two species. Additionally, melanophores responded to mercuric chloride and sodium arsenite, similar to B. splendens erythrophores, suggesting that melanophores can be used as detectors for environmental toxicants. This report highlights the potential of O. tshawytscha melanophores to be used as cell-based biosensors to address environmental toxicity, and warrants a continued investigation to strengthen this technology and its applications.

Erythrophore cell response to food-associated pathogenic bacteria: implications for detection.

Cell-based biosensors have been proposed for use as function-based detectors of toxic agents. We report the use of Betta splendens chromatophore cells, specifically erythrophore cells, for detection of food-associated pathogenic bacteria. Evaluation of erythrophore cell response, using Bacillus spp., has revealed that this response can distinguish pathogenic Bacillus cereus from a non-pathogenic B. cereus ΔplcR deletion mutant and a non-pathogenic Bacillus subtilis. Erythrophore cells were exposed to Salmonella enteritidis, Clostridium perfringens and Clostridium botulinum. Each bacterial pathogen elicited a response from erythrophore cells that was distinguished from the corresponding bacterial growth medium, and this observed response was unique for each bacterial pathogen. These findings suggest that erythrophore cell response has potential for use as a biosensor in the detection and toxicity assessment for food-associated pathogenic bacteria.

Exopolysaccharide expression in Lactococcus lactis subsp. cremoris Ropy352: evidence for novel gene organization.

Lactococcus lactis subsp. cremoris Ropy352 produces two distinct heteropolysaccharides, phenotypically described as ropy and mucoid, when cultured in nonfat milk. One exopolysaccharide precipitated with 50% ethanol as a series of elongated threads and was composed of glucose and galactose in a molar ratio of 3:2. The second exopolysaccharide precipitated with 75% ethanol as a fine flocculant and consisted of galactose, glucose, and mannose with a molar ratio of 67:21:12. A mutant strain, L. lactis subsp. cremoris EK240, lacking the ropy phenotype did not produce the exopolysaccharide that precipitated with 50% ethanol; however, it produced the exopolysaccharide that precipitated with 75% ethanol, indicating that the former exopolysaccharide is essential for the ropy phenotype. Cultures of L. lactis subsp. cremoris Ropy352 in 10% nonfat milk reached a viscosity of 25 Pa-s after 24 h, while those of the nonropy L. lactis subsp. cremoris EK240 mutant did not change. A mutation abolishing ropy exopolysaccharide expression mapped to a region on a plasmid containing two open reading frames, epsM and epsN, encoding novel glycosyltransferases bordered by ISS1 elements oriented in the same direction. Sequencing of this plasmid revealed two other regions involved in exopolysaccharide expression, an operon located between partial IS981 and IS982 elements, and an independent gene, epsU. Two and possibly three of these regions are involved in L. lactis subsp. cremoris Ropy352 exopolysaccharide expression and are arranged in a novel fashion different from that of typical lactococcal exopolysaccharide loci, and this provides genetic evidence for exopolysaccharide gene reorganization and evolution in Lactococcus.

Responses of fish chromatophore-based cytosensor to a broad range of biological agents.

A cytosensor based on living chromatophores from Betta splendens Siamese fighting fish was used to test several classes of biologically active agents. Tested agents include neurotransmitters, adenyl cyclase activators, cytoskeleton effectors, cell membrane effectors and protein synthesis inhibitors. Characteristic cell responses were analyzed, and potential cytosensor applications were considered. Streptococcus pyogenes toxins streptolysin S and streptolysin O, Clostridium tetani tetanolysin, Staphylococcus aureus alpha-toxin and Vibrio parahemolyticus hemolysin, all bacterial toxins that act on cell membranes, elicited a strong response from chromatophores. A comparison of purified toxin to actual bacterial culture from Vibrio parahemolyticus demonstrated a nearly identical chromatophore cell response pattern. This suggests that the cytosensor response is reflective of bacterial toxin production.

Blind and naive classification of toxicity by fish chromatophores.

Cellular and molecular pathways involved in the ability of animals to change color have been studied previously as biosensors and cytosensors of active and toxic agents, but such studies generally have been limited to just a few standardized agents. Here we describe the performance of cultured chromatophore pigment cells from the fin tissue of Siamese fighting fish as sensors of toxic agents under blind sampling conditions at the September 2002 EILATox-Oregon Workshop. Detection was accomplished by monitoring motor protein-mediated movements of cellular pigment in chromatophores at both the gross population level as well as in singly imaged cells. Pigment responses were recorded both during the exposure of chromatophores to each blind sample as well as afterwards when the cells were examined for after-effects by challenging them with clonidine, an adrenergic drug that induces standardized pigment movements. After recording all results and upon breaking the key to reveal the identities of the toxic agents, it was found that all of the toxic samples in the study had been distinguished accurately from non-toxic controls that were included among the blind samples. Furthermore, it was revealed that most of the toxic agents detected had never before been tested or calibrated against chromatophores, demonstrating that detection can be achieved under naive conditions that have not been optimized for the analysis of any particular toxic agent. Finally, by organizing the results into categories of pigment responses, a binary classification tree was generated that distinguished each toxic agent as having a distinct response pattern from the others. Thus, chromatophore-based cytosensors can discover toxicity in the absence of prior knowledge of the agent in question, and the categories of responses of the cells can be used to distinguish one toxic agent from another.

Learning Microbiology Through Cooperation: Designing Cooperative Learning Activities that Promote Interdependence, Interaction, and Accountability.

A microbiology course and its corresponding learning activities have been structured according to the Cooperative Learning Model. This course, The World According to Microbes, integrates science, math, engineering, and technology (SMET) majors and non-SMET majors into teams of students charged with problem solving activities that are microbial in origin. In this study we describe development of learning activities that utilize key components of Cooperative Learning-positive interdependence, promotive interaction, individual accountability, teamwork skills, and group processing. Assessments and evaluations over an 8-year period demonstrate high retention of key concepts in microbiology and high student satisfaction with the course.