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Two commercial products, Biotize and Cycle, containing bacteria as an active ingredient were characterized for species identification and batch-to-batch variation by denaturing gradient gel electrophoresis (DGGE), total cellular fatty acid analysis (FAA), and a taxonomic DNA microarray. DGGE was useful at assessing the stability of consortia in different batches, and cluster analysis differentiated each batch even when only slight differences in species composition were observed. DGGE, FAA, and DNA microarray results indicated little batch-to-batch variation in Biotize and some batch variation in Cycle. The 3 methods agreed well with species identification in Biotize but generated conflicting results in the species composition of Cycle. This multi-method approach was useful in determining if the observed bacterial species present in the products matched the expected species composition. [ABSTRACT FROM AUTHOR]

In order to understand how lepidopteran insects react physiologically to Bacillus thuringiensis crystal toxin ingestion, transcriptional profiling of Choristoneura fumiferana larvae exposed to sublethal doses of Cry1Ab protoxin were monitored using a C. fumiferana-specific cDNA microarray derived from a protoxin-specific subtractive library. Differential gene expression occurred primarily between 2 and 5 h postingestion. Metabolic enzymes such as lipases and proteases were generally repressed, whereas genes involved in detoxification, immune system regulation or general stress response were upregulated. A similar protoxin-specific transcriptional pattern was also observed with Manduca sexta larvae, using three upregulated genes (serpin, cytochrome P450 and carboxyl/cholinesterase) and one downregulated gene (β-glucosidase), suggesting that a susceptible larval response to Cry toxin exposure might be universal among lepidopterous insects. [ABSTRACT FROM AUTHOR]

Bacillus thuringiensis is a microbial control agent active against Choristoneura fumiferana, a lepidopteran defoliator of North American forests. Although the B. thuringiensis insecticidal crystal protoxins have a relatively narrow host range, there is concern about their impact on non-target species where intoxication effects may not be overt. Larval toxicity effects can be assessed at the molecular level by determining altered transcriptional profiles in response to sublethal protoxin exposure in sensitive insects. Subtraction hybridization libraries were created using two larval populations, control and protoxin-fed and were characterized by sequencing 1091 clones. Differential mRNA expression of selected clones, as measured by quantitative polymerase chain reaction, identified a number of metabolic and stress-related genes that were either transcriptionally enhanced or repressed after protoxin exposure. [ABSTRACT FROM AUTHOR]

Abstract: Numerous waterborne pathogens are difficult to detect and enumerate with accuracy due to methodological limitations and high costs of direct culturing. The purity of DNA extracted from wastewater samples is an important issue in the sensitivity and the usefulness of molecular methods such as polymerase chain reaction (PCR) and hybridizations on DNA microarrays. Ten different DNA extraction procedures, including physical and chemical extraction and purification steps, were examined to ascertain their relative effectiveness for extracting bacterial DNA from wastewater samples. The quality of the differentially extracted DNAs was subsequently assessed by PCR amplification and microarray hybridization. Our results showed that great differences existed among the ten procedures and only a few of the methods gave satisfactory results when applied to bacterial pathogens. This observation suggested that the extraction method needed to be carefully selected to produce significant and confident results in the detection of pathogens from environmental samples. [Copyright &y& Elsevier]

Commercially formulated bioproducts containing a complex consortia of bacteria as an active ingredient pose a significant challenge for regulatory agencies and companies seeking to assess the safety and efficacy of these bioproducts. The main challenge stems from how to characterize the bacterial composition of these products, for which there is presently a lack of suitable methods. A prototype DNA microarray composed of oligonucleotide probes for functional genes, virulence factors, and taxonomic genes for a number of bacterial species was developed to examine the utility of microarray technology as a molecular tool for characterizing consortia bioproducts. The genomic DNA from four different products was extracted by two methods and examined with the microarray prototype and by denaturing gradient gel electrophoresis (DGGE). Although the identity of the consortial species remains unknown, the microarray assay provided unique and reproducible hybridization patterns for all four products, and agreed with the fingerprints generated by DGGE. The ability to differentiate between a variety of consortia products demonstrates that DNA microarrays have the potential to be a powerful tool in monitoring complex microbial communities. [Copyright &y& Elsevier]

DNA microarray analyses revealed that clusters of repetitive extragenic palindromic PCR-related Escherichia coli isolates were isogenic only within interstitial Lake Huron beach water samples and not in surrounding waters. This suggested that adaptation and growth occurred within the interstitial water sites tested. All isolates were nonpathogenic, and three lake isolates possessed tetracycline resistance genes. [ABSTRACT FROM AUTHOR]