Specific detection of bacillus anthracis using a TaqMan mismatch amplification mutation assay

Single nucleotide polymorphisms (SNPs) are increasingly recognized as important diagnostic markers for the detection and differentiation of Bacillus anthracis. The use of SNP markers for identifying B. anthracis DNA in environmental samples containing genetically similar bacteria requires the ability to amplify and detect DNA with single nucleotide specificity. We designed a TaqMan mismatch amplification mutation assay (TaqMAMA) around a SNP in the plcR gene of B. anthracis. The assay permits specific, low-level detection (25 fg DNA) of this B. anthracis-specific SNP, even in the presence of environmental DNA extracts containing a 20,000-fold excess of the alternate allele. We anticipate that the ability to selectively amplify and detect low copy number DNAs with single nucleotide specificity will represent a valuable tool in the arena of biodefense and microbial forensics.     

Expression of mutated cationic trypsinogen reduces cellular viability in AR4-2J cells

Mutations in the human cationic trypsinogen are associated with hereditary pancreatitis. The cDNA coding for human cationic trypsinogen was subcloned into the expression vector pcDNA3. The mutations R122H, N29I, A16V, D22G, and K23R were introduced by site directed mutagenesis. We constructed an expression vector coding for active trypsin by subcloning the cDNA of trypsin lacking the coding region for the trypsin activating peptide behind an appropriate signal peptide. Expression of protein was verified by Western blot and measurement of enzymatic activity. AR4-2J cells were transiently transfected with the different expression vectors and cell viability and intracellular caspase-3 activity were quantified. In contrast to wild-type trypsinogen, expression of active trypsin and mutated trypsinogens reduced cell viability of AR4-2J cells. Expression of trypsin and R122H trypsinogen induced caspase-3 activity. Acinar cells might react to intracellular trypsin activity by triggering apoptosis.     

The effects of controlled propagation on an endangered species: genetic differentiation and divergence in body size among native and captive populations of the Socorro Isopod (Crustacea: Flabellifera)

The endangered Socorro Isopod, Thermosphaeroma thermophilum, is endemic to a single thermal spring in Socorro, NM. This species is cannibalistic, with males more cannibalistic than females, and with females and juveniles more vulnerable than males as prey. In 1990, the New Mexico Department of Game and Fish, created the Socorro Isopod Propagation Facility (SIPF) near the natural habitat, Sedillo Spring (SS), to increase total population size and to examine the effects of habitat heterogeneity on population growth. We report the genetic and morphological effects of this experiment, using the natural population as a control. Captive subpopulations experienced bottlenecks of known intensity and duration, as well as different intensities of cannibalism. Using 57 AFLP markers, we show that in 6 years (1990–1996), captive subpopulations diverged significantly from the natural population. Also during this 6-year period, body lengths of captive isopods diverged nearly 2-fold from the natural population, evidently because cannibalism and thus selection favoring large size was more intense in captive subpopulations than in nature. This hypothesis is supported by the fact that cannibalism and the apparent response to selection on body size became variable among captive subpopulations when physical structure was added to three of the four SIPF pools in April 1997. As expected if cannibalism was the source of selection for large body size, by August 1998 (15 months = 7–8 generations), the rate at which body size increased became inversely proportional to the amount of physical structure within pools. Although we are unable to separate the specific effects of population subdivision and cannibalism, our results show that these conditions in combination caused rapid changes in genetic variation and the external morphology of these captive subpopulations. Our results have important implications for future attempts to manage and propagate endangered species.     

A horizon scan of global conservation issues for 2012

Our aim in conducting annual horizon scans is to identify issues that, although currently receiving little attention, may be of increasing importance to the conservation of biological diversity in the future. The 15 issues presented here were identified by a diverse team of 22 experts in horizon scanning, and conservation science and its application. Methods for identifying and refining issues were the same as in two previous annual scans and are widely transferable to other disciplines. The issues highlight potential changes in climate, technology and human behaviour. Examples include warming of the deep sea, increased cultivation of perennial grains, burning of Arctic tundra, and the development of nuclear batteries and hydrokinetic in-stream turbines.     

Genome Sequence of “Candidatus Microthrix parvicella” Bio17-1, a Long-Chain-Fatty-Acid-Accumulating Filamentous Actinobacterium from a Biological Wastewater Treatment Plant

“Candidatus Microthrix” bacteria are deeply branching filamentous actinobacteria which occur at the water-air interface of biological wastewater treatment plants, where they are often responsible for foaming and bulking. Here, we report the first draft genome sequence of a strain from this genus: “Candidatus Microthrix parvicella” strain Bio17-1.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates

We examined 154 Norwegian B. cereus and B. thuringiensis soil isolates (collected from five different locations), 8 B. cereus and 2 B. thuringiensis reference strains, and 2 Bacillus anthracis strains by using fluorescent amplified fragment length polymorphism (AFLP). We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria. Both B. anthracis strains analyzed were closely related and affiliated with a B. cereus milk isolate (ATCC 4342) and a B. cereus human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including B. anthracis, were more closely related to one another than to the environmental isolates. Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups. This analysis was consistent with the AFLP analysis, although of much lower resolution. The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future.     

Identification, structure, and differential expression of members of a BURP domain containing protein family in soybean.

Expressed sequence tags (ESTs) exhibiting homology to a BURP domain containing gene family were identified from the Glycine max (L.) Merr. EST database. These ESTs were assembled into 16 contigs of variable sizes and lengths. Consistent with the structure of known BURP domain containing proteins, the translation products exhibit a modular structure consisting of a C-terminal BURP domain, an N-terminal signal sequence, and a variable internal region. The soybean family members exhibit 35-98% similarity in a -100-amino-acid C-terminal region, and a phylogenetic tree constructed using this region shows that some soybean family members group together in closely related pairs, triplets, and quartets, whereas others remain as singletons. The structure of these groups suggests that multiple gene duplication events occurred during the evolutionary history of this family. The depth and diversity of G. max EST libraries allowed tissue-specific expression patterns of the putative soybean BURPs to be examined. Consistent with known BURP proteins, the newly identified soybean BURPs have diverse expression patterns. Furthermore, putative paralogs can have both spatially and quantitatively distinct expression patterns. We discuss the functional and evolutionary implications of these findings, as well as the utility of EST-based analyses for identifying and characterizing gene families.