Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis.

The potential use of Bacillus anthracis as a weapon of mass destruction poses a threat to humans, domesticated animals, and wildlife and necessitates the need for a rapid and highly specific detection assay. We have developed a real-time PCR-based assay for the specific detection of B. anthracis by taking advantage of the unique nucleotide sequence of the B. anthracis rpoB gene. Variable region 1 of the rpoB gene was sequenced from 36 Bacillus strains, including 16 B. anthracis strains and 20 other related bacilli, and four nucleotides specific for B. anthracis were identified. PCR primers were selected so that two B. anthracis-specific nucleotides were at their 3' ends, whereas the remaining bases were specific to the probe region. This format permitted the PCR reactions to be performed on a LightCycler via fluorescence resonance energy transfer (FRET). The assay was found to be specific for 144 B. anthracis strains from different geographical locations and did not cross-react with other related bacilli (175 strains), with the exception of one strain. The PCR assay can be performed on isolated DNA as well as crude vegetative cell lysates in less than 1 h. Therefore, the rpoB-FRET assay could be used as a new chromosomal marker for rapid detection of B. anthracis.

Use of long-range repetitive element polymorphism-PCR to differentiate Bacillus anthracis strains.

The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105 B. anthracis strains of diverse geographical origins. All B. anthracis strains produced fingerprints comprising seven to eight bands, referred to as "skeleton" bands, while one to three "diagnostic" bands differentiated between B. anthracis strains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracis strains.

Transcriptional activation of the Aspergillus nidulans gpdA promoter by osmotic signals.

A differentially expressed gpdA cDNA clone was isolated from NaCl-adapted Aspergillus nidulans (FGSC359) and identified as glyceraldehyde-3-phosphate dehydrogenase (gpdA) on the basis of its nucleotide sequence. The level of gpdA RNA substantially increased in cultures gradually adapted to NaCl but was greatly reduced in cultures exposed briefly to a high concentration of NaCl. A pyrG auxotroph of A. nidulans (A773) was cotransformed with a gpdA-uidA construct and a plasmid containing the Neurospora crassa pyr4 gene as a selectable marker. One pyrG+ beta-glucuronidase-positive (GUS+) transformant was selected, and stable integration of the gpdA-uidA construct into the genome was confirmed by Southern blot analysis. Gradual adaptation to increasing concentrations of NaCl resulted in an increase in GUS activity to 2.7-fold. GUS activity was reduced after a 2-h exposure of an unadapted culture to 2 M NaCl but gradually increased to a maximum of twofold after 24 h. GUS activity also increased by 8.4-fold in Na2SO4-adapted cultures, 4.9-fold in polyethylene glycol-adapted cultures, and 7.5-fold in KCl-adapted cultures. These results are consistent with the hypothesis that the A. nidulans gpdA promoter is transcriptionally activated by osmotic signals.

DNA fingerprinting of Candida rugosa via repetitive sequence-based PCR.

A repetitive sequence-based PCR (rep-PCR) technique was developed to characterize the genotypic relatedness among Candida rugosa isolates. Two repetitive sequences, viz., Care-2 and Com29 from Candida albicans, were used to design primers Ca-21, Ca-22, and Com-21, respectively. When used alone or in combination, these primers generated discriminatory fingerprints by amplifying the adjacent variable regions of the genome. Twenty-three isolates from burn patients, eight from other human sources, and four C. rugosa isolates pathogenic in animals were placed into nine fingerprinting groups. Different primers placed these isolates into identical groups, indicating that rep-PCR is a specific and reproducible technique for molecular characterization of C. rugosa. Moreover, these primers unequivocally discriminated among other important Candida species such as C. albicans, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. kefyr, and C. lusitaniae. These data confirm the conservation of repetitive sequences in Candida species. Because of its ease and sensitivity, rep-PCR offers a relatively rapid and discriminatory method for molecular typing of C. rugosa in outbreaks.

Isolation of differentially expressed cDNA clones from salt-adapted Aspergillus nidulans.

Differentially expressed cDNA clones were isolated from salt-adapted Aspergillus nidulans (FGSC #359). Poly (A)+ RNA from adapted mycelia was used to construct a lambda Uni-ZAP cDNA library. The library was screened with mixed subtracted cDNA probes. Three-hundred and fifty-seven positive plaques were isolated in the primary screening. Sixty-two randomly selected plaques were purified and placed into eight different cross-hybridization groups. A representative cDNA from each group was used to study expression under unadapted, salt-adapted and salt-shock conditions. These clones, representing eight different genes, displayed enhanced expression under salt stress. Exploratory nucleotide sequencing was performed, and the predicted amino-acid sequence was compared with known gene sequences in the data-bank. Five of the cDNA clones were identified as a mitochondrial (mt) ATPase beta subunit, a mt ATPase subunit 9, a mt transport protein, a ubiquitin-extension protein and a ribosomal protein. Three cDNA clones could not be identified due to lack of adequate homology with known sequences. These results suggest that at least five genes with known function in cellular processes like ATP generation and protein synthesis, and three other genes of unknown identity, are greatly induced in salt-adapted conditions.

Biosynthetic pathways of glycerol accumulation under salt stress in Aspergillus nidulans.

A culture of Aspergillus nidulans (FGSC 359) was gradually adapted for growth in media containing up to 2 M NaCl or was exposed to a salt shock with 2 M NaCl. The intracellular glycerol level increased by about 7.9-fold in salt-adapted and 2.4-fold in salt-shocked cultures when compared to the unadapted culture. The biosynthetic pathway involved in the accumulation of glycerol was investigated under long-term salt adaptation and short-term salt shock. Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) was induced 1.4-fold in salt-shocked but not in salt-adapted cultures. An alternate enzymatic pathway involving glycerol dehydrogenase (NADP(+)-dependent) utilizing dihydroxyacetone (DHA) and/or DL-glyceraldehyde (DL-GAD) was induced by NaCl. DHA-dependent glycerol dehydrogenase activity was induced about 6.3-fold in salt-adapted and 1.35-fold in salt-shocked cultures, while DL-GAD-dependent activity was induced about 6.1-fold in salt-adapted and 1.2-fold in salt-shocked cultures. However, the level of glycerol dehydrogenase activity with DL-GAD as substrate was 7% of the DHA-dependent activity. We conclude that a salt-inducible NADP(+)-dependent glycerol dehydrogenase activity electrophoretically indistinguishable from previously described glycerol dehydrogenase I results in glycerol accumulation in salt-stressed A. nidulans.