Trace detection of meglumine and diatrizoate from Bacillus spore samples using liquid chromatography/mass spectrometry.

Following the September 11, 2001 terrorist attacks, letters containing Bacillus anthracis were distributed through the United States postal system killing five people. A complex forensic investigation commenced to identify the perpetrator of these mailings. A novel liquid chromatography/mass spectrometry protocol for the qualitative detection of trace levels of meglumine and diatrizoate in dried spore preparations of B. anthracis was developed. Meglumine and diatrizoate are components of radiographic imaging products that have been used to purify bacterial spores. Two separate chromatographic assays using multiple mass spectrometric analyses were developed for the detection of meglumine and diatrizoate. The assays achieved limits of detection for meglumine and diatrizoate of 1.00 and 10.0 ng/mL, respectively. Bacillus cereus T strain spores were effectively used as a surrogate for B. anthracis spores during method development and validation. This protocol was successfully applied to limited evidentiary B. anthracis spore material, providing probative information to the investigators.

An in vivo assay for conjugation-mediated recombination yields novel results for Streptomyces plasmid pIJ101.

Efficient transmission of circular plasmids in Streptomyces spp. proceeds by an uncharacterized mechanism that requires a cis-acting locus of transfer (clt) and often only a single plasmid-encoded protein. For circular plasmids from other bacteria, site- and strand-specific nicking takes place at the cis-acting oriT locus via the plasmid-encoded relaxase protein prior to single-strand transfer. Using an assay originally designed to demonstrate that conjugative transfer of plasmids containing tandem oriT loci results in the formation of a single composite oriT locus, we show here that an analogous construct involving the pIJ101 clt locus apparently does not undergo such a conjugation-mediated event during plasmid transfer. Our results, which imply that streptomycete plasmids are transferred by a functionally distinct mechanism compared to oriT-containing plasmids, are complementary to other recent evidences that support a novel double-stranded model for streptomycete circular plasmid transfer.

Common and distinguishing regulatory and expression characteristics of the highly related KorB proteins of streptomycete plasmids pIJ101 and pSB24.2.

The conjugative plasmid pIJ101 of the spore-forming bacterium Streptomyces lividans contains a regulatory gene, korB, whose product is required to repress potentially lethal expression of the pIJ101 kilB gene. The KorB protein also autoregulates korB gene expression and may be involved in control of pIJ101 copy number. KorB (pIJ101) is expressed as a 10-kDa protein in S. lividans that is immediately processed to a mature 6-kDa repressor molecule. The conjugative Streptomyces cyanogenus plasmid pSB24.1 is deleted upon entry into S. lividans to form pSB24.2, a nonconjugative derivative that contains a korB gene nearly identical to that of pIJ101. Previous evidence that korB of pSB24.2 is capable of overriding pIJ101 kilB-associated lethality supported the notion that pIJ101 and pSB24.2 encode highly related, perhaps even identical conjugation systems. Here we show that KorB (pIJ101) and KorB (pSB24.2) repress transcription from the pIJ101 kilB promoter equally well, although differences exist with respect to their interactions with kilB promoter sequences. Despite high sequence and functional similarities, KorB (pSB24.2) was found to exist as multiple stable forms ranging in size from 10 to 6 kDa both in S. lividans and S. cyanogenus. Immediate processing of KorB (pIJ101) exclusively to the 6-kDa repressor form meanwhile was conserved between the two species. A feature common to both proteins was a marked increase in expression or accumulation upon sporulation, an occurrence that may indicate a particular need for increased quantities of this regulatory protein upon spore germination and resumption of active growth of plasmid-containing cells.