ORF 2 from the Bacillus cereus linear plasmid pBClin15 encodes a DNA binding protein.

AIMS: To isolate and identify DNA-binding protein(s) with affinity for the mobile chromosomal repeat element bcr1 in Bacillus cereus group bacteria. METHODS AND RESULTS: A biotinylated bcr1 element was immobilized to streptavidin-coated magnetic beads and used to pull out a 20 kDa DNA-binding protein from a whole cell protein extract of B. cereus ATCC 14579. The protein was identified as the product of ORF 2 encoded by the bacteriophage-related autonomously replicating linear genetic element pBClin15 carried by the strain. DNA binding was not bcr1-specific. By Northern blotting ORF 2 was co-transcribed with ORF 1, and also in certain instances with ORF 3 by transcriptional readthrough of the terminator located between ORF 2 and ORF 3. CONCLUSIONS: ORF 2 from pBClin15 encodes a DNA-binding protein. ORF 2 is co-transcribed with its upstream gene ORF 1, and in a subset of the transcripts also with the downstream gene ORF 3 through alternative transcription termination. SIGNIFICANCE AND IMPACT OF THE STUDY: The B. cereus group contains bacterial species of medical and economic importance. Bacteriophages or phage-encoded proteins from these bacteria have been suggested as potential therapeutic agents. Understanding the biology of bacteriophage-related genetic elements through functional characterization of their genes is of high relevance.

The Bacillus cereus group: novel aspects of population structure and genome dynamics.

AIMS: To provide new insights into the population and genomic structure of the Bacillus cereus group of bacteria. METHODS AND RESULTS: The genetic relatedness among B. cereus group strains was assessed by multilocus sequence typing (MLST) using an optimized scheme based on seven chromosomal housekeeping genes. A set of 48 strains from different clinical sources was included, and six clonal complexes containing several genetically similar isolates from unrelated patients were identified. Interestingly, several clonal groups contained strains that were isolated from similar human sources. Furthermore, comparative whole genome sequence analysis of 16 strains led to the discovery of novel ubiquitous genome features of the B. cereus group, such as atypical group II introns, IStrons, and hitherto uncharacterized repeated elements. CONCLUSIONS: The B. cereus group constitutes a coherent population unified by the presence of ubiquitous and specific genetic elements which do not show any pattern, either in their sequences or genomic locations, which allows to differentiate between the member species of the group. Nevertheless, the population is very dynamic, as particular lineages of clinical origin can evolve to form clonal complexes. At the genome level, the dynamic behaviour is indicated by the presence of numerous mobile and repeated elements. SIGNIFICANCE AND IMPACT OF THE STUDY: The B. cereus group of bacteria comprises species that are of medical and economic importance. The MLST data, along with the primers and protocols used, will be available in a public, web-accessible database (http://mlstoslo.uio.no).

Characterization of a broad range antimicrobial substance from Bacillus cereus.

AIMS: The aim of this research was to isolate and characterize an antimicrobial substance from the Bacillus cereus type strain ATCC 14579. METHODS AND RESULTS: A substance with antimicrobial activity was isolated from B. cereus ATCC 14579. The substance was produced during late exponential growth and well into the stationary phase with a maximum 9 h after inoculation. The inhibitory substance was purified by reverse-phase HPLC and shown to be highly active against closely related Bacillus spp. Clinically relevant species such as Staphylococcus aureus and Micrococcus luteus were also inhibited. The substance was characterized as a bacteriocin-like inhibitory substance (BLIS) with a molecular mass of ca 3.4 kDa. The BLIS was very heat stable, and sensitive only to pronase E and proteinase K. Antimicrobial activity was stable and high in the pH range of 2.0-9.0, and relatively unaffected by organic chemicals. CONCLUSIONS: An antimicrobial substance produced by the B. cereus type strain ATCC 14579 was characterized, with a wide spectrum of activity and the potential to be applied as a control agent against pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first report of a substance with antimicrobial activity from the B. cereus type strain.

Bacillus cereus cellulitis from contaminated heroin.

Concern exists over recent unexplained deaths among intravenous drug users. This report describes a patient with crepitant cellulitis who was admitted complaining of severe pain in the right forearm. Ultrasonography demonstrated gas in the tissues and he was referred for early surgical debridement of the arm. He was treated with intravenous benzyl penicillin, gentamicin and metronidazole and made a full recovery. Aspirate samples grew Bacillus cereus, morphologically similar to the isolate obtained from a sample of the patient's own heroin. Antibiogram and API 50CHB profiles were also similar. Further typing included 'H' flagellar serotyping, which found both blood and heroin strains to be non-typable, and amplified fragment polymorphism analysis, which showed that the strains were indistinguishable. Genotyping of two selected genes from B. cereus confirmed almost certain identity between the two strains. This case illustrates the potential virulence of B. cereus when inoculated into tissues, and to our knowledge, is the first report to demonstrate a conclusive microbiological link between contaminated heroin and serious sepsis in a drug user due to B. cereus.

Localization of putative virulence genes on a physical map of the bacillus thuringiensis subsp. gelechiae chromosome

The insect pathogen Bacillus thuringiensis (Bt) has earlier been shown to possess virulence factors in addition to the crystal toxins. Bt subsp. gelechiae strain Bt13 lacks crystals but is still virulent to lepidopteran insects. Among the virulence co-expressed genes are two phospholipases; phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-degrading phospholipase C (PC-PLC), flagellin, and beta-lactamase I. In addition to these putative virulence factors the toxic neutral metalloprotease immune inhibitor A (InA) has been identified. In this paper we report a circular 5.9 Mb combined physical and genetic map of the of the Bt subsp. gelechiae chromosome. The genes encoding PI-PLC, PC-PLC, InA, flagellin, and beta-lactamase I are shown to be scattered over the chromosome. The PLC-encoding genes have been cloned from Bt13, and DNA sequencing showed that the Bt subsp. gelechiae PLC genes are >90% identical to their previously cloned equivalents from Bt or B. cereus. An HD-1 crystal toxin (cryIA) gene probe was found to hybridize to the Bt13 chromosome, but not to extrachromosomal elements.