ABSTRACT Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore-forming rod-shaped bacteria that have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and death in humans and other animal species. A collection of 174 C. botulinum strains was examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine the genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT/A to BoNT/G). Analysis of the16S rRNA gene sequences confirmed previous identifications of at least four distinct genomic backgrounds (groups I to IV), each of which has independently acquired one or more BoNT genes through horizontal gene transfer. AFLP analysis provided higher resolution and could be used to further subdivide the four groups into subgroups. Sequencing of the BoNT genes from multiple strains of serotypes A, B, and E confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven toxin genes of the serotypes were compared and showed various degrees of interrelatedness and recombination, as was previously noted for the nontoxic nonhemagglutinin gene, which is linked to the BoNT gene. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for the treatment of botulism.

ABSTRACT Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.

ABSTRACT A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensis and Burkholderia cepacia.

ABSTRACT 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.

ABSTRACT The complete sequencing and annotation of the 181.7-kb Bacillus anthracis virulence plasmid pXO1 predicted 143 genes but could only assign putative functions to 45. Hybridization assays, PCR amplification, and DNA sequencing were used to determine whether pXO1 open reading frame (ORF) sequences were present in other bacilli and more distantly related bacterial genera. Eighteen Bacillus species isolates and four other bacterial species were tested for the presence of 106 pXO1 ORFs. Three ORFs were conserved in most of the bacteria tested. Many of the pXO1 ORFs were detected in closely related Bacillus species, and some were detected only in B. anthracis isolates. Three isolates, Bacillus cereus D-17, B. cereus 43881, and Bacillus thuringiensis 33679, contained sequences that were similar to more than one-half of the pXO1 ORF sequences examined. The majority of the DNA fragments that were amplified by PCR from these organisms had DNA sequences between 80 and 98% similar to that of pXO1. Pulsed-field gel electrophoresis revealed large potential plasmids present in both B. cereus 43881 (341 kb) and B. thuringiensis ATCC 33679 (327 kb) that hybridized with a DNA probe composed of six pXO1 ORFs.

A very fast and ultrasensitive method has been developed for the detection and quantitation of specific nucleic and sequences of bacterial origin in solution. The method is based on a two-color, single fluorescent molecule detection technique developed in our laboratory. The technique was applied to the detection of Bacillus anthracis DNA in solution.

The Bacillus anthracis Sterne plasmid pXO1 was sequenced by random, "shotgun" cloning. A circular sequence of 181,654 bp was generated. One hundred forty-three open reading frames (ORFs) were predicted using GeneMark and GeneMark.hmm, comprising only 61% (110,817 bp) of the pXO1 DNA sequence. The overall guanine-plus-cytosine content of the plasmid is 32.5%. The most recognizable feature of the plasmid is a "pathogenicity island," defined by a 44.8-kb region that is bordered by inverted IS1627 elements at each end. This region contains the three toxin genes (cya, lef, and pagA), regulatory elements controlling the toxin genes, three germination response genes, and 19 additional ORFs. Nearly 70% of the ORFs on pXO1 do not have significant similarity to sequences available in open databases. Absent from the pXO1 sequence are homologs to genes that are typically required to drive theta replication and to maintain stability of large plasmids in Bacillus spp. Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species. Among the remaining ORFs, there are three sequences that may encode enzymes responsible for the synthesis of a polysaccharide capsule usually associated with serotype-specific virulent streptococci.